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. 2023 Jan 17;15(2):263.
doi: 10.3390/v15020263.

Expanding the Pathogen Panel in Wastewater Epidemiology to Influenza and Norovirus

Rudolf Markt  1   2 , Fabian Stillebacher  1 , Fabiana Nägele  1 , Anna Kammerer  1 , Nico Peer  1 , Maria Payr  1 , Christoph Scheffknecht  3 , Silvina Dria  3 , Simon Draxl-Weiskopf  1 , Markus Mayr  1 , Wolfgang Rauch  4 , Norbert Kreuzinger  5 , Lukas Rainer  6 , Florian Bachner  6 , Martin Zuba  6 , Herwig Ostermann  6 , Nina Lackner  2 , Heribert Insam  1 , Andreas Otto Wagner  1
Affiliations

Expanding the Pathogen Panel in Wastewater Epidemiology to Influenza and Norovirus

Rudolf Markt et al. Viruses. .

Abstract

Since the start of the 2019 pandemic, wastewater-based epidemiology (WBE) has proven to be a valuable tool for monitoring the prevalence of SARS-CoV-2. With methods and infrastructure being settled, it is time to expand the potential of this tool to a wider range of pathogens. We used over 500 archived RNA extracts from a WBE program for SARS-CoV-2 surveillance to monitor wastewater from 11 treatment plants for the presence of influenza and norovirus twice a week during the winter season of 2021/2022. Extracts were analyzed via digital PCR for influenza A, influenza B, norovirus GI, and norovirus GII. Resulting viral loads were normalized on the basis of NH4-N. Our results show a good applicability of ammonia-normalization to compare different wastewater treatment plants. Extracts originally prepared for SARS-CoV-2 surveillance contained sufficient genomic material to monitor influenza A, norovirus GI, and GII. Viral loads of influenza A and norovirus GII in wastewater correlated with numbers from infected inpatients. Further, SARS-CoV-2 related non-pharmaceutical interventions affected subsequent changes in viral loads of both pathogens. In conclusion, the expansion of existing WBE surveillance programs to include additional pathogens besides SARS-CoV-2 offers a valuable and cost-efficient possibility to gain public health information.

Keywords: PEG-precipitation; digital PCR; influenza; inpatients; norovirus; surveillance; wastewater; wastewater-based epidemiology.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Map of Austria and its political districts. Colored districts correspond to those containing the 6 WWTPs monitored in Salzburg (blue) and the 5 WWTPs in Vorarlberg (red), covering in total 0.7 Mio inhabitants.
Figure 2
Figure 2
Normalized virus loads for (A) influenza A, (B) norovirus GII, and (C) norovirus GI per WWTP. More intense color corresponds to higher loads in wastewater. Each plot is scaled separately due to large discrepancy of absolute loads between pathogens. WWTP V001–V005 correspond to the federal state Vorarlberg and WWTPs Z001–Z012 correspond to the federal state Salzburg.
Figure 3
Figure 3
Seasonal occurrence of (red line) influenza virus and (blue line) norovirus infections in inpatients aggregated per month across Austria. The dashed black line marks the date of the first clinical COVID-19 case in Austria.
Figure 4
Figure 4
Comparison between viral loads in wastewater and number of infected inpatients from September 2021–March 2022 including time points for non-pharmaceutical intervention for IAV (A) and NoV GII (B). (Blue dots) Viral loads and (red dots) number of inpatients were aggregated at a weekly basis. Smoothing lines with 95% confidence bands correspond to LOWESS-smoothing. Six events increasing or decreasing the impact of NPIs on the spread of the COVID-19 pandemic were set from September 2021 to March 2022: September 9–school opens, (I) November 15—lockdown for those unvaccinated, (II) November 22nd—complete lockdown, (III) December 12—partial opening, (IV) January 11—Omicron measures, (V) March 5—complete opening.
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