EU surveys insights: analytical tools, future directions, and the essential requirement for reference materials in wastewater monitoring of SARS-CoV-2, antimicrobial resistance and beyond.

BACKGROUND: Wastewater surveillance (WWS) acts as a vigilant sentinel system for communities, analysing sewage to protect public health by detecting outbreaks and monitoring trends in pathogens and contaminants. To achieve a thorough comprehension of present and upcoming practices and to identify challenges and opportunities for standardisation and improvement in WWS methodologies, two EU surveys were conducted targeting over 750 WWS laboratories across Europe and other regions. The first survey explored a diverse range of activities currently undertaken or planned by laboratories. The second survey specifically targeted methods and quality controls utilised for SARS-CoV-2 surveillance. RESULTS: The findings of the two surveys provide a comprehensive insight into the procedures and methodologies applied in WWS. In Europe, WWS primarily focuses on SARS-CoV-2 with 99% of the survey participants dedicated to this virus. However, the responses highlighted a lack of standardisation in the methodologies employed for monitoring SARS-CoV-2. The surveillance of other pathogens, including antimicrobial resistance, is currently fragmented and conducted by only a limited number of laboratories. Notably, these activities are anticipated to expand in the future. Survey replies emphasise the collective recognition of the need to enhance the accuracy of results in WWS practices, reflecting a shared commitment to advancing precision and effectiveness in WWS methodologies. CONCLUSIONS: These surveys identified a lack of standardised common procedures in WWS practices and the need for quality standards and reference materials to enhance the accuracy and reliability of WWS methods in the future. In addition, it is important to broaden surveillance efforts beyond SARS-CoV-2 to include other emerging pathogens and antimicrobial resistance to ensure a comprehensive approach to protecting public health.

Detection of Monkeypox Virus DNA in Airport Wastewater, Rome, Italy.

Environmental surveillance can be a complementary tool for detecting pathogens circulating in communities. We detected monkeypox virus DNA in wastewater from Italy's largest airport by using real-time PCR assays targeting the G2R region and F3L and N3R genes and sequencing. Wastewater surveillance can be quickly adapted to investigate emerging threats.

An Italian survey on the microbiological safety of toys containing aqueous media.

AIMS: The purpose of the present investigation is to fill the current gap in information regarding the microbiological quality of toys containing aqueous media and the related risks for users. METHODS: Over 18 years, a total of 491 sealed toys containing aqueous media were analysed using conventional microbial culture methods. In addition, molecular methods (PCR/nested RT-PCR, followed by Sanger sequencing) were employed to test for enteric viruses (enteroviruses and adenoviruses) in a subset of toys; subsequently, the infectivity of the positive samples was tested on cell cultures. RESULTS: Of the examined toys, 23.8% were noncompliant with the limits of the European guideline. The most frequently exceeded limits were those for Aerobic bacteria (84.6%), and Pseudomonas aeruginosa (29.9%). Other opportunistic bacterial species that were frequently detected were Stenotrophomonas maltophilia, Pseudomonas fluorescens, Burkholderia cepacia, Sphingomonas paucimobilis and Comamonas acidovorans. In a subset of 28 samples, adenovirus (25%) and enterovirus (11%) genome was also found to be present, although the samples with viral positivity did not show infectivity after inoculation on appropriate cell monolayers. CONCLUSIONS: The results indicate a condition of microbial exposure related to the use of toys containing aqueous media. SIGNIFICANCE AND IMPACT OF STUDY: The investigation highlights the need for more stringent monitoring during the production, packaging and storage of toys containing aqueous matrices in order to safeguard children's health.

Cytotoxicity and internalization analysis of silicon nanowires in Buffalo Green Monkey cells: a preliminary study to evaluate the possibility of carrying viruses inside the cells.

Silicon nanowires (SiNWs) are attractive functional nanomaterials for biomedical applications. The ability to easily tune their size and density, potential biocompatibility, and knowledge of the chemical activation of SiNWs surface make them natural tools to interact with biological materials. We evaluated the possibility of exploiting SiNWs as carriers to introduce organic compounds into cells. The cellular toxicity and the internalization capacity of free-standing and label-free SiNWs were tested on Buffalo Green Monkey cells (BGM). Confocal fluorescent observation of SiNWs conjugated with fluorescein-polyethylene imine (PEI) confirmed the internalization of the NWs into the Buffalo Green Monkey Cells (BGM).

