drugresistanttuberculosis 100227020029 phpapp01
DESCRIPTION
MDR TB –Great Human Concern Covering drug developement, dots, drugs used in treatmentTRANSCRIPT
Drug ResistantDrug Resistant TuberculosisTuberculosis
A Great Human ConcernA Great Human Concern
DrTVRao MDDrTVRao MD
MDR TB ndashGreat Human ConcernMDR TB ndashGreat Human Concern
HISTORY ofHISTORY ofTuberculosisTuberculosis
Tuberculosis Is an Tuberculosis Is an Ancient Disease Ancient Disease Identified as Spinal Identified as Spinal Tuberculosis in Tuberculosis in Egyptian Mummies Egyptian Mummies History dates to History dates to 1550 ndash 1080 BC 1550 ndash 1080 BC Identified Identified by PCRby PCR
A Tribute to Robert Koch A Tribute to Robert Koch Discoverer of MycobacteriumDiscoverer of Mycobacterium
TuberculosisTuberculosis
Global StatusGlobal Status
Nine million people suffer from Nine million people suffer from tuberculosis tuberculosis
Two million people die each Two million people die each yearyear
Tuberculosis accounts for one-Tuberculosis accounts for one-third of third of AIDSAIDS deaths world deaths world wide every yearwide every year
USAIDUSAID Report on Tuberculosis Report on Tuberculosis in Indiain India
India has more new tuberculosis (TB) cases India has more new tuberculosis (TB) cases annually than any other country ranking first annually than any other country ranking first among the 22 high-burden TB countries among the 22 high-burden TB countries worldwide according to the World Health worldwide according to the World Health Organizationrsquos (WHOrsquos) Global TB Report 2009 Organizationrsquos (WHOrsquos) Global TB Report 2009 TB remains one of the leading infectious causes TB remains one of the leading infectious causes of mortality in India causing more than 331000 of mortality in India causing more than 331000 deaths in 2007 There were approximately 196 deaths in 2007 There were approximately 196 million new TB cases in India in 2007 million new TB cases in India in 2007 representing more than 21 percent of all TB representing more than 21 percent of all TB cases worldwide cases worldwide
Nobody is absolutely Immune to Nobody is absolutely Immune to TuberculosisTuberculosis
1908-1920 (Calmette and Guerin)1908-1920 (Calmette and Guerin) Vaccine (BCG)Vaccine (BCG)
Attenuated strain Mycobacterium Bovis Attenuated strain Mycobacterium Bovis
19431943 Streptomycin developedStreptomycin developed
20th November 1944 20th November 1944 Critically ill TB patient injected dramatically Critically ill TB patient injected dramatically
recoveredrecovered
Pharmacological discoveriesPharmacological discoveries
Selman Abraham WaksmanSelman Abraham Waksman Nobel Nobel Prize for his discovery in 1952Prize for his discovery in 1952
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MDR TB ndashGreat Human ConcernMDR TB ndashGreat Human Concern
HISTORY ofHISTORY ofTuberculosisTuberculosis
Tuberculosis Is an Tuberculosis Is an Ancient Disease Ancient Disease Identified as Spinal Identified as Spinal Tuberculosis in Tuberculosis in Egyptian Mummies Egyptian Mummies History dates to History dates to 1550 ndash 1080 BC 1550 ndash 1080 BC Identified Identified by PCRby PCR
A Tribute to Robert Koch A Tribute to Robert Koch Discoverer of MycobacteriumDiscoverer of Mycobacterium
TuberculosisTuberculosis
Global StatusGlobal Status
Nine million people suffer from Nine million people suffer from tuberculosis tuberculosis
Two million people die each Two million people die each yearyear
Tuberculosis accounts for one-Tuberculosis accounts for one-third of third of AIDSAIDS deaths world deaths world wide every yearwide every year
USAIDUSAID Report on Tuberculosis Report on Tuberculosis in Indiain India
India has more new tuberculosis (TB) cases India has more new tuberculosis (TB) cases annually than any other country ranking first annually than any other country ranking first among the 22 high-burden TB countries among the 22 high-burden TB countries worldwide according to the World Health worldwide according to the World Health Organizationrsquos (WHOrsquos) Global TB Report 2009 Organizationrsquos (WHOrsquos) Global TB Report 2009 TB remains one of the leading infectious causes TB remains one of the leading infectious causes of mortality in India causing more than 331000 of mortality in India causing more than 331000 deaths in 2007 There were approximately 196 deaths in 2007 There were approximately 196 million new TB cases in India in 2007 million new TB cases in India in 2007 representing more than 21 percent of all TB representing more than 21 percent of all TB cases worldwide cases worldwide
Nobody is absolutely Immune to Nobody is absolutely Immune to TuberculosisTuberculosis
1908-1920 (Calmette and Guerin)1908-1920 (Calmette and Guerin) Vaccine (BCG)Vaccine (BCG)
Attenuated strain Mycobacterium Bovis Attenuated strain Mycobacterium Bovis
19431943 Streptomycin developedStreptomycin developed
20th November 1944 20th November 1944 Critically ill TB patient injected dramatically Critically ill TB patient injected dramatically
recoveredrecovered
Pharmacological discoveriesPharmacological discoveries
Selman Abraham WaksmanSelman Abraham Waksman Nobel Nobel Prize for his discovery in 1952Prize for his discovery in 1952
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
HISTORY ofHISTORY ofTuberculosisTuberculosis
Tuberculosis Is an Tuberculosis Is an Ancient Disease Ancient Disease Identified as Spinal Identified as Spinal Tuberculosis in Tuberculosis in Egyptian Mummies Egyptian Mummies History dates to History dates to 1550 ndash 1080 BC 1550 ndash 1080 BC Identified Identified by PCRby PCR
A Tribute to Robert Koch A Tribute to Robert Koch Discoverer of MycobacteriumDiscoverer of Mycobacterium
TuberculosisTuberculosis
Global StatusGlobal Status
Nine million people suffer from Nine million people suffer from tuberculosis tuberculosis
Two million people die each Two million people die each yearyear
Tuberculosis accounts for one-Tuberculosis accounts for one-third of third of AIDSAIDS deaths world deaths world wide every yearwide every year
USAIDUSAID Report on Tuberculosis Report on Tuberculosis in Indiain India
India has more new tuberculosis (TB) cases India has more new tuberculosis (TB) cases annually than any other country ranking first annually than any other country ranking first among the 22 high-burden TB countries among the 22 high-burden TB countries worldwide according to the World Health worldwide according to the World Health Organizationrsquos (WHOrsquos) Global TB Report 2009 Organizationrsquos (WHOrsquos) Global TB Report 2009 TB remains one of the leading infectious causes TB remains one of the leading infectious causes of mortality in India causing more than 331000 of mortality in India causing more than 331000 deaths in 2007 There were approximately 196 deaths in 2007 There were approximately 196 million new TB cases in India in 2007 million new TB cases in India in 2007 representing more than 21 percent of all TB representing more than 21 percent of all TB cases worldwide cases worldwide
