Methodic aspects of influenza and respiratory syncytial virus detection in raw wastewater and presence in treatment plants in southeastern Germany.

During the COVID-19 pandemic, wastewater-based epidemiology (WBE) has been developed as an additional tool to follow epidemiological trends in the catchment area of treatment plants. Meanwhile, further viral agents of respiratory infections were included in monitoring programs. However, differences in sample processing may impair the results comparison among studies. With identical virus strains, we investigated different concentration methods, RNA isolation kits and primer/probe combinations for detection of influenza virus (IV) A/B and respiratory syncytial virus (RSV) A/B. For procedure optimization, virus enrichment and RNA extraction methods were found to be of relevance. Conversely, the detection step was identified to have a relatively low influence. Using standardized protocols, 24 h composite samples from eight wastewater treatment plants (WWTP) in southeast Germany (2x/week) were analyzed (January 2024 to December 2024) for IVA/B and RSV-A/B. The following rates of virus-positive samples were determined: 20.5% (IVA including 2.2% H1N1pdm09), 4.5% (IVB), 32.6% (RSV-A) and 2.4% (RSV-B). IV and RSV detections showed a matching trend when compared to reported cases. This study contributes to a better understanding of factors influencing the detection of IV and RSV in wastewater as well as in the epidemiological significance of virus monitoring in WWTPs.