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Diagnosis of TB: what is new from the laboratory
Daniela Maria Cirillo
San Raffaele Scientific Institute
Milano, Italy
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Topics
• Introduction: role of the laboratory in TB diagnosis• Integrated laboratory network • Biosafety guidelines• Quality Assurance in the diagnosis of tuberculosis• Laboratory diagnosis of tuberculosis • Diagnostic algorithms• Conclusion
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Introduction
• Care of TB patients starts with a QA diagnosis
• A robust network of Tb laboratories is required:– Adaquate biosafety– Modern diagnostics– SOPs– QAs
• Integrated laboratory network
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Integrated laboratory network
An integrated laboratory network is:
able to provide all primary diagnostic services without requiring patients to go to different laboratory facilities for specific tests
ONE STOP SHOP
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The past
• Weakest component of health systems for:– slow policy change and constant
underfunding – Inappropriate technology transfer– Inadequate infrastructure and biosafety– Staff skills and number– Inadequate lab strengthening plans– Insufficient technical assistance
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The present
• Joint effort to improve and expand TB lab capacity integrated into a global health system strengthening
http://www.stoptb.org/wg/gli
• Research on new diagnostics tools accelerated and diagnostic pipeline rapidly growing ( expected point of care by 2015)
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Quality Assurance Programme
System designed to continuously improve the reliability and efficiency of laboratory services
Three main components:
• Quality control (QC - IQA)
• External Quality Assurance (EQA)
» On site evaluation and review
» Proficiency Testing / Panel testing (PT)
» Blind rechecking
• Quality improvement (QI)
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EQA Benefits• Allows comparison of performance and results
• Serves as an early warning-system for problems– Identifies systematic problems
• Provides objective evidence of laboratory quality
• Serves as an indicator of where to direct improvement efforts
• Identifies training needs
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Laboratory biosafety
• Mtb is a class 3 risk pathogen• All biosafety strategies ( minimum requirements) should
be based on procedures risk assessment• Based on:
– Bacillary load of samples and workload– Viability of bacilli– Aeroosol generation– Typology of the patients– Fitness of the staff
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MICROSCOPY
Rapid test
Inexpensive
Specificity: >95%
Sensitivity: 25-65% (90 % of infectious cases)
Positive Predictive Value for TB depends on epidemiological situation
1st AFB smear 80-82 %
2nd AFB smear 10-14 %
3rd AFB smear 5-8 %
Fluorescence Ziehl-Neelsen staining
Does not allow species identification
Not applicable to all samples
Excellent diagnostic test for TB control in high-incidence settings
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LED Fluorescence Microscopy
Advantages:
• increase in performance
• increase in lamp lifetime
• reduces initial, operating and maintenance costs (adaptable to existing microscopes)
• No need for dark room
• Batteries operated
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New Policy and Smear microscopy definition of a TB case
• New definition in 2007*:
“person with al least one smear-positive sample (1 AFB is sufficient) out of a total of two examined”
• 2 samples regardless the collection time*The definition/policy can be applied to countries performing microscopy under
satisfactory quality assurance programmes
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Same day approach
same-day-diagnosis approach (microscopy of two consecutive spot-spot sputum specimens) is equivalent, in terms of diagnostic accuracy, to conventional case-finding strategies by microscopy
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WHO recommends that:
Countries that have successfully implemented the two- specimen case-finding strategy consider a switch to the same-day-diagnosis approach, especially in settings where patients are likely to default from the diagnostic process
Countries that are still using the three-specimen case-finding strategy consider a gradual change to the same-day-diagnosis approach.
