Test Method Validation

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Thomas Haupt M.S. Wisconsin Division of Public
Health 608-266-5326 hauptte_at_dhfs.state.wi.us
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Arbovirus Surveillance -Human Surveillance

• Goals
• Assess local, state, and national public health
  impact of WNV disease and other arboviruses
• Demonstrate need for public health intervention
  and allocate resources
• ID risk factors for infection and high-risk
  population groups

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2003 Arbovirus Activity in Wisconsin, USA, Human
West Nile 17 9862 La Crosse 15
123 EEE 0 14 WEE 0 0 SLE
0 32 Cache Valley 1 1 Dengue 1 2
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WNV Human Infection Iceberg
10 fatal (lt0.1 of total infections)
1 CNS disease case 150 total infections
lt1 CNS disease
Very crude estimates
20 West Nile Fever
80 Asymptomatic
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Human West Nile Virus Cases Wisconsin 2003 n17
Milwaukee
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Human West Nile Cases in the U.S., 2003
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Case Definition of Arbovirus Infections
(Human) EEE, LAC, SLE, WNND (Excluding West Nile
fever) Clinical Description Meningitis
characterized by fever, headache, stiff neck,
and pleocytosis or encephalitis characterized by
fever, headache, and altered mental status
ranging from confusion to coma with or without
additional signs of brain dysfunction
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• Case Definition of Arbovirus Infections (Human)
• EEE, LAC, SLE, WNVND (Excluding West Nile fever)
• Laboratory criteria for diagnosis
• Virus-specific IgM antibodies demonstrated in
  CSF by
• antibody-capture enzyme immunoassay (EIA), or
• Virus-specific IgM antibodies demonstrated in
  serum by
• antibody-capture EIA and confirmed by
  demonstration of
• virus-specific serum immunoglobulin G (IgG)
  antibodies in
• the same or a later specimen by another
  serologic assay (e.g., PRNT).
• Fourfold or greater change in virus-specific
  serum antibody titer, or
• Isolation of virus from or demonstration of
  specific viral antigen

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Case Definition of Arbovirus Infections
(Human) EEE, LAC, SLE, WNND (Excluding West Nile
fever) Case Classification Confirmed case An
encephalitis or meningitis case occurring during
a period when arboviral transmission is likely
that is laboratory confirmed by one of the four
previously mentioned test methods. Probable
case An encephalitis or meningitis case
occurring during a period when arboviral
transmission is likely, with laboratory
confirmation of serum IgM antibodies detected by
antibody-capture EIA but with no available
results of a confirmatory testing.
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• Case Definition of West Nile fever
• Clinical
  Description
• A non-specific, self-limited, febrile illness
  caused by infection
• with West Nile virus, characterized by the acute
  onset of
• fever, headache, arthralgias, myalgias, and
  fatigue.
• Laboratory Criteria for diagnosis
  
• West Nile virus-specific IgM antibodies
  demonstrated in serum
• by antibody-capture enzyme immunoassay and
  confirmed by
• demonstration of West Nile virus-specific
  serum neutralizing
• antibodies in the same or a later specimen.
• Fourfold or greater change in West Nile
  virus-specific serum
• antibody titer.
• Isolation of West Nile virus from or
  demonstration of specific

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Case Definition of West Nile fever
Case
Classification Confirmed A clinically
compatible illness, that is laboratory confirmed
by one of the three previously mentioned test
methods. Probable Case A clinically
compatible illness with laboratory confirmation
of serum IgM antibodies detected by
antibody-capture EIA but with no available
results of a confirmatory testing.
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• Fee-exempt testing
• Fee-exempt testing at the WSLH for arbovirus
  infection
• will be offered to clinicians whose patients
  meet one of
• the following criteria
• Confirmatory testing of positive test results
  performed
• at laboratories other than the WSLH
• The patient is over 65 years old with signs and
  
• symptoms of meningitis or encephalitis with
  no
• other laboratory diagnosis or
• The patient has a diagnosis of Guillain-Barré
• syndrome and no other laboratory diagnosis.
• A local health department request if the
  case-patient
• lacks insurance coverage or the ability to
  pay.

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Arbovirus Surveillance - Sick/Dead Wild Bird
Surveillance

• Goal - utilize WNV bird mortality as a means of
  detecting WNV activity in a location
• Some wild bird species in North America, such as
  crows, are highly susceptible to WNV
• Crows can be used as a sentinel species
• Detection of WNV in wild birds not a predictor of
  human risk of WNV

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• Corvid Surveillance in Wisconsin 2003
• gt1500 were reported
• 387 were collected and tested for WNV
• 164 were positive for WNV.
• 48/72 counties reported positive cases

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Avian West Nile Virus Cases Wisconsin 2003 n 164
Cases by County
0 Cases 1-5 Cases 6-10 Cases gt10 Cases
Milwaukee
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Avian West Nile Cases in the U.S., 2003
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Arbovirus Surveillance - Veterinary Surveillance

