Institutionalising wastewater surveillance systems to minimise the impact of COVID-19: cases of Indonesia, Japan and Viet Nam.

This mini review describes the current status and challenges regarding institutionalisation of wastewater surveillance systems against COVID-19. Monitoring SARS-CoV-2 in wastewater has been proposed to be a potential tool to understand the actual prevalence of COVID-19 in the community, and it could be an effective approach to monitor the trend during the COVID-19 pandemic. However, challenges to institutionalise wastewater surveillance systems are still abundant and unfolding at a rapid rate given that the international understanding regarding the scientific knowledge and socio-political impacts of COVID-19 are in the developing stages. To better understand the existing challenges and bottlenecks, a comparative study between Japan, Viet Nam, and Indonesia was carried out in the present study. Through gaining a better understanding of common issues as well as issues specific to each country, we hope to contribute to building a robust multistakeholder system to monitor SARS-CoV-2 in wastewater as an effective disease surveillance system for COVID-19.

Validation of host-specific Bacteroidales quantitative PCR assays and their application to microbial source tracking of drinking water sources in the Kathmandu Valley, Nepal.

AIMS: To validate host-specific Bacteroidales assays to identify faecal-source contamination of drinking water sources in the Kathmandu Valley, Nepal. METHODS AND RESULTS: A total of 54 composite faecal-source samples were collected from human sewage, ruminants, pigs, dogs, chickens and ducks, which were analysed by quantitative polymerase chain reaction using human-specific (BacHum, HF183 SYBR, gyrB and HF183 TaqMan), ruminant-specific (BacCow and BacR), pig-specific (Pig2Bac and PF163) and dog-specific assays (BacCan SYBR). The BacHum, BacR and Pig2Bac assays were judged the best performing human-specific, ruminant-specific and pig-specific assays respectively. The BacCan SYBR assay highly cross-reacted with other species, resulting in poor performance. Furthermore, these validated assays were applied to microbial source tracking (MST) of 74 drinking water samples. Out of these, 20, 12 and 4% samples were judged contaminated by human, ruminant and pig faeces respectively. Detection ratios of human and ruminant faecal markers were relatively higher in built-up and agricultural areas respectively. CONCLUSION: BacHum, BacR and Pig2Bac assays were found suitable for MST and both, human and animal faecal contaminations of drinking water sources were common in the valley. SIGNIFICANCE AND IMPACT OF THE STUDY: MST could be an effective tool for preparing the faecal pollution strategies as these are site specific.

Temporal variations in genotype distribution of human sapoviruses and Aichi virus 1 in wastewater in Southern Arizona, United States.

AIMS: To investigate the molecular epidemiology, especially temporal variations in genotype distribution, of sapoviruses and Aichi virus 1 (AiV-1) in Arizona, United States, by examining wastewater. METHODS AND RESULTS: A total of 26 wastewater samples (13 influent and 13 effluent) were collected monthly from a wastewater treatment plant and viral strains were identified through nested reverse transcription-PCR followed by cloning and sequencing analysis. Identified sapovirus strains were classified into seven genotypes belonging to three genogroups (GI, GII, and GV): GI.1, GI.2, GI.3, GII.1, GII.2, GII.8 and GV.1, with a clear temporal shift. The majority of AiV-1 strains identified from the wastewater samples were classified into genotype B, and genotype A strains were identified in only two samples. CONCLUSIONS: We identified a number of sapovirus and AiV-1 strains belonging to multiple genotypes in wastewater samples collected over a 13-month period. Our results suggested a temporal shift in prevalent genotypes in the community. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study elucidating the genotype distribution of human sapoviruses and AiV-1 in wastewater in the United States. Wastewater surveillance is especially useful for understanding molecular epidemiology of viruses that are less commonly tested in clinical diagnosis, including sapoviruses and AiV-1.

Assessing the infection risk of enteropathogens from consumption of raw vegetables washed with contaminated water in Kathmandu Valley, Nepal.

AIMS: To assess diarrhoeal risks from enteropathogenic Escherichia coli, Giardia and Cryptosporidium from consuming raw spinach, cabbage, carrots and tomatoes in Kathmandu Valley, Nepal. METHODS AND RESULTS: The annual infection risk was quantified using the probabilistic Quantitative Microbial Risk Assessment approach, which considered 12 vegetable washing combinations. A new model was used to estimate dose of pathogens per exposure comprising parameters such as pathogen concentration in vegetable wash water before selling and eating, vegetable consumption rate, remaining pathogen ratio after washing, remaining water on vegetables after washing and water treatment removal efficiency. When all washing combinations were considered, high infection risks above the acceptable level of -4 log10 infection per person per year were obtained, whereas the risk was reduced when other sources excluding river water were used. Assuming use of water treated with ceramic filters by all consumers, a 0-2 log10 reduction in the estimated risks was obtained, which was insufficient to achieve the required risk level. CONCLUSION: High risk of diarrhoea prevails among raw vegetable consumers in the valley. SIGNIFICANCE AND IMPACT OF THE STUDY: It is needed to protect vegetable washing water sources and establish advanced water treatment methods to achieve the required level of public health risk.

