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Hema Kapoor MD. SM (NRM) Virology Section manager Bureau of laboratories Michigan Department of community Health Lansin

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 Michigan Department of community Health Lansin

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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. IgM Capture ELISA HRP HRP • Coat With Goat anti-Human IgM • 4° Overnight • Add Patient Serum @ 1:400 • 37° 1 Hour • Add West Nile Recombinant Antigen • 4° Overnight • Add HRP anti-Flavivirus McAb • 37° 1 Hour Ref: 4th National WNV Conf. 2003 www.cdc.gov

  11. WN Serological Data Typical Human WN Case • In primary flavivirus infections ; • Martin et al 2002: IgM P/N to WN is 3-5X greater than SLE. • 2002 data: Use 2X criteria WN to SLE ratio: only 1 exception in 417 WN confirmed cases. Ref: 4th National WNV Conf. 2003 www.cdc.gov

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

  13. 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 • Identifying viremic / antibody negative “window phase” patients • Low levels WNV RNA found in clinically ill, immuno- competent patients (i. e., WN fever)

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

  15. Weekly WNV Testing (Aug-Nov 02) Total Specimens (Jan, 02-Dec, 02)-2910

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

  17. 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

  18. 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)

  19. 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

  20. 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

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

  22. Getting Ready for summer of2003

  23. MDCH - The Michigan Department of Community Health MDA- Michigan Department of Agriculture MDNR- Michigan Department of Natural Resources MSU- Michigan State University AHDL- Animal Health Diagnostic Laboratory

  24. 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

  25. Testing reagents for 2003 CDC Reagent Production • No change in personnel or policy • Commercial Partners – patent license agreements for WN antigen production.

  26. Manufacturers with WNV Antibody Assays in development • Focus Technologies • Ortho Clinical Diagnostics • Abbott Laboratories • Chiron (recombinant antigens) • Pan -Bio

  27. Future of new assays for WNV Synergy between • Industry • Regulatory agencies • Public and • Private laboratories

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

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

  30. 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

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

  32. Bird Testing in 2003 • Strict IATA regulations • Availability of a field assay-Vec Test • Validation of IHC and PCR-2002 • IHC vs Vec Test at MSU lab in 2003 • Implementation of vec test ??

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

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