Nobody is absolutely Immune to Nobody is absolutely Immune to TuberculosisTuberculosis
1908-1920 (Calmette and Guerin)1908-1920 (Calmette and Guerin) Vaccine (BCG)Vaccine (BCG)
Attenuated strain Mycobacterium Bovis Attenuated strain Mycobacterium Bovis
19431943 Streptomycin developedStreptomycin developed
20th November 1944 20th November 1944 Critically ill TB patient injected dramatically Critically ill TB patient injected dramatically
recoveredrecovered
Pharmacological discoveriesPharmacological discoveries
Selman Abraham WaksmanSelman Abraham Waksman Nobel Nobel Prize for his discovery in 1952Prize for his discovery in 1952
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
A Tribute to Robert Koch A Tribute to Robert Koch Discoverer of MycobacteriumDiscoverer of Mycobacterium
TuberculosisTuberculosis
Global StatusGlobal Status
Nine million people suffer from Nine million people suffer from tuberculosis tuberculosis
Two million people die each Two million people die each yearyear
Tuberculosis accounts for one-Tuberculosis accounts for one-third of third of AIDSAIDS deaths world deaths world wide every yearwide every year
USAIDUSAID Report on Tuberculosis Report on Tuberculosis in Indiain India
India has more new tuberculosis (TB) cases India has more new tuberculosis (TB) cases annually than any other country ranking first annually than any other country ranking first among the 22 high-burden TB countries among the 22 high-burden TB countries worldwide according to the World Health worldwide according to the World Health Organizationrsquos (WHOrsquos) Global TB Report 2009 Organizationrsquos (WHOrsquos) Global TB Report 2009 TB remains one of the leading infectious causes TB remains one of the leading infectious causes of mortality in India causing more than 331000 of mortality in India causing more than 331000 deaths in 2007 There were approximately 196 deaths in 2007 There were approximately 196 million new TB cases in India in 2007 million new TB cases in India in 2007 representing more than 21 percent of all TB representing more than 21 percent of all TB cases worldwide cases worldwide
Nobody is absolutely Immune to Nobody is absolutely Immune to TuberculosisTuberculosis
1908-1920 (Calmette and Guerin)1908-1920 (Calmette and Guerin) Vaccine (BCG)Vaccine (BCG)
Attenuated strain Mycobacterium Bovis Attenuated strain Mycobacterium Bovis
19431943 Streptomycin developedStreptomycin developed
20th November 1944 20th November 1944 Critically ill TB patient injected dramatically Critically ill TB patient injected dramatically
recoveredrecovered
Pharmacological discoveriesPharmacological discoveries
Selman Abraham WaksmanSelman Abraham Waksman Nobel Nobel Prize for his discovery in 1952Prize for his discovery in 1952
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Global StatusGlobal Status
Nine million people suffer from Nine million people suffer from tuberculosis tuberculosis
Two million people die each Two million people die each yearyear
Tuberculosis accounts for one-Tuberculosis accounts for one-third of third of AIDSAIDS deaths world deaths world wide every yearwide every year
USAIDUSAID Report on Tuberculosis Report on Tuberculosis in Indiain India
India has more new tuberculosis (TB) cases India has more new tuberculosis (TB) cases annually than any other country ranking first annually than any other country ranking first among the 22 high-burden TB countries among the 22 high-burden TB countries worldwide according to the World Health worldwide according to the World Health Organizationrsquos (WHOrsquos) Global TB Report 2009 Organizationrsquos (WHOrsquos) Global TB Report 2009 TB remains one of the leading infectious causes TB remains one of the leading infectious causes of mortality in India causing more than 331000 of mortality in India causing more than 331000 deaths in 2007 There were approximately 196 deaths in 2007 There were approximately 196 million new TB cases in India in 2007 million new TB cases in India in 2007 representing more than 21 percent of all TB representing more than 21 percent of all TB cases worldwide cases worldwide
Nobody is absolutely Immune to Nobody is absolutely Immune to TuberculosisTuberculosis
1908-1920 (Calmette and Guerin)1908-1920 (Calmette and Guerin) Vaccine (BCG)Vaccine (BCG)
Attenuated strain Mycobacterium Bovis Attenuated strain Mycobacterium Bovis
19431943 Streptomycin developedStreptomycin developed
20th November 1944 20th November 1944 Critically ill TB patient injected dramatically Critically ill TB patient injected dramatically
recoveredrecovered
Pharmacological discoveriesPharmacological discoveries
Selman Abraham WaksmanSelman Abraham Waksman Nobel Nobel Prize for his discovery in 1952Prize for his discovery in 1952
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
USAIDUSAID Report on Tuberculosis Report on Tuberculosis in Indiain India
India has more new tuberculosis (TB) cases India has more new tuberculosis (TB) cases annually than any other country ranking first annually than any other country ranking first among the 22 high-burden TB countries among the 22 high-burden TB countries worldwide according to the World Health worldwide according to the World Health Organizationrsquos (WHOrsquos) Global TB Report 2009 Organizationrsquos (WHOrsquos) Global TB Report 2009 TB remains one of the leading infectious causes TB remains one of the leading infectious causes of mortality in India causing more than 331000 of mortality in India causing more than 331000 deaths in 2007 There were approximately 196 deaths in 2007 There were approximately 196 million new TB cases in India in 2007 million new TB cases in India in 2007 representing more than 21 percent of all TB representing more than 21 percent of all TB cases worldwide cases worldwide
Nobody is absolutely Immune to Nobody is absolutely Immune to TuberculosisTuberculosis
1908-1920 (Calmette and Guerin)1908-1920 (Calmette and Guerin) Vaccine (BCG)Vaccine (BCG)
Attenuated strain Mycobacterium Bovis Attenuated strain Mycobacterium Bovis
19431943 Streptomycin developedStreptomycin developed
20th November 1944 20th November 1944 Critically ill TB patient injected dramatically Critically ill TB patient injected dramatically
recoveredrecovered
Pharmacological discoveriesPharmacological discoveries
Selman Abraham WaksmanSelman Abraham Waksman Nobel Nobel Prize for his discovery in 1952Prize for his discovery in 1952
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Nobody is absolutely Immune to Nobody is absolutely Immune to TuberculosisTuberculosis
1908-1920 (Calmette and Guerin)1908-1920 (Calmette and Guerin) Vaccine (BCG)Vaccine (BCG)
Attenuated strain Mycobacterium Bovis Attenuated strain Mycobacterium Bovis
19431943 Streptomycin developedStreptomycin developed
20th November 1944 20th November 1944 Critically ill TB patient injected dramatically Critically ill TB patient injected dramatically
recoveredrecovered
Pharmacological discoveriesPharmacological discoveries
