Monitoring Enteroviruses and SARS-CoV-2 in Wastewater Using the Polio Environmental Surveillance System in Japan

FIG 1. Summary of the frequencies of virus isolation from wastewater and the reported numbers of cases associated with enterovirus in sentinel sites of an anonymous prefecture in Japan from 2019 to 2021. (A) Frequencies of isolation of nonpolio enteroviruses (NPEV) with serotypes and nonenterovirus (NEV). (B) Graph of clinical surveillance (pediatric sentinel surveillance) showing enterovirus-associated cases of hand-foot-and-mouth disease (HFMD), herpangina, and aseptic meningitis. Adenovirus (AdV) and mammalian orthoreovirus (MRV) were not typed (NT). No poliovirus was isolated. CB, coxsackievirus B; Echo, echovirus.

FIG 2. Summary of the numbers of new COVID-19 cases and SARS-CoV-2 RNA concentrations in wastewater samples obtained from WWTP-A and WWTP-B. The numbers of new COVID-19 cases per week in each catchment area are included. Data on RNA concentrations are presented separately based on the virus recovery method (solid based and PEG precipitation) and primer/probe sets (CDC N1/N2 and NIID N2). The blue squares and red triangles indicate results from the 1-step and 2-step RT-qPCR procedures, respectively. The limits of quantification (LOQs) for wastewater based on RT-qPCR assays are as follows: 200 gc/L for solid and 600 gc/L for PEG in WWTP-A and 250 gc/L for solid and 600 gc/L for PEG in WWTP-B.

FIG 3. (A to C) Scatterplots representing the RNA concentrations in wastewater samples (log10 gene copies [gc] per liter). Virus recovery methods (solid based and PEG precipitation) (A), primer/probe sets (CDC N1/N2 and NIID N2) (B), and RT-qPCR procedures (1 step and 2 steps) (C) were compared for 520 assays. Geometric means and geometric standard deviations are presented. Values at the bottom of the graphs represent the ratio of the number of positive samples to the total number of analyzed samples. (D) Detection rates for each method (solid based and PEG precipitation), primer/probe set (CDC N1/N2 and NIID N2), and RT-qPCR procedure (1 step and 2 steps).

FIG 4. Scatterplots representing the concentrations of PMMoV RNA as an internal control in wastewater samples in log10 gene copies (gc) per well by RT-qPCR assays. All tested RNA (1-step) and cDNA (2-step) samples examined using the SARS-CoV-2 assays were subjected to parallel analysis using the PMMoV assays. Geometric means and geometric standard deviations are presented.

FIG 5. Correlation between the reported number of new COVID-19 cases and SARS-CoV-2 RNA concentrations, analyzed using Spearman’s rank correlation test. Spearman’s r values are presented. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001. CI, confidence interval.