Identification of multiple arbovirus infections/exposure in Northeastern Brazil using a multiplex microsphere...

Several mosquito-borne arboviruses, including dengue (DENV), Zika (ZIKV), West Nile (WNV), yellow fever (YFV), and chikungunya (CHIKV) viruses, have caused disease outbreaks of public health concern in the tropics and subtropics. The overlap distribution of these arboviruses and cross-reactivities of antibodies to orthoflavivirus envelope protein underscore the need for a reliable and convenient serological test to distinguish these arboviruses in endemic countries. We developed a multiplex IgG microsphere immunoassay (MIA) using 13 antigens to test 8 panels of samples with well-documented arbovirus infections or vaccination (n = 374). We further employed this assay, combined with previously reported neutralization test and Western blot analysis to test serum samples (n = 300) collected from Saúde, a town in the Northeastern state of Bahia, Brazil. The sensitivity/specificity of mixed DENV1-4, ZIKV, WNV, and YFV nonstructural protein 1 (NS1), and CHIKV virus-like particles (VLP) IgG MIAs were 96.0%/98.3%, 100%/79.9%, 94.4%/73.4%, 52.2%/94.4%, and 100%/99.7%, respectively. The overall low specificity of ZIKV and WNV NS1 was due to cross-reactivities from panels with repeated orthoflavivirus infections, including secondary DENV and DENV + ZIKV infections. In Saúde, we found seropositivity of 70.3%, 22.3%, 39.7%, and 5.7% to DENV, ZIKV, YFV, and CHIKV, respectively, and the majority with multiple arbovirus infections or exposure including DENV + CHIKV, DENV + ZIKV, DENV + YFV, DENV + ZIKV + YFV, and/or CHIKV. Our findings highlight the importance of confirmatory tests for the NS1-based multiplex IgG MIA. This high-throughput assay, combined with confirmatory tests, provides a labor-saving and validated tool for serodiagnosis and serosurveillance of pathogenic arbovirus infections/exposure in endemic regions.IMPORTANCESeveral multiplex microsphere immunoassays (MIA) have been reported to detect pathogenic arbovirus infections, including dengue (DENV), Zika (ZIKV), West Nile (WNV), yellow fever (YFV), and chikungunya (CHIKV) viruses. None of them included sufficient control panels to validate the sensitivity/specificity of each antigen, confirmatory tests for equivocal samples, or comparison with available commercial kits to advance this technology from research to practical use. We employed eight panels of samples with known arbovirus infections or vaccination (n = 374) to evaluate the sensitivity/specificity of our multiplex IgG MIA. We reported high sensitivity of nonstructural protein 1 (NS1)-based IgG MIA (94.4%-100% except YFV NS1) and overall low specificity of ZIKV and WNV NS1 due to cross-reactivities from repeated orthoflavivirus infections, including secondary DENV and DENV + ZIKV infections. Using our algorithm and confirmatory tests, we reported multiple arbovirus infections or exposure (DENV + ZIKV, DENV + YFV, DENV + ZIKV + YFV, and/or CHIKV) in the majority of participants from a town in Northeastern Brazil.