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

Abstract

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 in untreated wastewaters has been reported in several countries. This study investigated the presence and persistence of viral RNA in treated and untreated wastewaters in Padua, Italy. An urban experimental network of sampling sites was tested for prospective surveillance activities.

Methods: Seven sampling sites (i.e. wastewater pumping stations, plant inlets and outlets) were selected from the two main municipal wastewater treatment plant systems. Eleven grab samples (9 untreated, 2 treated wastewaters) were collected on 2 dates. All samples were tested at t0 for SARS-CoV-2 RNA and t1 = 24 h to investigate its persistence, at room temperature and under refrigerated conditions. Overall, 33 sub-samples were concentrated by 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 refrigerated subsamples were positive as well. The two RT-qPCR assays showed differential sensitivity, with the N assay detecting 90% of successful amplifications.

Conclusions: SARS-CoV-2 RNA was detected in untreated and treated wastewaters. Its persistence after 24 h was demonstrated in subsamples kept at 4 °C. Hospitalization data suggested an approximate WBE detection power of 1 COVID-19 case per 531 inhabitants. The possible role of WBE in COVID-19 environmental surveillance is strongly supported by our findings. WBE can also provide precious support in the decision-making process of restriction policies during the epidemic remission phase. Optimization and standardization of laboratory methods should be sought in the short term, so that results from different studies can be compared with reliability.

Keywords: COVID-19; Environmental surveillance; SARS-CoV-2; Sewage; WBE.

Graphical abstract

Fig. 1

Map of sampling sites. The figure reports a simplified scheme of Padua sewerage system. The city main wastewater treatment plant (WWTP-A) has two separate inlets. The red and pink areas (city center) are served by two district wastewater pumping stations (WPSs), A-U-1 and A-U-2. Especially, A-U-1 drains sewage from the city hospital district. Wastewater from A-U-1 and A-U-2 then merge and reach WWTP-A as a unique influent (A-U-3). The second WWTP_A influent originates from the orange area (site A-U-4). The separate Southern blue area is served by WWTP_B. The plant influent and effluent were sampled (B-U-1 and B-T-1). (Figure adapted from Acegas-Aps-Amga technical sewerage plan, personal communication, 20 April 2020). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Graphical summary of COVID-19 epidemiological data for Padua Municipality. The figure shows a time frame of ±15 days from sampling dates (i.e. April 23rd and May 5th), reporting daily COVID-19 cases and the total cumulative case number, as reference for the local epidemic trend. Point prevalence was of 202 and 145 cases per 100,000 inhabitants on the first and on the second sampling date, that is 427 and 306 COVID-19 cases in the municipality, respectively (clinical surveillance data derived from: Protezione Civile, 2020).