Tracking community infection dynamics of COVID-19 by monitoring SARS-CoV-2 RNA in wastewater, counting positive reactions by qPCR.

Wastewater-based epidemiology has proved useful for monitoring the COVID-19 infection dynamics in communities. However, in regions of low prevalence, low concentrations of SARS-CoV-2 RNA in wastewater make this difficult. Here, we used real-time reverse-transcription PCR (RT-qPCR) to monitor SARS-CoV-2 RNA in wastewater from October 2020 to December 2022 during the third, fourth, fifth, sixth, seventh, and eighth waves of the COVID-19 outbreak in Japan. Viral RNA was below the limit of detection in all samples during the third and fourth waves. However, by counting the number of positive replicates in qPCR of each sample, we found that the positive ratio to all replicates in wastewater was significantly correlated with the number of clinically confirmed cases by the date of symptom onset during the third, fourth, and fifth waves. Time-step analysis indicated that, for 2 days either side of symptom onset, COVID-19 patients excreted in their feces large amounts of virus that wastewater surveillance could detect. We also demonstrated that the viral genome copy number in wastewater, as estimated from the positive ratio of SARSA-CoV-2 RNA, was correlated with the number of clinically confirmed cases. The positive count method is thus useful for tracing COVID-19 dynamics in regions of low prevalence.

Occurrence of E. coli and antibiotic-resistant E. coli in the southern watershed of Lake Biwa, including in wastewater treatment plant effluent and inflow rivers.

The emergence of antibiotic-resistant bacteria (ARB) and their antibiotic resistance genes (ARGs) poses a serious challenge to human, animal, and environmental health worldwide. ARB can spread into the environment via various sources and routes. In this study, we investigated the occurrence of antibiotic-resistant E. coli in the southern watershed of Lake Biwa. Two-year monitoring of antibiotic-resistant E. coli was carried out in the southern part of Lake Biwa and inflow rivers and at three WWTPs around the southern part of the lake. Concentrations of E. coli in waters that are resistant to ampicillin (AMP), cefotaxime (CTX), ceftazidime (CAZ), levofloxacin (LVFX), tetracycline (TC), and amikacin (AMK) were measured using the culture method. Of these antibiotic-resistant E. coli, AMP-resistant E. coli were found at the highest prevalence, followed by LVFX, CTX, CAZ, TC, and AMK-resistant in both the influent and effluent of WWTPs. These resistance patterns in wastewater are the same as those in clinical samples in Japan. The numbers of antibiotic-resistant E. coli decreased by around a factor of 1000 during the wastewater treatment processes, but the rates clearly increased, suggesting that selection for antibiotic resistance might occur during the wastewater treatment process. AMP-resistant and TC-resistant E. coli were also detected in Lake Biwa and inflow rivers, which suggests that antibiotic resistance might come from not only WWTPs but also livestock farms and small-scale wastewater treatment facilities located in the river catchment.

Comparison of five polyethylene glycol precipitation procedures for the RT-qPCR based recovery of murine hepatitis virus, bacteriophage phi6, and pepper mild mottle virus as a surrogate for SARS-CoV-2 from wastewater.

Polyethylene glycol (PEG) precipitation is one of the conventional methods for virus concentration. This technique has been used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. The procedures and seeded surrogate viruses were different among implementers; thus, the reported whole process recovery efficiencies considerably varied among studies. The present study compared five PEG precipitation procedures, with different operational parameters, for the RT-qPCR-based whole process recovery efficiency of murine hepatitis virus (MHV), bacteriophage phi6, and pepper mild mottle virus (PMMoV), and molecular process recovery efficiency of murine norovirus using 34 raw wastewater samples collected in Japan. The five procedures yielded significantly different whole process recovery efficiency of MHV (0.070%-2.6%) and phi6 (0.071%-0.51%). The observed concentration of indigenous PMMoV ranged from 8.9 to 9.7 log (8.2 × 108 to 5.6 × 109) copies/L. Interestingly, PEG precipitation with 2-h incubation outperformed that with overnight incubation partially due to the difference in molecular process recovery efficiency. The recovery load of MHV exhibited a positive correlation (r = 0.70) with that of PMMoV, suggesting that PMMoV is the potential indicator of the recovery efficiency of SARS-CoV-2. In addition, we reviewed 13 published studies and found considerable variability between different studies in the whole process recovery efficiency of enveloped viruses by PEG precipitation. This was due to the differences in operational parameters and surrogate viruses as well as the differences in wastewater quality and bias in the measurement of the seeded load of surrogate viruses, resulting from the use of different analytes and RNA extraction methods. Overall, the operational parameters (e.g., incubation time and pretreatment) should be optimized for PEG precipitation. Co-quantification of PMMoV may allow for the normalization of SARS-CoV-2 RNA concentration by correcting for the differences in whole process recovery efficiency and fecal load among samples.

Characterization of nitrosamines and nitrosamine precursors as non-point source pollutants during heavy rainfall events in an urban water environment.

N-nitrosodimethylamine (NDMA) and N-nitrosomorpholine (NMOR) and their specific precursors (N,N-dimethylformamide [DMF] for NDMA and morpholine [MOR] for NMOR) were widely identified in runoff of urban area around the southern Lake Biwa basin, Japan. It was thought that this runoff might constitute a non-point source of the four compounds in rivers and sewage treatment plants (STPs) during heavy rainfall events. We investigated the spatiotemporal patterns of NDMA, NMOR, DMF and MOR in runoff and rivers in rainy days. NDMA and NMOR were detected in concentrations of up to 295 ng/L, while DMF and MOR were detected in concentrations of up to 33.7 µg/L. Continuous sequential sampling over periods of 24 or 48 h at the largest STP in the study area revealed that the four compounds in the primary effluent (PE) each had higher mass fluxes during heavy rainfall events than on dry days. This phenomenon might be contributed to non-point sources (e.g., runoff) from infiltration/inflow related to rainwater into sanitary sewers. Moreover, the four compounds were confirmed to have higher mass fluxes in the final effluent of the STP during periods of PE bypass (1.3-1.7 times for NDMA, NMOR, and MOR; over 200 times for DMF; on average) than that on dry days because of increasing inflow during heavy rain than during periods without PE bypass in dry weather.