Laboratory Tests for WNV:
The symptoms of infection with WNV are similar to those observed for a variety of other diseases, such as enterovirus or other arboviral infection. Laboratory testing is required for a confirmed diagnosis.

The most common test for WNV infection is to measure antibodies in blood (serum) and/or cerebrospinal fluid (CSF, the fluid surrounding the brain and spinal cord). There are two types of antibody tests:

1. Detection of IgM antibodies. IgM is produced shortly after symptoms occur, and both serum and CSF are positive in most WNV-infected people within 8 days of onset of illness. The University Hygienic Laboratory (UHL) uses the WNV IgM-antibody test developed by CDC. This test has been shown to be highly specific and there are very few false positive results.
2. Detection of IgG antibodies. IgG is produced later in an infection. IgG may also be present in blood for reasons not related to a recent WNV infection, such as Yellow fever vaccination, infection with a virus related to WNV, or evidence of a past exposure to a related virus. This test is less specific, i.e. may produce false-positive results, and cannot be used to confirm WNV infection without further testing.

What is the best way to test for WNV Infection?

1. Submit the best sample: Serum and/or CSF collected 8 days from symptom onset. A second blood sample collected 21 days from the onset of illness is required in most cases to confirm infection.
2. Use the best test: The IgM test performed at UHL and the CDC is currently the most reliable test. Results from commercial laboratories may need to be confirmed at UHL, especially if the disease prevalence in Iowa is low at the time of the positive result.
3. Submit with UHL submission and patient history form.

WNV can also be detected by demonstration of specific viral antigen or genomic sequences using RT-PCR. These tests are typically used by UHL on mosquitoes and dead birds for surveillance. UHL performs RT-PCR on all CSF samples, but it is less sensitive than the IgM test due to a short viremic stage in humans. However, RT-PCR may be a useful test in immunosuppressed patients.

Specimens Accepted

UHL is currently testing specimens for presence of IgM and IgG antibodies against West Nile virus. An ELISA will be performed on all submitted specimens. This test generally takes at least 2 days to complete.

• Allowable specimens include acute serum, convalescent serum, and cerebrospinal fluid (CSF).
• Acute serum should be collected between 5-14 days from the onset of symptoms.
  • Best results at 8 days from symptom onset.
• Convalescent serum should be collected 21 days after onset of illness.
• CSF should be collected 3-10 days from symptom onset
  • Best results at 8 days from symptom onset
  • Keep CSF cold.
• Please keep specimens cold and ship on ice packs, as this increases the sensitivity of the test.
• Testing will be performed Monday-Thursday during peak season.

Interpretation of Test Results:

• Demonstration of WNV IgM antibody in CSF is considered diagnostic confirmation of WNV infection and strongly suggests central nervous system infection.
• Demonstration of WNV IgM antibody in serum is diagnostic, however, false positives and cross reactivity can occur especially in patients recently vaccinated against or infected with related Flaviviruses (e.g., St. Louis, Yellow fever, Japanese encephalitis, Dengue).
• WNV-specific IgM has persisted in patients for >500 days. Positive serologic tests must be considered in relation to clinical presentation.
• Due to low specificity, IgG antibody tests are not useful in the diagnosis of acute WNV infection. An IgG positive test result, either alone or in conjunction with a IgM negative test, is not diagnostic for acute infections and is NOT considered a case of WNV.
• An equivocal test result suggest a questionable presence of West Nile Virus IgM/IgG antibody detected. Repeat testing in 10-14 days may be helpful.
• What does an indeterminate result mean? In some situations a cross reaction can be due to heterophile antibodies, in which case the result is reported as indeterminate. Heterophile antibodies are non-specific antibodies that react with antigens in the test kit. If the clinical condition warrants, it may be appropriate to obtain an additional sample for retesting.

Request for Convalescent Specimens:

• Convalescent specimens will be requested on patients that submit an acute sample that is less than 8 days post symptom onset and test negative for IgM antibody with patient reporting clinical symptoms consistent with WNV infection. Other request for convalescent specimens include patients that have an equivocal or indeterminate test result after repeat testing at UHL.