Seasonality of respiratory, enteric, and urinary viruses revealed by wastewater genomic surveillance.

Wastewater surveillance can reveal population-level infectious disease burden and emergent public health threats can be reliably assessed through wastewater surveillance. While molecular methods for wastewater monitoring of microorganisms have traditionally relied on PCR-based approaches, next-generation sequencing (NGS) can provide deeper insights via genomic analyses of multiple diverse pathogens. We conducted a year-long sequencing surveillance of 1,408 composite wastewater samples collected from 12 neighborhood-level access points in the greater Tempe area, Arizona, USA, and show that variation in wastewater viruses is driven by seasonal time and location. The temporal dynamics of viruses in wastewater were influenced cyclically, with the most dissimilarity between samples 23 weeks apart (i.e., winter vs summer, spring vs fall). We identified diverse urinary and enteric viruses including polyomaviruses, astroviruses, and noroviruses, and showed that their genotypes/subtypes shifted across seasons. We show that while wastewater data of certain respiratory viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly correlate with clinical case rates, laboratory-reported case incidences were discordant with surges of high viral load in wastewater for other viruses like human coronavirus 229E. These results demonstrate the utility of wastewater sequencing for informing decision-making in public health.IMPORTANCEWastewater surveillance can provide insights into the spread of pathogens in communities. Advances in next-generation sequencing (NGS) methodologies allow for more precise detection of viruses in wastewater. Long-term wastewater surveillance of viruses is an important tool for public health preparedness. This system can act as a public health observatory that gives real-time early warning for infectious disease outbreaks and improved response times.

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.

Simultaneous detection and characterization of common respiratory pathogens in wastewater through genomic sequencing.

Genomic surveillance of SARS-CoV-2 has given insight into the evolution and epidemiology of the virus and its variant lineages during the COVID-19 pandemic. Expanding this approach to include a range of respiratory pathogens can better inform public health preparedness for potential outbreaks and epidemics. Here, we simultaneously sequenced 38 pathogens including influenza viruses, coronaviruses and bocaviruses, to examine the abundance and seasonality of respiratory pathogens in urban wastewater. We deployed a targeted bait capture method and short-read sequencing (Illumina Respiratory Virus Oligos Panel; RVOP) on composite wastewater samples from 8 wastewater treatment plants (WWTPs) and one associated hospital site. By combining seasonal sampling with whole genome sequencing, we were able to concurrently detect and characterise a range of common respiratory pathogens, including SARS-CoV-2, adenovirus and parainfluenza virus. We demonstrated that 38 respiratory pathogens can be detected at low abundances year-round, that hospital pathogen diversity is higher in winter vs. summer sampling events, and that significantly more viruses are detected in raw influent compared to treated effluent samples. Finally, we compared detection sensitivity of RT-qPCR vs. next generation sequencing for SARS-CoV-2, enteroviruses, influenza A/B, and respiratory syncytial viruses. We conclude that both should be used in combination; RT-qPCR allowed accurate quantification, whilst genomic sequencing detected pathogens at lower abundance. We demonstrate the valuable role of wastewater genomic surveillance and its contribution to the field of wastewater-based epidemiology, gaining rapid understanding of the seasonal presence and persistence for common respiratory pathogens. By simultaneously monitoring seasonal trends and early warning signs of many viruses circulating in communities, public health agencies can implement targeted prevention and rapid response plans.

Assessing environmental exposure to viruses in wastewater treatment plant and swine farm scenarios with next-generation sequencing and occupational risk approaches.

