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.

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 environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan.

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.

Reduction of Pathogenic and Indicator Viruses at a Drinking Water Treatment Plant in Southern Louisiana, USA.

Monthly sampling was conducted at a drinking water treatment plant (DWTP) in Southern Louisiana, USA from March 2017 to February 2018 to determine the prevalence and reduction efficiency of pathogenic and indicator viruses. Water samples were collected from the DWTP at three different treatment stages (raw, secondary-treated, and chlorinated drinking water) and subjected to quantification of seven pathogenic viruses and three indicator viruses [pepper mild mottle virus (PMMoV), tobacco mosaic virus (TMV), and crAssphage] based on quantitative polymerase chain reaction. Among the seven pathogenic viruses tested, only Aichi virus 1 (AiV-1) (7/12, 58%) and noroviruses of genogroup II (NoVs-GII) (2/12, 17%) were detected in the raw water samples. CrAssphage had the highest positive ratio at 78% (28/36), and its concentrations were significantly higher than those of the other indicator viruses for all three water types (P < 0.05). The reduction ratios of AiV-1 (0.7 ± 0.5 log10; n = 7) during the whole treatment process were the lowest among the tested viruses, followed by crAssphage (1.1 ± 1.9 log10; n = 9), TMV (1.3 ± 0.9 log10; n = 8), PMMoV (1.7 ± 0.8 log10; n = 12), and NoVs-GII (3.1 ± 0.1 log10; n = 2). Considering the high abundance and relatively low reduction, crAssphage was judged to be an appropriate process indicator during drinking water treatment. To the best of our knowledge, this is the first study to assess the reduction of crAssphage and TMV during drinking water treatment.

Reduction of Human Enteric and Indicator Viruses at a Wastewater Treatment Plant in Southern Louisiana, USA.

This study assessed wastewater quality through the quantification of four human enteric viruses and the applicability of pepper mild mottle virus (PMMoV) and tobacco mosaic virus (TMV) as indicators of viral reduction during wastewater treatment. Thirty-three samples were collected from three steps of a wastewater treatment plant in Southern Louisiana, USA for a year between March 2017 and February 2018. Noroviruses of genogroup I were the most prevalent human enteric viruses in influent samples. The concentrations of PMMoV in influent samples (5.9 ± 0.7 log10 copies/L) and biologically treated effluent samples (5.9 ± 0.5 log10 copies/L) were significantly higher than those of TMV (P < 0.05), and the reduction ratio of PMMoV (1.0 ± 0.8 log10) was found comparable to those of TMV and Aichi virus 1. Because of the high prevalence, high correlations with human enteric viruses, and lower reduction ratios, PMMoV was deemed an appropriate indicator of human enteric viral reduction during wastewater treatment process.

Applicability of crAssphage, pepper mild mottle virus, and tobacco mosaic virus as indicators of reduction of enteric viruses during wastewater treatment.

This study was conducted to evaluate the applicability of crAssphage, pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV) as indicators of the reduction of human enteric viruses during wastewater treatment. Thirty-nine samples were collected from three steps at a wastewater treatment plant (raw sewage, secondary-treated sewage, and final effluent) monthly for a 13-month period. In addition to the three indicator viruses, eight human enteric viruses [human adenoviruses, JC and BK polyomaviruses, Aichi virus 1 (AiV-1), enteroviruses, and noroviruses of genogroups I, II, and IV] were tested by quantitative PCR. Indicator viruses were consistently detected in the tested samples, except for a few final effluents for crAssphage and TMV. The mean concentrations of crAssphage were significantly higher than those of most tested viruses. The concentrations of crAssphage in raw sewage were positively correlated with the concentrations of all tested human enteric viruses (p <0.05), suggesting the applicability of crAssphage as a suitable indicator to estimate the concentrations of human enteric viruses in raw sewage. The reduction ratios of AiV-1 (1.8 ± 0.7 log10) were the lowest among the tested viruses, followed by TMV (2.0 ± 0.3 log10) and PMMoV (2.0 ± 0.4 log10). Our findings suggested that the use of not only AiV-1 and PMMoV but also TMV as indicators of reductions in viral levels can be applicable during wastewater treatment.

Evaluation of Human- and Animal-Specific Viral Markers and Application of CrAssphage, Pepper Mild Mottle Virus, and Tobacco Mosaic Virus as Potential Fecal Pollution Markers to River Water in Japan.

