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Hema Kapoor MD. SM (NRM) Virology Section manager Bureau of laboratories

Laboratory Issues and West Nile Virus. Hema Kapoor MD. SM (NRM) Virology Section manager Bureau of laboratories Michigan Department of community Health Lansing Michigan. Laboratory Issues and West Nile Virus. Arboviruses Laboratory Diagnosis

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Hema Kapoor MD. SM (NRM) Virology Section manager Bureau of laboratories

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  1. Laboratory Issues and West Nile Virus Hema Kapoor MD. SM (NRM) Virology Section manager Bureau of laboratories Michigan Department of community Health Lansing Michigan

  2. Laboratory Issues and West Nile Virus • Arboviruses • Laboratory Diagnosis • Investigation of 2002 WNV outbreak in Michigan • Plans for 2003-Lab perspective

  3. The arboviruses • Arthropod-borne viruses(mosquitoes, sand-flies, fleas, ticks, lice, etc) • Enveloped RNA viruses -4 families • Flaviviridae-WNV isolated in 1937 in west Nile district of Uganda in Eastern Africa.

  4. Arboviral encephalitides Mosquito • eastern equine encephalitis (EEE) • western equine encephalitis (WEE) • St. Louis encephalitis (SLE) • La Crosse (LAC) Ticks • Powassan

  5. WNV Transmission Dead end host New modes of transmission Dead end host

  6. Laboratory Issues and West Nile Virus • Arboviruses • Laboratory Diagnosis • Investigation of 2002 WNV outbreak in Michigan • Plans for 2003-Lab perspective

  7. Immune Response in WNV Infection ELISA P/N #pfu/ml IgM 150 IgG WN viremia -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 DAYS POST ONSET illness Ref: 4th National WNV Conf. 2003 www.cdc.gov

  8. Laboratory Diagnosis of Human cases • Specimens • Cerebrospinal fluid (CSF) • CSF and Serum combination • Sera- Acute and Convalescent (obtained at least 8 d and 22 d post onset respectively) • Least preferred single serum sample Ref: Antibody Capture ELISA (IgM & IgG) Protocol. CDC Fort Collins, colorado CDC Neutralization Test Protocol. CDC Fort Collins, colorado

  9. Laboratory Diagnosis of Human cases contd • Laboratory Tests • Capture enzyme-linked immunosorbent assay (MAC-ELISA-IgM). • Capture enzyme-linked immunosorbent assay (MAC-ELISA-IgG) and • Plaque Reduction Neutralization Test (PRNT) Ref: Antibody Capture ELISA (IgM & IgG) Protocol. CDC Fort Collins, colorado CDC Neutralization Test Protocol. CDC Fort Collins, colorado

  10. Serological Testing Criteria at Michigan • MAC-ELISA IgM performed in Singlet. Positive MAC-ELISA repeated in duplicate

  11. West Nile Virus Nucleic Acid Amplification Tests (NAAT) Diagnostic Testing • Higher persistent levels of WNV RNA found in immuno-compromised patients • Improved sensitivity with better quality of diagnostic samples or virus concentration methods? • WNV RNA(-) results are meaningless

  12. West Nile Virus Nucleic Acid Amplification Tests (NAAT) Diagnostic Testing • Identifying viremic / antibody negative “window phase” patients • Low levels WNV RNA found in clinically ill, immuno- competent patients (i. e., WN fever)

  13. Laboratory Issues and West Nile Virus • Arboviruses • Laboratory Diagnosis • Investigation of 2002 WNV outbreak in Michigan • Plans for 2003-Lab perspective

  14. Weekly WNV Testing (Aug-Nov 02) Total Specimens (Jan, 02-Dec, 02)-2910 June 3, 2003 June 3, 2003

  15. How we handled the 2002 outbreak in the laboratory ? Triaged the samples Virology section and intersection staff worked collectively 6 days a week

  16. CSF at MDCH 0 day Notification Report Via EPIC to Neg Pos IgM ELISA 1st Run via Submitter, EPIC LDH & EPI pos 4 day Quantity Sufficient QNS To report a Probable case IgM ELISA 2nd Run Investigation request a serum sample pos Equivocal Confirmed positive reports Via Request a convalescent serum 5 6day sample EPIC

  17. Neg Notification via EPIC to Sub, LHD & EPI Pos IgM ELISA 1st run Pos IgM ELISA 2nd run Report Via EPIC Pos Reported a presumptive positive (LAB) & probable case (EPI) EPIC with a request for a convalescent serum Via Investigation Convalescent serum No 4x No Yes increase PRNT on acute serum PRNT on pair Arboviral Panel (Convalescent sample not possible ) 4x rise in titer Pos Confirmed positive reports Via EPIC Acute Serum at MDCH (0day)

  18. Single serum with documented CNS symptoms or paired sera without 4X increase in titer IgM ELISA (+) PRNT (-) Test for SLE, EEE and CGV Neg Pairedsera Single serum collected too early (0-8 d) post onset No Yes Yes Probable WNV Case No WNV Case

  19. Laboratory Issues and West Nile Virus • Arboviruses • Laboratory Diagnosis • Investigation of 2002 WNV outbreak in Michigan • Plans for 2003-Lab perspective

  20. Plans for 2003 Human Surveillance • No triaging of samples • Test whole panel • Two LT positions in virology for WNV testing • Cross training • IgM capture ELISA testing on CSF and Paired serum • Attempt culture on for WNV

  21. Getting Ready for summer of 2003 contd Issues withPersistence of IgM antibodies

  22. Longevity of Human WN Virus-Reactive IgM in Serum Ref: 4th National WNV Conf. 2003 www.cdc.gov

  23. 2003 Testing Acute Serum at MDCH No Yes CNS symptoms + No 4x increase Arboviral Panel PRNT (IgG) on pair Approval of Virology Lab Manger Neg Report IgM ELISA 1st Run Pos 4x rise in titer IgM ELISA 2nd Run Pos PRNT on acute serum Confirmed positive reports Via EPIC Pos Hold and request for theConvalescent serum

  24. Persistence of IgM antibodies • In CSF-No studies Published-47d MDCH observations-Three cases with igM positive • 110d • 141d • 199d

  25. Over wintering adult Culex mosquitoes Human Cases ? To Sum Up…….

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