Occupational exposure to pathogens can pose health risks. This study investigates the viral exposure of workers in a wastewater treatment plant (WWTP) and a swine farm by analyzing aerosol and surfaces samples. Viral contamination was evaluated using quantitative polymerase chain reaction (qPCR) assays, and target enrichment sequencing (TES) was performed to identify the vertebrate viruses to which workers might be exposed. Additionally, Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate the occupational risk associated with viral exposure for WWTP workers, choosing Human Adenovirus (HAdV) as the reference pathogen. In the swine farm, QMRA was performed as an extrapolation, considering a hypothetical zoonotic virus with characteristics similar to Porcine Adenovirus (PAdV). The modelled exposure routes included aerosol inhalation and oral ingestion through contaminated surfaces and hand-to-mouth contact. HAdV and PAdV were widespread viruses in the WWTP and the swine farm, respectively, by qPCR assays. TES identified human and other vertebrate viruses WWTP samples, including viruses from families such as Adenoviridae, Circoviridae, Orthoherpesviridae, Papillomaviridae, and Parvoviridae. In the swine farm, most of the identified vertebrate viruses were porcine viruses belonging to Adenoviridae, Astroviridae, Circoviridae, Herpesviridae, Papillomaviridae, Parvoviridae, Picornaviridae, and Retroviridae. QMRA analysis revealed noteworthy risks of viral infections for WWTP workers if safety measures are not taken. The probability of illness due to HAdV inhalation was higher in summer compared to winter, while the greatest risk from oral ingestion was observed in workspaces during winter. Swine farm QMRA simulation suggested a potential occupational risk in the case of exposure to a hypothetical zoonotic virus. This study provides valuable insights into WWTP and swine farm worker's occupational exposure to human and other vertebrate viruses. QMRA and NGS analyses conducted in this study will assist managers in making evidence-based decisions, facilitating the implementation of protection measures, and risk mitigation practices for workers.

Sequencing and Variant Detection of Eight Abundant Plant-Infecting Tobamoviruses across Southern California Wastewater.

Tobamoviruses are agriculturally relevant viruses that cause crop losses and have infected plants in many regions of the world. These viruses are frequently found in municipal wastewater, likely coming from human diet and industrial waste across wastewater catchment areas. As part of a large wastewater-based epidemiology study across Southern California, we analyzed RNA sequence data from 275 influent wastewater samples obtained from eight wastewater treatment plants with a catchment area of approximately 16 million people from July 2020 to August 2021. We assembled 1,083 high-quality genomes, enumerated viral sequencing reads, and detected thousands of single nucleotide variants from eight common tobamoviruses: bell pepper mottle virus, cucumber green mottle mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, tomato brown rugose fruit virus, tomato mosaic virus, tomato mottle mosaic virus, and tropical soda apple mosaic virus. We show that single nucleotide variants had amino acid-altering consequences along with synonymous mutations, which represents potential evolution with functional consequences in genomes of these viruses. Our study shows the importance of wastewater sequencing to monitor the genomic diversity of these plant-infecting viruses, and we suggest that our data could be used to continue tracking the genomic variability of such pathogens. IMPORTANCE Diseases caused by viruses in the genus Tobamovirus cause crop losses around the world. As with other viruses, mutation occurring in the virus's genomes can have functional consequences and may alter viral infectivity. Many of these plant-infecting viruses have been found in wastewater, likely coming from human consumption of infected plants and produce. By sequencing RNA extracted from influent wastewater obtained from eight wastewater treatment plants in Southern California, we assembled high-quality viral genomes and detected thousands of single nucleotide variants from eight tobamoviruses. Our study shows that Tobamovirus genomes vary at many positions, which may have important consequences when designing assays for the detection of these viruses by agricultural or environmental scientists.

Averting an Outbreak of SARS-CoV-2 in a University Residence Hall through Wastewater Surveillance.

A wastewater surveillance program targeting a university residence hall was implemented during the spring semester 2021 as a proactive measure to avoid an outbreak of COVID-19 on campus. Over a period of 7 weeks from early February through late March 2021, wastewater originating from the residence hall was collected as grab samples 3 times per week. During this time, there was no detection of SARS-CoV-2 by reverse transcriptase quantitative PCR (RT-qPCR) in the residence hall wastewater stream. Aiming to obtain a sample more representative of the residence hall community, a decision was made to use passive samplers beginning in late March onwards. Adopting a Moore swab approach, SARS-CoV-2 was detected in wastewater samples just 2 days after passive samplers were deployed. These samples also tested positive for the B.1.1.7 (Alpha) variant of concern (VOC) using RT-qPCR. The positive result triggered a public health case-finding response, including a mobile testing unit deployed to the residence hall the following day, with testing of nearly 200 students and staff, which identified two laboratory-confirmed cases of Alpha variant COVID-19. These individuals were relocated to a separate quarantine facility, averting an outbreak on campus. Aggregating wastewater and clinical data, the campus wastewater surveillance program has yielded the first estimates of fecal shedding rates of the Alpha VOC of SARS-CoV-2 in individuals from a nonclinical setting. IMPORTANCE Among early adopters of wastewater monitoring for SARS-CoV-2 have been colleges and universities throughout North America, many of whom are using this approach to monitor congregate living facilities for early evidence of COVID-19 infection as an integral component of campus screening programs. Yet, while there have been numerous examples where wastewater monitoring on a university campus has detected evidence for infection among community members, there are few examples where this monitoring triggered a public health response that may have averted an actual outbreak. This report details a wastewater-testing program targeting a residence hall on a university campus during spring 2021, when there was mounting concern globally over the emergence of SARS-CoV-2 variants of concern, reported to be more transmissible than the wild-type Wuhan strain. In this communication, we present a clear example of how wastewater monitoring resulted in actionable responses by university administration and public health, which averted an outbreak of COVID-19 on a university campus.

