With the awareness of other modes of transmission of WNV (blood transfusion, organ transplantation, transplacental and breast milk) as well as concern for travel by Michigan residents to endemic areas where vectors are active year-round, BOL will test for WNV only on specimens submitted May 1 through October 31.  Samples submitted outside of this time period must be sent to CDC for testing.  Please include a completed CDC test request form for samples submitted from November 1 through April 30. 

*Powassan: This viral infection has been seen only along the eastern coast of US, although Michigan Department of Community Health (MDCH) reported its first Laboratory confirmed case in April, 2002.

Who should be tested for arboviral disease?

Hospitalized patients with any of the following syndromes:

1. Viral encephalitis , a clinical diagnosis characterized by:

   1. Fever > 38ºC or 100ºF; and

   2. CNS involvement, including altered mental status (altered level of consciousness, confusion, agitation, or lethargy) or other cortical signs (cranial nerve palsies, paresis or paralysis, or convulsions); and

   3. An abnormal CSF profile suggesting a viral etiology (a negative bacterial stain and culture with a pleocytosis [WBC between 5 and 1500 cells] and/or an elevated protein level > 40 mg/dL).

2. Viral meningitis, without recovery in 72 hours (aseptic meningitis due to enterovirus is typically of short duration and has a benign clinical course).

3. Guillain-Barre syndrome, especially with atypical features, such as fever, altered mental status, and/or a pleocytosis.

4. Patients presenting with febrile illness of sudden onset often accompanied by malaise, anorexia, nausea, vomiting, headache, myalgia, rash, lymphadenopathy and eye pain also need testing for West Nile Fever.

Which Diagnostic Test to order?

1. Arbovirus IgM antibody (Microsphere Immunoassay [MIA] or IgM Capture ELISA [MAC-ELISA]).

• Cerebrospinal fluid - This is the preferred specimen for IgM testing. The most efficient diagnostic method for WNV is the detection of IgM anitbody in cerebral spinal fluid (CSF) collected at least 8 days post onset of illness. Since IgM antibody does not cross the blood-brain barrier, antibody in CSF strongly suggests central nervous system infection.  WNV, SLE, and EEE IgM are tested at MDCH by MIA.  CGV IgM by MAC-ELISA.

• Serum - A single serum specimen for IgM MAC ELISA is NOT a preferred specimen. Presence of IgM antibodies in a single serum sample will not confirm a recent infection, because there has been a documented evidence of presence of IgM antibodies in serum for up to 500 days post-onset. Patients who have been recently vaccinated against or recently infected with related arboviruses (e.g., yellow fever, Japanese encephalitis, dengue) or those who had a WNV infection in the 2002 outbreak might have positive WNV MAC-ELISA results unrelated to recent WNV infection.

     2.  IgG antibody enzyme-linked immunosorbent assay (IgG-ELISA)

• Due to decreased funding for WNV testing, MDCH no longer performs IgG testing for any arbovirus.  Testing is available at CDC.  Please submit an acute and convalescent sample with a completed CDC test request form. 

     3.  Plaque Reduction Neutralization Test (PRNT)

• CSF or Paired Sera - Done as a confirmatory test on CSF when the MIA or MAC-ELISA can not distinguish between flavivirus infections or on paired sera when the CSF was equivocal by the MIA or MAC-ELISA.  The results on this test are reported as neutralizing antibody present or absent for the respective arbovirus.

Comment:

• Paired Serum samples: Acute sample drawn at least 8 days post onset and a convalescent specimen drawn at least 22 days post onset.

• Single serum specimens from cases which have CNS symptoms with documented lumbar puncture failure will be accepted for testing for IgM only after consultation with the Virology Section Manager.

How can testing be arranged?

Testing for patients with clinical symptoms suggesting WNV encephalitis/meningitis/fever can be obtained through submission of specimens by laboratories or physician offices to MDCH.

Kits for submission of human specimens for arbovirus panel (unit # 8) can be requested from MDCH by faxing a request to 517-335-9039 or by calling 517-335-9867. Forms are included in the collection kits or can be printed of the web site (http://www.michigan.gov/documents/DCH-0583TEST_REQUEST_7587_7.pdf).

