The identification of West Nile fever cases in a country where dengue is endemic presents us with many challenges. These challenges are due to the extensive cross-reactivity of flavivirus antigen in serological assays. Without the identification of the infecting virus either through nucleic acid or virus isolation, serology results often cannot with confidence provide a correct diagnosis when more than one flavivirus is circulating in the population. In order to resolve this limitation, the laboratory testing algorithm was modified in this study to include PRNT90 with IgG depletion and an NS1 antigen test. The PRNT90 with IgG depletion allowed for the differentiation of 17 (74%) samples that were initially designated uninterpretable due to cross-reactivity in the MAC ELISA, as well as of 14 (70%) WNV samples that were false positive for WNV in the MAC ELISA ( and ). The dengue NS1 antigen ELISA further confirmed dengue infection in 8 (19%) of the total 43 samples.
NS1 antigen detection has been shown to be useful as a tool for the diagnosis of an acute dengue infection. Previous studies have demonstrated that the NS1 antigen ELISA is specific for DENV rather than for other flaviviruses, such as WNV, Japanese encephalitis virus (JEV), and yellow fever virus (2
). The advantage of NS1 antigen detection is that it appears as early as 1 day after the onset of symptoms (DPO) and up to 18 DPO and may bridge the gap in which viral nucleic acid and IgM antibody detection is less likely to be positive (DPO = 4 to 5) (24
). Moreover, a previous study evaluating commercially available NS1 detection systems demonstrated the excellent sensitivity (83.2%) and specificity (100%) of the Bio-Rad Platelia dengue NS1 antigen test (2
). Because the NS1 test has high specificity for DENV, it can be utilized for differential diagnosis in cases in which the antigen for the IgM ELISA cross-reacts and the infecting virus cannot be identified by conventional RT-PCR.
Cross-reactivity between DENV and WNV was not observed in previous years of WNV surveillance in Puerto Rico (2003 to 2006). This may have been because the earlier surveillance system was a neuroinvasive WNV reporting system, whereas the WNV fever surveillance system, which began in July of 2007, tested cases that were suspected of having WNV regardless of encephalitic symptoms. In 2007, the American Red Cross (ARC) screened blood donations in Puerto Rico for WNV and confirmed WNV transmission in humans (5
). The goal of this study was to identify WNV cases using serological techniques that can differentiate between DENV and WNV despite the dengue immune background in the Puerto Rican population.
The background IgG reactivity of past DENV infections often cannot be distinguished from the current or most recent infection because of the high avidity of this antibody. A serosurvey conducted in 1982 in the municipality of Florida, Puerto Rico, indicated that 68 to 80% of individuals had past exposure to dengue; however, recent unpublished CDC data indicate that these levels are now much higher, demonstrating a high prevalence of dengue in Puerto Rico (22
). In the case of multiple arboviral infections over time, the sequence of infecting viral species can be an important determinant in the resulting serological responses detected by immunological assays. Secondary flavivirus infections where DENV infection is followed by an infection with a nondengue flavivirus (e.g., WNV) are hypothesized to result in less common epitopes based on the neutralization test; however, the epitopes are not well understood in the secondary flavivirus infections. The anamnestic response to the second flavivirus infection is believed to be less potent than that to sequential DENV infections. Alternatively, previous studies in Asia demonstrated that exposure to Japanese encephalitis virus (a flavivirus in the same serocomplex group as WNV) followed by a natural DENV infection caused a positive serological response to both viruses, although DENV neutralization titers were measurably higher than those for JEV (10
). However, sequential flavivirus infection in which the individual has been exposed to DENV on one or more occasions followed by a nondengue flavivirus results in a different immune response. In an animal study by Edelman et al. (9
), animals subjected to sequential infections with multiple DENV and JEV infections responded with high neutralizing titers to both JEV and DENV. Fifty animals displayed “original antigenic sin” in which the highest neutralizing titer was to DENV despite the most recent infection being caused by JEV (9
). In that study, the incidence of cases in which original antigenic sin occurred varied and was dependent on the first acquired flavivirus infection (14
The PRNT is the gold standard for differential diagnosis of flavivirus infections in humans. However, due to background DENV IgG reactivity in a population in which the disease is endemic, this test is not always a useful tool for differential diagnosis of secondary flavivirus infections. IgM antibodies are more specific and less cross-reactive than IgG (9
). For example, for an individual who acquired a laboratory infection of DENV4 after previous vaccination with JEV, when the IgM fraction of the individual's serum was neutralized, DENV4-specific results were obtained, while the IgG was broadly reactive (3
). In our study, the PRNT90
with IgG depletion used IgM antibodies to neutralize, and this resulted in the differentiation of 74% of the samples that were cross-reactive in the MAC ELISA for both WNV and DENV. The combination of PRNT90
with IgG depletion and the NS1 antigen test differentiated 36 (84%) of the samples in this study.
Cocirculation of flaviviruses such as DENV and WNV presents difficult challenges in the interpretation of results from standard serological assays, often complicating the diagnosis of the current infection. Frequently a series of confirmatory diagnostic tests is necessary, and our proposed testing algorithm was effective in determining the infecting virus in samples with a cross-reactive result for WNV and DENV in the MAC ELISA. Our findings show that the PRNT90 with IgG depletion, although technically difficult and time-consuming, is useful for differentiation of WNV and DENV, especially when used in combination with the NS1 antigen ELISA. Further evaluation of this algorithm is necessary for more complete information on the antibody response during the course of infection through the convalescent phase following a WNV infection in an individual that has past single or multiple exposures to DENV.