Comparative performance of electronegative membrane filtration and automated concentrating pipette for detection of antibiotic resistance genes and microbial markers in river water samples.

The target viral and bacterial concentrations in river water are essential for environmental monitoring and public health studies. Filtration-based methods are commonly employed, yet challenges arise due to recoverability and filter pore size. This study aimed to compare the performance of electronegative membrane filtration (EMF) and automated Concentrating Pipette (CP) Select (InnovaPrep) methods for quantifying antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and bacterial and viral markers in river water samples. Fifty-four river water samples were collected from upstream and downstream locations in a river in Japan. The CP Select method was modified by adding MgCl2 and using different tips. The recovery efficiencies for total coliforms and Escherichia coli were assessed, and class 1 integron-integrase gene (intI1), 16S rRNA, gene encoding sulfonamide resistance (sul1), cross-assembly phage (crAssphage), pepper mild mottle virus (PMMoV), and Escherichia coli gene (sfmD) were detected. CP Select showed recovery efficiencies of 45 %-63 % for total coliforms and 17 %-35 % for E. coli. The intI1, 16S rRNA, sul1, crAssphage, PMMoV, and sfmD concentrations using the modified CP Select method were 10.1 ± 0.5, 8.7 ± 0.2, 7.7 ± 0.2, 6.7 ± 0.2, 5.4 ± 0.2, and 3.5 ± 0.5 log10 copies/L, respectively. Higher intI1 and sul1 concentrations were observed downstream, with the highest contribution percentage (22 % and 21 %) using CP Select or EMF. The modified CP Select method with 0.05 µm tips yielded more quantifiable results for all target genes and greater PMMoV concentrations (p < 0.05). Positive correlations were found among bacterial, ARG/MGE, and viral markers (Spearman's ρ = 0.71 for 16S rRNA and sfmD, 0.88 for intI1 and sul1, and 0.64 for PMMoV and crAssphage). The modified CP Select method demonstrated effective recovery of bacteria and quantification of ARGs, MGEs, and microbial markers in river water. Further studies are required to validate these methods and confirm their applicability in diverse environmental contexts.

Beyond COVID-19: Wastewater-based epidemiology for multipathogen surveillance and normalization strategies.

Wastewater-based epidemiology (WBE) is a critical tool for monitoring community health. Although much attention has focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of coronavirus disease 2019 (COVID-19), other pathogens also pose significant health risks. This study quantified the presence of SARS-CoV-2, influenza A virus (Inf-A), and noroviruses of genogroups I (NoV-GI) and II (NoV-GII) in wastewater samples collected weekly (n = 170) from July 2023 to February 2024 from five wastewater treatment plants (WWTPs) in Yamanashi Prefecture, Japan, by quantitative PCR. Inf-A RNA exhibited localized prevalence with positive ratios of 59 %-82 % in different WWTPs, suggesting regional outbreaks within specific areas. NoV-GI (94 %, 160/170) and NoV-GII (100 %, 170/170) RNA were highly prevalent, with NoV-GII (6.1 ± 0.8 log10 copies/L) consistently exceeding NoV-GI (5.4 ± 0.7 log10 copies/L) RNA concentrations. SARS-CoV-2 RNA was detected in 100 % of the samples, with mean concentrations of 5.3 ± 0.5 log10 copies/L in WWTP E and 5.8 ± 0.4 log10 copies/L each in other WWTPs. Seasonal variability was evident, with higher concentrations of all pathogenic viruses during winter. Non-normalized and normalized virus concentrations by fecal indicator bacteria (Escherichia coli and total coliforms), an indicator virus (pepper mild mottle virus (PMMoV)), and turbidity revealed significant positive associations with the reported disease cases. Inf-A and NoV-GI + GII RNA concentrations showed strong correlations with influenza and acute gastroenteritis cases, particularly when normalized to E. coli (Spearman's ρ = 0.70-0.81) and total coliforms (ρ = 0.70-0.81), respectively. For SARS-CoV-2, non-normalized concentrations showed a correlation of 0.61, decreasing to 0.31 when normalized to PMMoV, suggesting that PMMoV is unsuitable. Turbidity normalization also yielded suboptimal results. This study underscored the importance of selecting suitable normalization parameters tailored to specific pathogens for accurate disease trend monitoring using WBE, demonstrating its utility beyond COVID-19 surveillance.