Selman Abraham WaksmanSelman Abraham Waksman Nobel Nobel Prize for his discovery in 1952Prize for his discovery in 1952
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
1908-1920 (Calmette and Guerin)1908-1920 (Calmette and Guerin) Vaccine (BCG)Vaccine (BCG)
Attenuated strain Mycobacterium Bovis Attenuated strain Mycobacterium Bovis
19431943 Streptomycin developedStreptomycin developed
20th November 1944 20th November 1944 Critically ill TB patient injected dramatically Critically ill TB patient injected dramatically
recoveredrecovered
Pharmacological discoveriesPharmacological discoveries
Selman Abraham WaksmanSelman Abraham Waksman Nobel Nobel Prize for his discovery in 1952Prize for his discovery in 1952
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Selman Abraham WaksmanSelman Abraham Waksman Nobel Nobel Prize for his discovery in 1952Prize for his discovery in 1952
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
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ow
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esig
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ply
the e
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ssion o
f any o
pin
ion
whatso
ever o
n th
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f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
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et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Pharmacological discoveriesPharmacological discoveries
1956-1960 1956-1960 Combination therapy of INH and PZA cures TBCombination therapy of INH and PZA cures TB
1955 Cycloserine1955 Cycloserine 1962 Ethambutol 1962 Ethambutol 1963 Rifampicin1963 Rifampicin 1970-19771970-1977
Combination of Rifampicin and Combination of Rifampicin and IIsoniazid adopted as soniazid adopted as International regime for treatment of TBInternational regime for treatment of TB
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
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or a
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nce
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f its frontie
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there
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WH
O 2
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ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
IntroductionIntroduction
Tuberculosis is an ancient disease amp it Tuberculosis is an ancient disease amp it remains the leading cause of death of remains the leading cause of death of human being human being
It is mainly caused by It is mainly caused by Mycobacterium Mycobacterium tuberculosistuberculosis
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Typical tubercle bacilliTypical tubercle bacilli
Human type Human type MtuberculosisMtuberculosis
Bovine type Bovine type MbovisMbovis
Vole type Vole type MmicrotiMmicroti
Human type Human type MafricanumMafricanum
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Multi Drug Resistant Multi Drug Resistant TuberculosisTuberculosis
MDR-TBMDR-TB
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
DefinitionDefinition
MDR-TB caused by strains of MDR-TB caused by strains of Mycobacterium Tuberculosis resistant Mycobacterium Tuberculosis resistant both both Rifampicin and IsoniazidRifampicin and Isoniazid with or with or without resistance to other drugswithout resistance to other drugs
Single Isoniazid or Rifampicin Single Isoniazid or Rifampicin resistance is not resistance is not MDR - TBMDR - TB
MDR TB is a laboratory diagnosisMDR TB is a laboratory diagnosis
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MDR-TB amp XDR-TBMDR-TB amp XDR-TBTHE 2008 REPORTTHE 2008 REPORT
of MDR-TB among new TB cases 1994-2007 of MDR-TB among new TB cases 1994-2007
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Classification of DrugsClassification of Drugs 3 Groups depending upon the degree of 3 Groups depending upon the degree of
effectiveness and potential side effectseffectiveness and potential side effects First Line (Primary agents)First Line (Primary agents)
are the most effective and have lowest toxicity are the most effective and have lowest toxicity IsoniazidIsoniazid RifampinRifampin
Second LineSecond Line Less effective and more toxic effectsLess effective and more toxic effects include (in no particular order) p-amino include (in no particular order) p-amino
salicylic acid Streptomycin Ethambutolsalicylic acid Streptomycin Ethambutol Third LineThird Line
are least effective and most toxicare least effective and most toxic Amikacin Amikacin Kanamycin Capreomycin Viomycin Kanamycin Capreomycin Viomycin Kanamycin CycloserineKanamycin Cycloserine
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Several Drugs becoming Several Drugs becoming resistantresistant
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Basic concepts ndash Keep factsBasic concepts ndash Keep facts
Primary (Initial) resistancePrimary (Initial) resistance TB patientrsquos initial TB patientrsquos initial Mycobacterium Mycobacterium
tuberculosis tuberculosis population resistant population resistant to drugsto drugs
Secondary (Acquired) resistanceSecondary (Acquired) resistance Drug-resistant Drug-resistant M tuberculosis M tuberculosis in initial in initial
population selected by inappropriate drug use population selected by inappropriate drug use (inadequate treatment or non-adherence(inadequate treatment or non-adherence))
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
When to suspect MDR TBWhen to suspect MDR TB
Re-treatment patients whorsquos sputum Re-treatment patients whorsquos sputum smear remains positive after three monthsrsquo smear remains positive after three monthsrsquo of intensive therapyof intensive therapy
Treatment failure and interruption casesTreatment failure and interruption cases Close contacts of MDR tuberculosis casesClose contacts of MDR tuberculosis cases Positive diagnoses with Positive diagnoses with
TB culture and susceptibility testingTB culture and susceptibility testing
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
What is extensively drug What is extensively drug resistant tuberculosis (XDR TB)resistant tuberculosis (XDR TB)
Extensively drug resistant TB (XDR TB) is Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB XDR TB a relatively rare type of MDR TB XDR TB is defined as TB which is resistant to is defined as TB which is resistant to isoniazid and rifampin plus resistant to isoniazid and rifampin plus resistant to any any fluoroquinolonefluoroquinolone and at least one of and at least one of three injectable second-line drugs three injectable second-line drugs (ie (ie amikacin kanamycin or capreomycin)amikacin kanamycin or capreomycin)
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Why XDR - TB a grave concernWhy XDR - TB a grave concern
Because XDR TB is resistant to first-line and Because XDR TB is resistant to first-line and secondline drugs patients are left with treatment secondline drugs patients are left with treatment options that are much less effective options that are much less effective