RNA Viromics of Southern California Wastewater and Detection of SARS-CoV-2 Single-Nucleotide Variants.

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the coronavirus disease 2019 (COVID-19) pandemic to track disease burden through analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the health care system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven wastewater treatment plants (WTPs), collected from August 2020 to January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single-nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater, and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically important SARS-CoV-2 variants along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. IMPORTANCE Wastewater-based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the coronavirus disease 2019 (COVID-19) pandemic to track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without adding burden to health care systems. In this study, we used metatranscriptomics and reverse transcription-droplet digital PCR (RT-ddPCR) to assay RNA viruses across Southern California wastewater from August 2020 to January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single-nucleotide variants through sequencing. Likewise, wastewater treatment plants (WTPs) harbored different viromes, and we detected other human pathogens, such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

Assessment of a Program for SARS-CoV-2 Screening and Environmental Monitoring in an Urban Public School District.

Importance: Scalable programs for school-based SARS-CoV-2 testing and surveillance are needed to guide in-person learning practices and inform risk assessments in kindergarten through 12th grade settings. Objectives: To characterize SARS-CoV-2 infections in staff and students in an urban public school setting and evaluate test-based strategies to support ongoing risk assessment and mitigation for kindergarten through 12th grade in-person learning. Design, Setting, and Participants: This pilot quality improvement program engaged 3 schools in Omaha, Nebraska, for weekly saliva polymerase chain reaction testing of staff and students participating in in-person learning over a 5-week period from November 9 to December 11, 2020. Wastewater, air, and surface samples were collected weekly and tested for SARS-CoV-2 RNA to evaluate surrogacy for case detection and interrogate transmission risk of in-building activities. Main Outcomes and Measures: SARS-CoV-2 detection in saliva and environmental samples and risk factors for SARS-CoV-2 infection. Results: A total of 2885 supervised, self-collected saliva samples were tested from 458 asymptomatic staff members (mean [SD] age, 42.9 [12.4] years; 303 women [66.2%]; 25 Black or African American [5.5%], 83 Hispanic [18.1%], 312 White [68.1%], and 35 other or not provided [7.6%]) and 315 students (mean age, 14.2 [0.7] years; 151 female students [48%]; 20 Black or African American [6.3%], 201 Hispanic [63.8%], 75 White [23.8%], and 19 other race or not provided [6.0%]). A total of 46 cases of SARS-CoV-2 (22 students and 24 staff members) were detected, representing an increase in cumulative case detection rates from 1.2% (12 of 1000) to 7.0% (70 of 1000) among students and from 2.1% (21 of 1000) to 5.3% (53 of 1000) among staff compared with conventional reporting mechanisms during the pilot period. SARS-CoV-2 RNA was detected in wastewater samples from all pilot schools as well as in air samples collected from 2 choir rooms. Sequencing of 21 viral genomes in saliva specimens demonstrated minimal clustering associated with 1 school. Geographical analysis of SARS-CoV-2 cases reported district-wide demonstrated higher community risk in zip codes proximal to the pilot schools. Conclusions and Relevance: In this study of staff and students in 3 urban public schools in Omaha, Nebraska, weekly screening of asymptomatic staff and students by saliva polymerase chain reaction testing was associated with increased SARS-CoV-2 case detection, exceeding infection rates reported at the county level. Experiences differed among schools, and virus sequencing and geographical analyses suggested a dynamic interplay of school-based and community-derived transmission risk. Collectively, these findings provide insight into the performance and community value of test-based SARS-CoV-2 screening and surveillance strategies in the kindergarten through 12th grade educational setting.

Early Warning Diagnostics for Emerging Infectious Diseases in Developing into Late-Stage Pandemics.

