Detection of SARS-CoV-2 RNA in wastewater, river water, and hospital wastewater of Nepal

The applicability of wastewater-based epidemiology (WBE) has been extensively studied throughout the world with remarkable findings. This study reports the presence and reduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at two wastewater treatment plants (WWTPs) of Nepal, along with river water, hospital wastewater (HWW), and wastewater from sewer lines collected between July 2020 and February 2021. SARS-CoV-2 RNA was detected in 50%, 54%, 100%, and 100% of water samples from WWTPs, river hospitals, and sewer lines, respectively, by at least one of four quantitative PCR assays tested (CDC-N1, CDC-N2, NIID_2019-nCOV_N, and N_Sarbeco). The CDC-N2 assay detected SARS-CoV-2 RNA in the highest number of raw influent samples of both WWTPs. The highest concentration was observed for an influent sample of WWTP A (5.5 +/- 1.0 log(10) genome copies/L) by the N_Sarbeco assay. SARS-CoV-2 was detected in 47% (16/34) of the total treated effluents of WWTPs, indicating that biological treatments installed at the tested WWTPs are not enough to eliminate SARS-CoV-2 RNA. One influent sample was positive for N501Y mutation using the mutation-specific qPCR, highlighting a need for further typing of water samples to detect Variants of Concern. Furthermore, crAssphage-normalized SARS-CoV-2 RNA concentrations in raw wastewater did not show any significant association with the number of new coronavirus disease 2019 (COVID-19) cases in the whole district where the WWTPs were located, suggesting a need for further studies focusing on suitability of viral as well as biochemical markers as a population normalizing factor. Detection of SARS-CoV-2 RNA before, after, and during the peaking in number of COVID-19 cases suggests that WBE is a useful tool for COVID-19 case estimation in developing countries.