Quantification and genetic diversity of Hepatitis E virus in wild boar (Sus scrofa) hunted for domestic consumption in Central Italy.

Hepatitis E is an emerging disease in industrialized countries. The food-borne transmission of hepatitis E virus (HEV) is associated principally with products derived from the domestic pig, the wild boar, and deer; however, few quantitative data are available on HEV loads in animals used in food production. This study assessed HEV occurrence, viral load and genetic variability in wild boar hunted for domestic consumption in the district of Viterbo (Central Italy) where high anti-HEV IgG seroprevalence values are reported in humans. A total of 332 liver and 69 intestine samples were obtained from wild boar hunted between 2011 and 2014. The liver tissue in 54 of the animals (16.3%) was HEV-positive. Viral loads in quantifiable liver samples (n = 29) ranged between 3.2 × 102 and 3.8 × 105 genome copies (g.c.)/g with a mean value of 1.85 × 104 g.c./g. A statistically significant positive correlation was found between viral concentration in liver and intestinal tissue, though mean viral load in the intestines was lower (3.13 × 103 g.c./g). Twenty-six samples were characterized molecularly as genotype 3 (G3) and four subtypes (a, c, f and l) were detected. Finally, twelve samples with near identical sequences were identified as G3 but could not be assigned to any of the known subtypes, and could therefore represent a potentially new subtype.

Hepatitis E in Italy: 5 years of national epidemiological, virological and environmental surveillance, 2012 to 2016.

Increasing numbers of hepatitis E cases are being reported in several European countries, including Italy, but the burden of hepatitis E virus (HEV) infection is largely unknown in the latter. To gain a better understanding of HEV epidemiology at national level in Italy, we piloted a strengthened and integrated human (epidemiological and virological) and environmental HEV surveillance system between 2012 and 2016. Over the 5-year period, 169 confirmed hepatitis E cases were identified, with a national annual incidence of 0.72 cases per 1,000,000. Of 65 HEV-RNA positive samples of sufficient quality for molecular analysis, 66% were genotype HEV3, 32% HEV1 and 1% HEV4. The most frequent risk factor reported by all HEV3 infected cases, was the consumption of undercooked pork and sausage. For the environmental surveillance, 679 urban sewage samples were collected from 53 wastewater treatment plants and HEV-RNA was detected in 38/679 of the samples. Among these, 25 (66%) were genotype HEV3 and the remaining were HEV1. We demonstrate that autochthonous transmission and environmental circulation of genotype HEV3 is adding to travel-related HEV transmission in Italy. We recommend the 'One Health' approach to integrated surveillance, and to include HEV-related messages within health information campaigns focussing on food security.

High prevalence of anti-hepatitis E virus antibodies among blood donors in central Italy, February to March 2014.

Prevalence of anti-hepatitis E virus (HEV) antibodies is highly variable in developed countries, which seems partly due to differences in assay sensitivity. Using validated sensitive assays, we tested 313 blood donors attending a hospital transfusion unit in central Italy in January and February 2014 for anti-HEV IgG and IgM and HEV RNA. Data on HEV exposure were collected from all donors. Overall anti-HEV IgG prevalence was 49% (153/313). Eating raw dried pig-liver sausage was the only independent predictor of HEV infection (adjusted prevalence rate ratio = 2.14; 95% confidence interval: 1.23-3.74). Three donors were positive for either anti-HEV IgM (n = 2; 0.6%) or HEV RNA (n = 2; 0.6%); they were completely asymptomatic, without alanine aminotransferase (ALT) abnormalities. Of the two HEV RNA-positive donors (both harbouring genotype 3), one was anti-HEV IgG- and IgM-positive, the other was anti-HEV IgG- and IgM-negative. The third donor was positive for anti-HEV IgG and IgM but HEV RNA-negative. HEV infection is therefore hyperendemic among blood donors (80% men 18-64 years-old) from central Italy and associated with local dietary habits. Nearly 1% of donors have acute or recent infection, implying potential transmission to blood recipients. Neither ALT nor anti-HEV IgM testing seems useful to prevent transfusion-transmitted HEV infection.

A large spectrum of alpha and beta papillomaviruses are detected in human stool samples.

