For Critical Care Decisions

Saving Lives – Preventing Disabilities

THE XCYTON SYNDROME EVALUATION SYSTEM

Critical Infections• High Mortality • Most of the deaths in first 72 - 96 hours of

admission in to the hospital• Transplant recipients and febrile

neutropenics• Loss of function • Blindness or visual impairment in 96 hours

after the start of first symptom. • Residual damage in the form of paralysis or

sensory loss, epilepsy in 96 hours• Septicaemia leaves behind residual Renal &

hepatic damage and Arthritis

Diagnostic Challenges of Critical infectionsDiagnosis needed in the first 24 hours of

admissionInfection is localized. No evidence of

infection in Blood / serum / plasmaClinical specimen available for eye

infectionsCorneal scrapings – a few cellsConjunctival swab – a few cellsAqueous humor – 50 LVitreous fluid - 50 L

Clinical specimen for CNS infectionsCSF- 1 mL

Current Diagnostic solutions for Critical infections

Bacterial & Fungal Cultures:• Take 72- 96 hours Not useful.• Sample is too small for Culture

– Ten to fifteen bacteria or fungal particles in eye samples and about 50 particles in CSF

• Anaerobes cannot be cultured• Mycobacteria take too long to grow 12-14

days in BacT Alert)• Over all success rate is less than 15% (7000

blood cultures in CMC 1100 positive and 840 are pathogens)

Diagnostic Challenges of Critical infections

Viral IdentificationCulture takes 7 daysRequires additional techniques such as

immuno-fluorescence for diagnostic validityImmunodiagnosis: serology: Antibody

detection; Useful only after 5 days of infection

Immunodiagnosis: antigen Detection: Very Low sensitivity useful in 15% of Pyogenic Meningitis or Cryptococcal meningitis.

Diagnostic Challenges of Critical infections

PCR and / or Other Nucleic Acid Based Detection tests: Very High SensitivityLow volume of clinical specimen to Perform multiple

PCR ReactionsConventional PCR read out is Gel Electrophoresis –

identification basis is size of the gene fragment amplified – not sequence – leading to unacceptable level of false alarms

Diagnostic Challenges of Critical infectionsXCyton’s Solution- A Paradigm Shift

Disease Based Diagnosis

Sequentially looking for one organism after the other

A B C D……N

Syndrome Evaluation System

Simultaneously looking for all pathogens that could probably cause a disease

A, B, C, D…..N all tested

What is available?DNA Micro Chips with about 60,000 features

defining pathogen sequencesEnd read out is not a definite “yes” or “No”Enormous data on signal to noise ratios and

probabilities Not useful in clinical settingWhy would I test a fever in a child in India against

Ebola or Marburg viruses?

What is available?

Multiplex PCR for bacterial Meningitis for three bacteriaMultiplex Real Time PCR for all Herpes Viruses using a

single set of primers and three probesMultiplex to distinguish Mycobacterium tuberculosis and

avium intracellulareNo multiplex System that can simultaneously detect

more than one class of pathogens such as viruses or bacteria

We intended to create…..

Simultaneous detection of all probable pathogens causing a syndrome irrespective of bacteria, fungi, parasites, RNA viruses and DNA viruses

Syndrome Syndrome Signature Signature Specific Specific

amplificatioamplificationn

Syndrome Syndrome Signature Signature Specific Specific

amplificatioamplificationn

The amplified product is introduced onto a

Syndrome signature Evaluation Protocol

Then it undergoes a process called Signature specific Hybridization, Then an enzymatic

reaction occurs and a colored spot appears..

Product SpecificationsTotal process time (DNA Extraction, amplification and

Hybridization) less than 7 hours allowing same day reporting

“Yes” or “No” answersNo quantification of the pathogenEnd read out to be by naked eye without loss of clinical

sensitivityNo use of fluorescence signals and fluorescent scannersUsable at even district level hospitals

The choice of genes of pathogens

Genes which are virulence associated. (Avoided 16s rRNA genes or 23s RNA genes)

Specific to Each pathogenBut conserved among all strains and subtypes

of that pathogenShould confer high sensitivity to amplification

How many genes per pathogen?Some pathogens require multiple genes to be

amplified to get adequate clinical sensitivityHSV Required three GenesCMV Required three GenesVZV Required only two genes