Genogroup distribution of F-specific coliphages in wastewater and river water in the Kofu basin in Japan.

AIMS: To determine the genogroup distribution of F-specific coliphages in aquatic environments using the plaque isolation procedure combined with genogroup-specific real-time PCR. METHODS AND RESULTS: Thirty water samples were collected from a wastewater treatment plant and a river in the Kofu basin in Japan on fine weather days. F-specific coliphages were detected in all tested samples, 187 (82%) of 227 phage plaques isolated were classified into one of the 4 F-specific RNA (F-RNA) coliphage genogroups and 24 (11%) plaques were F-specific DNA coliphages. Human genogroups II and III F-RNA coliphages were more abundant in raw sewage than animal genogroups I and IV, excluding one sample that was suspected to be heavily contaminated with sporadic heavy animal faeces. The secondary-treated sewage samples were highly contaminated with genogroup I F-RNA coliphages, probably because of different behaviours among the coliphage genogroups during wastewater treatment. The river water samples were expected to be mainly contaminated with human faeces, independent of rainfall effects. CONCLUSIONS: A wide range of F-specific coliphage genogroups were successfully identified in wastewater and river water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results clearly show the usefulness of the genogroup-specific real-time PCR for determining the genogroups of F-specific coliphages present in aquatic environments.

Molecular detection and genotyping of human noroviruses in influent and effluent water at a wastewater treatment plant in Japan.

AIMS: To investigate the prevalence, seasonality and genotype distribution of human noroviruses (NoVs) in wastewater in Japan. METHODS AND RESULTS: Influent and effluent water samples were collected monthly for a year from a wastewater treatment plant and examined for the presence of genogroups I and II (GI and GII) NoVs. Using real-time reverse transcription (RT)-PCR assays, 12 (100%) influent and six (50%) effluent samples were positive for both GI and GII NoV genomes, with a higher prevalence in winter. A total of 152 different NoV strains, comprising 84 GI and 68 GII strains, were identified using seminested RT-PCR assays followed by cloning and sequence analysis. These strains were classified into nine GI genotypes (GI/1, 2, 4, 5, 8, 9, 11, 12 and 14) and 13 GII genotypes (GII/1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15 and 16), showing considerable genetic diversity. CONCLUSIONS: Based on the partial capsid gene sequences, we identified a great number of NoV strains belonging to many genotypes, demonstrating that genetically diverse NoV strains are co-circulating in aquatic environments and human populations. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results clearly demonstrate the seasonal trend and genetic diversity of NoVs in wastewater.

Genetic diversity of genogroup IV noroviruses in wastewater in Japan.

AIMS: To investigate the prevalence, seasonality and genetic diversity of genogroup IV noroviruses (GIV NoVs) in wastewater in Japan. METHODS AND RESULTS: Untreated and treated wastewater samples were collected monthly for a year from a wastewater treatment plant in Japan. The concentrated wastewater samples were examined for the presence of GIV NoV genomes with seminested RT-PCR assay targeting partial capsid gene. Among 12 untreated and 12 treated wastewater samples tested, GIV NoV genomes were detected in three (25%) untreated and two (17%) treated wastewater samples with a high positive ratio in winter season. Genetic analysis revealed that the GIV NoVs in the wastewater samples were genetically diverse and were classified into three different genetic clusters. CONCLUSIONS: Frequent detection of GIV NoVs in winter season, which is a common epidemic period of human NoVs in Japan, indicates that GIV NoVs exhibit temporal trends similar to GI and GII NoVs. Based on the partial capsid gene sequences, we identified several unique GIV NoV strains belonging to the novel genetic cluster, demonstrating that GIV NoVs are more genetically diverse than previously appreciated. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings provide novel evidence of considerable genetic diversity among the GIV NoV strains.

Chlorine inactivation of human norovirus, murine norovirus and poliovirus in drinking water.

AIMS: To evaluate the reduction of human norovirus (HuNoV) by chlorine disinfection under typical drinking water treatment conditions. METHODS AND RESULTS: HuNoV, murine norovirus (MNV) and poliovirus type 1 (PV1) were inoculated into treated water before chlorination, collected from a drinking water treatment plant, and bench-scale free chlorine disinfection experiments were performed for two initial free chlorine concentrations, 0.1 and 0.5 mg l(-1). Inactivation of MNV reached more than 4 log(10) after 120 and 0.5 min contact time to chlorine at the initial free chlorine concentrations of 0.1 and 0.5 mg l(-1), respectively. CONCLUSIONS: MNV was inactivated faster than PV1, and there was no significant difference in the viral RNA reduction rate between HuNoV and MNV. The results suggest that appropriate water treatment process with chlorination can manage the risk of HuNoV infection via drinking water supply systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study would be useful for assessing or managing the risk of HuNoV infections from drinking water exposure.

Detection of genogroup IV norovirus in wastewater and river water in Japan.