Human papillomaviruses (HPVs) have been detected in urban wastewaters, demonstrating that epitheliotropic viruses can find their way into sewage through the washing of skin and mucous membranes. Papillomavirus shedding through faeces is still an unexplored issue. The objective of the present study was to investigate the presence of HPVs in stool samples. We analysed 103 faecal specimens collected from hospitalized patients with diarrhoea using validated primers able to detect α, ß and γ HPVs. PCR products underwent sequencing analysis and sequences were aligned to reference genomes from the Papillomavirus Episteme database. A total of 15 sequences were characterized from the faecal samples. Thirteen samples (12.6 %) were positive for nine genotypes belonging to the α and ß genera: HPV32 (LR, α1), HPV39 (HR, α7), HPV44 (LR, α10), HPV8 (ß1), HPV9, HPV23, HPV37, HPV38 and HPV120 (ß2). Two putative novel genotypes of the ß genus, species 1 and 2, were also detected. The tissue(s) of origin is unknown, since faeces can collect HPVs originating from or passing through the entire digestive system. To our knowledge, this is the first investigation on the occurrence and diversity of HPVs in faecal samples. Results from this study demonstrate that HPVs can find their way into sewage as a consequence of shedding in the faeces. This highlights the need for further studies aimed at understanding the prevalence of HPV in different water environments and the potential for waterborne transmission.

Molecular characterisation of human hepatitis E virus from Italy: comparative analysis of five reverse transcription-PCR assays.

BACKGROUND: Hepatitis E (HEV) is an important public-health concern as a major cause of enterically transmitted hepatitis worldwide. In industrialised countries it is considered rare, and largely confined to travellers returning from endemic areas. However, autochthonous (locally acquired) HEV infection is also emerging in these regions. The infection is caused by different genotypes, depending on whether it is travel-related or autochthonous. Conventional RT-PCR followed by sequencing of PCR products can identify HEV genotype and, depending on the region, the subtype, thus helping in defining the origin of infection and tracing the source of contamination. METHODS: We re-analysed a collection of serum samples previously confirmed as hepatitis E positive by anti-HEV IgM and IgG assays as well as by Real-Time PCR, with the aim to compare the performances of five different broad range RT-PCR assays that could be provided for molecular characterisation of HEV. This approach is certainly valuable to investigate the molecular epidemiology of acute hepatitis E in countries where co-circulation of different genotypes occurs, like Italy. RESULTS: Samples were analyzed by five assays targeting the ORF1, ORF2, and ORF2/3 regions. The sensitivity of these assays varied significantly, depending on the target region. Only 46% of samples tested positive by nested PCR; moreover, no single method was able to detect all positive samples. Most sequences originated from patients who had travelled to endemic areas (genotype 1), while the minority originated from Italian patients with no travel history (genotype 3). CONCLUSION: Broad range methods for molecular characterization of HEV still need to be improved to detect all circulating strains.

Surveillance of hepatitis A virus in urban sewages and comparison with cases notified in the course of an outbreak, Italy 2013.

BACKGROUND: Over the past 20 years, Hepatitis A notifications in Italy have been in decline. Since the beginning of 2013 however, Italy has been experiencing a foodborne hepatitis A outbreak caused by genotype IA, involving hundreds of cases. Consumption of frozen mixed berries was deemed the potential vehicle of infection.We aimed to investigate the spread of hepatitis A virus (HAV) in Italy through the monitoring of urban sewages collected at Wastewater Treatment Plants (WTPs) and a subsequent comparison of environmental surveillance data with data from the clinical surveillance performed during the epidemic. METHODS: The study covered 15 months, from July 2012 to September 2013, comprising the outbreak and the preceding six months. Environmental surveillance consisted of the analysis of urban sewage samples collected at 19 WTPs in seven of the Italian regions most affected by the epidemic. HAV isolates were detected and typed using a nested RT-PCR targeting the VP1/2A junction. Parallel clinical surveillance was performed by the sentinel surveillance system for acute viral hepatitis (SEIEVA) and by the ministerial Central Task Force on Hepatitis A, established with the purpose of determining the source of the outbreak and adopting appropriate outbreak control strategies. RESULTS: A total of 38/157 wastewater samples (24.2%) were positive for HAV, 16 collected in 2012 and 22 in 2013. Several HAV strains were detected, including the IA variant implicated in the outbreak and isolated from clinical cases over the same period. The vast majority of sequences belonged to genotype IB. Interestingly however, although these included variants related to strains that had been involved in past Italian epidemics, none were detected in recent clinical samples, probably due to underreporting or asymptomatic circulation. Conversely, a number of sequences were identified in clinical samples that were not found in wastewaters. CONCLUSIONS: The percentage of sewage samples detected as HAV-positive in this study are consistent with the classification of Italy as a country with low/intermediate endemicity. A combined environmental/clinical surveillance is able to provide a more complete picture of the spread of HAV and of the genotypes circulating in the population, allowing a better understanding of changes in disease trends.

