Analysing community wastewater in sub-sewersheds for the early detection of SARS-CoV-2 variants in a metropolitan area in Germany. wastewater-pilot

Background: Wastewater surveillance of SARS-CoV-2 offers advantages for different use cases, including the identification of emerging or novel virus variants. Previous studies focused either on wastewater treatment plants (WWTP) that treat wastewater of several hundred thousand people or at building level, with both approaches posing distinct challenges regarding small-scale intra-urban inferences, and therefore hampering early warning of emergence of novel virus variants. Objectives: This study tested the early detection potential of SARS-CoV-2 virus variants by analysing community wastewater at a small-scale sub-sewershed-level. It further examined spatial and temporal differences in the occurrence of SARS-CoV-2 RNA and viral single genomic mutations in community wastewater within a metropolitan wastewater system. Methods: To make an informed selection of sampling points within the wastewater system, we created a wastewater network and estimated relevant population- and environment-related variables. SARS-CoV-2 RNA in community wastewater from three sub-sewersheds and the receiving WWTP was quantified by real-time-qPCR and sequenced via whole genome sequencing. Results: High inter- and intravariability in the occurrence of SARS-CoV-2 single genomic mutations characterized the wastewater samples in the sub-sewersheds and the WWTP. Identified single mutations reflected the dominant variant of concern (VOC) Delta. We further observed area-specific viral single genomic mutations in each sub-sewershed; a VOC Omicron-specific genomic mutation was identified in one sub-sewershed, while not being found at the WWTP, and even earlier than in previous wastewater studies at the WWTP-level in the same area. Discussion: The detection of an Omicron-specific single genomic mutation at the sub-sewershed-level creates a strong argument for the small-scale approach employed in our study as it would have been missed if only sampled at the WWTP. The results of this project suggest that the small-scale approach may prove particularly useful for the early detection and description of spatially confined emerging or existing virus variants circulating in the population.