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Since it was first detected in hospitalized patients in the New York City area in 1999, West Nile virus (WNV) has spread across the United States, and is now considered to be endemic throughout the contiguous U.S., with the possible exception of high-altitude regions. Serologic surveys have found that symptoms develop in approximately 20% of infected people, most of whom develop West Nile fever, with fever, headache, fatigue, and occasionally rash, swollen lymph glands, and eye pain. Severe neuroinvasive disease occurs in about 1 in 150 infected people, and manifests as meningitis or encephalitis in about 60% to 75% of affected patients. Mortality ranges from 3% to 15%. Poliomyelitis, a flaccid paralysis syndrome, can also develop, but occurs less frequently than meningitis and encephalitis. The risk of neuroinvasive disease and death increases with age.
MAC-ELISA: WNV Test for Severe Disease and Captive-Bird Surveillance
West Nile virus is a positive single-stranded RNA Flavivirus belonging to the Japanese encephalitis virus antigenic complex. It primarily infects birds and mosquitoes, with humans, horses, and other vertebrates serving as incidental hosts. One to three days following infection by mosquito bite, there is a transient viremia that lasts a mean of six days (range, 1–11 days). Viremia resolves with the appearance of IgM and with the onset of clinical symptoms.
The 2012 guidance from the Centers for Disease Control and Prevention (CDC) requires laboratory testing for a confirmed diagnosis in a patient suspected to have WNV infection. The most efficient diagnostic method is detection of IgM antibody in serum or cerebrospinal fluid, respectively collected within 8–14 days or within 8 days of the onset of illness, using an IgM antibody-capture, enzyme-linked immunosorbent assay (MAC-ELISA). Because of cross-reactivity with other flaviviruses, a positive result should be confirmed by a plaque reduction neutralization test (PRNT) if infection with another Flavivirus needs to be ruled out. ELISAs, including the MAC-ELISA, are also included in CDC recommendations for screening serum samples collected from sentinel chickens. Positive tests must be confirmed by PRNT.
Diagnostic Automation/Cortez Diagnostics Inc. (Calabasas, CA) offers a West Nile IgM capture ELISA test for the qualitative detection of IgM antibodies in human serum to West Nile recombinant antigen (WNRA). This West Nile ELISA test is intended for the presumptive clinical laboratory diagnosis of WNV infection in patients with clinical symptoms consistent with meningoencephalitis. The test consists of an enzymatically amplified two-step sandwich-type immunoassay. Controls and test sera in two sets of duplicate samples are incubated in microtiter wells coated with antihuman IgM antibodies. After washing, one set of wells is incubated with WNRA and one set with normal cell antigen (NCA), followed by washing. The wells are then treated with a WNRA-specific antibody conjugated with horseradish peroxidase (HRP), followed by chromogenic substrate. Absorbance is measured at 450 nm. There are three wash steps, and the total incubation time is about 3 hr and 45 min. Above a certain threshold, the ratio of the absorbances of the WNRA wells and the control wells presumptively determines whether IgM antibodies to WNV are present. The test kit contains 96 microwells configured into 12 × 8 strips and is sufficient for 22 test specimens. The overall sensitivity and specificity are 96.2% and 98.4%, respectively (for details and confidence intervals, see product insert).
Focus Diagnostics, Inc. (Cypress, CA) offers the West Nile Virus DxSelect™ IgM Capture ELISA kit for the qualitative detection of IgM antibodies to WNV in human serum, and is indicated for patients with symptoms of meningoencephalitis. The 96-microwell assay is in a 12 × 8-strip format, and has FDA clearance and CE Mark approval. The ELISA utilizes wells coated with rabbit antihuman IgM, to which diluted samples are added. After incubation and washing, recombinant WNV antigen is added. Detection is by means of a mouse antiflavivirus conjugated with horseradish peroxidase and chromogenic substrate. Absorbance is measured at 450 nm. There are three wash steps, and the total incubation time is about 3 hr and 45 min. Positive samples must be tested a second time using the background-subtract procedure to rule out false positives caused by rheumatoid factor and heterophilic antibodies. For the background-subtract procedure, diluted sample is added to each of two wells, followed by incubation and washing. WNV antigen is added to one of the two wells and diluent only is added to the other well instead of antigen. The assay is then completed as usual. If both wells are positive, the sample is considered negative for the presence of WNV IgM. In addition, the Focus Diagnostics Reference Laboratory offers WNV testing, including antibody-capture ELISAs for serum and CSF, and RT-PCR for the detection of viral RNA in CSF, plasma, or serum.
