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Water treatment and reuse is gaining acceptance as a strategy to fight against water contamination and scarcity, but it usually requires complex treatments to ensure safety. Consequently, the electrochemical advanced processes have emerged as an effective alternative for water remediation. The main objective here is to perform a systematic study that quantifies the efficiency of a laboratory-scale electrochemical system to inactivate bacteria, bacterial spores, protozoa, bacteriophages and viruses in synthetic water, as well as in urban wastewater once treated in a wetland for reuse in irrigation. A Ti|RuO

The COVID-19 pandemic placed hygiene at the centre of disease prevention. Yet, access to the levels of water supply that support good hand hygiene and institutional cleaning, our understanding of hygiene behaviours, and access to soap are deficient in low-, middle- and high-income countries. This paper reviews the role of water, sanitation and hygiene (WaSH) in disease emergence, previous outbreaks, combatting COVID-19 and in preparing for future pandemics. We consider settings where these factors are particularly important and identify key preventive contributions to disease control and gaps in the evidence base. Urgent substantial action is required to remedy deficiencies in WaSH, particularly the provision of reliable, continuous piped water on-premises for all households and settings. Hygiene promotion programmes, underpinned by behavioural science, must be adapted to high-risk populations (such as the elderly and marginalised) and settings (such as healthcare facilities, transport hubs and workplaces). WaSH must be better integrated into preparation plans and with other sectors in prevention efforts. More finance and better use of financing instruments would extend and improve WaSH services. The lessons outlined justify no-regrets investment by government in response to and recovery from the current pandemic; to improve day-to-day lives and as preparedness for future pandemics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel member of the Coronaviridae family, has been identified as the etiologic agent of an ongoing pandemic of severe pneumonia known as COVID-19.(1) To date there have been millions of cases of COVID-19 diagnosed in 184 countries with case fatality rates ranging from 1.8% in Germany to 12.5% in Italy.(2) Limited diagnostic testing capacity and asymptomatic and oligosymptomatic infections result in significant uncertainty in the estimated extent of SARS-CoV-2 infection.(3) Recent reports have documented that infection with SARS-CoV-2 is accompanied by persistent shedding of virus RNA in feces in 27%(4) to 89% of patients at densities from 0.8 to 7.5 log10 gene copies per gram.(5) The presence of SARS-CoV-2 RNA in feces raises the potential to survey sewage for virus RNA to inform epidemiological monitoring of COVID-19, which we refer to as wastewater-based epidemiology (WBE),(6) but is also known as environmental surveillance.(7) Several studies have reported the detection of SARS-CoV-2 RNA in wastewater in the early stages of local outbreaks, further supporting the technical viability of WBE.(8−10) WBE could be especially informative given that asymptomatic and oligosymptomatic infections are unlikely to be detected during clinical surveillance. In such instances, WBE can be used to determine the burden of undiagnosed infections at the population level, which is critical to refining estimates of case-fatality rates. Additionally, wastewater offers an aggregate sample from an entire community that is more easily accessible than pooled clinical samples.(11) Along with clinical data and other technological approaches, such as contact tracing, WBE could provide critical monitoring of SARS-CoV-2 transmission within a community including the beginning, tapering, or re-emergence of an epidemic (Figure 1). This approach mirrors previous efforts in environmental monitoring, for example poliovirus RNA, to inform mechanistic models of pathogen transmission dynamics.(12)

Wastewater based epidemiology was employed to track the spread of SARS-CoV-2 within the sewershed areas of 10 wastewater treatment plants (WWTPs) in Catalonia, Spain. A total of 185 WWTPs inflow samples were collected over the period consisting of both the first wave (mid-March to June) and the second wave (July to November). Concentrations of SARS-CoV-2 RNA (N1 and N2 assays) were quantified in these wastewaters as well as those of Human adenoviruses (HAdV) and JC polyomavirus (JCPyV), as indicators of human faecal contamination. SARS-CoV-2 N gene daily loads strongly correlated with the number of cases diagnosed one week after sampling i.e. wastewater levels were a good predictor of cases to be diagnosed in the immediate future. The conditions present at small WWTPs relative to larger WWTPs influence the ability to follow the pandemic. Small WWTPs (, This work was partially funded by the projects PCI2019-103643, RTI2018-097346-B-I00, and AGL2017-86797-C2-1-R projects, all funded by the Spanish Ministry of Science, Innovation and Universities. S. Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona. This study is performed with partial support from the Water Research Institute of the University of Barcelona. IDAEA-CSIC is a Centre of Excellence Severo Ochoa (Spanish Ministry of Science and Innovation, Project CEX2018-000794-S). ICRA authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124). ICRA is part of the CERCA program. We would like to give a big thanks to our families and colleagues, for their support and help behind the scenes.

