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A six-plex digital PCR assay for monitoring respiratory viruses in wastewater

Nature Water (2025)Cite this article

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Abstract

Wastewater surveillance can track trends in multiple pathogens simultaneously by leveraging efficient laboratory processing. In Switzerland, wastewater surveillance of four respiratory pathogens is conducted at 14 locations representing 2.3 million people. Trends in respiratory diseases are tracked using a six-plex digital PCR assay targeting influenza A, influenza B, respiratory syncytial virus and SARS-CoV-2 N1 and N2 regions and murine hepatitis virus for recovery efficiency control. Wastewater data were integrated with disease data from two reporting systems, and comparisons from July 2023 to July 2024 showed strong agreement for most targets. Lower correspondence for influenza B highlighted challenges in tracking disease dynamics during seasons without pronounced outbreaks. Wastewater monitoring further revealed that targeting N1 or N2 led to divergent estimates of SARS-CoV-2 loads, highlighting the impact of mutations in assay target regions. The study emphasizes the importance of an integrated wastewater monitoring programme as a complementary tool for public health surveillance, demonstrating clear concordance with clinical data.

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Fig. 1: Two-dimensional visualization of the partition classification of RESPV6 dPCR assay after measuring positive control.
Fig. 2: Linear regression of target concentrations determined by the RESPV6 dPCR assay versus a single-plex assay.
Fig. 3: SARS-CoV-2 RNA concentrations comparing N1 and N2 genes.
Fig. 4: SARS-CoV-2 N1 and N2 load in wastewater across 14 Swiss locations.
Fig. 5: IAV, IBV and RSV RNA loads in wastewater across 14 Swiss locations.
Fig. 6: Comparison of viral RNA loads in wastewater and number of cases from Sentinella system.

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Data availability

Digital PCR data are available for download from wise.ethz.ch. Only data that passed our described quality control processes are available here. All data are available on request. Weekly case data are available from the Federal Office of Public Health (FOPH) via a public API (https://api.idd.bag.admin.ch/swagger-ui/api-doc.html). Daily case data were specifically requested from the FOPH.

Code availability

All code used in the analysis is available via GitHub at https://github.com/EawagPHH/A-six-plex-digital-PCR-assay-for-monitoring-respiratory-viruses-in-wastewater/blob/main/Analysis.Rmd.

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Acknowledgements

We would like to thank present and past members of the Laboratory Monitoring Team for developing the methods, processing samples and generating and analysing data. Additionally, we thank present and past collaborators of the WISE (Wastewater-based Infectious disease Surveillance and Epidemiology) project for their support and valuable intellectual contributions and NEXUS for their setting up and supporting the WISE database (wisedb.ethz.ch) which facilitated our analysis. We thank the technical staff of each WWTP for promptly sending us the wastewater samples on a weekly basis, the FOPH for sharing case data with us and A.C. Ripolles and T. Kohn for providing us with MHV. This study was funded by the Swiss National Science Foundation (SNSF Sinergia grant number CRSII5_205933) and by the Swiss Federal Office of Public Health (grant numbers 142006108/334.0-101/26 and 142006655/334.0-107/12) granted to C.O. and T.R.J.

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Author notes

  1. These authors contributed equally: Melissa Pitton, Rachel E. McLeod.

Authors and Affiliations

  1. Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland

    Melissa Pitton, Rachel E. McLeod, Lea Caduff, Ayazhan Dauletova, Jolinda de Korne-Elenbaas, Charles Gan, Camille Hablützel, Aurélie Holschneider, Seju Kang, Guy Loustalot, Patrick Schmidhalter, Linda Schneider, Anna Wettlauffer, Daniela Yordanova, Timothy R. Julian & Christoph Ort

  2. Swiss Tropical and Public Health Institute, Allschwil, Switzerland

    Timothy R. Julian

  3. University of Basel, Basel, Switzerland

    Timothy R. Julian

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Contributions

C.O. and T.R.J. contributed equally. C.O. and T.R.J. conceptualized, supervised and provided funding. M.P. and R.E.M. designed the study, performed analyses, prepared visualizations and wrote the first draft of the manuscript. L.C., J.d.K.-E., C.G. and S.K. contributed invaluable scientific insights. P.S. provided statistical support. A.D., C.H., A.H., G.L., L.S., A.W. and D.Y. helped with sample processing, data generation and curation. We declare that all authors have seen and approved the manuscript and have contributed substantially to the work.

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Correspondence to Timothy R. Julian or Christoph Ort.

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Nature Water thanks Robert Delatolla, David Walker and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Tables 1–6, Figs. 1–5 and Texts 1 and 2.

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Supplementary Table 1

dMIQE2020 checklist for authors, reviewers and editors.

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Pitton, M., McLeod, R.E., Caduff, L. et al. A six-plex digital PCR assay for monitoring respiratory viruses in wastewater. Nat Water (2025). https://doi.org/10.1038/s44221-025-00503-x

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