Comparative assessment of combined concentration and extraction methods for Influenza A and B virus detection in wastewater

Influenza, caused by Influenza A and B viruses, represents a significant global health burden due to recurrent seasonal epidemics and the risk of pandemics. To gauge the large volume of seasonal influenza cases, it may be helpful to complement classical surveillance systems with additional approaches such as wastewater-based epidemiology (WBE), which can aid in the early trend assessment of seasonal epidemics. WBE has emerged as a promising tool for population-level surveillance, enabling the detection of viral nucleic acids in wastewater and offering unique advantages over individual-based surveillance. This study evaluates the performance of six different combinations of virus concentration and RNA extraction methods for the detection of Influenza A and B viruses in wastewater. Composite samples from four wastewater treatment plants in North Germany were analyzed using precipitation, filtration and automated extraction protocols. Method performance was evaluated by quantitative recovery of viral RNA and a spike-and-recovery experiment. The combination of PureYield™ filtration and Maxwell® RSC extraction (PYC_EX1) consistently demonstrated the highest recovery rates for both Influenza virus A and B, achieving recovery efficiencies of up to 80.4% and 72.3%, respectively. This method also enabled reliable detection of low viral loads, which is critical for an early detection of rising incidence. Our findings underscore the importance of rigorous method evaluation to optimize WBE for influenza surveillance. By providing robust, sensitive and reproducible protocols, this study highlights the potential of WBE to improve public health preparedness, enables timely interventions and reduces the spread of influenza viruses within communities.