Quantitative measurements of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in raw wastewater have been implemented worldwide since the beginning of the pandemic. Recent efforts are being made to evaluate different viral concentration methodologies to overcome supplier shortages during lockdowns. A set of 22-wastewater samples seeded with murine hepatitis virus (MHV), a member of the Coronaviridae family, and the bacteriophage MS2, were used to characterize and compare two ultrafiltration-based methods: a centrifugal ultrafiltration device (Centricon® Plus-70) and the automated concentrating pipette CP-Select™. Based on the recovery efficiencies, significant differences were observed for MHV, with Centricon® Plus-70 (24%) being the most efficient method. Nevertheless, concentrations of naturally occurring SARS-CoV-2, Human adenoviruses and JC polyomaviruses in these samples did not result in significant differences between methods suggesting that testing naturally occurring viruses may complement the evaluation of viral concentration methodologies. Based on the virus adsorption to solids and the necessity of a pre-centrifugation step to remove larger particles and avoid clogging when using ultrafiltration methods, we assessed the percentage of viruses not quantified after ultrafiltration. Around 23% of the detected SARS-CoV-2 would be discarded during the debris removal step. The CP-Select™ provided the highest concentration factor (up to 333×) and the lowest LoD (6.19 × 103 GC/l) for MHV and proved to be fast, automatic, highly reproducible and suitable to work under BSL-2 measures., Graphical abstract Unlabelled Image, Highlights • Centricon® and CP-Select™ performed equally for naturally occurring SARS-CoV-2 • Higher MHV recoveries were calculated using centrifugal ultrafiltration devices. • Naturally occurring viruses complement concentration methods comparison. • A 23% of detected SARS-CoV-2 adsorb to the solid fraction of wastewater. • CP-Select™ fits into a BSL-2 cabinet enabling to work under biosafety containment.

Acute infectious gastroenteritis is an important illness worldwide, especially on children, with viruses accounting for approximately 70% of the acute cases. A high number of these cases have an unknown etiological agent and the rise of next generation sequencing technologies has opened new opportunities for viral pathogen detection and discovery. Viral metagenomics in routine clinical settings has the potential to identify unexpected or novel variants of viral pathogens that cause gastroenteritis. In this study, 124 samples from acute gastroenteritis patients from 2012&ndash, 2014 previously tested negative for common gastroenteritis pathogens were pooled by age and analyzed by next generation sequencing (NGS) to elucidate unidentified viral infections. The most abundant sequences detected potentially associated to acute gastroenteritis were from Astroviridae and Caliciviridae families, with the detection of norovirus GIV and sapoviruses. Lower number of contigs associated to rotaviruses were detected. As expected, other viruses that may be associated to gastroenteritis but also produce persistent infections in the gut were identified including several Picornaviridae members (EV, parechoviruses, cardioviruses) and adenoviruses. According to the sequencing data, astroviruses, sapoviruses and NoV GIV should be added to the list of viral pathogens screened in routine clinical analysis.

Since the novel SARS-CoV-2 was detected in faeces, environmental researchers have been using centrifugal ultrafiltration, polyethylene glycol precipitation and aluminium hydroxide flocculation to describe its presence in wastewater samples. High recoveries (up to 65%) are described with electronegative filtration when using surrogate viruses, but few literature reports recovery efficiencies using accurate quantification of enveloped viruses. Considering that every single virus will have a different behaviour during viral concentration, it is recommended to use an enveloped virus, and if possible, a betacoronaviruses as murine hepatitis virus (MHV), as a surrogate. In this review we show new data from a new available technology that provides a quick ultrafiltration protocol for SARS-CoV-2. Wastewater surveillance is an efficient system for the evaluation of the relative prevalence of SARS-CoV-2 infections in a community, and there is the need of using reliable concentration methods for an accurate and sensitive quantification of the virus in water., Graphical abstract Image 1, Highlights • There are efficient methods to concentrate enveloped virus such as coronaviruses. • More data on the recovery of the specific pathogen of interest is needed. • Viral surrogates, ideally betacoronavirus, may be used as SARS-CoV-2 process control. • Recovery calculation needs an accurate quantification of the viral stock.

