Specimen Required

Serology: Suitable specimens are individual samples (human serum) obtained by standard laboratory techniques.

Specimen Minimum Volume

0.6ml

Transport Temperature

*The samples should be stored for not more than 48h at 2-8^°C.

**For longer delay, freeze at ≤ -20^°C and transport on dry ice.

Reject Due To

Useful For

Presumptive diagnosis of West Nile virus infection

Clinical Information

Most people who are infected with WNV will not have any type of illness. It is estimated that about 20% of those who become infected will develop West Nile fever with mild symptoms, including fever, headache, myalgia, and occasionally a skin rash on the trunk of the body. About 1 of 150 WNV infections (<1%) result in meningitis or encephalitis. Case fatality rates among patients hospitalized during recent outbreaks have ranged from 4% to 14%. Advanced age is the most important risk factor for death, and patients older than 70 years of age are at particularly high risk.

Laboratory diagnosis is best achieved by demonstration of specific IgG and IgM class antibodies in serum specimens. PCR can detect WNV RNA in specimens from patients with WNV infection when specific antibodies to the virus are not present. However, the likelihood of detection is relatively low as the sensitivity of PCR detection is approximately 55% in cerebrospinal fluid and approximately 10% in blood, from patients with known WNV infection.

West Nile virus (WNV) is a mosquito-borne flavivirus (single-stranded RNA) that primarily infects birds but occasionally infects horses and humans. WNV was first isolated in 1937 from an infected person in the West Nile district of Uganda. Until the viral infection was recognized in 1999 in birds in New York City, WNV was found only in the Eastern Hemisphere, with wide distribution in Africa, Asia, the Middle East, and Europe. In 2002, a total of 3,389 human cases of WNV infection were reported from 37 states (794 cases in Illinois); 2,354 (69%) presented with meningoencephalitis, 704 (21%) had West Nile fever, and 331 (10%) had an unspecified illness. Overall, the WNV epidemic in the United States was the largest arboviral meningoencephalitis outbreak documented in the Western hemisphere. In addition, 33 cases of probable WNV infection occurred among persons who had received blood components in the month before illness onset.

Reference Values

Non-reactive

Interpretation

Anti-WNV IgG

REACTIVE: Presence of specific IgG class antibodies in a serum specimen indicates past or current infection or exposure to WNV or another flavivirus. Antigenic cross-reactivity with cytomegalovirus (CMV), enterovirus (in children) and/or bunyaviruses can occur. Due to flavivirus cross-reactivity persons previously infected with or vaccinated against other flaviviruses (Japanese encephalitis virus, dengue virus, yellow fever virus) may yield REACTIVE results, interpret with caution.

NON-REACTIVE: Absence of IgG class antibodies is presumptive evidence that the patient was not infected with West Nile Virus or another flavivirus. Specimens drawn early in the disease progression may be negative for IgG-specific antibodies to WNV. If WNV infection is suspected, a second specimen drawn approximately 7 – 14 days later should be tested. By week
3 postinfection, virtually all infected persons should have developed IgG antibodies to WNV. If acute-phase infection is suspected, serum specimens drawn within approximately 7 days postinfection should be compared with a specimen
drawn approximately 14 to 21 days after infection to demonstrate rising IgG antibody levels between the 2 serum specimens (notify laboratory).

INDETERMINATE: The specimen tested near the cut-off. A follow-up specimen 2 – 3 weeks later should be submitted.

Anti-WNV IgM

REACTIVE: Presence of specific IgM class antibodies in a serum specimen is consistent with current or recent infection with West Nile virus (WNV) or another flavivirus (due to antigenic cross-reactivity). WNV IgM REACTIVE results in children should be interpreted with caution as enterovirus cross-reactivity may cause false-positive findings. By the 8th day of illness,
most infected persons will have detectable serum IgM antibody to WNV; in most cases it will be detectable for at least 1 to 2 months after onset of illness, in some cases it will be detectable for > 500 days post exposure.

NON-REACTIVE: Absence of IgM class antibodies to WNV is consistent with lack of acute-phase infection with this virus. Specimens drawn too early in the acute phase (eg, before 8 days postinfection) may be negative for IgM-specific antibodies to WNV. If WNV infection is suspected, a second specimen drawn approximately 7 – 14 days postinfection should be tested.

INDETERMINATE: The specimen tested near the cut-off. Immediate recollection and repeat testing should clarify the serostatus.

Clinical Reference

A. Manual of Clinical Microbiology, 9^th ed., vol. 2. ASM Press, American Society for Microbiology, Washington, DC. Vaughn, D. W., Barrett, A., and Solomon, T. 2010. Flaviviruses (Yellow Fever, Dengue, Dengue Hemorrhagic Fever, Japanese Encephalitis, West Nile Encephalitis, St. Louis Encephalitis, Tick-Borne Encephalitis), p. 2133-2156. In Mandell, D., Bennett,  J. E., and Dolin, R. Principles and practice of infectious diseases, 7^th ed., vol. 2. Churchill Livingstone, Elsevier, Philadelphia, PA.

Focus Diagnostics. 2004. West Nile Virus IgG DxSelect™, Enzyme-linked Immunosorbent Assay (ELISA): package insert. Focus Diagnostics, Cypress, CA.

Lanciotti, R. S., and Tsai, T. F. 2007. Arboviruses, p. 1486-1500. In Murray, P. R., Baron, E. J., Jorgensen, J. H., Landry, M. L., and Pfaller, M.

Method Description

Anti-WNV IgG

In the Focus Diagnostics West Nile Virus IgG DxSelect™ assay, the polystyrene microwells are coated with recombinant West Nile virus antigen. Diluted serum samples and controls are incubated in the wells to allow anti-WNV IgG antibody (if present in the sample) to react with the antigen. Nonspecific reactants are removed by washing and peroxidase-conjugated anti-human IgG is added that reacts with human IgG bound to the antigen. Excess conjugate is removed by washing. Enzyme substrate and chromogen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of optical density (OD). Sample optical density readings are compared with reference cut-off OD readings to determine results.

Anti-WNV IgM

In the Focus Diagnostics West Nile Virus IgM Capture DxSelect™, the polystyrene microwells are coated with anti-human antibody specific for IgM (μ-chain). Diluted specimen samples and controls are incubated in the wells, and IgM present in the sample binds to the anti-human antibody (IgM specific) in the wells. Non-specific reactants are removed by washing. Recombinant WNV antigen is then added to the wells and incubated; and, if anti-WNV IgM is present in the sample, the WNV antigen binds to the anti-WNV in the well. Unbound WNV antigen is then removed by washing the well Mouse anti-flavivirus conjugated with horseradish peroxidase (HRPO) is then added to the wells and incubated; and, if WNV antigen has been retained in the well by the anti-flavivirus in the sample, the mouse anti-flavivirus: HRPO binds to the WNV antigen in the wells.
Excess conjugate is removed by washing. Enzyme substrate and chromogen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is read by a spectrophotometer. The color intensity is compared to the Cut-off’s to determine if antigen-specific IgM is present in the sample.

Performing Laboratory Location

Newfoundland & Labrador Public Health Laboratory

St. John’s