West nile virus laboratory tests

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]

Overview

In 1999 in the U.S., the sensitivity of polymerase chain reaction (PCR) tests of CSF for the diagnosis of human WN encephalitis cases was only 57%; more recent statistics are currently unavailable. Thus, PCR for the diagnosis of WN viral infections of the human central nervous system (CNS) continues to be experimental and should not replace tests for the detection of WNV-specific antibody in CSF and serum, tests that are far more sensitive.

A high clinical suspicion for arboviral encephalitis should be encouraged among health care providers. When the diagnosis is in doubt, appropriate clinical specimens should be submitted to CDC or another laboratory capable of performing reliable serologic testing for antibodies to domestic arboviruses. Testing of CSF and paired acute- and convalescent-phase serum samples should be strongly encouraged to maximize the accuracy of serologic results.

Laboratory Tests

Appropriate selection of diagnostic procedures and accurate interpretation of findings requires information describing the patient and the diagnostic specimen. For human specimens, the following data must accompany sera, CSF or tissue specimens for results to be properly interpreted and reported: 1) Symptom onset date (when known) 2) Date of sample collection 3) Unusual immunological status of patient (e.g., immunosuppression) 4) State and county of residence 5) Travel history (especially in flavivirus-endemic areas) 6) History of prior vaccination (e.g., yellow fever, Japanese encephalitis, or Tick-borne encephalitis viruses) 7) Brief clinical summary including clinical diagnosis (e.g., encephalitis, aseptic meningitis).

Minimally, onset and sample collection dates are required to perform and interpret initial screening tests. The remaining information is required to evaluate any specimens positive on initial screening. If possible, a convalescent serum sample taken at least 14 days following the acute sample should be obtained to enable confirmation by serological testing

Serology

The front-line screening assay for laboratory diagnosis of human WNV infection is the IgM assay. Currently, the FDA has cleared four commercially-available test kits from different manufacturers, for detection of WNV IgM antibodies. These four kits are used in many commercial and public health laboratories in the United States. In addition the CDC-defined IgM and IgG ELISA can be used; protocols and reagents are available from the CDC DVBD Diagnostic Laboratory. There is also a microsphere-based immunoassay for the detection of IgM antibodies that can differentiate WNV from SLE. Because the IgM and IgG ELISA tests can cross-react between flaviviruses (e.g., SLE, dengue, yellow fever, WN), they should be viewed as screening tests only. For a case to be considered confirmed, serum samples that are antibody-positive on initial screening should be evaluated by a more specific test; currently the plaque reduction neutralization test (PRNT) is the recommended test for differentiating between flavivirus infections. Though WNV is the most common cause of arboviral encephalitis in the United States, there are several other arboviral encephalitides present in the country and in other regions of the world. Specimens submitted for WNV testing should also be tested by ELISA and PRNT against other arboviruses known to be active or be present in the area or in the region where the patient traveled.

Virus Detection Assay

Numerous procedures have been developed for detecting viable WNV, WNV antigen or WNV RNA in human diagnostic samples, many of which have been adapted to detecting WNV in other vertebrates and in mosquito samples. These procedures vary in their sensitivity, specificity, and time required to conduct the test. The VectorTest, Antigen Capture ELISA, and Rapid Analyte Measurement Platform were developed specifically for testing mosquitoes for WNV antigen, were subsequently adapted to testing bird and other vertebrate samples, and are not used for human diagnostic testing. Additional details about these tests are contained in the following sections on mosquito and bird diagnostic tests. The remaining tests have been used in various human diagnostic assays.

Among the most sensitive procedures for detecting WNV in samples are those using RT-PCR to detect WNV RNA in human CSF, serum and other tissues. Fluorogenic 5' nuclease techniques (real-time PCR) and nucleic acid sequence-based amplification (NASBA) methods have been developed and validated for specific human diagnostic applications and for detecting WNV RNA in blood donations. WNV presence can be demonstrated by isolation of viable virus from samples taken from clinically ill humans. Appropriate samples include CSF (serum samples may be useful very early in infection) and brain tissue (taken at biopsy or postmortem). Virus isolation should be performed in known susceptible mammalian (e.g., Vero) or mosquito cell lines (e.g., C6/36). Mosquito origin cells may not show obvious cytopathic effect and must be screened by immunofluorescence or RT-PCR. Appropriate samples for virus isolation from clinically ill humans include CSF (serum samples may be useful very early in infection) and brain tissue (taken at biopsy or postmortem). Confirmation of virus isolate identity can be accomplished by indirect immunofluorescence assay (IFA) using virus-specific monoclonal antibodies or nucleic acid detection. The IFA using well-defined murine monoclonal antibodies (MAbs) is an efficient, economical, and rapid method to identify flaviviruses. MAbs are available that can differentiate WNV and SLE virus from each other and from other flaviviruses. Incorporating MAbs specific for other arboviruses known to circulate in various regions will increase the rapid diagnostic capacities of state and local laboratories. Nucleic acid detection methods including RT-PCR, TaqMan and nucleic acid sequence based amplification (NASBA) methods may be used to confirm virus isolates as WNV

References

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