EFSA is requested to assess the safety of a broad range of biological agents in the context of notification for market authorisation as sources of food and feed additives, food enzymes and plant protection products. The qualified presumption of safety (QPS) assessment was developed to provide a harmonised generic pre‐assessment to support safety risk assessments performed by EFSA's scientific Panels. The safety of unambiguously defined biological agents (at the highest taxonomic unit appropriate for the purpose for which an application is intended), and the completeness of the body of knowledge are assessed. Identified safety concerns for a taxonomic unit are, where possible and reasonable in number, reflected as ‘qualifications’ in connection with a recommendation for a QPS status. The list of QPS recommended biological agents was reviewed and updated in the current opinion and therefore becomes the valid list. The 2016 update reviews previously assessed microorganisms including bacteria, yeasts and viruses used for plant protection purposes following an Extensive Literature Search strategy. The taxonomic units related to the new notifications received since the 2013 QPS opinion, were periodically evaluated for a QPS status and the results published as Statements of the BIOHAZ Panel. Carnobacterium divergens, Lactobacillus diolivorans, Microbacterium imperiale, Pasteuria nishizawae, Pediococcus parvulus, Bacillus flexus, Bacillus smithii, Xanthomonas campestris and Candida cylindracea were recommended for the QPS list. All taxonomic units previously recommended for the 2013 QPS list had their status reconfirmed as well their qualifications with the exception of Pasteuria nishizawae for which the qualification was removed. The exclusion of filamentous fungi and enterococci from the QPS evaluations was reconsidered but monitoring will be maintained and the status will be re‐evaluated in the next QPS Opinion update. Evaluation of bacteriophages should remain as a case‐by‐case procedure and should not be considered for QPS status., This publication is linked to the following EFSA Journal article: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2017.4663/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2017.e15031/full

According to the European Union, half of the human population will live in cities, one of the main water spend zones and focus of pollutants it is expected by 2050. Considering the growing pressure on water resources whole world, a better knowledge for its management is needed to face this situation.URBANWAT is a project funded by the EU Commission under the call “Closing the Water Cycle Gap” of the Water JPI Strategic Research and Innovation Agenda. The goal of this project is to come up with an improvement of tools and criteria for groundwater management in urban areas to guarantee the urban water resources sustainability, identify their potential uses and their risks related to groundwater use from both environmental and human health perspectives by an integral approach developing novel technologies and methodologies.The project will involve a multidisciplinary approach integrating the research of the natural state of the hydrological cycle and pollutants identification (general chemistry, pollutants of emerging concern (CECs) and microorganisms, with emphasis in viruses).To achieve that goal, URBANWAT proposes to use innovative approaches based on liquid chromatography high resolution mass spectrometry (HLC-RMS) to: (1) detect differences in degradation in different anoxic conditions employing CECs as indicators of contamination and their transformation products (TPs) as indicators of degradability; (i2) analyse fate and transport of chosen contaminants in the soil-plant as a remediation system utilizing picked infrastructures; (3) understand the contaminants movement applying encapsulated DNA nanoparticles; (4) scout the presence of new and emergent viruses in groundwater samples employing viral metagenomics. Viral concentration methods from water samples will be also optimized in this research. To do so, column experiments will be carried out.URBANWAT project will focus on Barcelona city. In parallel, several studies will be carried out at full-scale in the demonstration facility called ‘WaterStreet’ at TUDelft. The expected results will help to provide novel and cost-efficient technologies for urban groundwater management with beneficial environmental, economic and societal impacts for the European Union (EU) facilitating their application worldwide.

