Adapted methods for monitoring influenza virus and respiratory syncytial virus in sludge and wastewater

Wastewater-based surveillance constitutes a valuable methodology for the continuous monitoring of viral circulation, with the capacity to function as an early warning system. It holds particular significance in scenarios where respiratory viruses exhibit overlapping clinical presentations, as occurs with SARS-CoV-2, influenza virus (IV), and respiratory syncytial virus (RSV), and allows seasonal virus outbreaks to be distinguished from COVID19 peaks. Furthermore, sewage sludge, given it harbors concentrated human waste from a large population, serves as a substantial reservoir for pathogen detection. To effectively integrate wastewater-based epidemiology into infectious disease surveillance, the detection methods employed in wastewater samples must be adapted to the distinct characteristics of sludge matrices. In this study, we adapted and applied protocols for the detection of IV and RSV in sewage sludge, comparing their performance with the results obtained in wastewater. To assess the efficiency of these protocols, sludge and wastewater samples were spiked with IV and RSV RNA, either free or incorporated in lentiviral particles. Samples were concentrated using the aluminum hydroxide adsorption-precipitation method before viral RNA extraction. Absolute virus quantification was carried out by RT-qPCR, including an internal control to monitor potential inhibitory factors. Recovery efficiencies for both free IV and RSV RNA were 60 % in sludge, and 75 % and 71 % respectively in wastewater, whereas the values for IV and RSV RNA in lentiviral particles were 16 % and 10 % in sludge and 21 % and 17 % in wastewater respectively. Additionally, the protocol enabled the quantification of naturally occurring IV and RSV in wastewater and sludge samples collected from two wastewater treatment plants during the winter months, thus affirming the efficacy of the employed methodologies.