Characterisation of microbial communities and quantification of antibiotic resistance genes in Italian wastewater treatment plants using 16S rRNA sequencing and digital PCR.

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in humans, animals and environment is a growing threat to public health. Wastewater treatment plants (WWTPs) are crucial in mitigating the risk of environmental contamination by effectively removing contaminants before discharge. However, the persistence of ARB and ARGs even after treatment is a challenge for the management of water system. To comprehensively assess antimicrobial resistance dynamics, we conducted a one-year monitoring study in three WWTPs in central Italy, both influents and effluents. We used seasonal sampling to analyze microbial communities by 16S rRNA, as well as to determine the prevalence and behaviour of major ARGs (sul1, tetA, blaTEM, blaOXA-48, blaCTX-M-1 group, blaKPC) and the class 1 Integron (int1). Predominant genera included in order: Arcobacter, Acinetobacter, Flavobacterium, Pseudarcobacter, Bacteroides, Aeromonas, Trichococcus, Cloacibacterium, Pseudomonas and Streptococcus. A higher diversity of bacterial communities was observed in the effluents compared to the influents. Within these communities, we also identified bacteria that may be associated with antibiotic resistance and pose a significant threat to human health. The mean concentrations (in gene copies per liter, gc/L) of ARGs and int1 in untreated wastewater (absolute abundance) were as follows: sul1 (4.1 × 109), tetA (5.2 × 108), blaTEM (1.1 × 108), blaOXA-48 (2.1 × 107), blaCTX-M-1 group (1.1 × 107), blaKPC (9.4 × 105), and int1 (5.5 × 109). The mean values in treated effluents showed reductions ranging from one to three log. However, after normalizing to the 16S rRNA gene (relative abundance), it was observed that in 37.5 % (42/112) of measurements, the relative abundance of ARGs increased in effluents compared to influents. Furthermore, correlations were identified between ARGs and bacterial genera including priority pathogens. This study improves our understanding of the dynamics of ARGs and provides insights to develop more effective strategies to reduce their spread, protecting public health and preserving the future efficacy of antibiotics.

Wastewater-Based Epidemiology as a Tool to Detect SARS-CoV-2 Circulation at the Community Level: Findings from a One-Year Wastewater Investigation Conducted in Sicily, Italy.

Wastewater-based epidemiology is a well-established tool for detecting and monitoring the spread of enteric pathogens and the use of illegal drugs in communities in real time. Since only a few studies in Italy have investigated the correlation between SARS-CoV-2 in wastewater and the prevalence of COVID-19 cases from clinical testing, we conducted a one-year wastewater surveillance study in Sicily to correlate the load of SARS-CoV-2 RNA in wastewater and the reported cumulative prevalence of COVID-19 in 14 cities from October 2021 to September 2022. Furthermore, we investigated the role of SARS-CoV-2 variants and subvariants in the increase in the number of SARS-CoV-2 infections. Our findings showed a significant correlation between SARS-CoV-2 RNA load in wastewater and the number of active cases reported by syndromic surveillance in the population. Moreover, the correlation between SARS-CoV-2 in wastewater and the active cases remained high when a lag of 7 or 14 days was considered. Finally, we attributed the epidemic waves observed to the rapid emergence of the Omicron variant and the BA.4 and BA.5 subvariants. We confirmed the effectiveness of wastewater monitoring as a powerful epidemiological proxy for viral variant spread and an efficient complementary method for surveillance.

Exploring the Astrovirome of Shellfish Matrices Using Nanopore Sequencing.

