Protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using RT-qPCR and pepper mild mottle virus normalization.

Wastewater surveillance allows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection levels to be tracked in a community. Here, we present a protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using quantitative reverse-transcription PCR (RT-qPCR) and pepper mild mottle virus (PMMoV) normalization. We describe steps for the pasteurization of wastewater samples, solids separation, supernatant filtration, viral precipitation and concentration, and RNA extraction. We then detail procedures for RT-qPCR, viral concentration extrapolation, PMMoV normalization, and longitudinal analysis. This protocol has the potential to be used for surveillance of other microorganisms. For complete details on the use and execution of this protocol, please refer to Sanchez Jimenez et al.1.

Wastewater surveillance in the COVID-19 post-emergency pandemic period: A promising approach to monitor and predict SARS-CoV-2 surges and evolution.

On May 24, 2023, approximately 3.5 years into the pandemic, the World Health Organization (WHO) declared the end of the COVID-19 global health emergency. However, as there are still ∼3000 COVID-19 deaths per day in May 2023, robust surveillance systems are still warranted to return to normalcy in times of low risk and respond appropriately in times of high risk. The different phases of the pandemic have been defined by infection numbers and variants, both of which have been determined through clinical tests that are subject to many biases. Unfortunately, the end of the COVID-19 emergency threatens to exasperate these biases, thereby warranting alternative tracking methods. We hypothesized that wastewater surveillance could be used as a more accurate and comprehensive method to track SARS-CoV-2 in the post-emergency pandemic period (PEPP). SARS-CoV-2 was quantified and sequenced from wastewater between June 2022 and March 2023 to research the anticipated 2022/23 winter surge. However, in the 2022/23 winter, there was lower-than-expected SARS-CoV-2 circulation, which was hypothesized to be due to diagnostic testing biases but was confirmed by our wastewater analysis, thereby emphasizing the unpredictable nature of SARS-CoV-2 surges while also questioning its winter seasonality. Even in times of low baseline circulation, we found wastewater surveillance to be sensitive enough to detect minor changes in circulation levels ∼30-46 days prior to diagnostic tests, suggesting that wastewater surveillance may be a more appropriate early warning system to prepare for unpredictable surges in the PEPP. Furthermore, sequencing of wastewater detected variants of concern that were positively correlated with clinical samples and also provided a method to identify mutations with a high likelihood of appearing in future variants, necessary for updating vaccines and therapeutics prior to novel variant circulation. Together, these data highlight the effectiveness of wastewater surveillance in the PEPP to limit the global health burden of SARS-CoV-2 due to increases in circulation and/or viral evolution.