Rotavirus A (RVA) is the most common virus associated with infantile gastroenteritisworldwide, being a public health threat, as it is excreted in large amounts in stool and can persist inthe environment for extended periods. In this study, we performed the detection of RVA and humanadenovirus (HAdV) by TaqMan qPCR and assessed the circulation of RVA genotypes in threewastewater treatment plants (WWTPs) between 2015 and 2016 in Catalonia, Spain. RVA wasdetected in 90% and HAdV in 100% of the WWTP samples, with viral loads ranging between 3.96 &times, 104 and 3.30 &times, 108 RT‐PCR Units/L and 9.51 &times, 104 and 1.16 &times, 106 genomic copies/L, respectively. RVAVP7 and VP4 gene analysis revealed the circulation of G2, G3, G9, G12, P[4], P[8], P[9] and P[10].Nucleotide sequencing (VP6 fragment) showed the circulation of I1 and I2 genotypes, commonlyassociated with human, bovine and porcine strains. It is important to mention that the RVA strainsisolated from the WWTPs were different from those recovered from piglets and calves living in thesame area of single sampling in 2016. These data highlight the importance of monitoring watermatrices for RVA epidemiology and may be a useful tool to evaluate and predict possibleemergence/reemergence of uncommon strains in a region.

In Quito, the microbiological contamination of surface water represents a public health problem, mainly due to the lack of sewage treatment from urban wastewater. Contaminated water contributes to the transmission of many enteric pathogens through direct consumption, agricultural and recreational use. Among the different pathogens present in urban discharges, viruses play an important role on disease, being causes of gastroenteritis, hepatitis, meningitis, respiratory infections, among others. This study analyzes the presence of viruses in highly impacted surface waters of urban rivers using next-generation sequencing techniques. Three representative locations of urban rivers, receiving the main discharges from Quito sewerage system, were selected. Water samples of 500 mL were concentrated by skimmed-milk flocculation method and the viral nucleic acid was extracted and processed for high throughput sequencing using Illumina MiSeq. The results yielded very relevant data of circulating viruses in the capital of Ecuador. A total of 29 viral families were obtained, of which 26 species were associated with infections in humans. Among the 26 species identified, several were related to gastroenteritis: Human Mastadenovirus F, Bufavirus, Sapporovirus, Norwalk virus and Mamastrovirus 1. Also detected were: Gammapapillomavirus associated with skin infections, Polyomavirus 1 related to cases of kidney damage, Parechovirus A described as cause of neonatal sepsis with neurological affectations and Hepatovirus A, the etiologic agent of Hepatitis A. Other emergent viruses identified, of which its pathogenicity remains to be fully clarified, were: Bocavirus, Circovirus, Aichi Virus and Cosavirus. The wide diversity of species detected through metagenomics gives us key information about the public health risks present in the urban rivers of Quito. In addition, this study describes for the first time the presence of important infectious agents not previously reported in Ecuador and with very little reports in Latin America.

The application of next-generation sequencing (NGS) techniques for the identification of viruses present in urban sewage has not been fully explored. This is partially due to a lack of reliable and sensitive protocols for studying viral diversity and to the highly complex analysis required for NGS data processing. One important step towards this goal is finding methods that can efficiently concentrate viruses from sewage samples. Here the application of a virus concentration method based on skimmed milk organic flocculation (SMF) using 10 L of sewage collected in different seasons enabled the detection of many viruses. However, some viruses, such as human adenoviruses, could not always be detected using metagenomics, even when quantitative PCR (qPCR) assessments were positive. A targeted metagenomic assay for adenoviruses was conducted and 59.41% of the obtained reads were assigned to murine adenoviruses. However, up to 20 different human adenoviruses (HAdV) were detected by this targeted assay being the most abundant HAdV-41 (29.24%) and HAdV-51 (1.63%). To improve metagenomics' sensitivity, two different protocols for virus concentration were comparatively analysed: an ultracentrifugation protocol and a lower-volume SMF protocol. The sewage virome contained 41 viral families, including pathogenic viral species from families Caliciviridae, Adenoviridae, Astroviridae, Picornaviridae, Polyomaviridae, Papillomaviridae and Hepeviridae. The contribution of urine to sewage metavirome seems to be restricted to a few specific DNA viral families, including the polyomavirus and papillomavirus species. In experimental infections with sewage in a rhesus macaque model, infective human hepatitis E and JC polyomavirus were identified. Urban raw sewage consists of the excreta of thousands of inhabitants; therefore, it is a representative sample for epidemiological surveillance purposes. The knowledge of the metavirome is of significance to public health, highlighting the presence of viral strains that are circulating within a population while acting as a complex matrix for viral discovery. (c) 2017 Elsevier B.V. All rights reserved.

