1. Algorithms and Testing Results for West Nile/Arbovirus Testing (2002) used at Michigan Department of Community Health H. Kapoor, P. Clark, F. P. Downes, P. Somsel, R. Oesterle, M. Casey and D. Wilkinson Michigan Department of Community Health, Lansing Abstract Michigan reported the second highest count of human cases of West Nile Virus (WNV) during the 2002 outbreak in the US. A total of 2910 human specimens, primarily cerebrospinal fluids and sera, were received from January 1 until December 31, 2002, of which 575 tested positive. The first confirmed human case of WNV was during the week of Aug 11, 2002 with the volume of requests increasing dramatically thereafter and reaching a peak of 333 per week. In response to an unanticipated volume of specimen submissions the Bureau of Laboratories (BOL) at Michigan Department of Community Health (MDCH) developed algorithms jointly with the Bureau of Epidemiology (BOE) using the CDC case definition and guidelines for WNV/Arbovirus testing. These emergent algorithms were shared with local health departments. The first line test utilized for diagnosis of WNV was the IgM-capture enzyme-linked immunosorbent assay (MAC-ELISA). A positive IgM on CSF was sufficient to establish a confirmed case of WNV. Other tests employed were IgG ELISA and Plaque Reduction Neutralization Test (PRNT). A four-fold rise in IgG titers in paired sera drawn in acute and convalescent stages (at least 22 days apart) distinguished a recently acquired infection from a past infection. To rule out the cross- reactions between WNV and other Arbovirus infections endemic in Michigan (SLE, EEE and CGV), antibody specificity was confirmed by PRNT. Communications of the positive results were expedited using electronic reporting to local health departments, submitters and the BOE. The testing algorithms adopted at BOL, MDCH and results of serological assays are presented. Introduction The Michigan Department of Community Health (MDCH) started conducting WNV surveillance in a cooperative plan with the Michigan Department of Agriculture (MDA), Michigan Department of Natural Resources (MDNR), Michigan State University (MSU) Animal Health Diagnostic Laboratory (AHDL) and Department of Entomology since 2000. The group focused on collection and testing of dead crows and blue jays, beginning May through September of 2001. The surveillance resulted in the positive birds as well mosquito pools in Michigan. Though 159 human specimens were tested in a serologic panel for the arboviruses, which includes WNV, St. Louis encephalitis (SLE), Eastern equine encephalitis (EEE) and California group virus encephalitis (CGV), none was found positive for WNV during 2001. The surveillance guidelines were revised for the year 2002, with the addition of a hot line and a web site for reporting of dead bird sightings. Reports to the hot line alone in 2002 totaled over 35,000 calls; 73 out of 83 participating counties in the state submitted birds, which tested positive, the first collected on May 14. Requests for human specimen testing of arbovirus panel started rising from June, 2002 with the first laboratory confirmed case reported during the week of August 11, 2002. Materials and methods Specimens  Cerebrospinal fluid (CSF)  CSF and Serum combination  Sera- Acute and Convalescent (obtained 22 days post onset)  Least preferred single serum sample 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 Contact Information Hema Kapoor M.D. Virology Section Manager Bureau of Laboratories Michigan Department of Community Health phone: (517) 335-8099 email: kapoorhe@michigan.gov Total Specimens (Jan, 02-Dec, 02)-2910 Results Three algorithms were developed depending upon Algorithm-1Algorithm-2Algorithm-3 CSF specimen received for testing. P/N ratio of 10 and above was considered a positive WNV. P/N between 2-10 was reported equivocal and requested for a serum sample. Paired sera ( Ac. & convalescent ) P/N ratio of 5 and above was considered a presumptive WNV. P/N between 2-5 was reported equivocal. A four-fold rise in IgG titers in paired sera drawn in acute and convalescent stages (at least 22 days apart) was used to distinguish a recently acquired infection from a past infection. PRNT was performed on all specimens showing a four fold increase in IgG titer. Single serum sample received for testing. P/N ratio- same as in algorithm-2. A convalescent serum was requested on a specimen showing equivocal titers. To rule out the cross- reactions between WNV and other arbovirus infections (SLE, EEE and CGV), specificity of the antibody in the specimen was confirmed by PRNT. MAC-ELISA IgM performed in singlet. Positive MAC-ELISA repeated in duplicate. CSF at MDCH 0 day IgM ELISA 1 st Run Notification Via EPIC to Submitter, LDH & EPI 3-4 day Pos Neg Report via E PIC 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 Interpretations for the Algorithm 1 1.CSF was the highest priority specimen at MDCH. 2.If the result was negative, then the submitter and the local health department were informed via EPIC, the lab reporting system 3.If the result was positive on the first run, then this was considered a Presumptive Positive and a notification was made to the to the submitter, local health department and the Bureau of Epidemiology. 