The spread of infectious diseases due to travel and trade can be seen throughout history, whether from early settlers or traveling businessmen. Increased globalization has allowed infectious diseases to quickly spread to different parts of the world and cause widespread infection. Posthoc analysis of more recent outbreaks-SARS, MERS, swine flu, and COVID-19-has demonstrated that the causative viruses were circulating through populations for days or weeks before they were first detected, allowing disease to spread before quarantines, contact tracing, and travel restrictions could be implemented. Earlier detection of future novel pathogens could decrease the time before countermeasures are enacted. In this Account, we examined a variety of novel technologies from the past 10 years that may allow for earlier detection of infectious diseases. We have arranged these technologies chronologically from pre-human predictive technologies to population-level screening tools. The earliest detection methods utilize artificial intelligence to analyze factors such as climate variation and zoonotic spillover as well as specific species and geographies to identify where the infection risk is high. Artificial intelligence can also be used to monitor health records, social media, and various publicly available data to identify disease outbreaks faster than traditional epidemiology. Secondary to predictive measures is monitoring infection in specific sentinel animal species, where domestic animals or wildlife are indicators of potential disease hotspots. These hotspots inform public health officials about geographic areas where infection risk in humans is high. Further along the timeline, once the disease has begun to infect humans, wastewater epidemiology can be used for unbiased sampling of large populations. This method has already been shown to precede spikes in COVID-19 diagnoses by 1 to 2 weeks. As total infections increase in humans, bioaerosol sampling in high-traffic areas can be used for disease monitoring, such as within an airport. Finally, as disease spreads more quickly between humans, rapid diagnostic technologies such as lateral flow assays and nucleic acid amplification become very important. Minimally invasive point-of-care methods can allow for quick adoption and use within a population. These individual diagnostic methods then transfer to higher-throughput methods for more intensive population screening as an infection spreads. There are many promising early warning technologies being developed. However, no single technology listed herein will prevent every future outbreak. A combination of technologies from across our infection timeline would offer the most benefit in preventing future widespread disease outbreaks and pandemics.

Detection of SARs-CoV-2 in wastewater using the existing environmental surveillance network: A potential supplementary system for monitoring COVID-19 transmission.

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.

Stability of viral haemorrhagic septicaemia virus, infectious hematopoietic necrosis virus and cyprinid herpesvirus 3 in various water samples.

Rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) are the two most common species in traditional fish farming in Germany. Their aquaculture is threatened upon others by viruses that can cause a high mortality. Therefore, this work focuses on three viruses-viral haemorrhagic septicaemia virus, infectious hematopoietic necrosis virus and cyprinid herpesvirus 3 (CyHV-3)-that endanger these species. To prevent their spread and contain further outbreaks, it is essential to know how long they can outlast in environmental waters and what affects their infectivity outside the host. Hence, the stability of the target viruses in various water matrices was examined and compared in this work. In general, all three viruses were quite stable within sterile water samples (showing mostly ≤1 log reduction after 96 hr) but were inactivated faster and to a higher extent (up to five log steps within 96 hr) in unsterile environmental water samples. The inactivation of the viruses correlated well with the increasing bacterial load of the samples, suggesting that bacteria had the greatest effect on their stability in the examined samples. In comparison, CyHV-3 seemed to be the most sensitive and maintained its infectivity for the shortest period.

High abundance of human herpesvirus 8 in wastewater from a large urban area.

AIMS: This study assesses the diversity and abundance of Human Herpesviruses (HHVs) in the influent of an urban wastewater treatment plant using shotgun sequencing, metagenomic analysis and qPCR. METHODS AND RESULTS: Influent wastewater samples were collected from the three interceptors that serve the City of Detroit and Wayne, Macomb and Oakland counties between November 2017 to February 2018. The samples were subjected to a series of processes to concentrate viruses which were further sequenced and amplified using qPCR. All nine types of HHV were detected in wastewater. Human Herpesvirus 8 (HHV-8), known as Kaposi's sarcoma herpesvirus, which is only prevalent in 5-10% of USA population, was found to be the most abundant followed by HHV-3 or Varicella-zoster virus. CONCLUSIONS: The high abundance of HHV-8 in the Detroit metropolitan area may be attributed to the HIV-AIDS outbreak that was ongoing in Detroit during the sampling period. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach described in this paper can be used to establish a baseline of viruses secreted by the community as a whole. Sudden changes in the baseline would identify changes in community health and immunity.

Variations among Viruses in Influent Water and Effluent Water at a Wastewater Plant over One Year as Assessed by Quantitative PCR and Metagenomics.