Validation and application of high-throughput quantitative PCR for the simultaneous detection of microbial source tracking markers in environmental water.

No single microbial source tracking (MST) marker can be applied to determine the sources of fecal pollution in all water types. This study aimed to validate a high-throughput quantitative polymerase chain reaction (HT-qPCR) method for the simultaneous detection of multiple MST markers. A total of 26 fecal-source samples that had been previously collected from human sewage (n = 6) and ruminant (n = 3), dog (n = 6), pig (n = 6), chicken (n = 3), and duck (n = 2) feces in the Kathmandu Valley, Nepal, were used to validate 10 host-specific MST markers, i.e., Bacteroidales (BacHum, gyrB, BacR, and Pig2Bac), mitochondrial DNA (mtDNA) (swine, bovine, and Dog-mtDNA), and viral (human adenovirus, porcine adenovirus, and chicken/turkey parvovirus) markers, via HT-qPCR. Only Dog-mtDNA showed 100 % accuracy. All the tested bacterial markers showed a sensitivity of 100 %. Nine of the 10 markers were further used to identify fecal contamination in groundwater sources (n = 54), tanker filling stations (n = 14), drinking water treatment plants (n = 5), and river water samples (n = 6). The human-specific Bacteroidales marker BacHum and ruminant-specific Bacteroidales marker BacR was detected at a high ratio in river water samples (83 % and 100 %, respectively). The results of HT-qPCR were in agreement with the standard qPCR. The comparable performances of HT-qPCR and standard qPCR as well as the successful detection of MST markers in the fecal-source and water samples demonstrated the potential applicability of these markers for detecting fecal contamination sources via HT-qPCR.

High-throughput microfluidic quantitative PCR system for the simultaneous detection of antibiotic resistance genes and bacterial and viral pathogens in wastewater.

Comprehensive data on bacterial and viral pathogens of diarrhea and studies applying culture-independent methods for examining antibiotic resistance in wastewater are lacking. This study aimed to simultaneously quantify antibiotic resistance genes (ARGs), class 1 integron-integrase (int1), bacterial and viral pathogens of diarrhea, 16S rRNA, and other indicators using a high-throughput quantitative PCR (HT-qPCR) system. Thirty-six grab wastewater samples from a wastewater treatment plant in Japan, collected three times a month between August 2022 and July 2023, were centrifuged, followed by nucleic acid extraction, reverse transcription, and HT-qPCR. Fourteen targets were included, and HT-qPCR was performed on the Biomark X9™ System (Standard BioTools). For all qPCR assays, R2 was ≥0.978 and the efficiencies ranged from 90.5% to 117.7%, exhibiting high performance. Of the 36 samples, 20 (56%) were positive for Norovirus genogroup II (NoV-GII), whereas Salmonella spp. and Campylobacter jejuni were detected in 24 (67%) and Campylobacter coli in 13 (36%) samples, with mean concentrations ranging from 3.2 ± 0.8 to 4.7 ± 0.3 log10 copies/L. NoV-GII detection ratios and concentrations were higher in winter and spring. None of the pathogens of diarrhea correlated with acute gastroenteritis cases, except for NoV-GII, suggesting the need for data on specific bacterial infections to validate bacterial wastewater-based epidemiology (WBE). All samples tested positive for sul1, int1, and blaCTX-M, irrespective of season. The less explored blaNDM-1 showed a wide prevalence (>83%) and consistent abundance ranging from 4.3 ± 1.0 to 4.9 ± 0.2 log10 copies/L in all seasons. sul1 was the predominant ARG, whereas absolute abundances of 16S rRNA, int1, and blaCTX-M varied seasonally. int1 was significantly correlated with blaCTX-M in autumn and spring, whereas it showed no correlation with blaNDM-1, questioning the applicability of int1 as a sole indicator of overall resistance determinants. This study exhibited that the HT-qPCR system is pivotal for WBE.

SARS-CoV-2 detection in pediatric dental clinic wastewater reflects the number of local COVID-19 cases in children under 10 years old.

