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Ravi Kumar  Xcyton Innovative Diagnostic
 

Ravi Kumar Xcyton Innovative Diagnostic

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Ravi Kumar Xcyton Innovative Diagnostic

Ravi Kumar Xcyton Innovative Diagnostic

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    Ravi Kumar  Xcyton Innovative Diagnostic Ravi Kumar Xcyton Innovative Diagnostic Presentation Transcript

    • 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 infections
      • Diagnosis needed in the first 24 hours of admission
      • Infection is localized. No evidence of infection in Blood / serum / plasma
      • Clinical specimen available for eye infections
        • Corneal scrapings – a few cells
        • Conjunctival swab – a few cells
        • Aqueous humor – 50  L
        • Vitreous fluid - 50  L
      • Clinical specimen for CNS infections
        • CSF- 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 Identification
      • Culture takes 7 days
      • Requires additional techniques such as immuno-fluorescence for diagnostic validity
      • Immunodiagnosis: 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 Sensitivity
        • Low volume of clinical specimen to Perform multiple PCR Reactions
        • Conventional 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 infections XCyton’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 sequences
      • End read out is not a definite “yes” or “No”
      • Enormous data on signal to noise ratios and probabilities
      • Not useful in clinical setting
      • Why 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 bacteria
      • Multiplex Real Time PCR for all Herpes Viruses using a single set of primers and three probes
      • Multiplex to distinguish Mycobacterium tuberculosis and avium intracellulare
      • No 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 Signature Specific amplification 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 Specifications
      • Total process time (DNA Extraction, amplification and Hybridization) less than 7 hours allowing same day reporting
      • “ Yes” or “No” answers
      • No quantification of the pathogen
      • End read out to be by naked eye without loss of clinical sensitivity
      • No use of fluorescence signals and fluorescent scanners
      • Usable 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 pathogen
      • But conserved among all strains and subtypes of that pathogen
      • Should confer high sensitivity to amplification
    • How many genes per pathogen?
      • Some pathogens require multiple genes to be amplified to get adequate clinical sensitivity
        • HSV Required three Genes
        • CMV Required three Genes
        • VZV Required only two genes
    • Primer & Target Design issues
      • Match the thermal profile of primers of all pathogens being multiplexed
      • Thermal profiles matched for uniform hybridization
      • Experimentally: some of the primers lacked desired sensitivity in uniplex
      • Some interfered with sensitivity of the other primers (not predicted by Bio-informatics): Lack of sensitivity in multiplex
      • Lack of sensitivity in presence of human genome such as blood sample
      • Ultimate criteria: Lower Limit of Detection should be same in uniplex & multiplex
    • Sensitivity & Specificity
      • Sensitivity is conferred by
        • Nucleic acid amplification
        • Amplification of signal by the enzyme at the level of hybridization
      • Specificity:
        • Sequence specific Hybridization
    • Strategy for SES development
    • CNS INFECTIONS
    • PATHOGENS CAUSING AES Viruses 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
    • SENSITIVITY Virus 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 Acceptance Criteria Precision Qualitative 20 data points; one pos for 20 days or duplicates for 10 days. Extraction to detection CLSI EP12-A MM6-A ≥ 95% precision Precision Quantitative 20 data points at 2-3 conc. Within run, between run, between day. Extraction to detection CLSI EP5-A & MM6-A ≥ 95% precision
    • S. pneumoniae precision study
      • ATCC strain of S.pneumoniae was grown and quantitated using Mcfurland’s standards
      • Calculated 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 200 µl and was stored at -20 0 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 study
      • T.gondi standards was obtained form QCMD
      • Calculated 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 200 µl and was stored at -20 0 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 collected
      • 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 POSITIVE CSF BANK NEGATIVE
      • Antigen detection
      • Pyogenic
      • Cryptococi
      • Antibody detection
      • Toxoplasma
      +ve CSF BANK
      • +ve
      • CSF BANK
      -ve
      • Pyogenic
      • GNBs
      • Cryptococci
      • Filamentous Fungi
      • M. tuberculosis
      • Plasmodium falciparum
      PCR - ve AES –ve CSF Bank -ve
      • Culture
      • Pyogenic
      • GNBs
      • Cryptococci
      • M. tuberculosis
      • Filamentous Fungi
      +ve CSF BANK +ve CSF BANK +ve CSF BANK PCR - ve +ve CSF BANK -ve AES –ve CSF Bank
      • Pyogenic
      • M. tuberculosis
    • VIROLOGY AES SAMPLES- ALGORITHM FOR CATEGORIZATION OF CSF SAMPLES POSITIVE CSF BANK
      • POSITIVE
      • CSF BANK
      NEGATIVE
      • JE / Dengue / WN
      • HSV
      • Measles
      • Mumps
      • Chikungunya
      PCR NEGATIVE AES –ve CSF Bank ANTIBODY SCREENING ANTIGEN DETECTION NEGATIVE POSITIVE CSF BANK POSITIVE CSF BANK PCR NEGATIVE
      • Enteroviruses
      • HSV
      • JE / Dengue / WN
      • Measles
      Virus Isolation Rabies
      • POSITIVE
      • CSF BANK
      NEGATIVE AES –ve CSF Bank
    • n = 60
    • AES EVALUATION – PANEL 1 Diagnosis 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 - DISCORDANT Sample 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
    • ACURACY STUDY OF EYE SAMPLES CONDUCTED AT SHANKAR NETRALAYA, CHENNAI Specimen Category No. Tested No. Positive HSV Culture Positive HSV FAT Positive 05 18 13 CMV FAT Positive 13 13 Adenovirus Culture Positive Adenovirus Culture negative in conjunctivitis 02 07 05 Varicella zoster virus 03 03 Aspergillus( KOH Positive) Aspergillus (KOH Negative) 04 16 12 Propionibacterium(PCR positive) 22 22 Bacterial Endopthalmitis ( culture/smear positive) 25 25 Mycobacterium tuberculosis (PCR Positive) 10 10 Toxoplasma ( PCR positive) 8 8 Mycobacterium chelonae (PCR Positive) 7 7 Mycobacterium fortuitum (PCR positive) 3 3 Aqueous Humour obtained at cataract surgeries (Non-infectious controls) 30 0
    • SEPTICAEMIA FEBRILE NEUTROPENIA TRANSPLANT INFECTIONS PNEUMONIA
    • WE ASPIRE….
      • Save double the number of eyes currently being saved
      • Save double the number of lives from septicaemia
      • Reduce 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