SARS-CoV-2 variants are emerging worldwide, and monitoring them is key in providing early warnings. Here, we summarize the different analytical approaches currently used to study the dissemination of SARS-CoV-2 variants in wastewater and discuss their advantages and disadvantages. We also provide preliminary results of two sensitive and cost-effective approaches: variant-specific reverse transcription-nested PCR assays and a nonvariant-specific amplicon deep sequencing strategy that targets three key regions of the viral spike protein. Next-generation sequencing approaches enable the simultaneous detection of signature mutations of different variants of concern in a single assay and may be the best option to explore the real picture at a particular time. Targeted PCR approaches focused on specific signature mutations will need continuous updating but are sensitive and cost-effective., This study was partially supported by the Catalan Government through 2020PANDE00044, and CDTI project PROCEED, EXP - 00132149/COI-20201289. Sílvia Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona. Eva Forés is an APIF fellow at the University of Barcelona. Marta Itarte is a fellow of the Catalan Government ‘AGAUR’ (FI) at the University of Barcelona. We also gratefully acknowledge the authors, originating and submitting laboratories of the SARS-CoV-2 sequences from GISAID’s EpiCov Database (https://www.epicov.org/epi3/) used in the phylogenetic analyses; access to the individual isolates is facilitated through the GISAID website (https://www.gisaid.org/). Graphical Abstract was created with Biorender.com.

Special issue Next-Generation Sequencing and Emerging Technologies for the Identification and Control of Microbial Contaminants in the Food Chain.-- 21 pages, 2 figures, 5 tables, supplementary materials https://www.mdpi.com/article/10.3390/foods10081820/s1.-- Data Availability Statement: The datasets generated during the current study are available in Zenodo under the DOI number https://doi.org/10.5281/zenodo.4722797, Fresh fruits and vegetables are susceptible to microbial contamination at every stage of the food production chain, and as a potential source of pathogens, irrigation water quality is a critical factor. Next-generation sequencing (NGS) techniques have been flourishing and expanding to a wide variety of fields. However, their application in food safety remains insufficiently explored, and their sensitivity requires improvement. In this study, quantitative polymerase chain reaction (qPCR) assays showed low but frequent contamination of common circulating viral pathogens, which were found in 46.9% of samples of fresh produce: 6/12 lettuce samples, 4/12 strawberries samples, and 5/8 parsley samples. Furthermore, the application of two different NGS approaches, target enrichment sequencing (TES) for detecting viruses that infect vertebrates and amplicon deep sequencing (ADS), revealed a high diversity of viral pathogens, especially Norovirus (NoV) and Human Papillomavirus (HPV), in fresh produce and irrigation water. All NoV and HPV types found in fresh fruit and vegetable samples were also detected in irrigation water sources, indicating that these viruses are common circulating pathogens in the population and that irrigation water may be the most probable source of viral pathogens in food samples, This work was partially funded by a grant from the Spanish Ministry of Economy and Competitiveness (MINECO) AGL2017-86797-C2-1-R. IDEA-CSIC is a Centre of Excellence Severo Ochoa (Spanish Ministry of Science and Innovation (MICINN), Project CEX2018-000794-S), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

In the wake of the COVID-19 pandemic, the use of next generation sequencing (NGS) has proved to be an important tool for the genetic characterization of SARS-CoV-2 from clinical samples. The use of different available NGS tools applied to wastewater samples could be the key for an in-depth study of the excreted virome, not only focusing on SARS-CoV-2 circulation and typing, but also to detect other potentially pandemic viruses within the same family. With this aim, 24-hours composite wastewater samples from March and July 2020 were sequenced by applying specific viral NGS as well as target enrichment NGS. The full virome of the analyzed samples was obtained, with human Coronaviridae members (CoV) present in one of those samples after applying the enrichment. One contig was identified as HCoV-OC43 and 8 contigs as SARS-CoV-2. CoVs from other animal hosts were also detected when applying this technique. These contigs were compared with those obtained from contemporary clinical specimens by applying the same target enrichment approach. The results showed that there is a co-circulation in urban areas of human and animal coronaviruses infecting domestic animals and rodents. NGS enrichment-based protocols might be crucial to describe the occurrence and genetic characteristics of SARS-CoV-2 and other Coronaviridae family members within the excreted virome present in wastewater., This study was partially supported by the Ministry of Science, Innovation and Universities (AGL2017-86797-C2-1-R) through the University of Barcelona and the Direcció General de Recerca i Innovació en Salut (DGRIS) Catalan Health Ministry Generalitat de Catalunya through Vall d'Hebron Research Institute (VHIR). Sílvia Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona.