This was the first longitudinal study to analyze dental clinic wastewater to estimate asymptomatic SARS-CoV-2 infection trends in children. We monitored wastewater over a 14-month period, spanning three major COVID-19 waves driven by the Alpha, Delta, and Omicron variants. Each Saturday, wastewater was sampled at the Pediatric Dental Clinic of the only dental hospital in Japan's Saitama Prefecture. The relationship between the weekly number of cases in Saitama Prefecture among residents aged < 10 years (exposure) and wastewater SARS-CoV-2 RNA detection (outcome) was examined. The number of cases was significantly associated with wastewater SARS-CoV-2 RNA positivity (risk ratio, 5.36; 95% confidence interval, 1.72-16.67; Fisher's exact test, p = 0.0005). A sample from Week 8 of 2022 harbored the Omicron variant. Compared to sporadic individual testing, this approach allows continuous population-level surveillance, which is less affected by healthcare seeking and test availability. Since wastewater from pediatric dental clinics originates from the oral cavities of asymptomatic children, such testing can provide important information regarding asymptomatic COVID-19 in children, complementing clinical pediatric data.

Application of Skimmed-Milk Flocculation Method for Wastewater Surveillance of COVID-19 in Kathmandu, Nepal.

Wastewater surveillance (WS) has been used globally as a complementary tool to monitor the spread of coronavirus disease 2019 (COVID-19) throughout the pandemic. However, a concern about the appropriateness of WS in low- and middle-income countries (LMICs) exists due to low sewer coverage and expensive viral concentration methods. In this study, influent wastewater samples (n = 63) collected from two wastewater treatment plants (WWTPs) of the Kathmandu Valley between March 2021 and February 2022 were concentrated using the economical skimmed-milk flocculation method (SMFM). The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was tested by qPCR using assays that target the nucleocapsid (N) and envelope (E) genes. Overall, 84% (53/63) of the total samples were positive for SARS-CoV-2 according to at least one of the tested assays, with concentrations ranging from 3.5 to 8.3 log10 gene copies/L, indicating the effectiveness of the SMFM. No correlation was observed between the total number of COVID-19 cases and SARS-CoV-2 RNA concentrations in wastewater collected from the two WWTPs (p > 0.05). This finding cautions the prediction of future COVID-19 waves and the estimation of the number of COVID-19 cases based on wastewater concentration in settings with low sewer coverage by WWTPs. Future studies on WS in LMICs are recommended to be conducted by downscaling to sewer drainage, targeting a limited number of houses. Overall, this study supports the notion that SMFM can be an excellent economical virus-concentrating method for WS of COVID-19 in LMICs.

Group A Streptococcus pyogenes in wastewater: Applicability of wastewater-based epidemiology for monitoring the prevalence of GAS pharyngitis during the late COVID-19 pandemic phase.

Streptococcus pyogenes, Group A Streptococcus (GAS), is a human pathogen that causes a spectrum of diseases from mild to severe, including GAS pharyngitis, a common acute respiratory disease in developed countries. Although wastewater-based epidemiology (WBE) has been extensively used to monitor viral pathogens such as severe acute respiratory syndrome coronavirus 2, its applicability to S. pyogenes remains unexplored. This study was conducted to investigate the feasibility of detecting and quantifying S. pyogenes in wastewater by quantitative polymerase chain reaction (qPCR) and evaluate the applicability of WBE for monitoring the prevalence of GAS pharyngitis. A total of 52 grab influent samples were collected from a wastewater treatment plant in Japan once a week between March 2023 and February 2024. The samples were centrifuged, followed by nucleic acid extraction and qPCR for the S. pyogenes-specific genes speB and spy1258. Of the 52 samples, 90 % and 81 % were positive for speB and spy1258 genes, respectively, indicating the feasibility of S. pyogenes for wastewater surveillance. However, the percentage of quantifiable samples for speB gene was significantly higher in winter than in spring and summer. Similarly, the concentrations of both genes in wastewater samples were significantly higher in winter (speB, 4.1 ± 0.27 log10 copies/L; spy1258, 4.1 ± 0.28 log10 copies/L; One-way ANOVA, p < 0.01) than in spring and summer. Higher concentrations and detection ratios of S. pyogenes genes were observed during increased GAS pharyngitis cases in the catchment. Significant moderate correlations were observed between target gene concentrations and reported GAS pharyngitis cases. This study enhances the understanding role of WBE in monitoring and managing infectious diseases within communities.