Changes to a same-day-diagnosis strategy be preceded by a detailed situation assessment
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WHO recommendations on sputum smear microscopy (2010)
• ZN light microscopy performed on UNCONCENTRATED sputum is suitable for all laboratory service levels
• Concentration of sputum is NOT recommended in programmatic settings
• Fluorescence microscopy is recommended for increased sensitivity (add 10%)
• LED microscopy is recommended over conventional fluorescence
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TB Culture*
Advantages• Definitive diagnosis of TB• Increases case finding of
30-50%• Early detection of cases• Provide strains for DST
and epidemiological studies
Disadvantages• Complex and expensive
compared to microscopy• Requires complex
handling of specimens• Skilled technicians• Appropriate infrastructure
and biosafety levels
LIMITATIONS: need for decontamination and identification
*coverage 500.000/1000000
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Culture: solid/liquid
solid• Low cost for reagents, not
automated• Culture level infrastructure• Low contamination rate• Long time to positivity• Colony morphology• ID required
liquid• Complex and expensive can
be automated (MGIT)• Highest infrastructure and
biosafety levels• Case finding increased 10%
over solid• Diagnostic delay reduced to
days• ID required
Strip speciation tests needed for fast ID of TbcomplexMolecular test for speciation of most common mycobacteria
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TB Culture: solid/liquid
• solid • liquid
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Standardization of procedures still difficult to set up
The correct performance of DST requires the understanding of several steps such as:
Drug Susceptibility Testing (DST): Drug Susceptibility Testing (DST): the technical challenge still persiststhe technical challenge still persists
•Origin of resistance and interpretative criteria•Dosage and stability of the incorporated compounds•Anti-mycobacterial activity of the incorporate drug•Interpretation of results and data reporting
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DST• Definitive diagnosis of DRTB
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DST (2)
• Indirect methods suitable for regional/ national laboratories
• Adequate lab infrastructure and biosafety• Accuracy varies with the drugs tested:
– R, H most accurate– Second line :
• automated liquid DST are the gold standard• Not recommended in the absence of QA capacity (200 high
risk specimen per year)
• Formal link with Supranational Reference laboratories requested
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Second line DST
• Recommended in appropriate structures for:– Aminoglycosides, Fluoroquinolones,
Polypeptides
• NOT recommended on routine for ethionamide, prothionamide, cycloserine,clofazimine, amox-clav, clarithromycin, linezolid)
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WHO laboratory policies for culture and DST
• Automated liquid culture (2007) Use of liquid culture in the contest of comprehensive plan for strengthening lab capacity starting with national/central level
• Rapid speciation in combination with culture Second line DST (2008) to be conducted for selected drugs a central level
Available at http://www.who.int/tb.dots/laboratory/policy/en/print.html
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Novel technologies for rapid screening of anti-TB drug
resistance have become a priority in Tuberculosis research
The effective treatment of MDR-TB is a life-saving intervention
Early diagnosis of both TB and DR-TB are the key for an effective Tb control
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WHO policy on new diagnostics is evidence-driven
• Following new investments in research new diagnostics are finally moving
• Policy formulation needs to be an ongoing process at global and country level
• WHO asses policies by a systematic, structured process
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Drug-Resistant TB: Definitions
• Mono-resistant: resistance to a single drug • Poly-resistant: resistance to more than one
