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.

Application of wastewater-based epidemiology for monitoring COVID-19 in hospital and housing wastewaters.

An alternative and complementary diagnostic method of surveillance is provided by wastewater-based surveillance (WBS), particularly in low-income nations like Nepal with scant wastewater treatment facilities and clinical testing infrastructure. In this study, a total of 146 water samples collected from two hospitals (n = 63) and three housing wastewaters (n = 83) from the Kathmandu Valley over the period of March 2021-Febraury 2022 were investigated for Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using quantitative reverse transcription TaqMan PCR assays targeting the N and E genes. Of the total, 67 % (98/146) samples were positive for SARS-CoV-2 RNA either by using N or E gene assay, with concentrations ranging from 3.6 to 9.1 log10 copies/L. There was a significant difference found between positive ratio (Chi-square test, p < 0.05) and concentration (t-test, p = 0.009) of SARS-CoV-2 RNA detected from hospital wastewater and housing waters. Wastewater data are correlated with COVID-19 active cases, indicating significance in specific areas like the Hospital (APFH) (p < 0.05). According to the application of a bivariate linear regression model (p < 0.05), the concentrations of N-genes may be used to predict the COVID-19 cases in the APFH. Remarkably, SARS-CoV-2 RNA was detected prior to, during, and following clinical case surges, implying that wastewater surveillance could serve as an early warning system for public health decisions. The significance of WBS in tracking and managing pandemics is emphasized by this study, especially in resource-constrained settings.

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.

Survivability of Delta and Omicron variants of SARS-CoV-2 in wastewater.

Concerns of fecal-aerosol transmission of coronavirus disease 2019 (COVID-2019) coupled with increased transmissibility and disease severity of Delta and Omicron variants of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggest studies on survival of VOC in wastewater are warranted. To the best of our knowledge, this is the first study to investigate the survivability of Delta and Omicron VOC in filtered and unfiltered raw wastewater, and secondary effluent at room temperature (23 °C). The time required for 90 % inactivation (T90) of Delta and Omicron VOC in unfiltered raw wastewater was calculated as 17.7 and 15.3 h, respectively. Rapid inactivation of VOC in wastewater and inability to isolate SARS-CoV-2 in wastewater suggest risks from fecal-aerosol transmission are low. Nevertheless, high transmissibility of VOC cautions overruling fecal-aerosol transmission of COVID-19. Future studies on survival of SARS-CoV-2 in wastewater should attempt viral culture by spiking feces collected from COVID-19 infected patients into wastewater to match the real-world scenario.

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.

Antimicrobial resistance in southeast Asian water environments: A systematic review of current evidence and future research directions.

Antimicrobial resistance has been a serious and complex issue for over a decade. Although research on antimicrobial resistance (AMR) has mainly focused on clinical and animal samples as essential for treatment, the AMR situation in aquatic environments may vary and have complicated patterns according to geographical area. Therefore, this study aimed to examine recent literature on the current situation and identify gaps in the AMR research on freshwater, seawater, and wastewater in Southeast Asia. The PubMed, Scopus, and ScienceDirect databases were searched for relevant publications published from January 2013 to June 2023 that focused on antimicrobial resistance bacteria (ARB) and antimicrobial resistance genes (ARGs) among water sources. Based on the inclusion criteria, the final screening included 41 studies, with acceptable agreement assessed using Cohen's inter-examiner kappa equal to 0.866. This review found that 23 out of 41 included studies investigated ARGs and ARB reservoirs in freshwater rather than in seawater and wastewater, and it frequently found that Escherichia coli was a predominant indicator in AMR detection conducted by both phenotypic and genotypic methods. Different ARGs, such as blaTEM, sul1, and tetA genes, were found to be at a high prevalence in wastewater, freshwater, and seawater. Existing evidence highlights the importance of wastewater management and constant water monitoring in preventing AMR dissemination and strengthening effective mitigation strategies. This review may be beneficial for updating current evidence and providing a framework for spreading ARB and ARGs, particularly region-specific water sources. Future AMR research should include samples from various water systems, such as drinking water or seawater, to generate contextually appropriate results. Robust evidence regarding standard detection methods is required for prospective-era work to raise practical policies and alerts for developing microbial source tracking and identifying sources of contamination-specific indicators in aquatic environment markers.

