Hema Kapoor MD. SM (NRM) Virology Section manager Bureau of laboratories Michigan Department of...
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Transcript of Hema Kapoor MD. SM (NRM) Virology Section manager Bureau of laboratories Michigan Department of...
Hema Kapoor MD. SM (NRM)
Virology Section manager
Bureau of laboratories
Michigan Department of community Health
Lansing Michigan
Laboratory Issues and West Nile Virus
Laboratory Issues and West Nile Virus
ArbovirusesLaboratory DiagnosisInvestigation of 2002 WNV
outbreak in MichiganPlans for 2003-Lab perspective
The arboviruses
Arthropod-borne viruses(mosquitoes, sand-flies, fleas, ticks, lice, etc)
Enveloped RNA viruses -4 familiesFlaviviridae-WNV isolated in 1937 in
west Nile district of Uganda in Eastern Africa.
Arboviral encephalitides
Mosquitoeastern equine encephalitis (EEE)western equine encephalitis (WEE) St. Louis encephalitis (SLE) La Crosse (LAC) Ticks Powassan
WNV Transmission
Dead end host
Dead end host
New modes of transmission
Laboratory Issues and West Nile Virus
ArbovirusesLaboratory DiagnosisInvestigation of 2002 WNV
outbreak in MichiganPlans for 2003-Lab perspective
DAYS POST ONSETDAYS POST ONSET
1 2 3 4 5 6 7 8 9 10-5 -4 -3 -2 -1 0
IgM
IgG
ELISAELISAP/NP/N#pfu/ml#pfu/ml
WNviremia
150
illnessillness
Immune Response in WNV Infection
Ref: 4th National WNV Conf. 2003 www.cdc.gov
Laboratory Diagnosis of Human cases
• SpecimensCerebrospinal fluid (CSF)CSF and Serum combinationSera- 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
Laboratory Diagnosis of Human cases contd
Ref: Antibody Capture ELISA (IgM & IgG) Protocol. CDC Fort Collins, colorado
CDC Neutralization Test Protocol. CDC Fort Collins, colorado
•Laboratory Tests
Capture enzyme-linked immunosorbent assay (MAC-ELISA-IgM).
Capture enzyme-linked immunosorbent assay (MAC-ELISA-IgG) and
Plaque Reduction Neutralization Test (PRNT)
HRPHRP
1. Coat With Goat anti-Human IgM 4° Overnight
2. Add Patient Serum @ 1:400 37° 1 Hour
3. Add West Nile Recombinant Antigen 4° Overnight
4. Add HRP anti-Flavivirus McAb 37° 1 Hour
IgM Capture ELISAIgM Capture ELISA
Ref: 4th National WNV Conf. 2003 www.cdc.gov
WN Serological DataWN Serological DataTypical Human WN Case
Days IgM P/N IgG P/N PRNTSample post-onset WN SLE WN SLE WN SLE
Typical WN Caseacute serum 8 12.75 4.00 1.37 2.04 1:80 1:20conv. serum 31 11.35 4.21 6.38 5.76 1:1280 1:80
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
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 2-10 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
Identifying viremic / antibody negative “window phase” patients
Low levels WNV RNA found in clinically ill, immuno- competent patients (i. e., WN fever)
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
Laboratory Issues and West Nile Virus
ArbovirusesLaboratory DiagnosisInvestigation of 2002 WNV
outbreak in MichiganPlans for 2003-Lab perspective
55
94
240
266
319
270 270289
333
193
144
231
7055
422311 16 20 20
91
61 65
38 38
12 16 14 620
0
112
0
50
100
150
200
250
300
350
11-Aug
18-Aug
25-Aug
01-Sep
08-Sep
15-Sep
22-Sep
29-Sep
06-Oct
13-Oct
20-Oct
27-Oct
03-Nov
10-Nov
17-Nov
24-Nov
Weeks
Nu
mb
ers
total positive
Total Specimens (Jan, 02-Dec, 02)-2910
Weekly WNV Testing (Aug-Nov 02)
Virology section and intersection staff worked collectively 6 days a week
How we handled the 2002 outbreak in the laboratory ?
Triaged the samples
CSF at MDCH 0 day
IgM ELISA 1st Run Report
via EPIC
NegNotificationVia EPIC toSubmitter,
LDH & EPI 4 day
Pos
Investigation
Confirmed positive reportsVia
EPIC 5 6day
IgM ELISA 2nd Run
pos
Quantity Sufficient
pos
Equivocal
Request a convalescent serum sample
QNS
To report a Probable case
request a serum sample
Convalescent serumNo
PRNT on acute serum (Convalescent sample not possible )
Pos
Acute Serum at MDCH (0day)
NegIgM ELISA 1st runReport Via EPIC
No 4x increase
4x rise in titer
Yes
PRNT on pair
Confirmed positive reportsVia
EPIC
Arboviral
Panel
IgM ELISA 2nd run
Reported a presumptive positive (LAB) & probable case (EPI)Via EPIC with a request for a convalescent serum
Pos
Pos
Pos
Investigation
Notification via EPIC to
Sub, LHD & EPI
DAYS POST ONSETDAYS POST ONSET
1 2 3 4 5 6 7 8 9 10-5 -4 -3 -2 -1 0
IgM
IgG
ELISAELISAP/NP/N#pfu/ml#pfu/ml
WNviremia
150
illnessillness
Immune Response in WNV Infection
Ref: 4th National WNV Conf. 2003 www.cdc.gov
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
Laboratory Issues and West Nile Virus
ArbovirusesLaboratory DiagnosisInvestigation of 2002 WNV
outbreak in MichiganPlans for 2003-Lab perspective
Getting Ready for summer of
2003
W N V Surveillance in M ichigan
Hum an Surve i l lance
A rbovira l Pane l T es ing
Hum an T seting
BO E & BO L
M D C H
Equine Surve i l lance
M D A
W ild Bird Surve i l lance
M D NR
M osquito pools
M SU
M osquito pools
D ept. of Entem ology
Equine and Bird tes ting
A HD L /D C PA H
Cooperative plan
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
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
Testing reagents for 2003
No change in personnel or policy Commercial Partners – patent license agreements
for WN antigen production.
1995 - 1999 100 – 150 Reagent Requests/year
2002 560 reagent requests
CDC Reagent Production
Focus Technologies
Ortho Clinical Diagnostics
Abbott Laboratories
Chiron (recombinant antigens)
Pan -Bio
Manufacturers with WNV Antibody Assays in development
Future of new assays for WNV
Synergy between
Industry Regulatory agencies Public and Private laboratories
Issues with Persistence of IgM antibodies
Getting Ready for summer of 2003 contd
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 (3.0-10.8)
300-400
21 9 (43) 2 11 (52) 4.0 (31.-6.5)
500 12 5 (42) 2 6 (60) 5.0 (3.1-6.9)
Ref: 4th National WNV Conf. 2003 www.cdc.gov
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 RunPos
Pos
Report
Acute Serum at MDCH
2003 Testing
Yes
4x rise in titer
PRNT (IgG) on pair
Confirmed positive reportsVia
EPIC
No 4x increaseArboviral
Panel
Persistence of IgM antibodies
In CSF-No studies
Published-47d
MDCH observations-Three cases with igM positive
110d
141d
199d
Bird Testing in 2003
Strict IATA regulationsAvailability of a field assay-Vec TestValidation of IHC and PCR-2002IHC vs Vec Test at MSU lab in 2003Implementation of vec test ??
To Sum Up……. Human Cases ?
Over wintering
adult Culex mosquitoes