• Goals
• Equine health impact of WNV and other arboviruses
• ID risk factors for high-risk population groups
  or geographic areas
• Identify movement of arboviruses, particularly
  WNV and EEE, into mammal populations

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Equine West Nile Virus Cases Wisconsin 2003 n64
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Equine West Nile Cases in the U.S., 2003
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Arbovirus Surveillance -Mosquito Surveillance

• Goals
• Assess threat of human disease
• ID geographic areas of high risk
• Assess need for and timing of intervention events

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• Mosquito Surveillance in Wisconsin 2003
• 2500 pools tested
• 2 positive

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Mosquito Trapping Sites Wisconsin 2003 n11
Milwaukee
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Laboratory Testing for West Nile Virus

• Michael Doering
• Microbiologist
• Communicable Disease Division
• Wisconsin State Laboratory of Hygiene

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West Nile Virus Diagnostic Assays

• Serological Assays for WNV
• IgM ELISA (CDC, Focus, PanBio
• IgG ELISA (CDC, Focus)
• Plaque Reduction Neutralization (PRNT)
• IFA (PanBio)
• IgA ELISA (CDC)
• Microsphere Immunoassay (CDC, NYSDH)

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CDC Tests for WN Virus
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WN Virus Testing at WSLH

• Avian Testing
• LightCycler Real-Time RT-PCR
• Cloacal Swabs
• Human Testing
• IgM ELISA
• Serum dilution neutralization assay
• WSLH

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ELISA P/N
pfu/ml
IgM
150
IgG
WN viremia
1 2 3 4 5 6 7 8 9 10
-5 -4 -3 -2 -1 0
DAYS POST ONSET
illness
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CDC IgM Capture ELISA

• Coat With Goat anti-Human IgM
• 4 Overnight

• Add Patient Serum _at_ 1400
• 37 1 Hour

• Add West Nile Recombinant Antigen
• 4 Overnight

• Add HRP anti-Flavivirus McAb
• 37 1 Hour

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Interpretation of Results

• P/N O.D. patient serum/O.D. negative control
  serum.
• P/N gt 3 positive
• P/N lt 2 negative
• P/N 2-3 equivocal
• ELISA Assay must be standardized in each lab

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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.

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Analysis of 1,336 IgM Positive Serum Specimens
for WN to SLE Ratio
CDC Centers for Disease Control and Prevention
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Longevity of Human WN Virus-Reactive IgM in Serum
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CDC IgM ELISA Assay

• Good Points
• Sensitive
• Relatively Specific (WN SLE P/N ratio)
• Technology Transferable

• Bad Points
• Cross-reactivity among flaviviruses
• Limited utility in secondary infections
• Two day test
• IgM persistence

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Focus IgM Capture ELISA

• Precoated with rabbit anti-human IgM

• Add Patient Serum _at_ 1101
• 37 1 Hour

• Add West Nile Recombinant Antigen
• 37 2 Hours

• Add HRP anti-Flavivirus McAb
• 37 30 minutes

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Focus Technologies WNV IgM ELISA

• IgM (mµ) capture format
• WNV recombinant protein (lyophilyzed)
• Peroxidase labeled monoclonal antibody
• Specimen/Antigen diluent, TMB, stop solution
• Cutoff, negative control, positive control
• One day test
• Patient Index Value Patient specimen optical
  density (OD)
• Mean of
  the Calibrator-Cutoff OD
• gt1.10 Positive
• 0.90 1.10 Equivocal
• lt 0.90 Negative

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Plaque Reduction Neutralization Test

• Tested serum and live virus added to cell culture
• If there are antibodies against the virus in the
  test serum
• reduction in virus damage
• compared to control with no antibodies

• Best test for differentiating WNV from SLE,
  dengue or Yellow Fever
• however
• It requires handling cell cultures and live virus
• It takes several days to evaluate plaque
  reduction

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Serological Testing Algorithm for West Nile Virus
human serum/csf
National Case Definition Confirmed IgM pos
csf IgM pos serum PRNT gt4-fold increase PRNT
titer
IgM ELISA WN SLE
NEG
POS
STOP
Plaque reduction Neutralization test (PRNT)
with SLE, WN, (other flaviviruses)
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WN Human Serological Data
Lessons Learned 1999-2002

• IgM Detectable in serum csf by onset (99)
• 6 exceptions------ serum from 800 cases
• 10 exceptions----- csf from 800
• IgG Positive by day 7 Post-Onset
• P/N 3-5X Higher to WN than SLE
• IgM Persistence gt 1 Year
• Secondary Flavivirus Infections are Problematic

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WSLH Arbovirus Serology

• Arbovirus IgM CEIA Ab Diagnostic Panel
• West Nile Virus
• St. Louis Encephalitis
• LaCrosse Encephalitis
• Eastern Equine Encephalitis
• Panel Cost
• 120
• Single Test Cost
• 30
• CDD Requisition Form B

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Thank you to Dr. Rob Lanciotti and CDC for the
use of their slides and data