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West Nile Virus Screening and Diagnostics Technology Emerges In Step with North American Outbreak


Helen Londe, M.D.
Contributing Editor

The first human case of West Nile virus (WNV) for 2005 was reported to have occurred in Kansas in late June. The CDC has released few details on the patient but has confirmed this being 2005's first documented incidence of the mosquito borne disease in the US.

WNVis a positive single-stranded RNA flavivirus of about 11 kb. It primarily infects birds and mosquitoes, with humans, horses, and other vertebrates serving as incidental hosts. The virus was first isolated from a febrile patient in the West Nile region of Uganda in 1937. It remained a little-understood cause of febrile illness and sporadic encephalitis in parts of Africa, Europe, and Asia until the late summer of 1999, when outbreak surveillance identified 59 patients hospitalized with West Nile virus infection in the New York City area. The following year, the Centers for Disease Control and Prevention, in collaboration with regional health departments, established ArboNET to track WNV infections in the US. Data from ArboNet have documented the dramatic westward spread of WNV across North America. As of January, 2005, avian or animal infections have been reported from 47 states and Puerto Rico, and human infections have been reported in 40 states.

By October, 2004, ArboNET had received reports of WNV infection in 58 mosquito species and 284 bird species and had recorded 6690 cases of neuroinvasive WNV disease (meningitis, encephalitis, or acute flaccid paralysis) among persons in the United States. ArboNet has also recorded 629 deaths. Serologic surveys indicate that symptoms develop in approximately 20 percent of infected people and that neuroinvasive disease occurs in 1 in 140. The risk of neuroinvasive disease and death increases with age.

Following a WNV infected mosquito bite, transient viremia occurs during the incubation period and lasts a mean of 6 days. Viremia resolves with the appearance of IgM and with the onset of cl inical symptoms. These observations explain the rationale for screening asymptomatic blood donors with nucleic acid-amplification tests (NAT) and the use of IgM to detect disease in patients with clinical symptoms.

Although WNV is transmitted primarily by mosquitoes, transmission has occurred through transfused blood products. Since 2003 donor mini-pools have been tested by NAT utilizing two test manufacturers approved by the FDA under the investigational new drug mechanism (TaqScreen WNV test, Roche Molecular Systems, Inc.; Procleix WNV assay, GenProbe, Inc.). Although screening has substantially reduced the risk of transfusion associated transmission, there have been screening failures because of low levels of viremia in asymptomatic donors and the use of mini-pools.

The most recent guidelines for diagnosis in patients with suspected WNV infections include ELISA detection of IgM and IgG antibodies to WNV in serum or cerebrospinal fluid. Because of cross reactivity with other flaviviruses, positive results must be confirmed with a plaque reduction neutralization test (PRNT). FDA-approved ELISA kits for detection of antibody are available from Focus Diagnostics, PANBIO Limited and InBios International, Inc. A promising microsphere immunoassay that is more specific for WNV has been described in the literature, but is not available commercially.

The intended use of the FDA approved ELISAs offered by all 3 companies is similar: the assays are intended to qualitatively detect antibodies in sera of patients having symptoms of meningioencephalitis as an aid in the presumptive diagnosis of WNV infection. Positive results must be confirmed by the PRNT or using current CDC guidelines.

Focus Diagnostics offers the West Nile Virus IgM Capture DxSelect, and the West Nile Virus IgG DxSelect. The IgM capture ELISA procedure utilizes wells coated with rabbit anti-human IgM to which diluted samples are added. After incubation and washing, recombinan t WNV antigen is added. Detection is by means of mouse anti-flavivirus conjugated with horseradish peroxidase (HRP), and enzyme substrate plus chromogen. Absorbance is measured at 450 nm. There are 3 wash steps and the total incubation time is 3 hours and 45 minutes. Positive samples must be tested with the background subtract procedure (either on the initial test or on a repeat test) to rule out false positives caused by rheumatoid factor and heterophilic antibodies. The IgG ELISA utilizes wells coated with recombinant WNV antigen to which diluted samples are added. Detection is by means of HRP-conjugated goat anti-human IgG, and enzyme substrate plus chromogen. Absorbance is measured at 450 nm. There are 3 wash steps and the total incubation time is 1 hour and 45 minutes.

In addition, Focus Diagnostics Reference Laboratory offers WNV testing that includes the PRNT, which satisfies the CDC requirement for performing a WNV-specific serology test; antibody ELISAs for serum and CSF, an IgA ELISA for serum, and RT-PCR to detect viral RNA in CSF or serum.

PANBIO offers the West Nile Virus IgM Capture ELISA and the West Nile Virus IgG Indirect ELISA. The IgM capture ELISA procedure utilizes wells coated with polyclonal sheep anti-human IgM antibodies to which diluted samples are added. After incubation and washing, a solution of inactivated WNV antigen combined with HRP-conjugated monoclonal anti-flavirus is added. After incubation and washing, the substrate is added. Absorbance is measured at 450 nm. There are 2 wash steps and total incubation time is 2 hours and 10 minutes. The IgG ELISA utilizes wells coated with purified and inactivated WNV antigen to which diluted samples are added. Detection is by means of HRP-conjugated sheep anti-human IgG and substrate. Absorbance is measured at 450 nm. There are 2 wash steps and total incubation time is 1 hour and 10 minutes.

In addition PANBIO offers as analyte specific reagents: Panbio West Nil e Virus IFA Slides and West Nile Virus Positive Control, which are intended for the qualitative serologic detection of antibodies to West Nile virus.

InBios offers the West Nile Detect IgM Capture ELISA. The ELISA procedure utilizes wells coated with goat anti-human IgM to which diluted samples are added. After incubation and washing, WNV derived recombinant protein (WNRA) and a control consisting of normal cell antigen are each added to two of four replicate wells of sample. Detection is by means of HRP-conjugated anti-WNV monoclonal antibody and substrate. Absorbance is measured at 450 nm. Above a certain threshold, the ratio of the absorbance of the WNRA and the antigen control wells presumptively determines whether antibodies to WNV are present. There are 3 wash steps and total incubation time is 3 hours and 16 minutes.

In addition InBios offers as analyte specific reagents: West Nile Detect IgG Elisa, and antigen and reagents for both IgM and IgG WNV ELISAs.
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