Evaluation of the Novaplex^TM Tropical Fever Virus Assay

Arboviral infections represent a growing challenge for global public health due to vector expansion, globalization, and climate change. Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), and West Nile virus (WNV) often co-circulate and present overlapping clinical manifestations, making rapid and accurate etiological diagnosis difficult. In this context, multiplex real-time RT-PCR assays can support clinical management and epidemiological surveillance. In this study, analytical and preliminary retrospective comparisons of the Novaplex^TM Tropical Fever Virus Assay, a multiplex RT-PCR system designed for the simultaneous detection of DENV, ZIKV, CHIKV, and WNV, were performed. Analytical sensitivity was assessed using serial viral-stock dilutions and Probit regression analysis to estimate the limit of detection (LOD) at a 95% probability. The LOD was 4.41 Log copies/mL for WNV2, 3.67 for DENV2, 2.57 for ZIKV, and 3.43 for CHIKV in PBS. In spiked plasma or urine, LODs ranged from 2.04 to 4.79 Log copies/mL. No cross-reactivity with Usutu virus was observed under the conditions tested. In a retrospective comparison of 34 clinical specimens with singleplex assays, agreement was the highest for WNV, substantial for CHIKV, and limited for DENV. These findings support Novaplex as a complementary tool for arboviral diagnosis and surveillance, although further clinical studies are warranted.