In recent months, wastewater-based epidemiology (WBE) has been shown to be an important tool for early detection of SARS-CoV-2 circulation in the population. In this study, a detection methodology for SARS-CoV-2 RNA (wildtype and variants of concern) in wastewater was developed based on the detection of different target genes (E and ORF1ab) by polyethylene glycol (PEG) precipitation and digital droplet PCR. This methodology was used to determine the SARS-CoV-2 concentration and the proportion of N501Y mutation in raw sewage of the wastewater treatment plant of the city of Karlsruhe in south-western Germany over a period of 1 year (June 2020 to July 2021). Comparison of SARS-CoV-2 concentrations with reported COVID-19 cases in the catchment area showed a significant correlation. As the clinical SARS-CoV-2 official case report chain takes time, viral RNA titre trends appeared more than 12 days earlier than clinical data, demonstrating the potential of wastewater-based epidemiology as an early warning system. Parallel PCR analysis using seven primer and probe systems revealed similar gene copy numbers with E, ORF, RdRP2 and NSP9 assays. RdPP1 and NSP3 generally resulted in lower copy numbers, and in particular for N1 there was low correlation with the other assays. The occurrence of the N501Y mutation in the wastewater of Karlsruhe was consistent with the occurrence of the alpha-variant (B.1.1.7) in the corresponding individual clinical tests. In batch experiments SARS-CoV-2 RNA was stable for several days under anaerobic conditions, but the copy numbers decreased rapidly in the presence of dissolved oxygen. Overall, this study shows that wastewater-based epidemiology is a sensitive and robust approach to detect trends in the spread of SARS-CoV-2 at an early stage, contributing to successful pandemic management.

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In the context of the COVID-19 pandemic, wastewater-based epidemiology (WBE) emerged as a useful tool to account for the prevalence of SARS-CoV-2 infections on a population scale. In this study, we analyzed wastewater samples from three large (>300,000 people served) and four small (<25,000 people served) communities throughout southern Germany from August to December 2021, capturing the fourth infection wave in Germany dominated by the Delta variant (B.1.617.2). As dilution can skew the SARS-CoV-2 biomarker concentrations in wastewater, normalization to wastewater parameters can improve the relationship between SARS-CoV-2 biomarker data and clinical prevalence data. In this study, we investigated the suitability and performance of various normalization parameters. Influent flow data showed strong relationships to precipitation data; accordingly, flow-normalization reacted distinctly to precipitation events. Normalization by surrogate viruses CrAssphage and pepper mild mottle virus showed varying performance for different sampling sites. The best normalization performance was achieved with a mixed fecal indicator calculated from both surrogate viruses. Analyzing the temporal and spatial variation of normalization parameters proved to be useful to explain normalization performance. Overall, our findings indicate that the performance of surrogate viruses, flow, and hydro-chemical data is site-specific. We recommend testing the suitability of normalization parameters individually for specific sewage systems.