Astroviruses are important human enteric pathogens transmissible with contaminated food and water. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates. The genetic diversity of human and animal astroviruses poses a challenge for diagnostics and taxonomy. As a proof of concept, we used a panastrovirus consensus primer set, able to amplify in a nested RT-PCR protocol a 400-nt-long fragment of the RNA-dependent RNA polymerase of most members of the Astroviridae family, in conjunction with a nanopore sequencing platform, to generate information on the astrovirome in filter-feeding mollusks. Amplicons generated from bivalve samples were used to generate libraries for deep sequencing. In three samples, only one unique RdRp sequence type was obtained. However, in seven samples and in three barcodes with eleven pooled samples, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. In total, 37 different sequence contigs were generated. Avian-origin astrovirus sequences were predominant, likely due to contamination of shellfish harvesting waters by marine birds. Astroviruses of the aquatic eco-system were also identified, whereas human astroviruses were not detected.

Wastewater-based Epidemiology and SARS-CoV-2: Variant Trends in the Apulia Region (Southern Italy) and Effect of Some Environmental Parameters.

During the COVID-19 pandemic, wastewater monitoring has been used to monitor the levels of SARS-CoV-2 RNA entering the sewerage system. In Italy, the Istituto Superiore di Sanità coordinated the SARI project (Sorveglianza Ambientale Reflue in Italia) to detect SARS-CoV-2 and its variants. In this study, the concentration of SARS-CoV-2 and its variants in raw wastewater against COVID-19 cases was evaluated together with the effect of temperature and precipitation on virus spread. We validated a predictive model, proposed by De Giglio et al., 2021, to establish the number of COVID-19 cases/100,000 inhabitants. A receiver operating characteristic curve model was applied to predict the number of COVID-19 cases and Poisson regression was applied to study the effect of temperature and rainfall on viral load. In Apulia, from October 2021 to December 2022, we analyzed 1041 samples, of which 985 (94.6%) tested positive for SARS-CoV-2. Median atmospheric temperature was inversely proportional to viral load in wastewater; no correlation was found with precipitation. The predictive model confirmed that at least 11 cases/100,000 inhabitants would occur in the 15 days following the detection of the virus in wastewater. Environmental surveillance of SARS-CoV-2 can be used to map the virus and its variants.

Surveillance for rat hepatitis E in wastewater networks, Italy.

IMPORTANCE: Hepatitis E virus (HEV) infection constitutes a significant health problem worldwide. In recent years, in addition to the zoonotic HEV3 and HEV4, emerging highly divergent hepevirus of rat origin (rat HEV [RHEV]) has been associated with human acute and chronic hepatitis. As environmental surveillance can be a complementary tool to explore emerging viruses of human and rodent origin, we investigated the epidemiology and the genetic variability of RHEV targeting 14 wastewater treatment plants in an Italian geographic area considered a hot spot for HEV infection in humans. Our results revealed that RHEV is a significant component of the wastewater microbiota with viral RNA detected in 43.9% of the specimens tested, adding further evidence to the need to investigate more in depth the real burden of RHEV infections in humans.

SARS-CoV-2 RNA in Wastewater and Bivalve Mollusk Samples of Campania, Southern Italy.

SARS-CoV-2 can be detected in the feces of infected people, consequently in wastewater, and in bivalve mollusks, that are able to accumulate viruses due to their ability to filter large amounts of water. This study aimed to monitor SARS-CoV-2 RNA presence in 168 raw wastewater samples collected from six wastewater treatment plants (WWTPs) and 57 mollusk samples obtained from eight harvesting sites in Campania, Italy. The monitoring period spanned from October 2021 to April 2022, and the results were compared and correlated with the epidemiological situation. In sewage, the ORF1b region of SARS-CoV-2 was detected using RT-qPCR, while in mollusks, three targets-RdRp, ORF1b, and E-were identified via RT-dPCR. Results showed a 92.3% rate of positive wastewater samples with increased genomic copies (g.c.)/(day*inhabitant) in December-January and March-April 2022. In the entire observation period, 54.4% of mollusks tested positive for at least one SARS-CoV-2 target, and the rate of positive samples showed a trend similar to that of the wastewater samples. The lower SARS-CoV-2 positivity rate in bivalve mollusks compared to sewages is a direct consequence of the seawater dilution effect. Our data confirm that both sample types can be used as sentinels to detect SARS-CoV-2 in the environment and suggest their potential use in obtaining complementary information on SARS-CoV-2.

Characterization of SARS-CoV-2 Variants in Military and Civilian Personnel of an Air Force Airport during Three Pandemic Waves in Italy.