XDR TB is of special concern for persons with XDR TB is of special concern for persons with HIV infection or other conditions that can HIV infection or other conditions that can weaken the immune system These persons are weaken the immune system These persons are more likely to develop TB disease once they are more likely to develop TB disease once they are infected and also have a higher risk of death infected and also have a higher risk of death once they develop TBonce they develop TB
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Global Estimates
ClassificatClassificationion
Estimated Number Estimated Number of Casesof Cases
Estimated Estimated Number of Number of
DeathsDeaths
All forms All forms TBTB
88 million88 million 16 million16 million
MDR TBMDR TB 424000424000 116000116000
XDR TBXDR TB 2700027000 1600016000
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Extensively Drug-Resistant Extensively Drug-Resistant Mycobacterium tuberculosisMycobacterium tuberculosis India India
The first XDR TB cases in India The first XDR TB cases in India and the emergence of XDR TB is and the emergence of XDR TB is reported by reported by Rajesh Mondal and Rajesh Mondal and Amita JainAmita Jain King Georges Medical King Georges Medical University Lucknow IndiaUniversity Lucknow India Volume 13 Volume 13 Number 9ndashSeptember 2007 in Number 9ndashSeptember 2007 in Emerging Infectious Emerging Infectious DiseasesDiseases
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Global incidence of tuberculosis Still rising as a result of the growing epidemic in
Africa
0
100
200
300
400
500
600
1990 1995 2000 2005 2010 2015
Inci
den
ce p
er 1
000
00 p
er y
ear
World
Cent Euro
East Europe
Est Market
East Medit
Lat America
West Pacific
Sth East Asia
AFR high HIV
AFR low HIV
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Drug susceptible TBsect
MDR-TB
1990sect
XDR-TB
2006sect
Total DR
Resistance to HampR ndash
Treatable with 2nd line drugs
Resistance to 2nd line drugs ndashTreatment options seriously restricted
Resistance to all available drugs ndash
No treatment options
or limited resistance manageable with 4 drug regimen - DOTS
Are we Returning to a Pre-Are we Returning to a Pre-antibiotic Eraantibiotic Era
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
WHO Surveillance and Incidence of WHO Surveillance and Incidence of MDR TBMDR TB
CountryCountry MDR TB of all new cases MDR TB of all new cases
EstoniaEstonia 141141
LatviaLatvia 9090
China (non-DOTS)China (non-DOTS) 7777
China (DOTS)China (DOTS) 2828
RussiaRussia 6060
IndiaIndia 3434
IranIran 5858
DominicanDominican 6666
Ivory CostIvory Cost 5353
Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002Dye et al Global Burden of Multidrug-Resistant TB JID 185(8) 2002
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Genesis of MDR TBGenesis of MDR TB
Resistance is a Resistance is a man-made amplificationman-made amplification of a of a natural phenomenonnatural phenomenon
Inadequate drug delivery is main cause of Inadequate drug delivery is main cause of secondary drug resistancesecondary drug resistance
Secondary drug resistance is the main cause Secondary drug resistance is the main cause of primary drug resistance due to transmission of primary drug resistance due to transmission of resistant strainsof resistant strains
MDR due to spontaneous mutations is not possible MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are as the genes encoding resistance for anti TB are unlinkedunlinked
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Strains with genetic drug resistance
Wild M TB strain
Acquired drug resistance
Primary drug resistance
Spontaneous mutationSpontaneous mutation
Selection inadequate treatmentSelection inadequate treatment
TransmissionTransmission
Development of anti-tuberculosis drug resistanceDevelopment of anti-tuberculosis drug resistance
Pablos-Mendez et al WHO 1997Pablos-Mendez et al WHO 1997
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Factors Contributing to Development Factors Contributing to Development
and Spread of MDR and XDR TBand Spread of MDR and XDR TB Weak TB programs (DOTS) Weak TB programs (DOTS)
Low completioncure ratesLow completioncure rates Lack of treatment follow up and Lack of treatment follow up and
patient support patient support Unreliable drug supply Unreliable drug supply Diagnostic delay Diagnostic delay
Absent or inadequate infection control Absent or inadequate infection control measuresmeasures
Uncontrolled use of 2Uncontrolled use of 2ndnd line drugs line drugs
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
INHINH Chromosomally mediatedChromosomally mediated Loss of catalaseperoxidaseLoss of catalaseperoxidase Mutation in mycolic acid synthesisMutation in mycolic acid synthesis Regulators of peroxide responseRegulators of peroxide response
Mechanism of resistanceMechanism of resistance
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
RifampinRifampin Reduced binding to RNA polymeraseReduced binding to RNA polymerase
Clusters of mutations at ldquoRifampin Resistance Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)Determining Regionrdquo (RRDR)
Reduced Cell wall permeabilityReduced Cell wall permeability
Mechanism of resistanceMechanism of resistance
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Gene location associated Gene location associated Drug-Resistant Mtuberculosis Drug-Resistant Mtuberculosis
DrugDrug Gene Gene Isoniazid Isoniazid Kat G Inh A Kas A Kat G Inh A Kas A RifampicinRifampicin rpo B rpo B EthambutolEthambutol emb B emb B StreptomycinStreptomycin rps L rps L PyrazinamidePyrazinamide pnc A pnc A FluoroquinolonesFluoroquinolones gyr A gyr A
Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens Dubaniewicz A et al Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis Int J Infect Dis20004129-33in pulmonary tuberculosis Int J Infect Dis20004129-33
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Drug Susceptibility Drug Susceptibility TestingTesting
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Susceptibility TestingSusceptibility Testing
1048708 1048708 Direct and indirect testingDirect and indirect testing 1048708 1048708 Primary Drugs testingPrimary Drugs testing 1048708 1048708 IsoniazidIsoniazid 1048708 1048708 RifampicinRifampicin 1048708 1048708 Ethambutol ()Ethambutol () 1048708 1048708 Pyrizinamide ()Pyrizinamide ()
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Drug susceptibility testing (DST)Drug susceptibility testing (DST)
DST is recommended for all new cases for all DST is recommended for all new cases for all first line drugs with specimens taken before first line drugs with specimens taken before initiating treatmentinitiating treatment
Accuracy Accuracy is more important than is more important than speedspeed DST results should come from a small number DST results should come from a small number
