Dynamic analysis of non-revenue water in district metered areas under varying water consumption conditions owing to COVID-19.

Increasing water demands and high water losses have rendered securing safe water challenging in the 21st century. Although non-revenue water (NRW), as a percentage of system input, has been commonly used by water utilities worldwide, in-depth analyses on the influence of water consumption fluctuation on NRW has never been conducted; instead, taking one-year average NRW volume has been recommended. Thus, this study analyzed the influence of water consumption fluctuation on NRW using the data of five district metered areas (DMAs) in Colombo City, Sri Lanka, and also by the network simulation analysis. The results showed that percentage and volumetric NRWs are strongly correlated with water consumption (r = 0.9373 and 0.9121, respectively) and with each other (r = 0.9977) due to pressure changes in water supply networks caused by water consumption fluctuation. Therefore, dynamic analysis of NRW by plotting DMA inflow and NRW against water consumption was conducted using the aforementioned DMA data and long-term (1956-2021) water consumption and NRW data in Tokyo. This method identified two factors influencing NRW: water consumption fluctuation and network leakage changes, and the results were verified; thus, it can be applied to NRW analysis even under the influence of high water consumption fluctuations.

Detection of Rotavirus Vaccine Strains in Oysters and Sewage and Their Relationship with the Gastroenteritis Epidemic.

Rotavirus is one of the major causes of infectious gastroenteritis among infants and children, and live attenuated vaccines for rotavirus A (RVA), namely, Rotarix and RotaTeq, have recently become available in Japan. Rotavirus is known to be excreted from patients and accumulated in oysters similar to norovirus; however, the vaccine strains in aquatic environments or oysters have not yet been analyzed. In this study, we focused on wild-type RVA, which is highly important in considering the risk of infectious diseases. We quantified total RVA, Rotarix, and RotaTeq strains in oyster and sewage samples collected between September 2014 and July 2016 to assess the contamination levels of wild-type RVA by subtracting the quantitative value of rotavirus vaccine strains from that of total RVA. The positive rates of wild-type RVA, Rotarix, and RotaTeq in oysters were 54, 14, and 31%, respectively. These rates were comparable to those of wild-type RVA (57%) and RotaTeq (35%) in sewage; however, Rotarix was not detected in any sewage samples. The comparison of viral concentrations in oysters and sewage suggested more efficient accumulation of the vaccine strains in oysters than the wild-type RVA. The concentration of wild-type RVA in oysters was significantly correlated with that in sewage with a lag time of -6 to 0 weeks which is required for viral transportation from wastewater treatment plants to oysters. On the other hand, no significant correlation was observed between wild-type RVA concentration in sewage and the number of rotavirus-associated gastroenteritis cases, implying the existence of asymptomatic RVA-infected individuals.IMPORTANCE We quantified rotavirus A (RVA), Rotarix, and RotaTeq strains in oyster and sewage samples during two gastroenteritis seasons and revealed the exact contamination of wild-type RVA by subtracting the quantitative value of rotavirus vaccine strains from that of RVA. The concentration of wild-type RVA was significantly correlated between oysters and sewage, although no significant correlation was seen between wild-type RVA concentration in sewage and the number of rotavirus-associated gastroenteritis cases. This finding suggested the existence of asymptomatic patients and that monitoring of rotavirus vaccine strain could be useful to understand the trend of wild-type RVA and rotavirus outbreak in detail. We believe that our study makes a significant contribution to the literature because it reports the detection of rotavirus vaccine strains in oysters.

Weekly Variation of Rotavirus A Concentrations in Sewage and Oysters in Japan, 2014-2016.

Concentrations of rotavirus A, in sewage and oysters collected weekly from September 2014 to April 2016 in Japan, were investigated using RT-qPCR; results showed up to 6.5 log10 copies/mL and 4.3 log10 copies/g of digestive tissue (DT) in sewage and oysters, respectively. No correlation was found between rotavirus concentration in sewage and oysters and cases of rotavirus-associated gastroenteritis.

Weekly variations in norovirus genogroup II genotypes in Japanese oysters.

Increased levels of norovirus contamination in oysters were reportedly associated with a gastroenteritis epidemic occurring upstream of an oyster farming area. In this study, we monitored the norovirus concentration in oysters weekly between November 2014 and March 2015 and investigated the statistical relationship between norovirus genogroup II (GII) concentrations in oyster and sewage samples and the number of gastroenteritis cases in the area using cross-correlation analysis. A peak correlation coefficient (R = 0.76) at a time lag of +1 week was observed between the number of gastroenteritis cases and norovirus GII concentrations in oysters, indicating that oyster contamination is correlated with the number of gastroenteritis cases with a 1-week delay. Moreover, weekly variations in norovirus GII genotypes in oysters were evaluated using pyrosequencing. Only GII.3 was detected in November and December 2014, whereas GII.17 and GII.4 were present from January to March 2015. GII.17 Kawasaki 2014 strains were detected more frequently than GII.4 Sydney 2012 strains in oyster samples, as previously observed in stool and sewage samples collected during the same study period in Miyagi, Japan. Our observations indicate that there is a time lag between the circulation of norovirus genotypes in the human population and the detection of those genotypes in oysters.

Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters.

Detection and genotyping of pathogenic RNA viruses in human and environmental samples are useful for monitoring the circulation and prevalence of these pathogens, whereas a conventional PCR assay followed by Sanger sequencing is time-consuming and laborious. The present study aimed to develop a high-throughput detection-and-genotyping tool for 11 human RNA viruses [Aichi virus; astrovirus; enterovirus; norovirus genogroup I (GI), GII, and GIV; hepatitis A virus; hepatitis E virus; rotavirus; sapovirus; and human parechovirus] using a microfluidic device and next-generation sequencer. Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan); genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads. The detection limit of the 11 tested viruses ranged from 100 to 103 copies/µL in cDNA sample, corresponding to 101-104 copies/mL-sewage, 105-108 copies/g-human feces, and 102-105 copies/g-digestive tissues of oyster. The developed assay was successfully applied for simultaneous detection and genotyping of RNA viruses to samples of human feces, sewage, and artificially contaminated oysters. Microfluidic nested PCR followed by MiSeq sequencing enables efficient tracking of the fate of multiple RNA viruses in various environments, which is essential for a better understanding of the circulation of human pathogenic RNA viruses in the human population.

Reverse transcription-quantitative PCR assays for genotype-specific detection of human noroviruses in clinical and environmental samples.

Circulation of human noroviruses in water environments is suspected to be genotype-dependent, but the established primer and probe sets for noroviruses are usually genogroup-specific, which do not allow to compare the genotype-specific properties, such as persistence in water environments and resistance to disinfectants. In this study, quantitative PCR assays were designed for genotype-specific quantification of four epidemiologically important genotypes, GII.3, GII.4, GII.6, and GII.17. Developed assays were tested using norovirus positive stool samples which were previously confirmed to present target genotypes of this study. The results were 100% in accordance with the previous results. Effect of the co-existence of multiple genotypes in a sample on the target genotype quantification was evaluated using composite stool samples and wastewater samples containing multiple genotypes and the presence of non-target genotypes didn't affect the quantification of target genotype. Sensitivity and specificity was 100% for all four assays developed in this study with no cross-reactions between genotypes demonstrating the validity of our assays and their applicability to clinical and environmental samples.

Oyster Contamination with Human Noroviruses Impacted by Urban Drainage and Seasonal Flooding in Vietnam.

This study investigated the level of norovirus contamination in oysters collected at a lagoon receiving urban drainage from Hue City for 17 months (August 2015-December 2016). We also investigated the genetic diversity of norovirus GI and GII in oyster and wastewater samples by using pyrosequencing to evaluate the effect of urban drainage on norovirus contamination of oysters. A total of 34 oyster samples were collected at two sampling sites (stations A and B) in a lagoon. Norovirus GI was more frequently detected than GII (positive rate 79 vs. 41%). Maximum concentrations of GI and GII were 2.4 × 105 and 2.3 × 104 copies/g, respectively. Co-contamination with GI and GII was observed in 35% of samples. Norovirus GII concentration was higher at station A in the flood season than in the dry season (P = 0.04, Wilcoxon signed-rank test). Six genotypes (GI.2, GI.3, GI.5, GII.2, GII.3, and GII.4) were identified in both wastewater and oyster samples, and genetically similar or identical sequences were obtained from the two types of samples. These observations suggest that urban drainage and seasonal flooding contribute to norovirus contamination of oysters in the study area.

Environmental Surveillance of Norovirus Genogroups I and II for Sensitive Detection of Epidemic Variants.