How are specimens submitted?

1. Collection of samples

• CSF : at least 1.5 ml should be submitted in sterile, plastic tubes. Hold CSF at 2-8^oC until shipped. Note: the tubes included in the collection kit#8 are non-sterile. Please replace with a sterile one.

• Serum: collect in a red top tube or SST. After sufficient clotting (one hour at room temperature), centrifuge the whole blood to separate cells from serum. Transfer at least 2.5 ml of serum to 12 X 55 mm plastic tubes. Label with unique patient identifier. Specimens less than 2.5 mL or specimens submitted in glass tubes are unsatisfactory and will not be tested. Freeze sera at -20 degrees C until ready to ship to MDCH.

2. Complete the request form

Complete the MDCH Test Requisition Form and provide the following additional information.  Also include this information on the CDC form submitted.

3. Shipping

• Place no more than two labeled tubes, wrapped in absorbent material such as paper towel or tissue, into aluminum screw-capped can and tighten the cap. Specimens may be shipped at ambient temperature provided there are no anticipated delays in transport beyond 48 hours. If transport of > 48 hours are expected, please ship the specimens on ice packs.

• Place aluminum can with completed test requisitions(s) and additional information into screw-capped cardboard container and secure tightened cap with tape.

• Complete and apply return orange address and clinical specimen label to cardboard container and ship by the most rapid and convenient means available (e.g., First Class, Priority or US Mail and Express mail with O/N delivery etc.) to the BOL at MDCH.

How to interpret results of arbovirus testing?

1. IgM MIA or MAC-ELISA:

• A positive IgM in CSF: is sufficient to establish a confirmed case of WNV infection (IgM has been documented in CSF for up to 141 days post-onset).

• A negative IgM in serum or CSF: The absence of IgM antibody must be interpreted with caution. The timing of the specimen collected with the onset of disease is crucial. Specimen must be drawn at least 8 days post-onset for detectable levels of antibody to be present. A specimen drawn too early in disease may be falsely negative due to undetectable levels of antibodies.

2. IgG ELISA:

• A positive IgG in Serum: A four-fold increase or rising serum antibody titers between appropriately timed acute and convalescent sera are presumptive evidence of recent infection.  Currently only available at CDC.

3. Neutralizing antibodies

• Neutralizing antibodies present : A four-fold or greater rise in neutralizing antibody titer between appropriately timed acute and convalescent sera is sufficient to establish a confirmed case of arboviral infection.

Reporting by Physicians and Non-Public Health Laboratorians

Provision of resources to control this disease is dependent to a great extent upon information on the incidence and prevalence of WNV in MI, which can come only from the community. Physicians as well as laboratorians are required by law to report cases of WNV and other unusual disease occurrences, outbreaks or epidemics to their local health departments. But assistance is required beyond reporting cases. If CSF specimens are submitted to non-public health laboratories where testing will not meet CDC standards to establish a case (IgM MIA or MAC-ELISA), clinical laboratories in Michigan are requested to retain a sample of CSF for submission to MDCH for confirmation of positive results. Because of the transmission of WNV in the state and the demonstrated protracted persistence of WNV IGM antibody, results of testing a single serum are uninterpretable. Alternatively, the testing laboratory can be notified to forward positive CSF specimens to MDCH if their test methods do not meet CDC standards to establish a case.

For further information on any aspect of arboviral testing in Michigan, call 517-335-8099.

Link to "Algorithms and Testing Results for West Nile/Arbovirus Testing (2002) used at Michigan Department of Community Health (Power Point Format)"

Link to "Testing Algorithms used at Bureau of Laboratories, Michigan Department of Community Health (Power Point Format)"

Michigan Department of Community Health, Bureau of Laboratories Web Page

Link to the Journal of the American Medical Association article on the long term effects of West Nile Virus in humans

CDC FactSheet: Treating and Testing West Nile Virus in Humans

updated 6/15/2009