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Presence of SARS-coronavirus-2 (SARS-CoV-2) in wastewater sample has been documented in several countries. Wastewater-based epidemiology (WBE) is potentially effective for early warning of COVID-19 outbreak. The purpose of this study was to verify the detection limit of WBE for COVID-19. In total, 27 influent wastewater samples were collected from four wastewater treatment plants in Ishikawa and Toyama prefectures in Japan. During the study period, numbers of the confirmed COVID-19 cases in these prefectures increased from almost 0 to around 20 per 100,000 peoples. SARS-CoV-2 RNA in the samples were identified by several PCR-based assays. Among the 27 samples, 7 were positive for SARS-CoV-2 by at least one out of the three quantitative RT-PCR assays. These samples were also positive by RT-nested PCR assays. The detection frequency became higher when the number of total confirmed SARS-CoV-2 cases in 100,000 peoples became above 10 in each prefecture. However, SARS-CoV-2 could also be detected with a low frequency when the number was below 1.0. Considering that the number of the confirmed cases does not necessarily reflect the actual prevalence of the infection at the time point, data on the relationship between the number of infection cases and concentration in wastewater needs to be accumulated further.

Yearly discharge of antibiotic-resistant bacteria (ARB) from combined sewer overflow (CSO) was estimated. The volume of CSO was estimated from operating data of the pumping station. In the target sewer catchment, 23% of the total of the volume of combined sewage was discharged untreated as CSO. Combined sewage contained 3-log larger E. coli than secondary treatment effluent although the abundance of antibiotic-resistant E. coli was not significantly different. In the target-combined sewer catchment, a yearly total of 4.8 × 10 16 CFU of E. coli was discharged from 6.1 × 10 6 m 3 of CSO, while 1.3 × 10 12 CFU of E. coli from 2.1 × 10 7 m 3 of effluent from the wastewater treatment plant (WWTP). This E. coli discharge was equivalent to 7.9 × 10 9 CFU/m 3 from CSO, and 6.2 × 10 4 CFU/m 3 from WWTP effluent. Consequently, a yearly total discharge of antibiotic-resistant E. coli from CSO was 3.7-log larger than the WWTP effluent. The small-flow CSO events, which had hourly flow rate smaller than five times of the average dryweather flow, accounted for 43% of the total CSO volume, but 79% of the total discharge of antibiotic-resistant E. coli due to a small dilution factor with stormwater and frequent discharge. Reduction of small-flow CSO events would be important for effective reduction of ARB discharge from CSO.

Since SARS-CoV-2 RNA in wastewater is often present at low concentration or under detection limit, ensuring the reliability of detection processes using appropriate process controls is essential. The objective of this study was to evaluate applicability and limitations of candidate surrogate viruses as process controls under combinations of different virus concentration and RNA extraction methods. Detection efficiency of SARS-CoV-2 spiked in wastewater was compared with those of candidate surrogate viruses of bacteriophage ϕ6, pepper mild mottle virus (PMMoV), F-specific coliphage (F-phage), and murine norovirus (MNV). After inactivated SARS-CoV-2 and ϕ6 were spiked in two different wastewaters, the viruses in solid and liquid fractions of wastewater were concentrated by centrifuge and polyethylene glycol (PEG) precipitation, respectively. Viral RNA was extracted by using QIAamp Viral RNA Mini Kit and 3 other commercially available extraction kits, then quantified by reverse transcription-quantitative PCR using CDCN1 assay. Regardless of extraction kits, SARS-CoV-2 was consistently detected with good efficiency from both liquid (11–200%) and solid fractions (7.1–93%). Among the candidate process controls, PMMoV was widely detected at good efficiencies from both liquid and solid fractions regardless of selection of RNA extraction kits. F-phage and MNV also showed good detection efficiencies in most combinations of wastewater fractions and RNA extraction kits. An enveloped virus ɸ6 was found often undetected or to have very low detection efficiency (0.1–4.2%) even when SARS-CoV-2 spiked in wastewater was detected with good efficiency. Consequently, PMMoV is widely applicable as process control for detection of SARS-CoV-2 either in liquid fractions concentrated by PEG precipitation, or in solid fractions concentrated by centrifuge.

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