Sewage samples have been investigated to study the norovirus concentrations in sewage or the genotypes of noroviruses circulating in human populations. However, the statistical relationship between the concentration of the virus and the number of infected individuals and the clinical importance of genotypes or strains detected in sewage are unclear. In this study, we carried out both environmental and clinical surveillance of noroviruses for 3 years, 2013 to 2016. We performed cross-correlation analysis of the concentrations of norovirus GI or GII in sewage samples collected weekly and the reported number of gastroenteritis cases. Norovirus genotypes in sewage were also analyzed by pyrosequencing and compared with those identified in stool samples. The cross-correlation analysis found the peak coefficient (R = 0.51) at a lag of zero, indicating that the variation in the GII concentration, expressed as the log10 number of copies per milliliter, was coincident with that in the gastroenteritis cases. A total of 15 norovirus genotypes and up to 8 genotypes per sample were detected in sewage, which included all of the 13 genotypes identified in the stool samples except 2. GII.4 was most frequently detected in both sample types, followed by GII.17. Phylogenetic analysis revealed that a strain belonging to the GII.17 Kawasaki 2014 lineage had been introduced into the study area in the 2012-2013 season. An increase in GI.3 cases was observed in the 2015-2016 season, and sewage monitoring identified the presence of GI.3 in the previous season (2014-2015). Our results demonstrated that monitoring of noroviruses in sewage is useful for sensitive detection of epidemic variants in human populations.IMPORTANCE We obtained statistical evidence of the relationship between the variation in the norovirus GII concentration in sewage and that of gastroenteritis cases during the 3-year study period. Sewage sample analysis by a pyrosequencing approach enabled us to understand the temporal variation in the norovirus genotypes circulating in human populations. We found that a strain closely related to the GII.17 Kawasaki 2014 lineage had been introduced into the study area at least 1 year before its appearance and identification in clinical cases. A similar pattern was observed for GI.3; cases were reported in the 2015-2016 season, and closely related strains were found in sewage in the previous season. Our observation indicates that monitoring of noroviruses in sewage is useful for the rapid detection of an epidemic and is also sensitive enough to study the molecular epidemiology of noroviruses. Applying this approach to other enteric pathogens in sewage will enhance our understanding of their ecology.

Pyrosequencing Analysis of Norovirus Genogroup II Distribution in Sewage and Oysters: First Detection of GII.17 Kawasaki 2014 in Oysters.

Norovirus GII.3, GII.4, and GII.17 were detected using pyrosequencing in sewage and oysters in January and February 2015, in Japan. The strains in sewage and oyster samples were genetically identical or similar, predominant strains belonging to GII.17 Kawasaki 2014 lineage. This is the first report of GII.17 Kawasaki 2014 in oysters.

Comparative Evaluation of Real-Time PCR Methods for Human Noroviruses in Wastewater and Human Stool.

Selecting the best quantitative PCR assay is essential to detect human norovirus genome effectively from clinical and environmental samples because no cell lines have been developed to propagate this virus. The real-time PCR methods for noroviruses GI (4 assays) and GII (3 assays) were evaluated using wastewater (n = 70) and norovirus-positive stool (n = 77) samples collected in Japan between 2012 and 2013. Standard quantitative PCR assays recommended by the U.S. Environmental Protection Agency, International Organization for Standardization, and Ministry of Health, Labour and Welfare, Japan, together with recently reported assays were included. Significant differences in positive rates and quantification cycles were observed by non-parametric analysis. The present study identifies the best assay for norovirus GI and GII to amplify norovirus genomes efficiently.

Bactericidal and virucidal mechanisms in the alkaline disinfection of compost using calcium lime and ash.

In the present study, the bactericidal and virucidal mechanisms in the alkaline disinfection of compost with calcium lime and ash were investigated. Two indicator microorganisms, Escherichia coli and MS2 coliphage, were used as surrogates for enteric pathogens. The alkaline-treated compost with calcium oxide (CaO) or ash resulted primarily in damage to the outer membrane and enzyme activities of E. coli. The alkaline treatment of compost also led to the infectivity loss of the coliphage because of the partial capsid damage and RNA exteriorization due to a raised pH, which is proportional to the amount of alkaline agents added. These results indicate that the alkaline treatment of compost using calcium oxide and ash is effective and can contribute to the safe usage of compost from a mixing type dry toilet.

Ammonia as an In Situ Sanitizer: Influence of Virus Genome Type on Inactivation.

UNLABELLED: Treatment of human excreta and animal manure (HEAM) is key in controlling the spread of persistent enteric pathogens, such as viruses. The extent of virus inactivation during HEAM storage and treatment appears to vary with virus genome type, although the reasons for this variability are not clear. Here, we investigated the inactivation of viruses of different genome types under conditions representative of HEAM storage or mesophilic digestion. The goals were to characterize the influence of HEAM solution conditions on inactivation and to determine the potential mechanisms involved. Specifically, eight viruses representing the four viral genome types (single-stranded RNA [ssRNA], double-stranded RNA [dsRNA], single-stranded DNA [ssDNA], and double-stranded DNA [dsDNA]) were exposed to synthetic solutions with well-controlled temperature (20 to 35°C), pH (8 to 9), and ammonia (NH3) concentrations (0 to 40 mmol liter(-1)). DNA and dsRNA viruses were considerably more resistant than ssRNA viruses, resulting in up to 1,000-fold-longer treatment times to reach a 4-log inactivation. The apparently slower inactivation of DNA viruses was rationalized by the higher stability of DNA than that of ssRNA in HEAM. Pushing the system toward harsher pH (>9) and temperature (>35°C) conditions, such as those encountered in thermophilic digestion and alkaline treatments, led to more consistent inactivation kinetics among ssRNA and other viruses. This suggests that the dependence of inactivation on genome type disappeared in favor of protein-mediated inactivation mechanisms common to all viruses. Finally, we recommend the use of MS2 as a conservative indicator to assess the inactivation of ssRNA viruses and the stable ΦX174 or dsDNA phages as indicators for persistent viruses. IMPORTANCE: Viruses are among the most environmentally persistent pathogens. They can be present in high concentrations in human excreta and animal manure (HEAM). Therefore, appropriate treatment of HEAM is important prior to its reuse or discharge into the environment. Here, we investigated the factors that determine the persistence of viruses in HEAM, and we determined the main mechanisms that lead to their inactivation. Unlike other organisms, viruses can have four different genome types (double- or single-stranded RNA or DNA), and the viruses studied herein represent all four types. Genome type appeared to be the major determinant for persistence. Single-stranded RNA viruses are the most labile, because this genome type is susceptible to degradation in HEAM. In contrast, the other genome types are more stable; therefore, inactivation is slower and mainly driven by the degradation of viral proteins. Overall, this study allows us to better understand the behavior of viruses in HEAM.

