1. Laboratory Issues and West Nile Virus Hema Kapoor MD. SM (NRM)
April 9, 2003
Laboratory Issues and
West Nile Virus
Hema Kapoor MD. SM (NRM)
Virology Section manager
Bureau of laboratories
Michigan Department of community Health
Lansing Michigan
June 3, 2003

2. Laboratory Issues and West Nile Virus
Arboviruses
Laboratory Diagnosis
Investigation of 2002 WNV outbreak in Michigan
Plans for 2003-Lab perspective
June 3, 2003

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.
June 3, 2003

4. Arboviral encephalitides
Mosquito
eastern equine encephalitis (EEE)
western equine encephalitis (WEE)
St. Louis encephalitis (SLE)
La Crosse (LAC)
Ticks
Powassan
June 3, 2003

5. New modes of transmission
WNV Transmission
Dead end host
New modes of transmission
Dead end host
June 3, 2003

6. Laboratory Issues and West Nile Virus
Arboviruses
Laboratory Diagnosis
Investigation of 2002 WNV outbreak in Michigan
Plans for 2003-Lab perspective
June 3, 2003

7. Immune Response in WNV Infection
April 9, 2003
Immune Response in WNV Infection
ELISA
P/N
#pfu/ml
IgM
150
IgG WN
viremia
Igm-peak is reached from day3-8 days. Cases bled too early for WNV may show false negative results.
DAYS POST ONSET
illness
June 3, 2003
Ref: 4th National WNV Conf

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
June 3, 2003

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
June 3, 2003

10. Serological Testing Criteria at Michigan
CSF
Paired sera
(Ac. at least 8 days and conv at least 22 days post onset)
Single serum
P/N ratio >/= 10 positive
P/N equivocal and requested for a serum sample.
P/N ratio >/=5 presumptive.
P/N 2-5 equivocal.
IgG Tests -A four-fold rise in titre to distinguish a recently acquired infection from a past infection.
PRNT was performed on all specimens showing a four fold increase in IgG titer.
P/N ratio- same as in paired sera.
A convalescent serum requested on equivocals .
PRNT To rule out the cross- reactions between WNV and other arbovirus infections (SLE, EEE and CGV).
MAC-ELISA IgM performed in Singlet. Positive MAC-ELISA repeated in duplicate
June 3, 2003

11. WNV RNA(-) results are meaningless
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
June 3, 2003

12. Identifying viremic / antibody negative “window phase” patients
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)
June 3, 2003

13. Laboratory Issues and West Nile Virus
Arboviruses
Laboratory Diagnosis
Investigation of 2002 WNV outbreak in Michigan
Plans for 2003-Lab perspective
June 3, 2003

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

16. CSF at MDCH 0 day IgM ELISA 1st Run Neg Pos Investigation 5 6day
April 9, 2003
CSF at MDCH 0 day
IgM ELISA 1st Run
Report
via
EPIC
Neg
Notification
Via EPIC to
Submitter,
LDH & EPI
4 day
Pos
Investigation
Confirmed positive reports
Via
EPIC 5
6day
IgM ELISA 2nd Run
pos
Quantity Sufficient
QNS
To report a Probable case
request a serum sample
Equivocal
Request a convalescent serum
sample
June 3, 2003

17. Acute Serum at MDCH (0day)
IgM ELISA 2nd run
Reported a presumptive positive (LAB) & probable case (EPI)
Via
EPIC with a request for a convalescent serum
Pos
Investigation
Notification
via EPIC to
Sub, LHD & EPI
Neg
IgM ELISA 1st run
Report
Via
EPIC
Convalescent serum
No 4x
increase
4x rise in titer
Yes
PRNT on pair
Confirmed positive reports
Via
EPIC
Arboviral
Panel No
PRNT on acute serum
(Convalescent sample not possible )
Pos
June 3, 2003

18. Single serum collected too early
Single serum with documented CNS symptoms
or paired sera without 4X increase in titer
IgM ELISA (+) PRNT (-)
Test for SLE, EEE and CGV
Probable WNV Case
Neg
Yes
Single serum collected too early
(0-8 d) post onset No
Paired sera
Yes
No WNV Case
June 3, 2003

19. Laboratory Issues and West Nile Virus
Arboviruses
Laboratory Diagnosis
Investigation of 2002 WNV outbreak in Michigan
Plans for 2003-Lab perspective
June 3, 2003

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
June 3, 2003

21. Issues with Persistence of IgM antibodies
Getting Ready for summer of contd
Issues with Persistence of IgM antibodies
June 3, 2003

22. Longevity of Human WN Virus-Reactive IgM in Serum
Days P.I. N
Positive MAC-ELISA
Total (%)
Ave. P/N (Range)
Positive (%)
Equivocal
200 22
13 (60) 4
17 (77)
6.0
( ) 21
9 (43) 2
11 (52)
4.0
( )
500 12
5 (42)
6 (60)
5.0
( )
June 3, 2003
Ref: 4th National WNV Conf

23. Confirmed positive reports
Acute Serum at MDCH
2003 Testing
Hold and request for the Convalescent serum
PRNT on acute serum
Pos No
CNS symptoms +
Approval of Virology Lab Manger
Neg
IgM ELISA 1st Run
IgM ELISA 2nd Run
Report
Yes
4x rise in titer
PRNT (IgG) on pair
Confirmed positive reports
Via
EPIC
No 4x
increase
Arboviral
Panel
June 3, 2003

24. Persistence of IgM antibodies
In CSF-No studies
Published-47d
MDCH observations-Three cases with igM positive
110d
141d
199d
June 3, 2003

25. Over wintering adult Culex mosquitoes
Human Cases ?
To Sum Up…….
June 3, 2003