Influent wastewater and effluent wastewater at the Rya treatment plant in Gothenburg, Sweden, were continuously monitored for enteric viruses by quantitative PCR (qPCR) during 1 year. Viruses in effluent wastewater were also identified by next-generation sequencing (NGS) in samples collected during spring, early summer, and winter. Samples of incoming wastewater were collected every second week. Seasonal variations in viral concentrations in incoming wastewater were found for noroviruses GII, sapovirus, rotavirus, parechovirus, and astrovirus. Norovirus GI and GIV and Aichi virus were present in various amounts during most weeks throughout the year, while hepatitis A virus, enterovirus, and adenovirus were identified less frequently. Fluctuations in viral concentrations in incoming wastewater were related to the number of diagnosed patients. The viruses were also detected in treated wastewater, however, with a 3- to 6-log10 reduction in concentration. Seven different hepatitis E virus (HEV) strains were identified in the effluents. Five of these strains belonged to genotype 3 and have been isolated in Sweden from swine, wild boars, and humans and in drinking water. The other two strains were divergent and had not been identified previously. They were similar to strains infecting rats and humans. Surveillance of enteric viruses in wastewater is a tool for early detection and follow-up of gastroenteritis outbreaks in society and for the identification of new viruses that can cause infection in humans.IMPORTANCE Both influent wastewater and treated wastewater at a wastewater treatment plant (WWTP) contain a high variety of human viral pathogens with seasonal variability when followed for 1 year. The peak of the amount of 11 different viruses in the inlet wastewater preceded the peak of the number of diagnosed patients by 2 to 4 weeks. The treatment of wastewater reduced viral concentrations by 3 to 6 log10 Despite the treatment of wastewater, up to 5 log10 virus particles per liter were released from into the surrounding river. Hepatitis E virus (HEV) strains previously identified in drinking water and two new strains, similar to those infecting rats and humans, were identified in the treated wastewater released from the WWTP.

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.

Recomendaciones para el manejo de las aguas residuales municipales, 11 de mayo del 2020 / Recommendations for the Management of Municipal Wastewater, 11 May 2020

Esta ficha presenta recomendaciones generales para evitar la transmisión del SARS-COV-2 a través de aguas residuales; orientaciones para los operadores de agua y saneamiento; e indicaciones para el tratamiento, la desinfección y la reutilización de aguas residuales.

This technical note presents general recommendations to avoid the transmission of SARS-VOC-2 by wastewater, guidelines for water and sanitation operators, and indications for treatment and disinfection and reuse of wastewater.

First detection of SARS-CoV-2 in untreated wastewaters in Italy.

Several studies have demonstrated the advantages of environmental surveillance through the monitoring of sewage for the assessment of viruses circulating in a given community (wastewater-based epidemiology, WBE). During the COVID-19 public health emergency, many reports have described the presence of SARS-CoV-2 RNA in stools from COVID-19 patients, and a few studies reported the occurrence of SARS-CoV-2 in wastewaters worldwide. Italy is among the world's worst-affected countries in the COVID-19 pandemic, but so far there are no studies assessing the presence of SARS-CoV-2 in Italian wastewaters. To this aim, twelve influent sewage samples, collected between February and April 2020 from Wastewater Treatment Plants in Milan and Rome, were tested adapting, for concentration, the standard WHO procedure for Poliovirus surveillance. Molecular analysis was undertaken with three nested protocols, including a newly designed SARS-CoV-2 specific primer set. SARS-CoV-2 RNA detection was accomplished in volumes of 250 ml of wastewaters collected in areas of high (Milan) and low (Rome) epidemic circulation, according to clinical data. Overall, 6 out of 12 samples were positive. One of the positive results was obtained in a Milan wastewater sample collected a few days after the first notified Italian case of autochthonous SARS-CoV-2. The study confirms that WBE has the potential to be applied to SARS-CoV-2 as a sensitive tool to study spatial and temporal trends of virus circulation in the population.

Quantitative Detection of Human Adenovirus and Human Rotavirus Group A in Wastewater and El-Rahawy Drainage Canal Influencing River Nile in the North of Giza, Egypt.

