Detection and targeted sequencing of influenza A virus subtypes using Dutch wastewater samples from influenza season 2023-2024 and 2024-2025.

The substantial public health burden of influenza A viruses (IAV) requires continual monitoring of circulation to identify risks. Wastewater-based surveillance (WBS) enables non-invasive monitoring of IAV circulation in large populations. In this study, we use the WBS infrastructure set up in the Netherlands during the coronavirus disease 2019 pandemic to evaluate the practical application of monitoring IAV in wastewater. We employ RT-qPCR to identify trends in IAV positivity using 5618 wastewater samples taken at 40 treatment plants, which peaked during flu epidemics. Furthermore, custom primer panels were used to perform tiled-amplification and subsequent sequencing of the hemagglutinin (HA) and neuraminidase (NA) segments of the prevalent subtypes A(H1N1)pdm09 and A(H3N2) from 128 wastewater samples. This enabled (partial) recovery of IAV segments in 115 samples, which were used to determine relative subclade abundances and identify vaccine-resistant mutations, both of which corresponded well to clinical surveillance data. Although further improvements in sample virus concentration and assay sensitivity are required to consistently generate high-quality data and insights for public health professionals, we show that monitoring of IAV and associated subtypes in wastewater is feasible.