Serological Evaluation of Suspected West Nile Virus Human Cases following Its Introduction during a Dengue Outbreak in Puerto Rico in 2007

Abstract

A laboratory testing algorithm was evaluated to confirm West Nile virus (WNV) infection in human serum following the introduction of the virus in Puerto Rico in 2007. This testing algorithm used two standard diagnostic assays, the IgM antibody capture enzyme-linked immunosorbent assay (MAC ELISA) and real-time reverse transcriptase PCR (RT-PCR), along with two nonconventional assays, the nonstructural protein 1 (NS1) ELISA and a 90%-plaque-reduction neutralization test (PRNT 90 ) with IgG depletion for dengue virus (DENV) and WNV. A total of 2,321 serum samples from suspected WNV human cases were submitted for testing. Approximately one-third (867, 37%) were cross-reactive for DENV and WNV by MAC ELISA and had negative RT-PCR results for both viruses. Of a subset of 43 samples tested, 31 (72%) of these cases were identified as positive for DENV in the PRNT 90 with IgG depletion and 8 (19%) were positive in the DENV NS1 antigen ELISA. These two assays combined differentiated 36 (84%) of the samples that could not be diagnosed using the standard diagnostic testing methods.The introduction of West Nile virus (WNV) into the northeastern United States in 1999 and its subsequent rapid spread throughout the United States raised concerns about the potential for the introduction and spread of the virus in the Caribbean (4, 6, 7, 16). Since 1999, evidence of WNV transmission has been reported throughout the Caribbean, where diagnosis has been complicated by the cocirculation of other flaviviruses, including the dengue virus (DENV) (12). The continued spread of WNV through North America, Latin America, and the Caribbean has highlighted the need for disease-specific diagnostic tests for flaviviruses. Until recently, DENV has been the only circulating flavivirus in Puerto Rico; therefore, the surveillance system testing algorithm was not designed to detect other arboviruses. The first serological evidence of WNV in Puerto Rico was reported in wild birds in 2003. The first WNV isolate was obtained in mosquitoes in June 2007 in the municipalities of Ceiba and Naguabo along the northeastern coast of the island and coincided with the largest outbreaks of dengue since 1998 (Fig. 1) (1). The epidemic curve indicated that the dengue outbreak began May 2007, 1 week prior to the serological detection of WNV in sentinel chickens.The Centers for Disease Control and Prevention (CDC) and the Puerto Rico Health Department have jointly managed an island-wide WNV surveillance system for humans since 2003. The data presented in this study are an evaluation of the samples obtained from the WNV surveillance from July through December 2007 following the detection of WNV in sentinel chickens (1). The purpose of this study was to evaluate a new testing algorithm to differentiate between WNV and DENV cases in IgM-cross-reactive samples. A new testing algorithm was developed to evaluate suspected WNV-positive serum samples using a 90%-plaque-reduction neutralization test (PRNT 90 ) with IgG depletion. Further differentiation was achieved using the ...

Characterization of Puerto Rican West Nile Virus isolates in mice

“…Possible explanations for the differences in WNV disease observed in humans, horses and birds between North America compared virus strains and Latin America include: 1. The circulation of attenuated strains in Latin America and the Caribbean, 2. the natural resistance to WNV infection in birds in Latin America [ 40 ] 3. an increased avian diversity in the tropics [ 41 ] 4. the co-circulation of other flaviviruses such as dengue virus that may confer some cross-protective immunity in humans and 5. the limited viral isolates obtained due to non-sustainable WNV surveillance in humans and animals in Latin America [ 42 ]. Additional studies are needed to better understand the genotypes and their corresponding phenotypes of WNV strains circulating in PR and Latin America.…”

Diagnosis of West Nile Virus Human Infections: Overview and Proposal of Diagnostic Protocols Considering the Results of External Quality Assessment Studies

“…The detection of WNV IgG by EIAs is now standardized and these methods are widely used for the determination of immune status to WNV either in suspected patients or in healthy asymptomatic populations [65,99,100,101,102,103]. A major limitation of these tests is, firstly, their restricted clinical specificity due to extensive cross-reactions with flaviviruses.…”

Enhanced West Nile Virus Surveillance in a Dengue-Endemic Area—Puerto Rico, 2007

“…When the PRNT 90 yielded indeterminate results because of reactivity of more than two viruses (i.e., DENV-1, -2, -3, or -4, St. Louis Encephalitis virus [SLEV], or WNV), a PRNT 90 IgG depletion assay was used to determine the infecting virus. 12 Laboratory definitions. A laboratory-positive WNV case was defined as a case with any of the following four findings: detection of WNV nucleic acid in a specimen by RT-PCR, WNV IgM seroconversion from negative to positive by anti-WNV MAC-ELISA in paired specimens, a positive anti-WNV MAC-ELISA in a single specimen with a negative anti-DENV MAC-ELISA, or a PRNT 90 (or PRNT 90 IgG depletion assay) with a WNV titer at least four times higher than the titer of any of the four DENV types or SLEV.…”