Primer & Target Design issues

Match the thermal profile of primers of all pathogens being multiplexed

Thermal profiles matched for uniform hybridizationExperimentally: some of the primers lacked desired sensitivity

in uniplexSome interfered with sensitivity of the other primers (not

predicted by Bio-informatics): Lack of sensitivity in multiplexLack of sensitivity in presence of human genome such as blood

sampleUltimate criteria: Lower Limit of Detection should be same in

uniplex & multiplex

Sensitivity & Specificity

Sensitivity is conferred byNucleic acid amplificationAmplification of signal by the enzyme at

the level of hybridization Specificity:

Sequence specific Hybridization

Strategy for SES development

CNS INFECTIONS

PATHOGENS CAUSING AESViruses

Flaviviridae – JE, Dengue 1-4, West Nile

Paramyxoviridae – Nipah, Measles, Mumps

Enteroviridae – Polio, Coxsackie, Echo, Entero70-72

Rhabdoviridae – Rabies, Chandipura

Togaviridae – Rubella

Alphaviridae – Chikungunya

Herpesviridae – HSV, CMV, VZV, HHV-6

Polyomaviridae - JC

Bacteria

M.tuberculosis

S.pneumoniae

H.Influenzae

N.meningitidis

Fungi

C.neoformans

Aspergillus

Candida

Mucor

Rhizopus

Parasites

Toxoplasma gondii

P.falciparum – not found in CSF

SENSITIVITYVirus Sensitivity

(no. of viral particle /pfu/ml)

JEV 100

Measles 0.1

Rubella 0.1

Mumps 0.5

Chikungunya 1

Rabies 1

Nipah 2.57fg

Chandipura 1.47fgm

Enteroviridae 1

LIMIT OF DETECTION OF PATHOGENS IN AES DNA CHIP

Organism Sensitivity(organisms/ ml)

Cross reactivity observed

HSV 50 particles No

M.tuberculosis 50 particle No

H.influenzae 140 particles No

N.meningitidis 115 particles No

S.pneumoniae 400 particles No

HHV-6 50 particle No

T.gondii 50 particle No

CMV 250 particles No

VZV 4pfu No

C.neoformans 50 particles No

JC Not determined No

PRECISION STUDY

Verification

Criteria Reference AcceptanceCriteria

PrecisionQualitative

20 data points; one pos for 20 days or duplicates for 10 days. Extraction to detection

CLSI EP12-AMM6-A

≥ 95% precision

PrecisionQuantitative

20 data points at 2-3conc. Within run, between run, between day. Extraction to detection

CLSI EP5-A & MM6-A

≥95% precision

S. pneumoniae precision studyATCC strain of S.pneumoniae was grown

and quantitated using Mcfurland’s standardsCalculated quantity of the culture was

dissolved in CSF to get 400 org/ml and 800org/ml

Both the spiked CSF was made into 10 aliquots of 200µl and was stored at -200 C

DNA was extracted from these aliquots on 10 days and was followed by mPCR and hybridization

Spot intensities was graded by a single person for all the data points

T.gondii precision studyT.gondi standards was obtained form QCMDCalculated quantity of the particles was

dissolved in CSF to get 25 org/ml and 50org/ml

Both the spiked CSF was made into 10 aliquots of 200µl and was stored at -200 C

DNA was extracted from these aliquots on 10 days and was followed by mPCR and hybridization

Spot intensities was graded by a single person for all the data points

ACURACY STUDY ON POSTMORTEM PROVEN CASES OF CNS DISEASES

Sample Diagnosis Post mortem proven

No Tested No Positive

HSV Encephalitis 4 4

CMV Encephalitis 3 3

VZV Encephalitis 2 2

Toxoplasma Encephalitis 3 3

Tuberculous meningitis 3 3

Normal CSF’s obtained at spinal anesthesia

25 0

Number of CSF samples proposed to be Number of CSF samples proposed to be collectedcollected

100 CSF samples: Positive for any AES aetiological agent/agents by standard laboratory tests (60 collected and characterized)

100 CSF samples: Negative for AES aetiological agents by standard laboratory tests ( 112 collected and partially characterized)

50 CSF samples: “Normal”Control samples (to be collected from patients undergoing spinal anaesthesia for minor surgical procedures) (None Collected – Hospitals approached)