[EN] In this study, the use of skimmed milk flocculation (SMF) to simultaneously concentrate viruses, bacteria and protozoa was evaluated. We selected strains of faecal indicator bacteria and pathogens, such as Escherichia coli and Helicobacter pylori. The viruses selected were adenovirus (HAdV 35), rotavirus (RoV SA-11), the bacteriophage MS2 and bovine viral diarrhoea virus (BVDV). The protozoa tested were Acanthamoeba, Giardia and cryptosporidium. The mean recoveries with q(RT)PCR were 66% (HAdV 35), 24% (MS2), 28% (RoV SA-11), 15% (BVDV), 60% (E. coli), 30% (H. pylori) and 21% (Acanthamoeba castellanii). When testing the infectivity, the mean recoveries were 59% (HAdV 35), 12% (MS2), 26% (RoV SA-11) and 0.7% (BVDV). The protozoa Giardia lamblia and Cryptosporidium parvum were studied by immunofluorescence with recoveries of 18% and 13%, respectively. Although q(RT)PCR consistently showed higher quantification values (as expected), q(RT)PCR and the infectivity assays showed similar recoveries for HAdV 35 and RoV SA-11. Additionally, we investigated modelling the variability and uncertainty of the recovery with this method to extrapolate the quantification obtained by q(RT)PCR and estimate the real concentration. The 95% prediction intervals of the real concentration of the microorganisms inoculated were calculated using a general non-parametric bootstrap procedure adapted in our context to estimate the technical error of the measurements. SMF shows recoveries with a low variability that permits the use of a mathematical approximation to predict the concentration of the pathogen and indicator with acceptable low intervals. The values of uncertainty may be used for a quantitative microbial risk analysis or diagnostic purposes. (C) 2017 The Author(s). Published by Elsevier B.V., This study was funded by the Water Challenges for a Changing World Joint Programming Initiative (W2013-095-C03-01), the Spanish Ministry of Economy and Competitiveness (MINECO - AGL2014-55081-R) and the Grup de Recerca Consolidat: Virus, bacteris i protozous d'interes en salut publica, aigua y aliments (Generalitat de Catalunya, Virbap - 2014-SGR-914). Eloy Gonzales-Gustavson would like to acknowledge the Presidente de la República scholarship from the Peruvian Government, which supported a PhD grant.

[EN] Viruses (e.g., noroviruses and hepatitis A and E virus), bacteria (e.g., Salmonella spp. and pathogenic Escherichia coli) and protozoa (e.g., Cryptosporidium parvum and Giardia intestinalis) are well-known contributors to food-borne illnesses linked to contaminated fresh produce. As agricultural irrigation increases the total amount of water used annually, reclaimed water is a good alternative to reduce dependency on conventional irrigation water sources. European guidelines have established acceptable concentrations of certain pathogens and/or indicators in irrigation water, depending on the irrigation system used and the irrigated crop. However, the incidences of food-borne infections are known to be underestimated and all the different pathogens contributing to these infections are not known. Next-generation sequencing (NGS) enables the determination of the viral, bacterial and protozoan populations present in a water sample, providing an opportunity to detect emerging pathogens and develop improved tools for monitoring the quality of irrigation water. This is a descriptive study of the virome, bacteriome and parasitome present in different irrigation water sources. We applied the same concentration method for all the studied samples and specific metagenomic approaches to characterize both DNA and RNA viruses, bacteria and protozoa. In general, most of the known viral species corresponded to plant viruses and bacteriophages. Viral diversity in river water varied over the year, with higher bacteriophage prevalences during the autumn and winter. Reservoir water contained Enterobacter cloacae, an opportunistic human pathogen and an indicator of fecal contamination, as well as Naegleria australiensis and Naegleria clarki. Hepatitis E virus and Naegleria fowleri, emerging human pathogens, were detected in groundwater. Reclaimed water produced in a constructed wetland system presented a virome and bacteriome that resembled those of freshwater samples (river and reservoir water). Viral, bacterial and protozoan pathogens were occasionally detected in the different irrigation water sources included in this study, justifying the use of improved NGS techniques to get a comprehensive evaluation of microbial species and potential environmental health hazards associated to irrigation water., This work was supported through a grant funded by the Spanish Ministry of Economy and Competitiveness (MINECO) in the frame of the collaborative international consortium JPIW2013-095-C03-01, JPIW2013-095-C03-02 and JPIW2013-095-C03-03 of the Water Challenges for a Changing World Joint Programming Initiative (Water JPI) Pilot Call and partially by AGL2017-86797-C2-1-R. Silvia Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona.