Evaluating acute gastroenteritis-causing pathogen reduction in wastewater and the applicability of river water for wastewater-based epidemiology in the Kathmandu Valley, Nepal.

Rapid urbanization and population growth without the implementation of proper waste management are capable of contaminating water sources, which can lead to acute gastroenteritis. This study examined the detection and reduction of five gastroenteritis-causing enteropathogens, Salmonella, Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, and genogroup IV norovirus, and one respiratory pathogen, influenza A virus, in two municipal wastewater treatment plants (WWTP) using an oxidation ditch system (WWTP A; n = 20) and a stabilization pond system (WWTP B; n = 18) in the Kathmandu Valley, Nepal, collected between August 2017 and August 2019. All enteropathogens were detected in wastewater via quantitative PCR. The concentrations of the pathogens ranged from 5.7 to 7.9 log10 copies/L in WWTP A and from 4.9 to 8.1 log10 copies/L in WWTP B. The log10 reduction values of the pathogens ranged from 0.3 to 1.0 in WWTP A and from -0.1 to 0.2 in WWTP B. The association between the pathogen concentrations and the number of clinical cases in the corresponding week could not be evaluated; however, the consistent detection of pathogens in the wastewater despite low number of case reports suggested the use of wastewater-based epidemiology (WBE) for early warning of acute gastroenteritis (AGE) in the Kathmandu Valley. The pathogens were also detected in river water at approximately 7.0 log10 copies/L and exhibited no significant difference in concentration compared to wastewater, suggesting the applicability of river water for WBE of AGE. Insufficient treatment of all pathogens in the wastewater was observed, suggesting the need for full rehabilitation of the treatment plants. However, the influent may be utilized for early detection of AGE-causing pathogens in the city, whereas the river water may serve as an alternative in areas without connection to the WWTPs.

Detection of SARS-CoV-2 and Omicron variant RNA in wastewater samples from Manila, Philippines.

Manila, a highly urbanized city, is listed as one of the top cities with the highest recorded number of coronavirus disease 2019 (COVID-19) cases in the Philippines. This study aimed to detect and quantify the RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the Omicron variant in 51 wastewater samples collected from three locations in Manila, namely Estero de Santa Clara, Estero de Pandacan, which are open drainages, and a sewage treatment plant (STP) at De La Salle University-Manila, between July 2022 and February 2023. Using one-step reverse transcription-quantitative polymerase chain reaction, SARS-CoV-2 and Omicron variant RNA were detected in 78 % (40/51; 4.9 ± 0.5 log10 copies/L) and 60 % (24/40; 4.4 ± 0.3 log10 copies/L) of wastewater samples collected from all sampling sites, respectively. SARS-CoV-2 RNA was detected frequently at Estero de Santa Clara (88 %, 15/17); its highest concentration was at the STP (6.3 log10 copies/L). The Omicron variant RNA was present in the samples collected (4.4 ± 0.3 log10 copies/L) from all sampling sites, with the highest concentration at the STP (4.9 log10 copies/L). Regardless of normalization, using concentrations of pepper mild mottle virus RNA, SARS-CoV-2 RNA concentrations exhibited the highest positive correlation with COVID-19 reported cases in Manila 5 days after the clinical report. These findings revealed that wastewater-based epidemiology may aid in identifying and monitoring of the presence of pathogens in open drainages and STPs in the Philippines. This paper provides the first documentation on SARS-CoV-2 and the Omicron variant in wastewater from Manila.

Quantification of multiple respiratory viruses in wastewater in the Kathmandu Valley, Nepal: Potential implications of wastewater-based epidemiology for community disease surveillance in developing countries.