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.

Moving forward with COVID-19: Future research prospects of wastewater-based epidemiology methodologies and applications.

Wastewater-based epidemiology (WBE) has been demonstrated for its great potential in tracking of coronavirus disease 2019 (COVID-19) transmission among populations despite some inherent methodological limitations. These include non-optimized sampling approaches and analytical methods; stability of viruses in sewer systems; partitioning/retention in biofilms; and the singular and inaccurate back-calculation step to predict the number of infected individuals in the community. Future research is expected to (1) standardize best practices in wastewater sampling, analysis and data reporting protocols for the sensitive and reproducible detection of viruses in wastewater; (2) understand the in-sewer viral stability and partitioning under the impacts of dynamic wastewater flow, properties, chemicals, biofilms and sediments; and (3) achieve smart wastewater surveillance with artificial intelligence and big data models. Further specific research is essential in the monitoring of other viral pathogens with pandemic potential and subcatchment applications to maximize the benefits of WBE beyond COVID-19.

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.

Correlation between SARS-CoV-2 RNA concentration in wastewater and COVID-19 cases in community: A systematic review and meta-analysis.

Wastewater-based epidemiology (WBE) has been considered as a promising approach for population-wide surveillance of coronavirus disease 2019 (COVID-19). Many studies have successfully quantified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentration in wastewater (CRNA). However, the correlation between the CRNA and the COVID-19 clinically confirmed cases in the corresponding wastewater catchments varies and the impacts of environmental and other factors remain unclear. A systematic review and meta-analysis were conducted to identify the correlation between CRNA and various types of clinically confirmed case numbers, including prevalence and incidence rates. The impacts of environmental factors, WBE sampling design, and epidemiological conditions on the correlation were assessed for the same datasets. The systematic review identified 133 correlation coefficients, ranging from -0.38 to 0.99. The correlation between CRNA and new cases (either daily new, weekly new, or future cases) was stronger than that of active cases and cumulative cases. These correlation coefficients were potentially affected by environmental and epidemiological conditions and WBE sampling design. Larger variations of air temperature and clinical testing coverage, and the increase of catchment size showed strong negative impacts on the correlation between CRNA and COVID-19 case numbers. Interestingly, the sampling technique had negligible impact although increasing the sampling frequency improved the correlation. These findings highlight the importance of viral shedding dynamics, in-sewer decay, WBE sampling design and clinical testing on the accurate back-estimation of COVID-19 case numbers through the WBE 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.

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.

Wastewater monitoring in tourist cities as potential sentinel sites for near real-time dynamics of imported SARS-CoV-2 variants.

Wastewater-based epidemiology (WBE) complements the clinical surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants' distribution in populations. Many developed nations have established national and regional WBE systems; however, governance and budget constraints could be obstacles for low- and middle-income countries. An urgent need thus exists to identify hotspots to serve as sentinel sites for WBE. We hypothesized that representative wastewater treatment plants (WWTPs) in two international gateway cities, Bangkok and Phuket, Thailand, could be sentineled for SARS-CoV-2 and its variants to reflect the clinical distribution patterns at city level and serve as early indicators of new variants entering the country. Municipal wastewater samples (n = 132) were collected from eight representative municipal WWTPs in Bangkok and Phuket during 19 sampling events from October 2021 to March 2022, which were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the US CDC N1 and N2 multiplex and variant (Alpha, Delta, and Omicron BA.1 and BA.2) singleplex assays. The variant detection ratios from Bangkok and Phuket followed similar trends to the national clinical testing data, and each variant's viral loads agreed with the daily new cases (3-d moving average). Omicron BA.1 was detected in Phuket wastewater prior to Bangkok, possibly due to Phuket's WWTPs serving tourist communities. We found that the Omicron BA.1 and BA.2 viral loads predominantly drove the SARS-CoV-2 resurgence. We also noted a shifting pattern in the Bangkok WBE from a 22-d early warning in early 2021 to a near real-time pattern in late 2021. The potential application of tourist hotspots for WBE to indicate the arrival of new variants and re-emerging or unprecedented infectious agents could support tourism-dependent economies by complementing the reduced clinical regulations while maintaining public health protection via wastewater surveillance.