Development of a Single Thermal Cycling Multiplex RT-qPCR Assay for Rapid Arbovirus Detection

Background: The co-circulation of multiple arboviruses with similar clinical presentations poses a significant challenge for public health surveillance. This study aimed to develop and clinically validate a novel multiplex RT-qPCR assay for the simultaneous detection of five arboviruses in three reactions. Methods: A diagnostic accuracy study was conducted in Salvador, Brazil. Evaluation was performed using 254 prospective serum samples and 34 archived positive samples. The primers and probes for the assay were designed in silico, all targeting highly conserved regions of Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), Oropouche (OROV), and West Nile (WNV) viruses. The protocol was optimized in three reactions with a single thermal cycle, comprising a tetraplex reaction (DENV, CHIKV, ZIKV, and hRNAseP, an endogenous control) and two singleplex reactions (OROV and WNV). Results: In prospective analysis, the assay identified 45 DENV, 3 CHIKV, and 9 OROV-positive cases. Diagnostic accuracy for DENV and CHIKV was 96% (95% CI: 93.3-98.0%), successfully detecting four DENV cases missed by the reference standard. Furthermore, incorporating OROV enabled the identification of nine cases that conventional triplex protocols would have overlooked. Conclusion: This single-cycling multiplex RT-qPCR assay enhances epidemiological surveillance by expanding detection beyond traditional triplex targets to include OROV and WNV. This operational optimization reduces turnaround times and prevents the underdiagnosis of emerging arboviruses, serving as a robust diagnostic tool for endemic regions.