We investigated SARS-CoV-2 variants circulating, from November 2020 to March 2022, among military and civilian personnel at an Air Force airport in Italy in order to classify viral isolates in a potential hotspot for virus spread. Positive samples were subjected to Next-Generation Sequencing (NGS) of the whole viral genome and Sanger sequencing of the spike coding region. Phylogenetic analysis classified viral isolates and traced their evolutionary relationships. Clusters were identified using 70% cut-off. Sequencing methods yielded comparable results in terms of variant classification. In 2020 and 2021, we identified several variants, including B.1.258 (4/67), B.1.177 (9/67), Alpha (B.1.1.7, 9/67), Gamma (P.1.1, 4/67), and Delta (4/67). In 2022, only Omicron and its sub-lineage variants were observed (37/67). SARS-CoV-2 isolates were screened to detect naturally occurring resistance in genomic regions, the target of new therapies, comparing them to the Wuhan Hu-1 reference strain. Interestingly, 2/30 non-Omicron isolates carried the G15S 3CLpro substitution responsible for reduced susceptibility to protease inhibitors. On the other hand, Omicron isolates carried unusual substitutions A1803V, D1809N, and A949T on PLpro, and the D216N on 3CLpro. Finally, the P323L substitution on RdRp coding regions was not associated with the mutational pattern related to polymerase inhibitor resistance. This study highlights the importance of continuous genomic surveillance to monitor SARS-CoV-2 evolution in the general population, as well as in restricted communities.

Occurrence of Hepatitis A Virus in Water Matrices: A Systematic Review and Meta-Analysis.

Hepatitis A is a common form of viral hepatitis. It is usually transmitted through the ingestion of contaminated food and water. This systematic review was carried out to summarise the overall prevalence of Hepatitis A virus (HAV) in different water matrices: untreated and treated wastewater, surface water, groundwater, drinking water, and others (e.g., irrigation water and floodwater). The literature search was performed in four databases: PubMed, Web of Science, Global Index Medicus, and Excerpta Medica Database. Heterogeneity (I2) was assessed using the χ2 test on the Cochran Q statistic and H parameters. A total of 200 prevalence data from 144 articles were included in this meta-analysis. The overall prevalence of HAV in water matrices was 16.7% (95% CI: 13.4−20.3). The prevalence for individual matrix was as follows: 31.4% (95% CI: 23.0−40.4) untreated wastewater, 18.0% (95% CI: 9.5−28.2) treated wastewater, 15.0% (95% CI: 10.1−20.5) surface water, 2.3% (95% CI: 0.1−6.0) in groundwater, 0.3% (95% CI: 0.0−1.7) in drinking water, and 8.5% (95% CI: 3.1−15.6) in other matrices. The prevalence was higher in low-income economies (29.0%). Africa and Eastern Mediterranean were the regions with higher HAV prevalence values. This study showed a high heterogeneity (I2 > 75%) with a significant publication bias (p value Egger test < 0.001). The results of this review suggest that water matrices could be an important route of HAV transmission even in industrialized countries, despite the lower prevalence compared to less industrialized countries, and the availability of advanced water management systems. More effective water/wastewater treatment strategies are needed in developing countries to limit the environmental circulation of HAV.

Assessing the Prevalence of Astroviruses in Water Environments: A Systematic Review and Meta-analysis.

Astroviruses (AstVs) are a major cause of gastroenteritis, especially in children. They can be transmitted through various pathways, including environmental contamination via water matrices. This study aimed to investigate the prevalence of AstV in different types of water, such as untreated and treated wastewater, surface water (e.g., rivers, lakes, and seawater), groundwater, drinking water, and other water matrices (e.g., irrigation water, gray water, reservoir water, floodwater, and pig slaughterhouse effluents). The meta-analysis included 80 articles, and the overall prevalence of AstV in water matrices was 36.6% [95% confidence interval (CI) of 29.6-44.0]. The highest prevalence was found in untreated wastewater at 56.8% (95% CI of 41.5-71.5), followed by treated wastewater at 48.5% (95% CI of 30.6-66.5), surface water at 28.6% (95% CI of 21.1-36.7), other matrices at 9.8% (95% CI of 0.7-25.3), drinking water at 3.3% (95% CI of 0.2-8.7), and groundwater at 0.5% (95% CI of 0.0-3.4). The most frequent AstVs detected in water environments were human AstVs, but canine and feline AstVs were also detected. Our findings highlight the importance of water as a potential route for AstV transmission, even in high-income countries. Effective water surveillance and treatment measures are necessary to minimize AstV environmental circulation and human infection through water.