Ravi Kumar Xcyton Innovative Diagnostic
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For Critical Care Decisions
Saving Lives – Preventing Disabilities
THE XCYTON SYNDROME EVALUATION SYSTEM
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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
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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
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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)
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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.
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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
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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
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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?
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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
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We intended to create…..
Simultaneous detection of all probable pathogens causing a syndrome irrespective of bacteria, fungi, parasites, RNA viruses and DNA viruses
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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..
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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
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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
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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
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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
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Sensitivity & Specificity
Sensitivity is conferred byNucleic acid amplificationAmplification of signal by the enzyme at
the level of hybridization Specificity:
Sequence specific Hybridization
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Strategy for SES development
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CNS INFECTIONS
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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EYE INFECTIONS
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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
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SEPTICAEMIAFEBRILE NEUTROPENIATRANSPLANT INFECTIONSPNEUMONIA
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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
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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