Despite being the major cause of death, clinical surveillance of respiratory viruses at the community level is very passive, especially in developing countries. This study focused on the surveillance of three respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IFV-A), and respiratory syncytial virus (RSV)] in the Kathmandu Valley, Nepal, by implication of wastewater-based epidemiology (WBE). Fifty-one untreated wastewater samples were from two wastewater treatment plants (WWTPs) between April and October 2022. Among eight combinations of the pre-evaluated methods, the combination of concentration by simple centrifugation, pretreatment by DNA/RNA Shield (Zymo Research), and extraction by the QIAamp Viral RNA Mini Kit (QIAGEN) showed the best performance for detecting respiratory viruses. Using this method with a one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR), SARS-CoV-2 RNA was successfully detected from both WWTPs (positive ratio, 100 % and 81 %) at concentrations of 5.6 ± 0.6 log10 copies/L from each WWTP. Forty-six SARS-CoV-2 RNA-positive samples were further tested for three mutation site-specific one-step RT-qPCR (L452R, T478K, and E484A/G339D), where G339D/E484A mutations were frequently detected in both WWTPs (96 %). IFV-A RNA was more frequently detected in WWTP A (84 %) compared to WWTP B (38 %). RSV RNA was also detected in both WWTPs (28 % and 8 %, respectively). This is the first study on detecting IFV-A and RSV in wastewater in Nepal, showing the applicability and importance of WBE for respiratory viruses in developing countries where clinical data are lacking.

Estimation of Norovirus infections in Japan: An application of wastewater-based epidemiology for enteric disease assessment.

Noroviruses of genogroup I (NoV GI) and NoV GII are the primary causes of acute gastroenteritis (AGE) in developed countries. However, asymptomatic and untested NoV infections lead to an underestimation of AGE cases, and the lack of mandatory viral identification in clinical cases hinders precise estimation of NoV infections. Back estimation of NoV infections in the community using a wastewater-based epidemiology (WBE) approach can provide valuable insights into the disease's extent, progression, and epidemiology, aiding in developing effective control strategies. This study employed a one-step reverse transcription-quantitative PCR to quantify NoVs GI and GII in wastewater samples (n = 83) collected twice a week from June 2022 to March 2023 in Japan. All samples from the Winter-Spring (n = 27) tested positive for NoV GI and GII RNA, while 73 % and 88 % of samples from the Summer-Autumn (n = 56) were positive for NoV GI and NoV GII RNA, respectively. Significantly higher concentrations of NoV GI/GII RNA were found in the Winter-Spring season compared to the Summer-Autumn season. NoV RNA was consistently detected in wastewater throughout the year, demonstrating the persistence of AGE cases in the catchment, suggesting an endemic NoV infection. Estimates of NoV infection incorporated viral RNA concentrations, wastewater parameters, and signal persistence in a mass balance equation using Monte Carlo Simulation. The median estimated NoV GI infections per 100,000 population for Summer-Autumn was 133 and for the Winter-Spring season, it was 881. Estimated NoV GII infections were 1357 for Summer-Autumn and 11,997 for the Winter-Spring season per 100,000 population. The estimated NoV infections exceeded by 3.2 and 23.9 folds than the reported AGE cases in Summer-Autumn and Winter-Spring seasons, respectively. The seasonal trend of estimated NoV infections closely matched that of AGE cases, highlighting the utility of WBE in understanding the epidemiology of enteric infections.

Detection of enteroviruses related to hand foot and mouth disease in wastewater of Asian communities.

Hand, foot, and mouth disease (HFMD) is contagious and predominantly affects children below the age of five. HFMD-associated serotypes of Enterovirus A (EVA) family include EVA71, Coxsackievirus A type 6 (CVA6), 10 (CVA10), and 16 (CVA16). Although prevalent in numerous Asian countries, studies on HFMD-causing agents in wastewater are scarce. This study aimed to conduct wastewater surveillance in various Asian communities to detect and quantify serotypes of EVA associated with HFMD. In total, 77 wastewater samples were collected from Indonesia, the Philippines, Thailand, and Vietnam from March 2022 to February 2023. The detection ratio for CVA6 RNA in samples from Vietnam was 40 % (8/20). The detection ratio for CVA6 and EVA71 RNA each was 25 % (5/20) for the Indonesian samples, indicating the need for clinical surveillance of CVA6, as clinical reports have been limited. For the Philippines, 12 % (2/17) of the samples were positive for CVA6 and EVA71 RNA each, with only one quantifiable sample each. Samples from Thailand had a lower detection ratio (1/20) for CVA6 RNA, and the concentration was unquantifiable. Conversely, CVA10 and CVA16 RNAs were not detected in any of the samples. The minimum and maximum concentrations of CVA6 RNA were 2.7 and 3.9 log10 copies/L and those for EVA71 RNA were 2.5 and 4.9 log10 copies/L, respectively. This study underscores the importance of wastewater surveillance in understanding the epidemiology of HFMD-associated EVA serotypes in Asian communities. Long-term wastewater surveillance is recommended to monitor changes in dominant serotypes, understand seasonality, and develop effective prevention and control strategies for HFMD.