drug, but not to the combination of isoniazid and rifampicin
• Multidrug-resistant (MDR): resistance to at least isoniazid and rifampicin
• Extensively drug-resistant (XDR): MDR plus resistance to fluoroquinolones and at least 1 of the 3 injectable drugs (amikacin, kanamycin, capreomycin)
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Drug-Resistant TB: Definitions
MONORESISTANT
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Drug-Resistant TB: Definitions
POLIRESISTANT
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Drug-Resistant TB: Definitions
MULTI DRUG RESISTANT
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Drug-Resistant TB: Definitions
???MULTI DRUG RESISTANT
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Drug-Resistant TB: Definitions
EXTENSIVELY DRUG
RESISTANT
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Impact of Resistance on Outcome
Resistance pattern New Cases (%) Retreatment (%)
Pan-susceptible 4 10
Any Resistance 5 21
MDR 30 45
INH (not MDR) 6 23
RIF (not MDR) 13 29
Other 4 15
% of cases with failure or death, standard 4-drug regimen
Espinal MA, et al. JAMA. 2000;283(19):2537-45
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Mechanisms of resistance
• Production of enzymes that modify or destroy the antibiotic molecule
• Alteration of the target binding site
• Active escretion of the molecule (efflux pump)
• Membrane permeability reduction
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INH = 1 in 106
RIF = 1 in 108
EMB = 1 in 106
Str = 1 in 106
I + R = 1 in 1014
Frequency of Resistance Mutations
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Genes involved in drug-resistance for major Genes involved in drug-resistance for major anti-tubercular drugsanti-tubercular drugs
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PAGE | 36
RIF resistance as surrogate marker for MDR TB
• RIF resistance as a mono resistance not very frequent (5-15% of them)
• 80-95% of RIF resistant strains are also resistant to INH• Key-drug in the anti-TB regimen• Low rate of spontaneous mutations• Mutations affect an hot-spot region in the rpoB gene
Khue et al. A 10-year prospective surveillance of Mycobacterium tuberculosis drug resistance in France 1995–2004. Eur Respiratory J. 2007
Ideal candidate for the molecular detection of resistance to the drugIdeal candidate for the molecular
detection of resistance to the drug
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Commercial Line Probe AssaysCommercial Line Probe Assays
Hain LifescienceHain Lifescience
InnogeneticsInnogenetics
INNO-LiPA-INNO-LiPA-Rif.TBRif.TB
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GenoType® MTBDR
INNO-LiPA Rif.TB
Company Hain Lifescience
Innogenetics
M. tuberculosis detection Yes Yes
Detection of RMP Resistance in M. tb Complex
Yes Yes
Detection INH Resistance in M. tb Complex
Yes No
Strip Assay Yes Yes
PCR based Yes Yes
From liquid or solid culture Yes Yes
Direct assay Yes Yes (modified version)
TBCDetection: 23S-rRNA/16S-rRNA Yes Yes
RMP-Resistance: rpoB gene Yes Yes
INH-Resistance: katG gene/inhA gene Yes No
Universalcontrol Yes No
rpoB unicontrol Yes No
kat G/inHA unicontrol Yes No
Comparison GenoType® MTBDRplus and INNO-LiPA Rif.TB
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New generation of LiPA performs better in both Sm+ and Sm- samples
Miotto al. JCM 2008
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Molecular line probe assays for rapid screening of patients at risk of MDR-TB
Policy statement by WHO and Partners
June 27, 2008
Endorsement of the two commercial line probe assays for rifampicin resistance detection:Tests are CE marked and meet predefined performance targets in controlled evaluation studiesBoth tests are highly sensitive and specific for rifampicin resistance detection from TB strains
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Smear positive Smear negative
Rif/ Rif INH LiPA
MTB detected, INH(*) and RMP susceptible
MTB detected, INH and/or RMP resistant (MDR-TB)
•MTBC, INH(*) and RMP mutation non detected NON MDR•No culture
Start standard 1st line treatment
Culture (liquid/ solid)DST ( first and second line)
Report as negative OR if TB is highly suspected (HIV+,child..)
