Applying a multiplex electrochemiluminescence NS1 assay to detect and differentiate Zika and dengue virus exposures in long-term community cohorts in Brazil and Thailand
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Background
Distinguishing Zika virus (ZIKV) from Dengue virus (DENV) infection remains a major diagnostic challenge in co-endemic regions due to antigenic cross-reactivity. We evaluated a multiplex electrochemiluminescence Meso Scale Discovery System (MSD) NS1 assay to define ZIKV infection with high specificity across diverse immune backgrounds.
Methods
We analyzed longitudinal samples from PCR-confirmed ZIKV and DENV infections across multiple cohorts, assessed correlations between MSD NS1 signal intensities and PRNT 50 titres, and performed ROC analyses to derive seroconversion thresholds. We then applied these criteria to identify ZIKV seroconversions in Brazil (2015-2024) and Thailand (2015-2025)
Findings
ZIKV infections produced sustained NS1 responses, whereas primary DENV infections showed minimal ZIKV cross-reactivity and secondary DENV infections exhibited only transient, low-level increases. MSD NS1 signal intensities correlated with PRNT 50 titres for ZIKV and all four DENV serotypes (ρ = 0.54–0.84). ROC analyses identified a fold-change ≥ 10.96 as the optimal ZIKV seroconversion cutoff, with an AUC of 100% among DENV-naïve and 90.7% among DENV-exposed participants. Adding a second criterion — ZIKV/DENV fold-change ratio > 1 — further increased specificity. Application of this dual-threshold framework revealed high ZIKV incidence during the 2015–2016 epidemic in Brazil and very low incidence in the post-epidemic period. In the Thailand cohort, synchronous seroconversion among household members suggested potential intra-household transmission.
Interpretation
The MSD NS1 multiplex assay provides a scalable, quantitative, and highly specific approach for identifying ZIKV infection and estimating seroincidence in co-endemic settings. The dual-threshold framework (fold-change ≥ 10.96 and ZIKV/DENV ratio > 1) enhances diagnostic precision, offering a robust tool for surveillance and transmission studies in flavivirus-endemic regions.
Funding
Wellcome Trust Fund; The National Council for Scientific and Technological Development (CNPq; grant: 440891/2016–7, 311365/2021–3, and 303307/2026-9 to G.S.R.); the Bahia Foundation for Research Support (grant PET0022/2016 to G.S.R.); the Coordination for the Improvement of Higher Education Personnel, Brazilian Ministry of Education (grant 88881.130749/2016–01 to G.S.R.)
RESEARCH IN CONTEXT
Evidence before this study
We searched PubMed, Scopus, and Web of Science for articles published in English up to May 1, 2026, using combinations of the terms “Zika virus”, “dengue virus”, “NS1”, “serology”, “cross-reactivity”, “electrochemiluminescence”, “multiplex assay”, “seroincidence”, and “PRNT”. Previous studies demonstrated that serological discrimination between Zika virus (ZIKV) and dengue virus (DENV) infections is challenging because of extensive flavivirus cross-reactivity, particularly in DENV-endemic settings. Conventional ELISA-based assays frequently lack specificity, especially among individuals with prior flavivirus exposure. Plaque reduction neutralization testing (PRNT) remains the reference standard for differentiating flavivirus infections, but its low throughput, high cost, and technical complexity limit its application in large epidemiological studies. Recent studies have highlighted NS1-based assays as promising alternatives because NS1 antibodies appear more virus-specific than envelope-directed responses. Multiplex electrochemiluminescence platforms, including Meso Scale Discovery (MSD), have shown high analytical sensitivity and scalability for infectious disease serology, but evidence supporting their use for distinguishing ZIKV from DENV infections across distinct immune backgrounds and longitudinal cohort settings remains limited. Few studies have evaluated quantitative seroconversion thresholds for identifying ZIKV infection in populations with prior DENV exposure or explored their application for long-term seroincidence estimation and transmission inference.
Added value of this study
This study validates a multiplex electrochemiluminescence NS1 assay for differentiating ZIKV and DENV infections using multiple well-characterized cohorts from Brazil, Thailand, and the United States, including PCR-confirmed ZIKV and DENV infections, longitudinal community cohorts, and a controlled human dengue infection model. We identified a standardized serological threshold based on ZIKV NS1 fold-change (≥10·96) and demonstrated that combining this threshold with a second criterion—the ZIKV-to-DENV fold-change ratio > 1, substantially improves specificity in highly DENV-endemic populations. We additionally showed strong correlations between MSD signal intensities and PRNT 50 neutralization titres for ZIKV and all four DENV serotypes, supporting the use of the assay as a scalable surrogate for neutralization-based approaches. Application of this dual-threshold framework enabled estimation of ZIKV seroincidence over nearly a decade of follow-up in Brazil and identified patterns suggestive of intra-household transmission in Thailand. Together, these findings provide a practical high-throughput strategy for distinguishing flavivirus infections in epidemiological and surveillance settings.
Implications of all the available evidence
Accurate discrimination between ZIKV and DENV infections remains a major barrier for flavivirus surveillance, seroepidemiological studies, and vaccine evaluation in co-endemic regions. Our findings suggest that multiplex NS1 electrochemiluminescence assays combined with quantitative dual-threshold criteria can provide a scalable and standardized alternative to PRNT for identifying ZIKV infections in populations with heterogeneous flavivirus exposure histories. This framework could strengthen surveillance systems by improving estimates of ZIKV seroprevalence and seroincidence, supporting detection of silent or low-level transmission, and enabling more reliable interpretation of longitudinal immune responses in endemic settings. The approach may also facilitate vaccine studies and outbreak investigations in regions where multiple flaviviruses co-circulate.
Key messages
Strong and long-lasting ZIKV NS1 responses with minimal DENV cross-reactivity
The Meso Scale Discovery System (MSD) multiplex NS1 assay measured robust and durable ZIKV-specific antibody responses following PCR-confirmed infection, while primary and secondary DENV infections produced only minimal or transient ZIKV NS1 reactivity, demonstrating high antigenic specificity.
MSD NS1 signals correlate strongly with PRNT 50 neutralization titres
Across multiple cohorts, MSD signal intensities showed strong concordance with PRNT 50 titres for both ZIKV and DENV serotypes, validating the MSD assay as a reliable, scalable surrogate for functional neutralization assays.
A dual-threshold framework improves diagnostic accuracy
ROC analyses identified a fold-change ≥ 10.96 as a sensitive and specific marker of ZIKV seroconversion, further refined by a ZIKV/DENV fold-change ratio > 1 to enhance specificity and reduce misclassification in DENV-exposed populations.
Accurate detection of ZIKV seroincidence and transmission patterns
Application of the dual-threshold criteria revealed high ZIKV incidence during the 2015–2016 epidemic in Brazil and very low transmission in subsequent years. In a Thai household, spatiotemporal clustering of seroconversion among three members is suggestive of intra-household transmission, demonstrating the framework’s utility for detecting potential transmission clusters in seroepidemiological surveillance.