Optimization of the 5-plex digital PCR workflow for simultaneous monitoring of SARS-CoV-2 and other pathogenic viruses in wastewater.

Wastewater-based epidemiology is a valuable tool for monitoring pathogenic viruses in the environment, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). While quantitative polymerase chain reaction (qPCR) is widely used for pathogen surveillance in wastewater, it can be affected by inhibition and is limited to relative quantification. Digital PCR (dPCR) offers potential solutions to these limitations. In this study, a 5-plex dPCR workflow was optimized for the simultaneous detection of SARS-CoV-2, influenza A virus, enteroviruses (EnV), and noroviruses of genogroups I (NoV-GI) and GII (NoV-GII) in wastewater samples. Wastewater samples (n = 36) were collected from a wastewater treatment plant in Japan between August and October 2022. The optimization included the evaluation of singleplex and 5-plex dPCR assays, and two different concentration methods, extraction kits, and dPCR approaches. The performance of singleplex and 5-plex dPCR assays showed comparable linearity and reliability, with the 5-plex assays showing greater efficiency. The polyethylene glycol (PEG) precipitation method showed better performance over the centrifugation method, two-step reverse transcription (RT)-dPCR over the one-step RT-dPCR, and AllPrep PowerViral DNA/RNA Kit showed better performance than the QIAamp Viral RNA Mini Kit. The optimal workflow therefore included PEG precipitation, the AllPrep PowerViral DNA/RNA Kit, and two-step RT-dPCR. This workflow was selected to monitor the presence of SARS-CoV-2 and other pathogenic viruses in wastewater samples in a 5-plex dPCR approach, yielding promising results. SARS-CoV-2 RNA was detected in the majority of samples, with NoV-GI, NoV-GII, and EnV also being detected. The successful optimization and application of the 5-plex dPCR assay for pathogen surveillance in wastewater offers significant benefits, including enhanced community health assessment and more effective responses to public health threats.

Monitoring hand foot and mouth disease using long-term wastewater surveillance in Japan: Quantitative PCR assay development and application.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease that primarily affects children under five years of age. It is mainly caused by serotypes of Enterovirus A (EVA): EVA71, Coxsackievirus A types 6 (CVA6), 10 (CVA10), and 16 (CVA16). Despite being highly prevalent in Japan and other countries in the Asia-Pacific region, few studies have investigated HFMD pathogens in wastewater. The present study aimed to develop a highly sensitive and broadly reactive quantitative polymerase chain reaction (qPCR) assay of dominant serotype CVA6, to revise previously developed CVA6, CVA10, and CVA16 assays, and to test these assays in wastewater samples from Yamanashi Prefecture, Japan. The new-CVA6 qPCR assay was developed with maximal nucleotide percent identity among CVA6 isolates from Japan. The new-CVA6 and revised assays were highly sensitive and had the ability to quantify respective positive controls at levels as low as 1 copy/µL. Among the 53 grab influent samples collected between March 2022 and March 2023, EVA71, CVA10, and CVA16 RNA were not detected in any samples, whereas the new-CVA6 assay could detect CVA6 RNA in 38 % (20/53) of samples. CVA6 RNA was detected at a significantly higher concentration in the summer season (3.3 ± 0.8 log10 copies/L; 79 % (11/14)) than in autumn (2.7 ± 0.6 log10 copies/L; 69 % (9/13)). The seasonal trend of CVA6 RNA detection in wastewater aligned with the trend of HFMD case reports in the catchment of the wastewater treatment plant. This is the first study to report the detection and seasonal trends of the EVA serotypes associated with HFMD in wastewater samples in Japan. It provides evidence that wastewater-based epidemiology is applicable even for diseases that are prevalent only in specific population groups.