Five human-specific markers were detected in 59-74% of 27 human fecal-source samples collected in Yamanashi Prefecture, Japan. Similarly, potential human-specific markers, crAssphage, pepper mild mottle virus (PMMoV), and tobacco mosaic virus were detected in 96-100% of samples, with crAssphage showing the maximum concentration of 12.03 log copies/L. However, these markers were detected in 100% (3/3) of pig fecal-source samples, suggesting their applicability as general fecal pollution markers. Microbial source tracking analysis demonstrated that the rivers are contaminated by human and pig fecal sources. CrAssphage showed higher marker concentrations in river water samples than PMMoV, suggesting the preference of crAssphage to PMMoV as a marker of fecal pollution.

Detection of Pathogenic Viruses, Pathogen Indicators, and Fecal-Source Markers within Tanker Water and Their Sources in the Kathmandu Valley, Nepal.

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.

Performance Evaluation of Human-Specific Viral Markers and Application of Pepper Mild Mottle Virus and CrAssphage to Environmental Water Samples as Fecal Pollution Markers in the Kathmandu Valley, Nepal.

Monitoring of environmental water is crucial to protecting humans and animals from possible health risks. Although numerous human-specific viral markers have been designed to track the presence of human fecal contamination in water, they lack adequate sensitivity and specificity in different geographical regions. We evaluated the performances of six human-specific viral markers [Aichi virus 1 (AiV-1), human adenoviruses (HAdVs), BK and JC polyomaviruses (BKPyVs and JCPyVs), pepper mild mottle virus (PMMoV), and crAssphage] using 122 fecal-source samples collected from humans and five animal hosts in the Kathmandu Valley, Nepal. PMMoV and crAssphage showed high sensitivity (90-100%) with concentrations of 4.5-9.1 and 6.2-7.0 log10 copies/g wet feces (n = 10), respectively, whereas BKPyVs, JCPyVs, HAdVs, and AiV-1 showed poor performances with sensitivities of 30-40%. PMMoV and crAssphage were detected in 40-100% and 8-90%, respectively, of all types of animal fecal sources and showed no significantly different concentrations among most of the fecal sources (Kruskal-Wallis test, P > 0.05), suggesting their applicability as general fecal pollution markers. Furthermore, a total of 115 environmental water samples were tested for PMMoV and crAssphage to identify fecal pollution. PMMoV and crAssphage were successfully detected in 62% (71/115) and 73% (84/115) of water samples, respectively. The greater abundance and higher mean concentration of crAssphage (4.1 ± 0.9 log10 copies/L) compared with PMMoV (3.3 ± 1.4 log10 copies/L) indicated greater chance of detection of crAssphage in water samples, suggesting that crAssphage could be preferred to PMMoV as a marker of fecal pollution.

Co-Infection by Waterborne Enteric Viruses in Children with Gastroenteritis in Nepal.

Enteric viruses are highly contagious and a major cause of waterborne gastroenteritis in children younger than five years of age in developing world. This study examined the prevalence of enteric virus infection in children with gastroenteritis to identify risk factors for co-infections. In total, 107 stool samples were collected from patients with acute gastroenteritis along with samples of their household drinking water and other possible contamination sources, such as food and hand. The presence of major gastroenteritis-causing enteric virus species (group A rotaviruses, enteroviruses, adenoviruses, and noroviruses of genogroup I) in stool and water samples was examined using quantitative polymerase chain reaction. Among the 107 stool samples tested, 103 (96%) samples contained at least one of the four tested enteric viruses, and the combination of group A rotaviruses and enteroviruses was the most common co-infection (52%, n = 54/103). At least one viral agent was detected in 16 (16%) of 103 drinking water samples. Identical enteric viruses were detected in both the stool and water samples taken from the same patients in 13% of cases (n = 13/103). Group A rotaviruses were most frequently found in children suffering from acute diarrhea. No socio-demographic and clinical factors were associated with the risk of co-infection compared with mono-infection. These less commonly diagnosed viral etiological agents in hospitals are highly prevalent in patients with acute gastroenteritis.

Reduction of Cryptosporidium, Giardia, and Fecal Indicators by Bardenpho Wastewater Treatment.