of well-equipped experienced laboratories who of well-equipped experienced laboratories who participate and perform well in an participate and perform well in an international international DSTDST quality control scheme quality control scheme
The WHO Supranational Laboratory Quality The WHO Supranational Laboratory Quality Control Network offers the greatest global Control Network offers the greatest global coverage for thiscoverage for this
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Drug susceptibility TestingDrug susceptibility Testing
Assessment of grwoth inhibition on solid Assessment of grwoth inhibition on solid media containing various dilutions of the media containing various dilutions of the drug in comparison with the test strainsdrug in comparison with the test strains
As the method depend observation of As the method depend observation of grwoth grwoth Results are not available until several Results are not available until several weeks after isolation of the organismweeks after isolation of the organism
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Other accredited MethodsOther accredited Methods
Radiometric and Non radiometric methodsRadiometric and Non radiometric methods Nucleicacid technology ndash effective upto Nucleicacid technology ndash effective upto
95 in mutations to rifampicin resistance 95 in mutations to rifampicin resistance to gene rpoB geneto gene rpoB gene
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Drug susceptibility testing Drug susceptibility testing (DST(DST))
As a minimum laboratories As a minimum laboratories supplying DST data should supplying DST data should correctly identify resistance to correctly identify resistance to isoniazid and rifampicin in over isoniazid and rifampicin in over 90 of quality control samples 90 of quality control samples in two out of the last three in two out of the last three quality control roundsquality control rounds
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Detection of Rifampicin Drug Detection of Rifampicin Drug susceptibility testing (DST) is more susceptibility testing (DST) is more
importantimportant Early identification of mycobacterial growth as Early identification of mycobacterial growth as
M tuberculosis M tuberculosis complex and the identification of complex and the identification of rifampicin resistance should be the rifampicin resistance should be the first priority first priority as rifampicin resistance invalidates standard as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TBuseful marker in most countries for MDR-TB
Laboratories should aim to identify isolates as Laboratories should aim to identify isolates as M tuberculosis M tuberculosis complex and perform rifampicin complex and perform rifampicin resistance in resistance in 9090 of isolates within 1-2 working of isolates within 1-2 working days This is technologically feasible days This is technologically feasible
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Drug susceptibility testingDrug susceptibility testing
For DST laboratories modern molecular For DST laboratories modern molecular techniques permit the successful techniques permit the successful identification of isoniazid resistance in at identification of isoniazid resistance in at least least 7575 of mycobacterial cultures within of mycobacterial cultures within 1-2 working days and are useful 1-2 working days and are useful preliminary screens for isoniazid preliminary screens for isoniazid resistance resistance
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Secondary Drugs testing[lack of Secondary Drugs testing[lack of standardized methods]standardized methods]
Ofloxacin quinolonesOfloxacin quinolones EthionamideEthionamide
KanamycinKanamycin CapreomycinCapreomycin Ensure quality control and quality Ensure quality control and quality
assurance assurance
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MODSMODS MMicroscopicicroscopic OObservation ofbservation of DDrug Susceptibility rug Susceptibility TTestingesting
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MODS affordable Technically MODS affordable Technically Feasible Feasible
MODS arose during experiments conducted by MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Luz Caviedes under the guidance of Professor Robert GilmanRobert Gilman at Universidad Peruana at Universidad Peruana Cayetano Heredia in Lima Peru in the late Cayetano Heredia in Lima Peru in the late 1990s in which a colorimetric test for TB growth 1990s in which a colorimetric test for TB growth was being investigated The observation that was being investigated The observation that microcolonies could be seen under the microcolonies could be seen under the microscope long before a colour change microscope long before a colour change occurred prompted the development of MODS occurred prompted the development of MODS
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Review Article in Indian Journal Review Article in Indian Journal of Medical Microbiologyof Medical Microbiology
Caviedes L Moore Caviedes L Moore DA Introducing DA Introducing mods A low-cost mods A low-cost low-tech tool for high-low-tech tool for high-performance performance detection of detection of tuberculosis and tuberculosis and multidrug resistant multidrug resistant tuberculosis Indian J tuberculosis Indian J Med Microbial Med Microbial 20072587-8 20072587-8
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Observation of Grwoth in liquid Observation of Grwoth in liquid MediaMedia
MODS depends upon three key principles MODS depends upon three key principles (which have been known for decades) (1) (which have been known for decades) (1) Mycobacterium tuberculosisMycobacterium tuberculosis grows faster grows faster in liquid (broth) than on solid media (2) in in liquid (broth) than on solid media (2) in liquid cultures liquid cultures M tuberculosisM tuberculosis grows in a grows in a visually characteristic manner (tangles visually characteristic manner (tangles cording) which can be observed under the cording) which can be observed under the microscope long before the naked eye microscope long before the naked eye could visualize colonies on solid agarcould visualize colonies on solid agar
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Least time required for detection Least time required for detection of MDRof MDR
Incorporation of anti-Incorporation of anti-TB drugs into broth TB drugs into broth cultures at the outset cultures at the outset enables direct enables direct susceptibility testing susceptibility testing from sputum samplesfrom sputum samples
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MODS more streamlinedMODS more streamlined
Recently completed operational field Recently completed operational field studies have served to refine and studies have served to refine and streamline the methodology further and streamline the methodology further and importantly validate MODS as a test for TB importantly validate MODS as a test for TB detection and MDRTB detection directly detection and MDRTB detection directly from sputum from sputum
