Wastewater-based surveillance of norovirus GI and GII: Comparative performance of commercial and in-house RT-qPCR Assays

Noroviruses, the leading cause of nonbacterial acute gastroenteritis outbreaks, are widely detected in wastewater worldwide and reflect community-level viral circulation. These RNA viruses exhibit high mutation and recombination rates, favoring the emergence of novel variants with epidemiological relevance. In this study, wastewater-based epidemiology was applied to monitor the circulation of norovirus genogroups GI and GII in raw sewage samples collected biweekly throughout 2024 from a wastewater treatment plant serving the Metropolitan Region of Rio de Janeiro, Brazil. In parallel, the analytical performance of a commercial RT-qPCR kit was evaluated in comparison with an in-house RT-qPCR protocol for norovirus detection and quantification. A total of 24 samples were concentrated by ultracentrifugation, and viral nucleic acids were extracted using the Quick-DNA/RNA™ Viral MagBead kit. Norovirus GI and GII were detected and quantified using both the IBMP Biomol Rotavirus and Norovirus Kit and in-house assays. The IBMP kit showed higher detection rates for GI (54.2%) and GII (100%) compared with the in-house protocol (20.9% and 70.8%, respectively), as well as higher median viral concentrations for both genogroups. [GI (5.8 ± 0.7 vs. 4.8 ± 0.2 log10 gc µL-1) and GII (7.1 ± 0.7 vs. 6.6 ± 0.8 log10 gc µL-1)]. Molecular characterization based on partial ORF1 (RdRp) and ORF2 (VP1) regions identified genotypes GII.17[P17], GI.3[P3], and GI.3[P13], indicating the environmental circulation of globally disseminated strains previously associated with outbreaks. Overall, these findings support the applicability of wastewater-based surveillance for monitoring norovirus genotype circulation and highlight the usefulness of a commercial RT-qPCR assay for sensitive detection and quantification of noroviruses in environmental samples.