Multiplex dpcr detection of RSV and influenza viruses in urban wastewater: a proof-of-concept study

Aim of the study Among the most important respiratory viruses of public health concern are influenza virus and respiratory syncytial virus (RSV), both of which are responsible for severe seasonal epidemics with high morbidity and mortality rates, especially in vulnerable populations. Wastewater-based epidemiology (WBE) has recently emerged as a valuable tool for monitoring respiratory viruses in the population, in addition to enteric viruses. In this context, the new EU Urban Wastewater Directive (2024/3019) promotes the systematic monitoring of pathogens, including influenza viruses, in urban wastewater. The aim of this study was to provide a methodology for the analysis of respiratory viruses of public health interest in wastewater to support and complement clinical surveillance. Methods used A total of 60 wastewater samples were analysed, collected weekly from October 2023 to March 2024 from three wastewater treatment plants in the city of Rome. Samples were processed using PEG and NaCl precipitation; nucleic acids were extracted using commercial magnetic silica-based systems and purified using the OneStep PCR Inhibitor Removal Kit (Zymo Research). The samples were analysed using multiplex RT-digital PCR (RT-dPCR) assays optimised for the detection and quantification of Influenza A/B and RSV A/B viral targets in wastewater. Results and conclusions Of the 60 wastewater samples analysed, 25% tested positive for influenza A virus (IAV), 1.7% for influenza B virus (IBV), 40% for respiratory syncytial virus A (RSV A) and 10% for RSV B. IAV was detected in December, January and February, with the highest positivity rate of 11.7% in both December and January. IBV was detected in only one sample in February. RSV A was detected continuously from October 2023 to March 2024, showing a gradual increase and peaking at 13.3% in February. RSV B was detected in October, January, February and March, with a peak of 5% in January and a minimum positivity rate of 1.7% in the other months. Virus concentrations ranged from 1.64 × 10³ to 1.06 × 10⁴ genome copies per litre (gc/L) for IAV, with the highest value recorded on 28 December. For IBV, the only positive sample in February showed a concentration of 1.64 × 10³ gc/L. RSV A concentrations ranged from 1.60 × 10³ to 1.18 × 10⁴ gc/L, while RSV B concentrations ranged from 1.58 × 10³ to 3.14 × 10³ gc/L. Overall, with this approach the environmental data suggested to be consistent with the human surveillance trends observed during the study period in the country. The study confirms the support of wastewater-based epidemiology (WBE) in monitoring seasonal respiratory viruses such as Influenza A/B and RSV A/B. The results support the use of WBE as a complementary tool to clinical surveillance in line with the new EU Urban Wastewater Treatment Directive.