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Inverted Microscope a minimal Inverted Microscope a minimal needneed
Characteristic ldquo Characteristic ldquo tangles ldquo of tangles ldquo of Mtuberculosis can Mtuberculosis can be visualised under be visualised under microscope long microscope long before colonies to before colonies to the naked eyethe naked eye
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MODS for detection of MODS for detection of MDR - TBMDR - TB
The scientific observations have proved The scientific observations have proved that a single MODS culture of sputum that a single MODS culture of sputum
sample offers more rapid and sensitive sample offers more rapid and sensitive detection of tuberculosis and Multidrug-detection of tuberculosis and Multidrug-resistant tuberculosis than the existing resistant tuberculosis than the existing
gold standard methods usedgold standard methods used
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Advantages of MODS methodology in MDR detection
bull All the chemical ingredients are available locally except few which can be acquired easily
bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
bull Risk to technician handling the specimens is minimal there is no absolute need to obtain grade III safety cabinets
bull Technology transfer is easier all the new technical manpower can be trained easily
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Performing MODS AssayPerforming MODS Assay
The MODS assay was performed as The MODS assay was performed as described in standard protocolsdescribed in standard protocols
Broth cultures were prepared in 24 well Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) tissue culture plates ( Becton Dickinson) each containing decontaminant 7H9 broth each containing decontaminant 7H9 broth (Becton Dickinson) oxalic acid albumin (Becton Dickinson) oxalic acid albumin dextrose and catlase (OADC) (Becton dextrose and catlase (OADC) (Becton Dickinson) and Dickinson) and Polymyxin Amphotericin B Polymyxin Amphotericin B Nalidixic acidtrimethoprim and azlocillinNalidixic acidtrimethoprim and azlocillin (PANTA) (PANTA)
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MODS Assay ( Contd)MODS Assay ( Contd)
For each sample 12 wells were usedFor each sample 12 wells were used Four in control wells no drug was used Four in control wells no drug was used
and each of the remaining 8 wells and each of the remaining 8 wells contained one of the four drugs at one of contained one of the four drugs at one of the two concentrations testedthe two concentrations tested
The cultures were examined under an inverted The cultures were examined under an inverted light microscope at magnification of 40x every light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15 on day ( except weekends ) from 4 to day 15 on alternative days from 16 today 25 and twice alternative days from 16 today 25 and twice weekly from 26 to 40day weekly from 26 to 40day
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Sample layout on MODS plate Sample layout on MODS plate (2 samples per plate)(2 samples per plate)
No plate contained 2 samples from the same No plate contained 2 samples from the same patientpatient
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Drug susceptibility TestingDrug susceptibility TestingIn MODSIn MODS
Drug susceptibility testing was performed Drug susceptibility testing was performed with the use of MODS assay with the use of MODS assay
Growth in the drug free control wells but Growth in the drug free control wells but not in drug containing wells indicates not in drug containing wells indicates susceptibilitysusceptibility
The drug concentration were as follows The drug concentration were as follows Isoniazid 01 and 04 microgmilliliterIsoniazid 01 and 04 microgmilliliter Rifampicin 1 and 2 microg per millilitreRifampicin 1 and 2 microg per millilitre
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Differentiation from Typical and Differentiation from Typical and Atypical MycobacteriumAtypical Mycobacterium
Nontuberculous Nontuberculous mycobacteria (NTM) mycobacteria (NTM) were recognized by were recognized by their lack of cording or their lack of cording or (in the case of (in the case of Mycobacterium Mycobacterium chelonae that uniquely chelonae that uniquely among NTM does among NTM does form cords) rapid form cords) rapid overgrowth of wells by overgrowth of wells by day 5day 5
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
In MODS growth is In MODS growth is identified by cording on identified by cording on
MicroscopyMicroscopy
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MODS assay ( Contd)MODS assay ( Contd) To minimize cross To minimize cross
contamination and contamination and occupational occupational exposure plates exposure plates were permanently were permanently sealed inside plastic sealed inside plastic zip lock bags after zip lock bags after inoculation and were inoculation and were subsequently subsequently examined with in the examined with in the bagbag
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Observation of growth in Observation of growth in MODSMODS
Positive cultures Positive cultures were identified by were identified by cord formation cord formation characteristic of characteristic of Mtuberculosis Mtuberculosis grwothin liquid grwothin liquid medium in drug medium in drug free control wellsfree control wells
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MODS in Atypical MODS in Atypical MycobacteriumMycobacterium
Non tuberculous Non tuberculous mycobacterium mycobacterium were recognised by were recognised by their lack of cording their lack of cording or for Mchelonae ( or for Mchelonae ( which forms cords) which forms cords) by rapid by rapid overgrwoth by day overgrwoth by day 55
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Contamination in MODS Contamination in MODS AssayAssay
Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wellsclouding in wells
If contamination was If contamination was detected the original detected the original samples was cultured samples was cultured again after being again after being decontaminated once decontaminated once moremore
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Honduras study comparing the Honduras study comparing the LJ mediumLJ medium
Per specimen there was concordance Per specimen there was concordance between MODS and LJ culture in 942 between MODS and LJ culture in 942 MODS tests were also less prone to MODS tests were also less prone to contamination than LJ cultures 62 [38] contamination than LJ cultures 62 [38] vs (95 [58] of 1639 samples vs (95 [58] of 1639 samples respectively (P le001) respectively (P le001)
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
PCR Molecular susceptibility PCR Molecular susceptibility testingtesting
RMP resistance
INH resistance
HainGenotypeMTBDR
INNO-LiPARifTBassay
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Confirming Confirming MODSMODS results results