Increased demand for water reuse and reclamation accentuates the importance for optimal wastewater treatment to limit protozoa in effluents. Two wastewater treatment plants utilizing advanced Bardenpho were investigated over a 12-month period to determine the incidence and reduction of Cryptosporidium, Giardia, Cyclospora, and fecal indicators. Results were compared to facilities that previously operated in the same geographical area. Protozoa (oo)cysts were concentrated using an electronegative filter and subsequently detected by fluorescent microscopy and/or PCR methods. Cryptosporidium and Giardia were frequently detected in raw sewage, but Cyclospora was not detected in any wastewater samples. Facilities with Bardenpho treatment exhibited higher removals of (oo)cysts than facilities utilizing activated sludge or trickling filters. This was likely due to Bardenpho systems having increased solid wasting rates; however, this mechanism cannot be confirmed as sludge samples were not analyzed. Use of dissolved-air-flotation instead of sedimentation tanks did not result in more efficient removal of (oo)cysts. Concentrations of protozoa were compared with each other, Escherichia coli, somatic coliphage, and viruses (pepper mild mottle virus, Aichi virus 1, adenovirus, and polyomaviruses JC and BK). Although significant correlations were rare, somatic coliphage showed the highest potential as an indicator for the abundance of protozoa in wastewaters.

Presence of Human Enteric Viruses, Protozoa, and Indicators of Pathogens in the Bagmati River, Nepal.

Quantification of waterborne pathogens in water sources is essential for alerting the community about health hazards. This study determined the presence of human enteric viruses and protozoa in the Bagmati River, Nepal, and detected fecal indicator bacteria (total coliforms, Escherichia coli, and Enterococcus spp.), human-fecal markers (human Bacteroidales and JC and BK polyomaviruses), and index viruses (tobacco mosaic virus and pepper mild mottle virus). During a one-year period between October 2015 and September 2016, a total of 18 surface water samples were collected periodically from three sites along the river. Using quantitative polymerase chain reaction, all eight types of human enteric viruses tested-including adenoviruses, noroviruses, and enteroviruses, were detected frequently at the midstream and downstream sites, with concentrations of 4.4-8.3 log copies/L. Enteroviruses and saliviruses were the most frequently detected enteric viruses, which were present in 72% (13/18) of the tested samples. Giardia spp. were detected by fluorescence microscopy in 78% (14/18) of the samples, with a lower detection ratio at the upstream site. Cryptosporidium spp. were detected only at the midstream and downstream sites, with a positive ratio of 39% (7/18). The high concentrations of enteric viruses suggest that the midstream and downstream regions are heavily contaminated with human feces and that there are alarming possibilities of waterborne diseases. The concentrations of enteric viruses were significantly higher in the dry season than the wet season (p < 0.05). There was a significant positive correlation between the concentrations of human enteric viruses and the tested indicators for the presence of pathogens (IPP) (p < 0.05), suggesting that these IPP can be used to estimate the presence of enteric viruses in the Bagmati River water.

A review on recent progress in the detection methods and prevalence of human enteric viruses in water.

Waterborne human enteric viruses, such as noroviruses and adenoviruses, are excreted in the feces of infected individuals and transmitted via the fecal-oral route including contaminated food and water. Since viruses are normally present at low concentrations in aquatic environments, they should be concentrated into smaller volumes prior to downstream molecular biological applications, such as quantitative polymerase chain reaction (qPCR). This review describes recent progress made in the development of concentration and detection methods of human enteric viruses in water, and discusses their applications for providing a better understanding of the prevalence of the viruses in various types of water worldwide. Maximum concentrations of human enteric viruses in water that have been reported in previous studies are summarized to assess viral abundances in aquatic environments. Some descriptions are also available on recent applications of sequencing analyses used to determine the genetic diversity of viral genomes in water samples, including those of novel viruses. Furthermore, the importance and significance of utilizing appropriate process controls during viral analyses are discussed, and three types of process controls are considered: whole process controls, molecular process controls, and (reverse transcription (RT)-)qPCR controls. Although no standards have been established for acceptable values of virus recovery and/or extraction-(RT-)qPCR efficiency, use of at least one of these appropriate control types is highly recommended for more accurate interpretation of observed data.

Virological Quality of Irrigation Water Sources and Pepper Mild Mottle Virus and Tobacco Mosaic Virus as Index of Pathogenic Virus Contamination Level.