MICROBIOLOGY AES SAMPLES- ALGORITHM FOR CATEGORIZATION OF CSF SAMPLES

MICROSCOPY

POSITIVECSF BANK

NEGATIVE

Antigen detection Pyogenic Cryptococi

Antibody detection

Toxoplasma

+veCSF BANK

+veCSF BANK

-ve

PyogenicGNBsCryptococciFilamentous FungiM. tuberculosisPlasmodium

falciparum

PCR

-ve

AES –ve CSF Bank

-ve

CulturePyogenicGNBsCryptococciM. tuberculosisFilamentous Fungi

+veCSF BANK

+veCSF BANK

+veCSF BANK

PCR

-ve

+veCSF BANK

-ve AES –ve CSF Bank

Pyogenic M.

tuberculosis

VIROLOGY AES SAMPLES- ALGORITHM FOR CATEGORIZATION OF CSF SAMPLES

POSITIVECSF BANK

POSITIVECSF BANK

NEGATIVE

JE / Dengue / WNHSVMeaslesMumpsChikungunya

PCR

NEGATIVE

AES –ve CSF Bank

ANTIBODY SCREENING ANTIGEN DETECTION

NEGATIVE

POSITIVECSF BANK

POSITIVECSF BANK

PCR

NEGATIVE

EnterovirusesHSVJE / Dengue / WNMeasles

Virus Isolation

Rabies

POSITIVECSF BANK

NEGATIVE AES –ve CSF Bank

ETIOLOGICAL BREAK UP OF POSITIVE SAMPLES BANKED

Pneumococcus9% H.Inluenza

5%Meningococcus

8%

M.Tuberculosis8%

Filamentous Fungi5%

Cryptococcus8%Toxoplasmosis

8%

JEV8%

HSV18%

JCV3%

CMV5%

VZV5%

Rabies5%

Measles5%

n = 60

AES EVALUATION – PANEL 1Diagnosis at NIMHANS No Tested No. XCytoScreen

+ve

HSV 2 2

TB 3 2 +1 (CMV)

Cryptococcus 4 2 + 1 (CMV) + 1(Toxo)

Streptococcus pneumoniae 3 3

Neisseria meningitidis 2 2

Toxoplasma gondii 1 1

AES with no aetiological agent found

45 15

Spinal anaesthesia samples 50 0

CLINICAL SAMPLES - DISCORDANTSample ID NIMHANS XDPL

31 Neg Tb+SP

35 Neg HSV

54 Neg HSV

62 Neg CMV

64 Neg HSV

68 Neg HSV,HHV-6

56 Neg SP

1005 Neg Sp

1006 Neg SP

1004 Neg SP

99 Neg VZV+TB+SP

100 Neg TB

96 Neg Tb+SP

311 Neg HSV

97 Neg CMV+ SP

EYE INFECTIONS

Specimen Category No. Tested No. Positive

HSV Culture Positive

HSV FAT Positive

05

1813

CMV FAT Positive 13 13Adenovirus Culture Positive

Adenovirus Culture negative in conjunctivitis

02

0705

Varicella zoster virus 03 03Aspergillus( KOH Positive)

Aspergillus (KOH Negative)

04

1612

Propionibacterium(PCR positive) 22 22Bacterial Endopthalmitis ( culture/smear positive) 25 25

Mycobacterium tuberculosis (PCR Positive) 10 10Toxoplasma ( PCR positive) 8 8Mycobacterium chelonae (PCR Positive) 7 7Mycobacterium fortuitum (PCR positive) 3 3Aqueous Humour obtained at cataract surgeries (Non-infectious controls)

30 0

ACURACY STUDY OF EYE SAMPLES CONDUCTED AT SHANKAR NETRALAYA, CHENNAI

SEPTICAEMIAFEBRILE NEUTROPENIATRANSPLANT INFECTIONSPNEUMONIA

WE ASPIRE….Save double the number of eyes currently

being savedSave double the number of lives from

septicaemiaReduce the disability by half in Acute

Encephalitis

OUR EXTENDED FAMILY

Primier Scientific Institutes

Indian Institute of Science (IISc),

Bangalore

International Centre for Genetic

Engineering and Biotechnology

(ICGEB), New Delhi

Centre for Cellular and Molecular

Biology (CCMB), Hyderabad

Rajiv Gandhi Institute of

Biotechnology, Trivandrum

Primier Medical Institutions

NIMHANS , Bangalore

AIIMS , New Delhi

Shankaranetralaya, Chennai

L.V. Prasad Eye Institute, Hyderabad

Christian Medical College, Vellore

ACTREC, Mumbai

Cancer Foundation of India(CFI)

KMIO, Bangalore

St.John’s Medical College, Bangalore