Spacer Spacer Oligonucleotide typing Oligonucleotide typing SpoligotypingSpoligotyping polymerase chain polymerase chain reaction with multiple reaction with multiple primers or both were primers or both were applied to all isolates applied to all isolates from each of the three from each of the three types of cultures in types of cultures in order to confirm the order to confirm the presence of presence of MtuberculosisMtuberculosis
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MODS and MDR detection
bull The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR
bull In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
bull Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Why MODS is a better Why MODS is a better method for MDR TB method for MDR TB
detectiondetection If a MODS culture was negative on day If a MODS culture was negative on day
15there is 997 chance that the 15there is 997 chance that the sample is truly culture negativesample is truly culture negative
The negative MODS cultures can be The negative MODS cultures can be discarded after 3 weeksdiscarded after 3 weeks
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Biosafety concerns in MODS Biosafety concerns in MODS technologytechnology
Legitimate concerns about biosafety with Legitimate concerns about biosafety with other liquid culture systems do not really other liquid culture systems do not really apply to MODS indeed the converse is apply to MODS indeed the converse is the case After inoculation with the case After inoculation with decontaminated sample the MODS plates decontaminated sample the MODS plates are permanently sealed in ziplock are permanently sealed in ziplock polythene bags through which the polythene bags through which the microscopic examination is made thus microscopic examination is made thus spillage of the mycobacterial soup spillage of the mycobacterial soup cannot occur cannot occur
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Lower Grade Biosafety is Lower Grade Biosafety is adequateadequate
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
N 95 mask protects from BiohazardN 95 mask protects from Biohazard
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
No transfer of Materials needed No transfer of Materials needed in MODSin MODS
As no secondary sub-culture is needed As no secondary sub-culture is needed (because this is direct and not indirect (because this is direct and not indirect susceptibility testing) no further susceptibility testing) no further manipulation is required - this zero manipulation is required - this zero potential for aerosolisation or accident potential for aerosolisation or accident compares favourably with the hazard compares favourably with the hazard associated with preparation of a associated with preparation of a standardized inoculum for indirect DST standardized inoculum for indirect DST
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Computer pattern Computer pattern recognitionrecognition
of of Mycobacterium Mycobacterium tuberculosistuberculosis
in MODS culturein MODS culture
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Automation in MODSAutomation in MODS
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
M tuberculosis in MODS x10 objective (sputum sample inoculation)
Day 6
Day 16 Day 17
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
MODS can be used in Extra pulmonary MODS can be used in Extra pulmonary TuberculosisTuberculosis
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Draw backs of MODSDraw backs of MODS
One possible drawback however could be One possible drawback however could be the inability of the laboratory technicians to the inability of the laboratory technicians to distinguish between TB and some NTM distinguish between TB and some NTM This could potentially have clinical impact This could potentially have clinical impact in settings where NTM prevalence is high in settings where NTM prevalence is high and not all mycobacteria respond to anti-and not all mycobacteria respond to anti-TB treatment TB treatment
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Other WHO-Endorsed ToolsOther WHO-Endorsed Tools
Liquid culture (eg MGIT BacTALERT)Liquid culture (eg MGIT BacTALERT)
Capilia TBCapilia TB Rapid strip test that detects a TB-specific Rapid strip test that detects a TB-specific
antigen from cultureantigen from culture
Molecular line probe assays (eg Molecular line probe assays (eg GenoType MTBDRplus INNO-LiPA RifTB)GenoType MTBDRplus INNO-LiPA RifTB) Strip test for detection of TB and drug-Strip test for detection of TB and drug-
resistance conferring mutationsresistance conferring mutations
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
WHO Controls the Tuberculosis WHO Controls the Tuberculosis related workrelated work
The laboratory methods for anti-The laboratory methods for anti-tuberculosis drug susceptibility testing tuberculosis drug susceptibility testing should be selected from among those that should be selected from among those that are WHO-recommended and all are WHO-recommended and all laboratory processes should be quality-laboratory processes should be quality-assured in cooperation with a partner assured in cooperation with a partner Supranational Reference Laboratory Supranational Reference Laboratory (SRL)(SRL)
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
XDR-TB in South AfricaXDR-TB in South AfricaAugust 2006August 2006
53 of 544 patients defined as XDR-TB 53 of 544 patients defined as XDR-TB casescases
bull bull 52 of the 53 patients died on average 52 of the 53 patients died on average within 25within 25
days including those on antiretroviral days including those on antiretroviral therapytherapy
bull bull Further investigations being carried outFurther investigations being carried out bull bull XDR-TB likely in bordering African XDR-TB likely in bordering African
countriescountries
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Molecular FingerprintingMolecular Fingerprinting
26 of 30 (87) XDR TB isolates found to 26 of 30 (87) XDR TB isolates found to be genetically similarbe genetically similar
Majority of patients had no previous history Majority of patients had no previous history of TB treatment Suggestive of recent of TB treatment Suggestive of recent infection with drug-resistant straininfection with drug-resistant strain
Additional cases identified in 28 of the 68 Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natalhospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
CDC Updates Guidelines for Nucleic AcidCDC Updates Guidelines for Nucleic AcidAmplification Techniques to Diagnose Amplification Techniques to Diagnose
TuberculosisTuberculosis NAAT results should be interpreted in NAAT results should be interpreted in
conjunction with the AFB smear conjunction with the AFB smear resultsresults
NAAT and smear positive start Rx NAAT and smear positive start Rx despite pending culture results PPV despite pending culture results PPV 9595