Irrigation water is a doorway for the pathogen contamination of fresh produce. We quantified pathogenic viruses [human adenoviruses, noroviruses of genogroups I and II, group A rotaviruses, Aichi virus 1 (AiV-1), enteroviruses (EnVs), and salivirus (SaliV)] and examined potential index viruses [JC and BK polyomaviruses (JCPyVs and BKPyVs), pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV)] in irrigation water sources in the Kathmandu Valley, Nepal. River, sewage, wastewater treatment plant (WWTP) effluent, pond, canal, and groundwater samples were collected in September 2014, and in April and August 2015. Viruses were concentrated using an electronegative membrane-vortex method and quantified using TaqMan (MGB)-based quantitative PCR (qPCR) assays with murine norovirus as a molecular process control to determine extraction-reverse transcription-qPCR efficiency. Tested pathogenic viruses were prevalent with maximum concentrations of 5.5-8.8 log10 copies/L, and there was a greater abundance of EnVs, SaliV, and AiV-1. Virus concentrations in river water were equivalent to those in sewage. Canal, pond, and groundwater samples were found to be less contaminated than river, sewage, and WWTP effluent. Seasonal dependency was clearly evident for most of the viruses, with peak concentrations in the dry season. JCPyVs and BKPyVs had a poor detection ratio and correspondence with pathogenic viruses. Instead, the frequently proposed PMMoV and the newly proposed TMV were strongly predictive of the pathogen contamination level, particularly in the dry season. We recommend utilizing canal, pond, and groundwater for irrigation to minimize deleterious health effects and propose PMMoV and TMV as indexes to elucidate pathogenic virus levels in environmental samples.

Distinct behaviors of infectious F-specific RNA coliphage genogroups at a wastewater treatment plant.

The present study aimed to determine the differences in the behaviors of four F-specific RNA (F-RNA) coliphage genogroups (GI-GIV) during wastewater treatment. Raw sewage, aeration tank effluent, secondary-treated sewage, and return activated sludge were collected from a wastewater treatment plant in Japan at monthly intervals between March and December 2011 (n=10 each). F-specific coliphages were detected by plaque assay in all tested samples, with a concentration ranging from -0.10 to 3.66 log10 plaque-forming units/ml. Subsequently, eight plaques were isolated from each sample, followed by genogroup-specific reverse-transcription quantitative PCR (qPCR) for F-RNA coliphages and qPCR for F-specific DNA (F-DNA) coliphages. GI F-RNA coliphages were the most abundant in the secondary-treated sewage samples (73% of the plaque isolates), while GII F-RNA coliphages were the most abundant in the other three sample types (41-81%, depending on sample type). Based on the results of the quantification and genotyping, the annual mean concentrations of each F-specific coliphage type were calculated, and their reduction ratios during wastewater treatment were compared with those of indicator bacteria (total coliforms and Escherichia coli) and enteric viruses (human adenoviruses and GI and GII noroviruses). The mean reduction ratio of GI F-RNA coliphages was the lowest (0.93 log10), followed by those of the indicator bacteria and enteric viruses (1.59-2.43 log10), GII-GIV F-RNA coliphages (>2.60-3.21 log10), and F-DNA coliphages (>3.41 log10). These results suggest that GI F-RNA coliphages may be used as an appropriate indicator of virus reduction during wastewater treatment.

Risk of diarrhoea from shallow groundwater contaminated with enteropathogens in the Kathmandu Valley, Nepal.

Shallow groundwater is the main water source among many alternatives in the Kathmandu Valley, Nepal, which has a rapidly growing population and intermittent piped water supply. Although human pathogens are detected in groundwater, its health effects are unclear. We estimated risk of diarrhoea from shallow groundwater use using quantitative microbial risk assessment. Escherichia coli, Giardia cyst and Cryptosporidium oocyst levels were analysed in dug and tube wells samples. E. coli concentrations were converted to those of enteropathogenic E. coli (EPEC). Risks from EPEC in dug wells and from Cryptosporidium and Giardia in both dug and tube wells were higher than the acceptable limit (<10⁻4 infections/person-year) for both drinking and bathing exposures. Risk from protozoan enteropathogens increased the total risk 10,000 times, indicating that ignoring protozoans could lead to serious risk underestimation. Bathing exposure considerably increased risk, indicating that it is an important pathway. Point-of-use (POU) water treatment decreased the risk six-fold and decreased risk overestimation. Because removal efficiency of POU water treatment has the largest impact on total risk, increasing the coverage and efficiency of POU water treatment could be a practical risk management strategy in the Kathmandu Valley and similar settings.

