SARS-CoV-2 RNA in Wastewater Was Highly Correlated With the Number of COVID-19 Cases During the Fourth and Fifth Pandemic Wave in Kobe City, Japan

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the current coronavirus disease 2019 (COVID-19) pandemic and associated respiratory infections, has been detected in the feces of patients. Therefore, determining SARS-CoV-2 RNA levels in sewage may help to predict the number of infected people within the area. In this study, we quantified SARS-CoV-2 RNA copy number using reverse transcription quantitative real-time PCR with primers and probes targeting the N gene, which allows the detection of both wild-type and variant strain of SARS-CoV-2 in sewage samples from two wastewater treatment plants (WWTPs) in Kobe City, Japan, during the fourth and fifth pandemic waves of COVID-19 between February 2021 and October 2021. The wastewater samples were concentrated via centrifugation, yielding a pelleted solid fraction and a supernatant, which was subjected to polyethylene glycol (PEG) precipitation. The SARS-CoV-2 RNA was significantly and frequently detected in the solid fraction than in the PEG-precipitated fraction. In addition, the copy number in the solid fraction was highly correlated with the number of COVID-19 cases in the WWTP basin (WWTP-A: r = 0.8205, p < 0.001; WWTP-B: r = 0.8482, p < 0.001). The limit of capturing COVID-19 cases per 100,000 people was 0.75 cases in WWTP-A and 1.20 cases in WWTP-B, respectively. Quantitative studies of RNA in sewage can be useful for administrative purposes related to public health, including issuing warnings and implementing preventive measures within sewage basins.