[EN] The wide diversity of irrigation water sources (i.e., drinking water, groundwater, reservoir water, river water) includes reclaimed water as a requested measure for increasing water availability, but it is also a challenge as pathogen exposure may increase. This study evaluates the level of microbial contamination in different irrigation waters to improve the knowledge and analyses management measures for safety irrigation. Over a one-year period, the occurrence of a set of viruses, bacteria and protozoa, was quantified and the performance of a wetland system, producing reclaimed water intended for irrigation, was characterized. Human fecal pollution (HAdV) was found in most of the irrigation water types analysed. Hepatitis E virus (HEV), an emerging zoonotic pathogen, was present in groundwater where porcine contamination was identified (PAdV). The skin-carcinoma associated Merkel cell polyomavirus (MCPyV), was found occasionally in river water. Noroviruses were detected, as expected, in winter, in river water and reclaimed water. Groundwater, river water and reservoir water also harboured potential bacterial pathogens, like Helicobacter pylori, Legionella spp. and Aeromonas spp. that could be internalized and viable inside amoebas like Acanthamoeba castellanii, which was also detected. Neither Giardia cysts, nor any Cryptosporidium oocysts were detected. The wetland system removed 3 Log(10) of viruses and 5 Log(10) of bacteria, which resembled the river water quality. Irrigation waters were prone to variable contamination levels and according to the European guidance documents, the E. coli (EC) levels were not always acceptable. Sporadic detection of viral pathogens as NoV GII and HAdV was identified in water samples presenting lower EC than the established limit (100MNP/100 mL). When dealing with reclaimed water as a source of irrigation the analysis of some viral parameters, like HAdV during the peak irrigation period (summer and spring) or NoV during the coldest months, could complement existing water management tools based on bacterial indicators., Thiswork was partially funded by a grant fromthe Spanish Ministry of Economy and Competitiveness (MINECO) in the frame of the collaborative international consortium JPIW2013-095-C03-01, JPIW2013095-C03-02 and JPIW2013-095-C03-03 of the Water Challenges for a Changing World Joint Programming Initiative (Water JPI) Pilot Call and partially by AGL2017-86797-C2-1-R. Silvia Bofill-Mas is a SerraHunter fellow at the University of Barcelona. Special thanks to E. Fores, E. Anfruns, and X. Romero from Granollers city council, for the valuable information and help during the sampling campaign.

NGS techniques are excellent tools to monitor and identify viral pathogens circulating among the population with some limitations that need to be overcome, especially in complex matrices. Sewage contains a high amount of other microorganisms that could interfere when trying to sequence viruses for which random PCR amplifications are needed before NGS. The selection of appropriate NGS tools is important for reliable identification of viral diversity among the population. We have compared different NGS methodologies (Untargeted Viral Metagenomics, Target Enrichment Sequencing and Amplicon Deep Sequencing) for the detection and characterisation of viruses in urban sewage, focusing on three important human pathogens: papillomaviruses, adenoviruses and enteroviruses. A full picture of excreted viruses was obtained by applying Untargeted Viral Metagenomics, which detected members of four different vertebrate viral families in addition to bacteriophages, plant viruses and viruses infecting other hosts. Target Enrichment Sequencing, using specific vertebrate viral probes, allowed the detection of up to eight families containing human viruses, with high variety of types within the families and with a high genome coverage. By applying Amplicon Deep Sequencing, the diversity of enteroviruses, adenoviruses and papillomaviruses observed was higher than when applying the other two strategies and this technique allowed the subtyping of an enterovirus A71 C1 strain related to a brainstem encephalitis outbreak occurring at the same time in the sampling area. From the data obtained, we concluded that the different strategies studied provided different levels of analysis: TES is the best strategy to obtain a broad picture of human viruses present in complex samples such as sewage. Other NGS strategies are useful for studying the virome of complex samples when also targeting viruses infecting plants, bacteria, invertebrates or fungi (Untargeted Viral Metagenomics) or when observing the variety within a sole viral family is the objective of the study (Amplicon Deep Sequencing).

