SARS-CoV-2 RNA detected in urban wastewater from Porto, Portugal: Method optimization and continuous 25-week monitoring

Research on the emerging COVID-19 pandemic is demonstrating that wastewater infrastructures can be used as public health observatories of virus circulation in human communities. Important efforts are being organized worldwide to implement sewage-based surveillance of SARS-CoV-2 that can be used for preventive or early warning purposes, informing preparedness and response measures. However, its successful implementation requires important and iterative methodological improvements, as well as the establishment of standardized methods. The aim of this study was to develop a continuous monitoring protocol for SARS-CoV-2 in wastewater, that could be used to model virus circulation within the communities, complementing the current clinical surveillance. Specific objectives included (1) optimization and validation of a method for virus quantification; (2) monitoring the time-evolution of SARS-CoV-2 in wastewater from two wastewater treatment plants (WWTPs) in the city of Porto, Portugal. Untreated wastewater samples were collected weekly from the two WWTPs between May 2020 and March 2021, encompassing two COVID-19 incidence peaks in the region (mid-November 2020 and mid-January 2021). In the first stage of this study, we compared, optimized and selected a sampling and analysis protocol that included virus concentration through centrifugation, RNA extraction from both liquid and solid fractions and quantification by reverse transcription quantitative PCR (RT-qPCR). In the second stage, we used the selected methodology to track SARS-CoV-2 in the collected wastewater over time. SARS-CoV-2 RNA was detected in 39 and 37 out of 48 liquid and solid fraction samples of untreated wastewater, respectively. The copy numbers varied throughout the study between 0 and 0.15 copies/ng RNA and a good fit was observed between the SARS-CoV-2 RNA concentration in the untreated wastewater and the COVID-19 temporal trends in the study region. We also analyzed eight samples from the treated effluent and found no SARS-CoV-2 RNA detection after tertiary treatment and UV disinfection. In agreement with the recent literature, the results from this study support the use of wastewater-based surveillance to complement clinical testing and evaluate temporal and spatial trends of the current pandemic.