Smear negative NAAT positive use Smear negative NAAT positive use clinical judgment to either treat or await clinical judgment to either treat or await cultureculture
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Selection from automated systems for Selection from automated systems for molecular andmolecular and
bacteriological rapid diagnosticsbacteriological rapid diagnostics
PCRPCR RocheCOBASreg Amplicorreg RocheCOBASreg Amplicorreg
amplification kitsamplification kits RocheCOBASreg LightCyclerreg (real-RocheCOBASreg LightCyclerreg (real-
time-PCR)time-PCR) RocheCOBASreg TaqMan 48regRocheCOBASreg TaqMan 48reg (increases the specificity of real-time-(increases the specificity of real-time-
PCR)PCR)
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Microscopy and Culturing still a Microscopy and Culturing still a top prioritytop priority
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Is PCR methods a solution Is PCR methods a solution
PCR cant yet PCR cant yet replace neither replace neither microscopy microscopy culturing and culturing and competent clinical competent clinical examinationexamination
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
No testing method replaces No testing method replaces clinical assessmentclinical assessment
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Extreme Drug resistant Extreme Drug resistant Tuberculosis (XDR-TB)Tuberculosis (XDR-TB)
Resistant to all first line drugs namely Resistant to all first line drugs namely Isoniazid Isoniazid and Rifampin and Rifampin and and
Three or more Three or more second line drugs (SLDrsquoS) that are second line drugs (SLDrsquoS) that are used to treat MDR-TBused to treat MDR-TB Thequinalones like OfloaxinThequinalones like Ofloaxin
OrOr Aminoglygocides like Capreomycin amp Kanamycin Aminoglygocides like Capreomycin amp Kanamycin
No third-line drugs available to treat XDR-TB No third-line drugs available to treat XDR-TB since none since none has been developed in the last 40 has been developed in the last 40 yearsyears
DrTVRao MDDrTVRao MD
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
XDR-TB 82306XDR-TB 82306
ldquo ldquoRapidly Fatal in South Africardquo Rapidly Fatal in South Africardquo Tugela Tugela Ferry KwaZulu-NatalFerry KwaZulu-Natal
10 isolates resistant to ALL 110 isolates resistant to ALL 1stst and and 22ndnd line agents line agents
5152 XDR dead in median 16 days 5152 XDR dead in median 16 days after first positive sputumafter first positive sputum
67 AIDS deaths w MDR TB67 AIDS deaths w MDR TB
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Czech Republic
The b
oundarie
s and n
am
es sh
ow
n a
nd th
e d
esig
natio
ns u
sed o
n th
is map d
o n
ot im
ply
the e
xpre
ssion o
f any o
pin
ion
whatso
ever o
n th
e p
art o
f the W
HO
conce
rnin
g th
e le
gal sta
tus o
f any co
untry
territo
ry city
or a
rea o
r of its a
uth
oritie
s or co
nce
rnin
g th
e d
elim
itatio
n o
f its frontie
rs or b
oundarie
s Dotte
d lin
es o
n m
aps re
pre
sent a
ppro
xim
ate
bord
er lin
es fo
r w
hich
there
may n
ot y
et b
e fu
ll agre
em
ent
WH
O 2
005 A
ll rights re
serv
ed
Ecuador
Georgia
Argentina
Bangladesh
Germany
Republic of Korea
Armenia
Russian Federation
South Africa
Portugal
Latvia
Mexico
Peru
USA
Brazil
UK
Sweden
Thailand
Chile
Based on information provided to WHO Stop TB Department 13 September 2007
SpainIslamic Republic of Iran
China Hong Kong SAR
France
Japan
NorwayCanada
Italy
Netherlands
Estonia
Lithuania
Ireland
Romania
Israel
Azerbaijan
Poland
Slovenia
India
Australia
Mozambique
Vietnam
Countries with confirmed XDR-TB cases as of September 2007
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Summary Summary Drug resistant TBDrug resistant TB
bull Drug-resistant TB poses a grave public health threat Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settingsespecially in high HIV prevalence settings
bull XDR-TB strains have been found in all regions of the world XDR-TB strains have been found in all regions of the world
bull XDR-TB occurs as a result inadequate TB control XDR-TB occurs as a result inadequate TB control programmesprogrammes
bull XDR-TB if identified early canXDR-TB if identified early can be treated and cured but be treated and cured but experience limited to low HIV prevalence settings experience limited to low HIV prevalence settings
bull Infection control measures must be strengthened Infection control measures must be strengthened
bull XDR-TB underlines the need for investment in basic TB XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics control plus development of new TB diagnostics treatments and vaccinestreatments and vaccines
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Health Care Workers and MDR Health Care Workers and MDR TBTB
Recognised risk for health care workersRecognised risk for health care workers Risk assessmentRisk assessment High risk ndash Prolonged closed contact with High risk ndash Prolonged closed contact with
infectious patientsinfectious patients Smear positive MDR TB patientsSmear positive MDR TB patients Medium risk ndashPrimary health care centres Medium risk ndashPrimary health care centres
involvedinvolved Sputum collection on TB suspectsSputum collection on TB suspects Low risk ndashHealth care support staff eg Low risk ndashHealth care support staff eg
cleanerscleaners Porters and admin staffPorters and admin staff
DrTVRao MDDrTVRao MD
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Koch failed to conquer tuberculosis which still Koch failed to conquer tuberculosis which still causes enormous health problems worldwide causes enormous health problems worldwide
100 years after his Nobel award100 years after his Nobel award The scientific academies The scientific academies
noted that the triumphant noted that the triumphant discovery of 1882 was discovery of 1882 was followed by a succession followed by a succession of failures first of all the of failures first of all the failed attempt to present failed attempt to present tuberculin as a remedy tuberculin as a remedy against tuberculosis in against tuberculosis in 1890-91 which severely 1890-91 which severely damaged Kochs damaged Kochs reputationreputation
Medical History 2001 45 1-32 Medical History 2001 45 1-32 CHRISTOPH GRADMANNCHRISTOPH GRADMANN
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Are there any solutions for Are there any solutions for effective Diagnosis in TB effective Diagnosis in TB
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Many more powerful hands needed Many more powerful hands needed to Control Tuberculosisto Control Tuberculosis
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Contribute your Knowledge Wisdom Contribute your Knowledge Wisdom to prevent spread and control of to prevent spread and control of
TuberculosisTuberculosis
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom
Created by DrTVRao MD for Created by DrTVRao MD for lsquoersquo learninglsquoersquo learning Programme Programme
EmailEmail
doctortvraogmailcomdoctortvraogmailcom