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.

Uncertainties in estimating SARS-CoV-2 prevalence by wastewater-based epidemiology

“…Recent studies indicate the SARS-CoV-2 was rather stable at 4°C for at least 14 days [64] , [91] . The persistence of SARS-CoV-2 RNA after 24 h storage at 4°C was demonstrated with the same positive ratio before and after storage though the actual concentration of RNA detected was not reported [77] . A storage time within 48-72 hours is highly recommended to avoid the decay of SARS-CoV-2 RNA in wastewater [86] .…”

“…With the recent development of detection methods, 13 available primer-probe sets, targeting regions and cycling parameters were further summarized (Table S2). Among these primer and probe sets, inconsistent performance between different primer-probe sets was commonly observed [27] , [75] , [77] , [80] , [98] . For instance, using the same wastewater samples, a higher positive ratio was detected with CDC N1 assays (19/63) than in E_Sarbeco assays (2/63) [75] .…”

Evaluating the presence of SARS-CoV-2 RNA in the particulate matters during the peak of COVID-19 in Padua, northern Italy

“…2.5 gc/μL. Nevertheless, some authors suggested a possible differential performance between the N and the Orf1b assay, with N showing a 10× sensitivity in both clinical and environmental samples ( Chu et al, 2020 ; Baldovin et al, 2020 ). The above described processes are graphically represented in the flow chart of Fig.…”

Effects of COVID-19 on the environment: An overview on air, water, wastewater, and solid waste

“…This, in return, has raised the concern of having the wastewater, if not properly treated, as a tool to transmit and hence increase the SARS-CoV-2 spread and infection ( Gonzalez et al, 2020 ). Baldovin et al have analyzed samples from different wastewater points such as pumping station, wastewater treatment plant inlet, and outlet and found that SARS-CoV-2 RNA was present in both untreated and treated wastewater, with persistence up to 24 h in samples kept at 4 o C ( Baldovin et al, 2020 ). The relatively long half-life span of the SARS-CoV-2 of about 3 days in sewage systems and 3-4 in solid feces have been the main concern, as it can result in the increased spread and infection rate ( Nghiem et al, 2020 ).…”