Long-term SARS-CoV-2 surveillance in wastewater and estimation of COVID-19 cases: An application of wastewater-based epidemiology.

The role of wastewater-based epidemiology (WBE), a powerful tool to complement clinical surveillance, has increased as many grassroots-level facilities, such as municipalities and cities, are actively involved in wastewater monitoring, and the clinical testing of coronavirus disease 2019 (COVID-19) is downscaled widely. This study aimed to conduct long-term wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Yamanashi Prefecture, Japan, using one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay and estimate COVID-19 cases using a cubic regression model that is simple to implement. Influent wastewater samples (n = 132) from a wastewater treatment plant were collected normally once weekly between September 2020 and January 2022 and twice weekly between February and August 2022. Viruses in wastewater samples (40 mL) were concentrated by the polyethylene glycol precipitation method, followed by RNA extraction and RT-qPCR. The K-6-fold cross-validation method was used to select the appropriate data type (SARS-CoV-2 RNA concentration and COVID-19 cases) suitable for the final model run. SARS-CoV-2 RNA was successfully detected in 67 % (88 of 132) of the samples tested during the whole surveillance period, 37 % (24 of 65) and 96 % (64 of 67) of the samples collected before and during 2022, respectively, with concentrations ranging from 3.5 to 6.3 log10 copies/L. This study applied a nonnormalized SARS-CoV-2 RNA concentration and nonstandardized data for running the final 14-day (1 to 14 days) offset models to estimate weekly average COVID-19 cases. Comparing the parameters used for a model evaluation, the best model showed that COVID-19 cases lagged 3 days behind the SARS-CoV-2 RNA concentration in wastewater samples during the Omicron variant phase (year 2022). Finally, 3- and 7-day offset models successfully predicted the trend of COVID-19 cases from September 2022 until February 2023, indicating the applicability of WBE as an early warning tool.

First quantitative detection of tomato brown rugose fruit virus in wastewater in Louisiana.

We investigated the occurrence of tomato brown rugose fruit virus (ToBRFV) at a conventional wastewater treatment plant in Louisiana over a 13-month period, from March 2017 to March 2018. Influent, secondary effluent, and final effluent wastewater samples were collected monthly, and viruses were concentrated by the adsorption-elution method using an electronegative filter, followed by the detection using quantitative polymerase chain reaction. ToBRFV was detected in 10 (77 %) of 13 influent samples, 9 (69 %) of 13 in secondary effluent, and 6 (50 %) of 12 final effluents. The concentrations of ToBRFV in the influent samples ranged from 3.5 to 6.1 log10 copies/L and it was always higher than those in secondary or final effluents. Wastewater samples showed a high positive ratio of ToBRFV during fall and winter months. The findings highlight that routine monitoring of new viral indicator such as ToBRFV is necessary to understand its environmental distribution and correlation with pathogenic viruses. This is the first study providing quantitative data on the occurrence of ToBRFV in wastewater.

Optimization and performance evaluation of an automated filtration method for the recovery of SARS-CoV-2 and other viruses in wastewater.

A rapid virus concentration method is needed to get high throughput. Reliable results of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection in wastewater are necessary for applications in wastewater-based epidemiology. In this study, an automated filtration method using a concentrating pipette (CP Select; Innovaprep) was applied to detect SARS-CoV-2 in wastewater samples with several modifications to increase its sensitivity and throughput. The performance of the CP Select method was compared to other concentration methods (polyethylene glycol precipitation and direct capture using silica column) to evaluate its applicability to SARS-CoV-2 detection in wastewater. SARS-CoV-2 RNA was successfully detected in six of eight wastewater samples using the CP Select method, whereas other methods could detect SARS-CoV-2 RNA in all wastewater samples. Enteric viruses, such as noroviruses of genogroups I (NoVs-GI) and II (NoVs-GII) and enteroviruses, were tested, resulting in 100 % NoVs-GII detection using all concentration methods. As for NoVs-GI and enteroviruses, all methods gave comparable number of detected samples in wastewater samples. This study showed that the optimized CP Select method was less sensitive in SARS-CoV-2 detection in wastewater than other methods, whereas all methods were applicable to detect or recover other viruses in wastewater.

Development of two microbial source tracking markers for detection of wastewater-associated Escherichia coli isolates.

