SARS-CoV-2 RNA detection and persistence in wastewater samples: An experimental network for COVID-19 environmental surveillance in Padua, Veneto Region (NE Italy)

Background: Clinical detection of SARS-CoV-2 RNA in stools supports the idea of wastewater-based epidemiology(WBE) as a precious tool for COVID-19 environmental surveillance. Successful detection of SARS-CoV-2 RNA inuntreated wastewaters has been reported in several countries. This study investigated the presence and persis-tence of viral RNA in treated and untreated wastewaters in Padua, Italy. An urban experimental network of sam-pling sites was tested for prospective surveillance activities. Methods: Seven sampling sites(i.e. wastewater pumping stations, plantinlets and outlets)wereselectedfrom thetwo main municipal wastewater treatment plant systems. Eleven grab samples (9 untreated, 2 treated wastewa-ters) were collected on 2 dates. All samples were tested at t0 for SARS-CoV-2 RNA and t1 = 24 h to investigate itspersistence, at room temperature and under refrigerated conditions. Overall, 33 sub-samples were concentratedby ultrafiltration and tested for molecular detection of viral RNA with two RT-qPCR assays. Results: At t0, positivity for at least one RT-qPCR assay was achieved by 4/9 untreated wastewater samples and 2/2 tertiary treated samples. A minimum SARS-CoV-2 titer of 4.8–4.9 log10 gc/L was estimated. At t1, three refrig-erated subsamples were positive as well. The two RT-qPCR assays showed differential sensitivity, with the Nassay detecting 90% of successful amplifications. Conclusions: SARS-CoV-2 RNA was detected in untreated and treated wastewaters. Its persistence after 24 h wasdemonstrated in subsamples kept at 4 °C. Hospitalization data suggested an approximate WBE detection powerof 1 COVID-19 case per 531 inhabitants. The possible role of WBE in COVID-19 environmental surveillance is strongly supported by ourfindings. WBE can also provide precious support in the decision-making process of re-striction policies during the epidemic remission phase. Optimization and standardization of laboratory methodsshould be sought in the short term, so that results from different studies can be compared with reliability.