AIMS: To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV). METHODS AND RESULTS: Influent and effluent samples from a wastewater treatment plant and the Tamagawa River water samples were collected monthly for a year. The water samples were concentrated by the adsorption-elution method, using an HA electronegative filter with acid rinse procedure, followed by quantitative detection of GIV NoV using TaqMan-based real-time RT-PCR. Both wastewater and river water samples showed a high positive ratio of GIV NoV during winter and spring. The highest concentration in wastewater and river water was 6.9 x 10(4) and 1.5 x 10(4) copies l(-1), respectively. CONCLUSIONS: Presence of GIV NoV in the environments demonstrates that not only GI and GII NoVs but also GIV strains are circulating and that routine monitoring of GIV NoV in water environments is recommended to understand its epidemics, environmental distribution and potential health risks. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study providing quantitative data on the occurrence of GIV NoV in environmental water over a 1-year period.

Quantitative detection of sapoviruses in wastewater and river water in Japan.

AIMS: To detect sapoviruses at a wastewater treatment plant (WWTP) and in a river in Japan, quantitatively. METHODS AND RESULTS: Influent and effluent samples at a WWTP and river water samples were collected monthly for 1 year. The water samples were subjected to virus concentration using an HA electronegative filter, followed by quantification of sapoviruses using real-time PCR. The concentration of sapoviruses in influent ranged from 2.8 x 10(3) to 1.3 x 10(5) copies per litre, showing a higher value in winter. Seven (58%) of 12 effluent samples were positive for sapoviruses, as were 23 (64%) of 36 river water samples collected from three sites along the Tamagawa River. CONCLUSIONS: Sapoviruses were abundant in the influent even in the nonepidemic period, suggesting that sporadic and asymptomatic infections occur throughout the year. Increasing concentration of sapoviruses was discharged into the river during the epidemic period winter. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study demonstrating the quantitative detection of sapoviruses in aquatic environments.

Quantitative analysis of human enteric adenoviruses in aquatic environments.

AIMS: The aim of this study was to determine human adenoviruses (HuAdVs) in aquatic environments by real-time polymerase chain reaction (PCR). METHODS AND RESULTS: In order to describe the ratio of enteric serotypes to the total HuAdVs, the primer set specific for the enteric serotypes 40 and 41 was used in parallel with the universal primer set for all 51 serotypes of HuAdVs. The enteric serotypes of HuAdVs were detected at the concentration of 7.3-1500 PCR-detection units (PDU) per ml in raw sewage (n = 17), 0.00060-4.1 PDU ml(-1) in secondary-treated sewage before chlorination (n = 17), 0.0018-7.0 PDU ml(-1) in river water (n = 36), and 0.032-6.1 PDU ml(-1) in seawater (n = 18). The concentration of HuAdVs, determined by the universal primer set, was equivalent to that of enteric serotypes in almost all the samples tested. CONCLUSIONS: Enteric serotypes were predominant among all serotypes of HuAdVs in the aquatic environments. SIGNIFICANCE AND IMPACT OF THE STUDY: The abundance of enteric serotypes of HuAdVs should be more emphasized than other serotypes in order to assess the risk of their infection via water.

Effects of rainfall on the occurrence of human adenoviruses, total coliforms, and Escherichia coli in seawater.

A two-month survey was conducted in order to evaluate the effects of rainfall on the fate of microorganisms in seawater in the Tokyo Bay, Japan. The seawater sample (1,000 mL) was applied to a method to concentrate virus, followed by a quantification of human adenoviruses using the real-time PCR. Total coliforms and E. coli, which were determined by the colony forming method, were detected in all 47 seawater samples, while human adenoviruses were detected in 38 (81%) of the samples. The concentration of tested microorganisms showed 1-2 log units increase after rainfall events, followed by the gradual decrease to the level before the rainfall within a few days.

Seasonal profiles of human noroviruses and indicator bacteria in a wastewater treatment plant in Tokyo, Japan.

The seasonal profiles of microorganisms in raw sewage, secondary-treated sewage, and final effluent at a wastewater treatment plant in Tokyo, Japan, were quantitatively determined each month for one year, from July 2003 to June 2004. Human noroviruses, which were determined by real-time PCR, in raw sewage varied from 0.17-260 copies/mL for genotype 1 and from 2.4-1900 copies/mL for genotype 2, showing much higher values in winter, the epidemic season. The concentration of total coliforms, Escherichia coli, or F-specific phages in raw sewage was almost constant throughout the year. Human noroviruses of genotype 2 were removed most effectively (3.69 log10 on average) at the wastewater treatment plant, followed by E. coli (3.37 log10), total coliforms (3.05 loglo), F-specific phages (2.81 log10), and human noroviruses of genotype 1 (2.27 log10). The removal ratio of human noroviruses was almost constant, independent of the initial concentration of the viruses in raw sewage, which led to the increasing concentration of human noroviruses in final effluent in winter. None of the tested bacteria was judged to be a reliable indicator of human noroviruses in final effluent.