Escherichia coli has been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identify E. coli microbial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewater E. coli genomes sequenced by us (n = 50) and RefSeq animal E. coli genomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewater E. coli genomes and 3318 RefSeq animal E. coli genomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associated E. coli isolates. These two markers showed higher performance than the previously developed human wastewater-associated E. coli markers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9 % and sensitivity of 25.7 %. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detecting E. coli isolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.

Monkeypox outbreak: Wastewater and environmental surveillance perspective.

Monkeypox disease (MPXD), a viral disease caused by the monkeypox virus (MPXV), is an emerging zoonotic disease endemic in some countries of Central and Western Africa but seldom reported outside the affected region. Since May 2022, MPXD has been reported at least in 74 countries globally, prompting the World Health Organization to declare the MPXD outbreak a Public Health Emergency of International Concern. As of July 24, 2022; 92 % (68/74) of the countries with reported MPXD cases had no historical MPXD case reports. From the One Health perspective, the spread of MPXV in the environment poses a risk not only to humans but also to small mammals and may, ultimately, spread to potent novel host populations. Wastewater-based surveillance (WBS) has been extensively utilized to monitor communicable diseases, particularly during the ongoing COVID-19 pandemic. It helped in monitoring infectious disease caseloads as well as specific viral variants circulating in communities. The detection of MPXV DNA in lesion materials (e.g. skin, vesicle fluid, crusts), skin rashes, and various body fluids, including respiratory and nasal secretions, saliva, urine, feces, and semen of infected individuals, supports the possibility of using WBS as an early proxy for the detection of MPXV infections. WBS of MPXV DNA can be used to monitor MPXV activity/trends in sewerage network areas even before detecting laboratory-confirmed clinical cases within a community. However, several factors affect the detection of MPXV in wastewater including, but not limited to, routes and duration time of virus shedding by infected individuals, infection rates in the relevant affected population, environmental persistence, the processes and analytical sensitivity of the used methods. Further research is needed to identify the key factors that impact the detection of MPXV biomarkers in wastewater and improve the utility of WBS of MPXV as an early warning and monitoring tool for safeguarding human health. In this review, we shortly summarize aspects of the MPXV outbreak relevant to wastewater monitoring and discuss the challenges associated with WBS.

Application of a high-throughput quantitative PCR system for simultaneous monitoring of SARS-CoV-2 variants and other pathogenic viruses in wastewater.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuously emerging, highlighting the importance of regular surveillance of SARS-CoV-2 and other epidemiologically significant pathogenic viruses in the current context. Reverse transcription-quantitative PCR (RT-qPCR) is expensive, time-consuming, labor-intensive, requires a large reagent volume, and only tests a few targets in a single run. High-throughput qPCR (HT-qPCR) utilizing the Biomark HD system (Fluidigm) can be used as an alternative. This study applied an HT-qPCR to simultaneously detect SARS-CoV-2, SARS-CoV-2 nucleotide substituted RNA, and other pathogenic viruses in wastewater. Wastewater samples were collected from the coronavirus disease 2019 (COVID-19) quarantine facility between October 2020 and February 2021 (n = 4) and from the combined and separated sewer lines of a wastewater treatment plant (WWTP) in Yokkaichi, Mie Prefecture, Japan, between March and August 2021 (n = 23 each). The samples were analyzed by HT-qPCR using five SARS-CoV-2, nine SARS-CoV-2 spike gene nucleotide substitution-specific, five pathogenic viruses, and three process control assays. All samples from the quarantine facility tested positive for SARS-CoV-2 and the nucleotide substitutions N501Y and S69-70 del (Alpha variant) were detected in the December 2020 sample, coinciding with the first clinical case in Japan. Only three WWTP samples were positive when tested with a single SARS-CoV-2 assay, whereas more than eight samples were positive when tested with all assays, indicating that using multiple assays increases the likelihood of detection. The nucleotide substitution L452R (Delta variant) was detected in the WWTP samples of Mie Prefecture in April 2021, but the detection of Delta variant from patients had not been reported until May 2021. Aichi virus 1 and norovirus GII were prevalent in WWTP samples. This study demonstrated that HT-qPCR may be the most time- and cost-efficient method for tracking COVID-19 and broadly monitoring community health.