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.

The emergence of new variants of SARS-CoV-2 associated with varying infectivity, pathogenicity, diagnosis, and effectiveness against treatments challenged the overall management of the COVID-19 pandemic. Wastewater surveillance (WWS), i.e., monitoring COVID-19 infections in communities through detecting viruses in wastewater, was applied to track the emergence and spread of SARS-CoV-2 variants globally. However, there is a lack of comprehensive understanding of the use and effectiveness of WWS for new SARS-CoV-2 variants. Here we systematically reviewed published articles reporting monitoring of different SARS-CoV-2 variants in wastewater by following the PRISMA guidelines and provided the current state of the art of this study area. A total of 80 WWS studies were found that reported different monitoring variants of SARS-CoV-2 until November 2022. Most of these studies (66 out of the total 80, 82.5%) were conducted in Europe and North America, i.e., resource-rich countries. There was a high variation in WWS sampling strategy around the world, with composite sampling (50/66 total studies, 76%) as the primary method in resource-rich countries. In contrast, grab sampling was more common (8/14 total studies, 57%) in resource-limited countries. Among detection methods, the reverse transcriptase polymerase chain reaction (RT-PCR)-based sequencing method and quantitative RT-PCR method were commonly used for monitoring SARS-CoV-2 variants in wastewater. Among different variants, the B1.1.7 (Alpha) variant that appeared earlier in the pandemic was the most reported (48/80 total studies), followed by B.1.617.2 (Delta), B.1.351 (Beta), P.1 (Gamma), and others in wastewater. All variants reported in WWS studies followed the same pattern as the clinical reporting within the same timeline, demonstrating that WWS tracked all variants in a timely way when the variants emerged. Thus, wastewater monitoring may be utilized to identify the presence or absence of SARS-CoV-2 and follow the development and transmission of existing and emerging variants. Routine wastewater monitoring is a powerful infectious disease surveillance tool when implemented globally.

Wastewater treatment plants (WWTPs) represent all known types of antibiotic resistance mechanisms and are considered as the critical points for the spread of antibiotic resistance genes (ARGs). The purpose of this study is to investigate the removal of a Class 1 integrase gene (intI1) and a selected set of ARGs (bla_TEM, ermF, mecA, and tetA) at two conventional WWTPs by using chlorination in Louisiana, USA. We collected 69 wastewater samples (23 each from influent, secondary effluent, and final effluent) and determined the concentrations of ARGs by using quantitative polymerase chain reaction. All tested ARGs, except for mecA, were detected in 83–96% and 30–65% of influent and final effluent samples, respectively. Although the ARGs underwent approximately 3-log₁₀ reduction, two WWTPs on an average still released 3.3 ± 1.7 log₁₀ copies/mL of total ARGs studied in the effluents. Chlorination was found to be critical in the significant reduction of total ARGs (p < 0.05). Correlation analysis and the ability of intI1 to persist through the treatment processes recommend the use of intI1 as a marker of ARGs in effluents to monitor the spread of antibiotic resistance in effluents. Our study suggests that conventional WWTPs using chlorination do not favor the proliferation of antibiotic resistance bacteria and ARGs during wastewater treatment.

Hospitals and wastewater treatment plants (WWTPs) are high-risk point sources of antibiotic-resistance genes (ARGs) and antibiotic-resistant bacteria. This study investigates the occurrence of clinically relevant ARGs (sul1, tet(B), bla_CTX-M, bla_NDM-1, qnrS) and a class one integron (intI1) gene in urban rivers, hospitals, and municipal wastewater in the Kathmandu Valley, Nepal. Twenty-five water samples were collected from three rivers, six hospitals, and a wastewater treatment plant to determine the concentrations of ARGs and intI1 using quantitative polymerase chain reactions. From the results, all tested ARGs were detected in the river water; also, concentrations of ARGs in WWTP and hospital effluents varied from 6.2 to 12.5 log₁₀ copies/L, highlighting the role of a WWTP and hospitals in the dissemination of ARGs. Except for bla_NDM-1, significant positive correlations were found between intI1 and other individual ARGs (r = 0.71–0.96, p < 0.05), indicating the probable implications of intI1 in the transfer of ARGs. Furthermore, this study supports the statement that the bla_NDM-1 gene is most likely to be spread in the environment through untreated hospital wastewater. Due to the interaction of surface water and groundwater, future research should focus on ARGs and factors associated with the increase/decrease in their concentration levels in drinking water sources of the Kathmandu Valley.

Urban rivers affected by anthropogenic activities can act as reservoirs of antibiotic resistance genes (ARGs). This study aimed to describe the occurrence of selected ARGs (bla_TEM, ermF, mecA, and tetA) and a class 1 integron (intI1) in an urban river in Nepal. A total of 18 water samples were collected periodically from upstream, midstream, and downstream sites along the Bagmati River over a 1-year period. All ARGs except mecA and intI1 were consistently detected by a quantitative polymerase chain reaction in the midstream and downstream sites, with concentrations ranging from 3.1 to 7.8 log copies/mL. ARG abundance was significantly lower at the upstream site (p < 0.05), reflecting the impact of anthropogenic activities on increasing concentrations of ARGs at midstream and downstream sites. Our findings demonstrate the presence of clinically relevant ARGs in the urban river water of Nepal, suggesting a need for mitigating strategies to prevent the spread of antibiotic resistance in the environment.

Tanker water is used extensively for drinking as well as domestic purposes in the Kathmandu Valley of Nepal. This study aimed to investigate water quality in terms of microbial contamination and determine sources of fecal pollution within these waters. Thirty-one samples from 17 tanker filling stations (TFSs) and 30 water tanker (WT) samples were collected during the dry and wet seasons of 2016. Escherichia coli was detected in 52% of the 31 TFS samples and even more frequently in WT samples. Of the six pathogenic viruses tested, enteroviruses, noroviruses of genogroup II (NoVs-GII), human adenoviruses (HAdVs), and group A rotaviruses were detected using quantitative PCR (qPCR) at 10, five, four, and two TFSs, respectively, whereas Aichi virus 1 and NoVs-GI were not detected at any sites. Index viruses, such as pepper mild mottle virus and tobacco mosaic virus, were detected using qPCR in 77% and 95% out of 22 samples, respectively, all of which were positive for at least one of the tested pathogenic viruses. At least one of the four human-associated markers tested (i.e., BacHum, HAdVs, and JC and BK polyomaviruses) was detected using qPCR in 39% of TFS samples. Ruminant-associated markers were detected at three stations, and pig- and chicken-associated markers were found at one station each of the suburbs. These findings indicate that water supplied by TFSs is generally of poor quality and should be improved, and proper management of WTs should be implemented.