Detection of Respiratory Viruses in Wastewater Samples Using Commercial Multiplex PCR Assays

Wastewater-based epidemiology (WBE) has emerged as a supplement to clinical investigations, facilitating the acquisition of timely and non-invasive epidemiological data. In this study, a one-year WBE study was conducted in Perugia (Umbria, Italy) to evaluate respiratory viruses using multiplex assays. Fifty-two wastewater samples were collected weekly between October 2023 and September 2024 and analyzed with the Allplex™ Respiratory Panel 2/3 (for Adenovirus (AdV), Enterovirus (EV), Metapneumovirus (MPV), Parainfluenza virus types 1/2/3/4 (PIV1-4), Bocavirus (BoV), Alphacoronavirus 229E, Alphacoronavirus NL63, Betacoronavirus OC43, and Rhinovirus (RV)) and FTD respiratory pathogens 21 (only for IFV-A and RSV). The results were descriptively compared with clinical data to explore temporal concordance between wastewater and clinical surveillance. Wastewater analysis revealed frequent detection of AdV, EV, and BoV, whereas RV was detected in 67.3% of samples. AdV, EV, and BoV were detected more frequently in wastewater than in clinical samples. RSV showed a seasonal pattern in clinical samples but was only sporadically detected in wastewater, whereas PIVs were detected more frequently in clinical samples than in wastewater. By descriptive comparison, IFV-A, MPV, and non-SARS-CoV-2 coronaviruses showed peaks occurring in similar periods in wastewater and clinical samples. These results support the potential of WBE as a complementary tool to traditional clinical surveillance and suggest that commercial multiplex assays may be useful for broad, exploratory screening of respiratory viral targets in wastewater.