Molecular Methods for West Nile Virus Testing
PCR testing for the diagnosis of WNV infections in humans is not sensitive, because of the low-level, transient viremia during the incubation period. However, PCR and another form of nucleic acid testing (NAT) are used to test blood donors. Although WNV is transmitted to humans primarily by mosquitoes, transmission has occurred through transfused blood products in a small number of confirmed cases. The Food and Drug Administration (FDA) in late 2009 recommended NAT of individual donor (ID) samples during periods of high WNV activity and minipool NAT testing at other times if ID-NAT is not feasible.
The COBAS® TaqScreen West Nile Virus Test (Roche Molecular Systems, Inc., Pleasanton, CA) is used with the COBAS S 201 system. It is a qualitative test intended for the direct detection of WNV RNA in the plasma of human donors, including donors of whole blood and blood components. The COBAS TaqScreen West Nile Virus Test uses a generic nucleic acid preparation technique on the COBAS AmpliPrep Instrument. WNV RNA is then detected by automated, real-time PCR amplification on the COBAS TaqMan® Analyzer. The test incorporates an internal control for monitoring test performance in each individual test, as well as the AmpErase enzyme to reduce potential contamination by previously amplified material.
The PROCLEIX® WNV Assay (Novartis Diagnostics, Emeryville, CA) is a qualitative in vitro nucleic acid CE-marked and FDA-approved assay system for the detection of WNV RNA in plasma specimens from human donors of whole blood and blood components. The assay can be performed on the semiautomated PROCLEIX modular platform or the automated PROCLEIX TIGRIS® System. The assay involves three main steps, which take place in a single tube: sample preparation, WNV RNA target amplification by transcription-mediated amplification (TMA), and detection of the amplification products by the hybridization protection assay (HPA). RNA is isolated from specimens via the use of target capture. The specimen is treated with a detergent to release viral genomic RNA. Capture oligonucleotides that are homologous to highly conserved regions of WNV are hybridized to the WNV RNA target in the test specimen. The hybridized target is then captured onto magnetic microparticles that are separated from the specimen in a magnetic field. After washing followed by TMA, detection is achieved by HPA using single-stranded nucleic acid probes with chemiluminescent labels. The Selection Reagent differentiates between hybridized and unhybridized probes by inactivating the label on unhybridized probes. During the detection step, the chemiluminescent signal produced by the hybridized probes is measured in a luminometer.
WNV and Global Warming
Global warming will lead to further spreading of West Nile virus and other mosquito-borne pathogens. The mosquitoes that transmit WNV thrive in hot and dry conditions, and pick up the virus more easily the hotter it gets. In the United States, epicenters of transmission have been linked closely to above-average summer temperatures. High temperatures also have been associated with the virus jumping to a mosquito (Culex pipiens) that thrives in urban habitats.
In 2012, the 3969 cases of WNV infections reported to the CDC through the first week of October were the highest number of cases reported by that date since 2003. Of these, 2010 (51%) were classified as neuroinvasive and 1959 (49%) as non-neuroinvasive disease. There were 163 deaths. Although it is difficult to attribute any single outbreak to climate change, the trend to repeated outbreaks of illness caused by WNV is clear. Although reversing climate change is the optimum solution, testing for WNV is here for the duration.
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