Quantitative detection of human enteric adenoviruses in river water by microfluidic digital polymerase chain reaction.

We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.

Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona.

We investigated the occurrence of Cryptosporidium, Giardia, and Cyclospora at two wastewater treatment plants (WWTPs) in Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent wastewater samples were collected monthly, and protozoan (oo)cysts were concentrated using an electronegative filter, followed by the detection of protozoa using fluorescent microscopy (Cryptosporidium oocysts and Giardia cysts) and PCR-based methods (Cryptosporidium spp., Giardia intestinalis, and Cyclospora cayetanensis). The concentration of Giardia cysts in the influent was always higher than that of Cryptosporidium oocysts (mean concentration of 4.8-6.4×10(3) versus 7.4×10(1)-1.0×10(2)(oo)cysts/l) with no clear seasonality, and log10 reduction of Giardia cysts was significantly higher than that of Cryptosporidium oocysts for both WWTPs (P<0.05). Log10 reduction of Giardia cysts at the WWTP utilizing activated sludge was significantly higher than the other WWTP using trickling filter (P=0.014), while no statistically significant difference between the two WWTPs was observed for the log10 reduction of Cryptosporidium oocysts (P=0.207). Phylogenetic analysis revealed that G. intestinalis strains identified in wastewater belonged to two assemblages, AII and B, which are potentially infectious to humans. C. cayetanensis was also detected from both influent and effluent using a newly developed quantitative PCR, with the highest influent concentration of 1.2×10(4)copies/l. Our results demonstrated that these protozoan pathogens are prevalent in the study area and that efficacy of the conventional wastewater treatment processes at physically removing (oo)cysts is limited.

Development of a novel method for simultaneous concentration of viruses and protozoa from a single water sample.

A novel method, electronegative membrane-vortex (EMV) method, was developed for simultaneous concentration of viruses and protozoa from a single water sample. Viruses and protozoa in a water sample were mixed with a cation solution and adsorbed on an electronegative membrane. Concentrated virus and protozoa samples were obtained as supernatant and pellet fractions, respectively, by vigorous vortex mixing of the membrane and centrifugation of the eluted material. The highest recovery efficiencies of model microbes from river water and tap water by this EMV method were obtained using a mixed cellulose ester membrane with a pore size of 0.45 µm (Millipore) as the electronegative membrane and MgCl(2) as the cation solution. The recovery was 27.7-86.5% for poliovirus, 25.7-68.3% for coliphage Qß, 28.0-60.0% for Cryptosporidium oocysts, and 35.0-53.0% for Giardia cysts. The EMV method detected successfully indigenous viruses and protozoa in wastewater and river water samples from the Kofu basin, Japan, showing an overall positive rate of 100% (43/43) for human adenovirus, 79% (34/43) for norovirus GI, 65% (28/43) for norovirus GII, 23% (10/43) for Cryptosporidium oocysts, and 60% (26/43) for Giardia cysts. By direct DNA sequencing, a total of four genotypes (AI, AII, B, and G) of Giardia intestinalis were identified in the water samples, indicating that the river water was contaminated with feces from various mammals, including humans.

One-year weekly survey of noroviruses and enteric adenoviruses in the Tone River water in Tokyo metropolitan area, Japan.

To investigate the actual fluctuations in the concentrations of noroviruses (NoVs) GI and GII, and enteric adenoviruses (EAdVs) in river water and its relationship with the number of acute infectious gastroenteritis patients, one-year weekly quantitative monitoring of NoVs GI and GII and EAdVs was performed in the Tone River in Japan where the surface water is utilized for the main production of drinking water for the Tokyo Metropolitan Area from October 2009 to September 2010. Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. The concentrations of NoVs GI and GII and EAdVs fluctuated strongly and were more abundant in winter and early spring. The concentration of NoVs GI was transiently greater than 10,000 copies/L. The number of acute infectious gastroenteritis patients in the upper river basin was highly correlated with all the viral concentrations, while general microbial indicator data such as turbidity and heterotrophic plate count were independent of viral concentration as suggested in previous studies. To the best of our knowledge, this is the first study that clearly shows the strong correlation of the number of gastroenteritis with virus contamination in lower river basin.