Influenza RNA fluxes monitoring in wastewater as a complementary epidemiological surveillance indicator: A four-year nationwide study in Luxembourg

Wastewater surveillance has demonstrated success in monitoring SARS-CoV-2 in communities, indicating potential for extension to other respiratory viruses. This study investigates influenza A and B viruses (IAV; IBV) in raw urban wastewater over a 4-year period, introducing two key concepts: the use of viral RNA fluxes instead of concentration measurements and the determination of epidemiological parameters directly from wastewater data. The estimation of daily fluxes, representing the number of viral genome copies per day per 100,000 inhabitants, offers an integrative approach that combines microbiological and hydrological measurements to better assess viral particle dynamics in a water system. A total of 1013 wastewater samples collected between March 2020 and March 2024 from Luxembourg's four largest wastewater treatment plants (covering about 52 % of the population) were analysed using RT-qPCR and RT-droplet digital PCR (RT-ddPCR), following concentration of viral particles by ultrafiltration. Data on the presence of IAV and IBV were expressed as either detection rates or fluxes. Significant correlations were observed between the number of laboratory-confirmed influenza cases and both wastewater detection rates (RT-qPCR: Spearman ρ = 0.52; RT-ddPCR: ρ = 0.61, p-value <10-13) and viral RNA fluxes (RT-ddPCR: ρ = 0.64, p-value <10-15). More importantly, our results demonstrated that critical influenza seasonality parameters (start, peak and end weeks of the epidemic) can be effectively determined from wastewater data. These findings establish wastewater surveillance as a cost-effective, non-invasive approach to support and complement existing influenza surveillance programs, with potential applications for other respiratory pathogens.