perform (liquid) culture
Culture AFB positive, TB morphology
Unsuccessful amplification
Perform (liquid) culture
Culture AFB positive
ID
Sputum samples
Monitoring sputum conversion
Standard MDR treatment
Individualized treatment
Treatment with 2nd-3rdDrugs available/isolation
monitoring
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Possible automation on LiPA
LiPAs require:Level II biosafety areasSkilled laboratory staffAmplicon Contamination control
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•simple 1-step external sample prep. •time-to-result < 2 h •throughput: >16 tests / day / module •no need for biosafety cabinet •integrated controls •true random access •specific for MTB •sensitivity similar to culture •detection of rif-resistance via rpoB gene •test cartridges for GeneXpert System •several GeneXpert modules can be combined in 1 workstation •~1 day technician training required
XPERT TB-MDR
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Simple Sample Processing – Direct Sputum
1. Add 2:1 Sample Buffer to sample
2. Shake then stand 10 minutes
3. Shake then stand further 5 minutes
4. Transfer 2mlto cartridge
Begin Test…
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Moving to “district” :Dharamsala, Delek Hospital
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Sensitivity (cfu/ml) of pulmonary TB diagnostics
Solidculture
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Advantages
• Simple to perform• Minimal training• Virtually cross contamination free• Minimal biosafety requirement• Higher sensitivity in paucibacillary samples
(HIV+)
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Proportion of TB cases detected
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Time to detection
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Time to RFP resistance detection
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Potential limits of Xpert MTB/RIF technology
• Unknown the performance at a district level• If RFP resistance is diagnosed at a low level MDR prevalence
environment, the assay needs to be confirmed • Need to perform a culture for DST to evaluate other drug resistance• Need to perform a culture for monitoring issue (culture conversion)• It requires uninterrupted and stable electronic power supplies and
yearly calibration • Size for storage issues
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New WHO recommendations
WHO RECOMMENDATION, December 8th, 2010
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New WHO recommendations
WHO RECOMMENDATION, December 8th, 2010
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New WHO recommendations
WHO RECOMMENDATION, December 8th, 2010
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GenoType® MTBDRplus
XpertTB-MDRplus
Company Hain Lifescience
Cepheid
M. tuberculosis detection Yes Yes
Detection of RMP Resistance in M. tb Complex
Yes Yes
Detection INH Resistance in M. tb Complex
Yes No
Fully automated /training No/Yes Yes/No
DNA tech PCR Mol Beacon
From liquid or solid culture Yes NA
Direct assay Yes Yes
Level of biosafety II microscopy
Time to results Same day 2h
Cost per test Low/Mod Mod/High
Universal control Yes Yes
Extraction control No Yes
Cost of MaintenanceContamination control
LowNo
HighYes
Comparison GenoType® MTBDRplus and XpertTB/MDR
Intermediate Reference labsPatients testing from Sm/ C+ (Rif)Fast tool Surveillance purposes
Potential to District level as fast patients diagnostic tool, needs evaluation at district level
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Smear positive Smear negative
Rif/ Rif INH LiPA
MTB detected, INH(*) and RMP susceptible
MTB detected, INH and/or RMP resistant (MDR-TB)
•MTBC, INH(*) and RMP mutation non detected NON MDR•No culture
Start standard 1st line treatment
Culture (liquid/ solid)DST ( first and second line)
Report as negative OR if TB is highly suspected (HIV+,child..)
perform (liquid) culture
Culture AFB positive, TB morphology
Unsuccessful amplification
Perform (liquid) culture
Culture AFB positive
ID
Sputum samples
Monitoring sputum conversion
Standard MDR treatment
Individualized treatment
Treatment with 2nd-3rdDrugs available/isolation
monitoring
Xpert-TB/MDR-TB
Contact tracing?
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Genes involved in drug-resistance for major Genes involved in drug-resistance for major anti-tubercular drugsanti-tubercular drugs
40-80%
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XDR molecular diagnosis: GenoType MTBDRsl (Hain Lifescience)
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Technologies and laboratory appropriateness
• Introducing new technology requires addressing of core elements:– Infrustructure, biosafety measures and maintenance– Equipment validation and maintenance– Specimen transport and referral mechanisms– Management of laboratory commodities and supplies– Laboratory information data and management system– Laboratory quality management– Strategies for HR development and retentionGLI road map at
:http://www.who.int/tb/dots/laboratory/policy/en
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“Point of care assay”
Closer if• Large scale innovation and delivery
will be fully supported by:–Scientists and industry–Large operational research trials–Large view policy makers and regulators–Retooling of existing resources