4.If there was sufficient CSF, the EIA test was repeated in duplicate, and a repeat positive result was reported out via EPIC 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 was made for convalescent serum. This case would be classified as a probable case of West Nile Virus encephalitis. 5.If there was insufficient CSF for a repeat test, the result was reported out as QNS (quantity not sufficient) and a request was made from the submitter for a serum sample. Algorithm 1 PRNT on pair IgM ELISA 2 nd Run Slide 3 Report Via EPIC Neg Pos 4x rise in titer Yes No PRNT on acute serum (Convalescent sample not possible ) Convalescent serum Confirmed positive reports Via EPIC Pos IgM ELISA 1 st Run Acute Serum at MDCH (0day) Reported a presumptive positive (LAB) & probable case (EPI) Via EPIC with a request for a convalescent serum Pos Notification via Remedy to LDH & EPI Investigation No 4x increase Slide 3 Algorithm 2 Interpretations for the algorithm 2 Serum specimens did not receive priority unless they were accompanied by clinical information which suggested CNS involvement. If the IgM EIA result was negative, then the submitter and the local health department were informed via EPIC, the lab reporting system. These were classified as No Case. When the IgM EIA result was positive then this was a preliminary positive and was reported to the local health department to initiate an investigation. Please note this was only preliminary positive and not a confirmation. This test result was not characterized as positive for WNV at this stage. The serum was then retested in duplicate by EIA. If this was again positive, then the test result was considered a presumptive positive and the case could be classified as a Probable Case of West Nile Virus. The submitter and the local health department were notified of the result via EPIC. The submitter was asked to submit a convalescent serum, taken at least 22 days after the acute specimen. If a convalescent serum was not available then a Plaque Reduction Neutralization test (PRNT) was performed on the acute specimen. If specific neutralizing antibodies were detected, the test result was considered positive and the case was classified as a Confirmed Case of WNV. If a convalescent serum was available it was tested along with the acute serum in a PRNT and the titers compared. A four-fold increase in titer was evidence of a recent infection and the test result was reported as positive and the case considered a Confirmed Case of WNV. If a four fold increase in titer was not observed, but specific neutralizing antibodies detected, the test result was reported as positive and the case considered a confirmed case of WNV. Single serum with Documented CNS symptoms or paired sera without 4 x increase in titer IgM ELISA (+) PRNT (-) No WNV Case Probable WNV Case Test for EEE and CGV Neg NoYes Single serum collected too early (0-8 d) after onset of symptoms Paired sera Yes Algorithm 3 Interpretations for the algorithm 3 In a few cases there were situations where results were more difficult to interpret. A single serum sample from a patient who had documentation of CNS symptoms, and who had a positive WNV IgM EIA and a negative WNV/SLE PRNT could have an encephalitis due to another flavivirus. The EIA could be positive because of the cross-reactivity that can occur with flaviviruses, whereas the PRNT is considerably more specific and can discriminate between the viruses. If the IgM and PRNT for EEE or the California group of encephalitic viruses (CGV) were positive, then the case could be reported and investigated as a EEE or other viral encephalitis. If the IgM & PRNT for EEE and CGV were negative, it might 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 was 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 and the case was classified as not a Case. Conclusions  We found these algorithms to be a handy tool for use by laboratory personnel, especially during the initial phases of WNV outbreak when the laboratory was overwhelmed with clinical specimens.  We had an easy and useful way of communicating laboratory results to the Epidemiologists, Local Health Departments and the submitters engaged in the investigation of the outbreak using these algorithms. Acknowledgements We wish to acknowledge the CDC for ELC (Epi/ Lab Cooperative agreement) grants (regular and supplemental #U50/CCU514403-05-2 and the following organizations and their staff who helped in completing the Arbovirus testing for this outbreak. MDCH Bureau of Laboratories. MDCH Bureau of Epidemiology. Michigan Department of Agriculture. Michigan Department of Natural Resources. Michigan State University Animal Health Diagnostic Laboratory and Department of Entomology.