Environmental monitoring is critical in a developing country like Egypt where there is an insufficient framework for recording and tracking outbreaks. In this study, the prevalence of human adenovirus (HAdV), rotavirus group A (RVA) was determined in urban sewage, activated sludge, drainage water, drainage sediment, Nile water, and Nile sediment, using quantitative polymerase chain reaction (qPCR) analysis. HAdV was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 genome copies/liter (GC/L), 33% of activated sludge with viral concentrations ranging from 103 to 107 GC/kilogram (GC/kg), 95% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 75% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, 50% of Nile water with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile sediment with viral concentrations ranging from 103 to 107 GC/kg. RVA was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 GC/L, 75% of activated sludge with viral concentrations ranging from 103 to 107 GC/L, 58% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 50% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile water with viral concentrations ranging from 103 to 107 GC/kg. In conclusion, Abu-Rawash WWTP acts as a source of HAdV and RVA, releasing them into El-Rahawy drain then to the River Nile Rosetta branch.

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.

Viral and Bacterial Fecal Indicators in Untreated Wastewater across the Contiguous United States Exhibit Geospatial Trends.

Cultivated fecal indicator bacteria such as Escherichia coli and enterococci are typically used to assess the sanitary quality of recreational waters. However, these indicators suffer from several limitations, such as the length of time needed to obtain results and the fact that they are commensal inhabitants of the gastrointestinal tract of many animals and have fate and transport characteristics dissimilar to pathogenic viruses. Numerous emerging technologies that offer same-day water quality results or pollution source information or that more closely mimic persistence patterns of disease-causing pathogens that may improve water quality management are now available, but data detailing geospatial trends in wastewater across the United States are sparse. We report geospatial trends of cultivated bacteriophage (somatic, F+, and total coliphages and GB-124 phage), as well as genetic markers targeting polyomavirus, enterococci, E. coli, Bacteroidetes, and human-associated Bacteroides spp. (HF183/BacR287 and HumM2) in 49 primary influent sewage samples collected from facilities across the contiguous United States. Samples were selected from rural and urban facilities spanning broad latitude, longitude, elevation, and air temperature gradients by using a geographic information system stratified random site selection procedure. Most indicators in sewage demonstrated a remarkable similarity in concentration regardless of location. However, some exhibited predictable shifts in concentration based on either facility elevation or local air temperature. Geospatial patterns identified in this study, or the absence of such patterns, may have several impacts on the direction of future water quality management research, as well as the selection of alternative metrics to estimate sewage pollution on a national scale.IMPORTANCE This study provides multiple insights to consider for the application of bacterial and viral indicators in sewage to surface water quality monitoring across the contiguous United States, ranging from method selection considerations to future research directions. Systematic testing of a large collection of sewage samples confirmed that crAssphage genetic markers occur at a higher average concentration than key human-associated Bacteroides spp. on a national scale. Geospatial testing also suggested that some methods may be more suitable than others for widespread implementation. Nationwide characterization of indicator geospatial trends in untreated sewage represents an important step toward the validation of these newer methods for future water quality monitoring applications. In addition, the large paired-measurement data set reported here affords the opportunity to conduct a range of secondary analyses, such as the generation of new or updated quantitative microbial risk assessment models used to estimate public health risk.

Field Performance of Two Methods for Detection of Poliovirus in Wastewater Samples, Mexico 2016-2017.

To enhance our ability to monitor poliovirus circulation and certify eradication, we evaluated the performance of the bag-mediated filtration system (BMFS) against the two-phase separation (TPS) method for concentrating wastewater samples for poliovirus detection. Sequential samples were collected at two sites in Mexico; one L was collected by grab and ~ 5 L were collected and filtered in situ with the BMFS. In the laboratory, 500 mL collected by grab were concentrated using TPS and the sample contained in the filter of the BMFS was eluted without secondary concentration. Concentrates were tested for the presence of poliovirus and non-poliovirus enterovirus (NPEV) using Global Poliovirus Laboratory Network standard procedures. Between February 16, 2016, and April 18, 2017, 125 pairs of samples were obtained. Collectors spent an average (± standard deviation) of 4.3 ± 2.2 min collecting the TPS sample versus 73.5 ± 30.5 min collecting and filtering the BMFS sample. Laboratory processing required an estimated 5 h for concentration by TPS and 3.5 h for elution. Sabin 1 poliovirus was detected in 37 [30%] samples with the TPS versus 24 [19%] samples with the BMFS (McNemar's mid p value = 0.004). Sabin 3 poliovirus was detected in 59 [47%] versus 49 (39%) samples (p = 0.043), and NPEV was detected in 67 [54%] versus 40 [32%] samples (p < 0.001). The BMFS method without secondary concentration did not perform as well as the TPS method for detecting Sabin poliovirus and NPEV. Further studies are needed to guide the selection of cost-effective environmental surveillance methods for the polio endgame.