First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan

Eiji Haramoto,Bikash Malla,Ocean Thakali,Masaaki Kitajima

doi:10.1016/j.scitotenv.2020.140405

Abstract

Wastewater-based epidemiology is a powerful tool to understand the actual incidence of coronavirus disease 2019 (COVID-19) in a community because severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can be shed in the feces of infected individuals regardless of their symptoms. The present study aimed to assess the presence of SARS-CoV-2 RNA in wastewater and river water in Yamanashi Prefecture, Japan, using four quantitative and two nested PCR assays. Influent and secondary-treated (before chlorination) wastewater samples and river water samples were collected five times from a wastewater treatment plant and three times from a river, respectively, between March 17 and May 7, 2020. The wastewater and river water samples (200–5000 mL) were processed by using two different methods: the electronegative membrane-vortex (EMV) method and the membrane adsorption-direct RNA extraction method. Based on the observed concentrations of indigenous pepper mild mottle virus RNA, the EMV method was found superior to the membrane adsorption-direct RNA extraction method. SARS-CoV-2 RNA was successfully detected in one of five secondary-treated wastewater samples with a concentration of 2.4 × 103 copies/L by N_Sarbeco qPCR assay following the EMV method, with sequence confirmation of the qPCR product, whereas all the influent samples were tested negative for SARS-CoV-2 RNA. This result could be attributed to higher limit of detection for influent (4.0 × 103–8.2 × 104 copies/L) with a lower filtration volume (200 mL) compared to that for secondary-treated wastewater (1.4 × 102–2.5 × 103 copies/L) with a higher filtration volume of 5000 mL. None of the river water samples tested positive for SARS-CoV-2 RNA. Comparison with the reported COVID-19 cases in Yamanashi Prefecture showed that SARS-CoV-2 RNA was detected in the secondary-treated wastewater sample when the cases peaked in the community. This is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in Japan.

Highlights

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing global pandemic of coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China, in December 2019 (World Health Organization, 2020a)
The reported proportion of asymptomatic infection of COVID-19 ranges from 18% to 31% (Mizumoto et al, 2020; Nishiura et al, 2020; Treibel et al, 2020), and 21% of COVID-19 patients showed diarrheal symptoms (Wan et al 2020), which means a large number of symptomatic and asymptomatic individuals shed the virus in stool, which reaches a wastewater treatment plant (WWTP) through sewage pipes
The log10 reduction ratios (mean ± standard deviation (SD)) of E. coli and pepper mild mottle virus (PMMoV) RNA based on their concentrations in influent and secondary-treated wastewater samples were 2.7 ± 0.3 log10 (n = 5) and 1.8 ± 0.2 log10 (n = 5), respectively

Summary

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing global pandemic of coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China, in December 2019 (World Health Organization, 2020a). This report suggests that SARS-CoV-2 actively replicates in enterocytes of human intestine and the virus is subsequently shed in feces. A previous study investigating a total of 4,243 COVID-19 patients reported that 17.6% of the patients exhibited gastrointestinal symptoms and SARS-CoV-2 RNA was detected in stool samples from higher proportion (48.1%) of the patients (Cheung et al, 2020). This result indicated that the virus could be shed in feces of infected individuals without gastrointestinal symptoms in addition to patients with diarrhea. Even after a patient recover from the respiratory symptoms, viruses can still be shed in feces for several days (Wu et al, 2020)

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