This study presents the results of systematic wastewater monitoring of SARS-CoV-2 RNA and basic wastewater parameters from four different wastewater treatment plants (WWTPs) in the Czech Republic over the 2020-2022 epidemic. Two-step reverse-transcription quantitative PCR targeting genes encoding the N and Nsp12 proteins was employed to detect SARS-CoV-2 RNA loading in 420 wastewater samples. The results obtained were used to evaluate the potential of wastewater analysis for describing the epidemiological situation in cities of different sizes and determining temporal differences based on the prevailing SARS-CoV-2 variant. Strong correlations between the number of active and hospitalised COVID-19 cases in each WWTP catchment area and the concentration of SARS-CoV-2 RNA detected in the wastewater clearly demonstrated the suitability of this wastewater-based epidemiological approach for WWTPs of different sizes and characteristics, despite differences in SARS-CoV-2 variant waves, with some WWTPs showing high predictive potential. This study demonstrated on the data from the Czech Republic that targeted systematic monitoring of wastewater provides sufficiently robust data for surveillance of viral loads in sample populations, and thus contributes to preventing the spread of infection and subsequent introduction of appropriate measures.

Charles University 1st Faculty of Medicine Institute of Hygiene and Epidemiology Studničkova 7 128 00 Prague Czech Republic

Masaryk University Faculty of Science Kotlářská 267 2 611 37 Brno Czech Republic

T G Masaryk Water Research Institute p r i Brno Branch Mojmírovo náměstí 16 612 00 Brno Czech Republic

T G Masaryk Water Research Institute Podbabská 30 160 00 Prague Czech Republic

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