Combining Immunoassays to Identify Zika Virus Infection in Dengue-Endemic Areas

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged as a global health threat. The rise in ZIKV infections has driven an increased incidence of neonates born with microcephaly or other neurological malformations. Therefore, screening for ZIKV infection can considerably impact pregnant women, especially during the first trimester. The majority of ZIKV infections are mild or asymptomatic, and clinical diagnosis is inaccurate. Moreover, given the high level of cross-reactivity among flaviviruses, serological approaches to distinguish ZIKV from dengue virus (DENV) infections are complicated. We used the combination of DENV and ZIKV nonstructural protein 1 (NS1) IgG enzyme-linked immunosorbent assay (ELISA) and ZIKV NS1 blockade-of-binding (BOB) ELISA to test the convalescent sera of non-flavivirus, primary DENV, secondary DENV, and ZIKV infections. Our findings indicate that primary testing using a ZIKV NS1 IgG ELISA, the test of choice for large-scale ZIKV serosurvey studies, provided relatively high sensitivity. Moreover, the confirmation of positive ELISA results using the ZIKV NS1 BOB ELISA increased average specificity to 94.59% across serum samples. The combined use of two simple ELISAs for ZIKV serosurveys and the monitoring of ZIKV infection during pregnancy can elucidate the epidemiology, pathogenesis, and complications of ZIKV in DENV-endemic areas.

Keywords: Zika virus; cross-reactivity; dengue virus; nonstructural protein 1; serological diagnosis.

Figure 1

The performance of serological assays using DENV infection samples in the convalescent phase. (A) anti-ZIKV IgG ELISA. (B) ZIKV NS1 IgG ELISA. (C) ZIKV NS1/DENV NS1 IgG ELISA. (D) ZIKV NS1 BOB ELISA. The dotted line represents the cut-off value of the P/N ratio or %BOB inhibition. ZIKV: Zika virus, DENV: dengue virus, ELISA: enzyme-linked immunosorbent assay, BOB: blockade-of-binding, NS1: nonstructural protein 1, P/N ratio: ratio of average OD₄₅₀ of test sample divided by the average OD₄₅₀ of the negative sample.

Figure 2

Performance of serological assay using ZIKV infection samples in the convalescent phase. (A) ZIKV NS1 ELISA. The dotted line represents the cut-off value of the P/N ratio. (B) ZIKV NS1 BOB ELISA. The dotted line represents the cut-off value of % BOB inhibition. (C) ZIKV NS1/DENV NS1 ELISA. The dotted line represents the cut-off value of the P/N ratio. (D–F) ZIKV NS1 BOB ELISA in a sequential annual blood sample of three ZIKV infection patients (01-0464, 07-0052, and 01-0493). The red arrow indicates the date of ZIKV RT-PCR positivity. The dotted lines represent the cut-off value of % BOB inhibition. ZIKV: Zika virus, DENV: dengue virus, ELISA: enzyme-linked immunosorbent assay, BOB: blockade-of-binding, NS1: nonstructural protein 1, P/N ratio: ratio of average OD₄₅₀ of test sample divided by the average OD₄₅₀ of the negative sample.