1. Testing algorithms used at Bureau of Labs Michigan Department of Community Health Information on testing algorithms for processing and reporting serological assays on arbovirus panel with specific reference to West Nile Virus testing

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

3. Notes to Slide 1 1. CSF is the preferred specimen to look for IgM antibodies to establish a diagnosis of WNV encephalitis. 2.If the result is negative, the submitter and the local health department will be informed via EPIC, the lab reporting system. 3.If the result is positive on the first run, this is considered a Presumptive Positive and a notification is made to the to the local health department and the submitter. Please note that this is not a confirmed positive and should not be characterized that way. 4.If there is sufficient CSF, the EIA test is repeated, and a repeat positive result is reported out on EPIC ( Electronic reporting system) to the submitter and the local health department as a confirmed positive result. This would meet the case definition of a confirmed case of West Nile Virus encephalitis for surveillance purposes. If the test result is equivocal, a request will be made for convalescent serum ( drawn at least 22 days post onset of illness).This case would be classified as a probable case of West Nile Virus encephalitis. 5.If there is insufficient CSF for a repeat test, the result is reported out as QNS (quantity not sufficient) and a request is made from the submitter for a serum sample.

4. Slide 2 Hold and request Convalescent serum Acute Serum at MDCH Yes 4x rise in titer PRNT (IgG) on pair Confirmed positive reports Via EPIC No 4x increase Slide 3 PRNT on acute serum Pos No CNS symptoms + Testing Approved by Lab Manager Neg IgM ELISA 1 st Run IgM ELISA 2 nd Run Pos Report

5. Notes to Slide 2 1.Acute serum sample when received at MDCH will be held and a request will be sent for a convalescent sample ( drawn at least 22 days post onset of the illness). This is due to the documentation of persistence of IgM antibodies in serum specimens up to 525 days post onset of illness. 2.If a convalescent serum is not available, contact the Virology Lab Manger. In cases where the history and clinical picture are suggestive of a recent WNV infection, a Plaque Reduction Neutralization test (PRNT) will be performed on the acute specimen. If this is confirmed, the test result will be considered positive and the case will be classified as a Confirmed Case of WNV. 3.If a convalescent serum is available it will be tested along with the acute serum in a PRNT and the titres compared. A four-fold increase in titre is evidence of a recent infection and the test will be reported as positive and the case considered a Confirmed Case of WNV. 4.The submitter, Epidemiology and the local health department will be notified of the results via EPIC.

6. Single serum with Documented CNS symptoms or paired sera without 4 x increase in titer IgM ELISA (+) Netralizing Antibody (-) No WNV Case Probable WNV Case Test for EEE, SLE and CGV Neg No Yes Slide 3 Single serum collected too early (0-8 d) after onset of symptoms Paired sera

7. Notes for Slide 3 In a few cases there will be results which are more difficult to interpret. A single serum sample from a patient who has documentation of CNS symptoms, but who has a positive EIA and a negative PRNT could have an encephalitis due to another flavivirus. The EIA could be positive because of the cross-reactivity that can occur with flaviviruses. The PRNT is considerably more specific and can discriminate between the viruses. If the tests for EEE and the California group of encephalitic viruses (CGV) are positive, the case should be reported and investigated as a EEE or other viral encephalitis If the tests for SLE, EEE and CGV are negative then it may be that the serum was collected too early for the generation of sufficient IgG antibody to be detected in the plaque reduction neutralization test. If the serum was collected within 8 days of symptom onset the case is classified as a Probable case of WNV If the serum was collected more than 8 days after the onset of symptoms the test result would not be considered positive. This would be classified as Not a Case.