Temporal dynamics of norovirus determined through monitoring of municipal wastewater by pyrosequencing and virological surveillance of gastroenteritis cases.

Norovirus is a leading etiological agent of viral gastroenteritis. Because of relatively mild disease symptoms and frequent asymptomatic infections, information on the ecology of this virus is limited. Our objective was to examine the genetic diversity of norovirus circulating in the human population by means of genotyping the virus in municipal wastewater. We investigated norovirus genogroups I and II (GI and GII) in municipal wastewater in Japan by pyrosequencing and quantitative PCR (qPCR) from November 2012 to March 2013. Virological surveillance for gastroenteritis cases was concurrently conducted in the same area. A total of fourteen distinct genotypes in total (GI.1, 3, 4, 6, 7, GII.2, 4, 5, 6, 7, 12, 13, 14, and 17), with up to eight genotypes detected per sample, were observed in wastewater using pyrosequencing; only four genotypes (GI.6, GII.4, 5, and 14) were obtained from clinical samples. Seventy-eight percent of norovirus-positive stool samples contained GII.4, but this genotype was not dominant in wastewater. The norovirus GII.4 Sydney 2012 variant, which appeared and spread during our study period, was detected in both the wastewater and clinical samples. These results suggest that an environmental approach using pyrosequencing yields a more detailed distribution of norovirus genotypes/variants. Thus, wastewater monitoring by pyrosequencing is expected to provide an effective analysis of the distribution of norovirus genotypes causing symptomatic and asymptomatic infections in human populations.

Ammonia as an in situ sanitizer: inactivation kinetics and mechanisms of the ssRNA virus MS2 by NH3.

Sanitizing human and animal waste (e.g., urine, fecal sludge, or grey water) is a critical step in reducing the spread of disease and ensuring microbially safe reuse of waste materials. Viruses are particularly persistent pathogens and can be transmitted through inadequately sanitized waste. However, adequate storage or digestion of waste can strongly reduce the number of viruses due to increases in pH and uncharged aqueous ammonia (NH3), a known biocide. In this study we investigated the kinetics and mechanisms of inactivation of the single-stranded RNA virus MS2 under temperature, pH and NH3 conditions representative of waste storage. MS2 inactivation was mainly controlled by the activity of NH3 over a pH range of 7.0­9.5 and temperatures lower than 40 °C. Other bases (e.g., hydroxide, carbonate) additionally contributed to the observed reduction of infective MS2. The loss in MS2 infectivity could be rationalized by a loss in genome integrity, which was attributed to genome cleavage via alkaline transesterification. The contribution of each base to genome transesterification, and hence inactivation, could be related to the base pKa by means of a Bronsted relationship. The Bronsted relationship in conjunction with the activity of bases in solution enabled an accurate prediction of MS2 inactivation rates.

A fate model of pathogenic viruses in a composting toilet based on coliphage inactivation.

A composting toilet using sawdust as a matrix has the potential to trap pathogens that might occasionally be contained in human feces. Therefore, care should be taken when handling the sawdust. It should also be noted that pathogenic viruses tend to have stronger tolerance than pathogenic bacteria. The fates of several species of coliphages, T4, lambda, Qbeta and MS2, in sawdust were investigated as a viral model. The fates of coliphages were significantly different among them, and they changed in response to temperature and the water content of the sawdust. As the results, T4 coliphage had the strongest tolerance and Qbeta had the weakest one in sawdust. It was estimated the days required to decrease virus to a safe level based on a risk assessment. According to the rates of Qbeta and T4, 15 days and 167 days were required respectively for a safe level of infection risk based on actually operated composting toilet condition. Thus, it was significantly different depending on the species and sawdust conditions.