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent. At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes. The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

The use of lagooning as a complementary natural method of treating secondary effluents of wastewater treatment plants has been employed as an affordable and easy means of producing reclaimed water. However, using reclaimed water for some purposes, for example, for food irrigation, presents some risks if the effluents contain microbial pathogens. Classical bacterial indicators that are used to assess faecal contamination in water do not always properly indicate the presence of bacterial or viral pathogens. In the current study, the presence of faecal indicator bacteria (FIB), heterotrophic bacterial counts (HBC), pathogens and opportunistic pathogens, such as Legionella spp., Aeromonas spp., Arcobacter spp., free-living amoeba (FLA), several viral indicators (human adenovirus and polyomavirus JC) and viral pathogens (noroviruses and hepatitis E virus) were analysed for 1 year in inlet and outlet water to assess the removal efficiency of a lagooning system. We observed 2.58 (1.17-4.59) and 1.65 (0.15-3.14) log reductions in Escherichia coli (EC) and intestinal enterococci (IE), respectively, between the inlet and outlet samples. Genomic copies of the viruses were log reduced by 1.18 (0.24-2.93), 0.64 (0.12-1.97), 0.45 (0.04-2.54) and 0.72 (0.22-2.50) for human adenovirus (HAdV), JC polyomavirus (JCPyV) and human noroviruses (NoV GI and GII), respectively. No regrowth of opportunistic pathogens was observed within the system. FLA, detected in all samples, did not show a clear trend. The reduction of faecal pathogens was irregular with 6 out of 12 samples and 4 out of 12 samples exceeding the EC and IE values, specified in the Spanish legislation for reclaimed water (RD 1620/2007). This data evidences that there is a need for more studies to evaluate the removal mechanisms of lagooning systems in order to optimize pathogen reduction. Moreover, surveillance of water used to irrigate raw edible vegetables should be conducted to ensure the fulfilment of the microbial requirements for the production of safe reclaimed water.

Outbreaks of hepatitis E virus (HEV) are typically associated with contaminated drinking water. A recent HEV outbreak (2012–14) in South Sudan affected ≥10 000 people. This study evaluated different water matrices and food during the last phase of the outbreak by analyzing HEV and human adenovirus (HAdV) as viral indicator of human contamination. The results revealed that the water sources analyzed were negative and that only household samples presented HAdV contamination, identifying a possible route of viral transmission within the population.

Highlights • Wastewater surveillance cheaper and less invasive than massive testing of individuals. • Useful early alert of viral circulation to prevent outbreaks and inform policy decisions. • Success of surveillance requires public legitimacy and trust of such measures., Worldwide, clinical data remain the gold standard for disease surveillance and tracking. However, such data are limited due to factors such as reporting bias and inability to track asymptomatic disease carriers. Disease agents are excreted in the urine and feces of infected individuals regardless of disease symptom severity. Wastewater surveillance – that is, monitoring disease via human effluent – represents a valuable complement to clinical approaches. Because wastewater is relatively inexpensive and easy to collect and can be monitored at different levels of population aggregation as needed, wastewater surveillance can offer a real-time, cost-effective view of a community's health that is independent of biases associated with case-reporting. For SARS-CoV-2 and other disease-causing agents we envision an aggregate wastewater-monitoring system at the level of a wastewater treatment plant and exploratory or confirmatory monitoring of the sewerage system at the neighborhood scale to identify or confirm clusters of infection or assess impact of control measures where transmission has been established. Implementation will require constructing a framework with collaborating government agencies, public or private utilities, and civil society organizations for appropriate use of data collected from wastewater, identification of an appropriate scale of sample collection and aggregation to balance privacy concerns and risk of stigmatization with public health preservation, and consideration of the social implications of wastewater surveillance.

The aim of this study is to investigate the occurrence of faecal indicator and microbial pathogens (bacteria and virus) in the shallow urban aquifer of the Besòs River Delta (NE Spain). To this end, human adenovirus (HAdV) and Norovirus of genogroups I and II (NoV GI and NoV GII) as well as the faecal indicator bacteria (FIB) Escherichia coli (EC) and faecal enterococci (FE) were monitored in groundwater and in the River Besòs in December 2013 and in July 2104. None of the targeted pathogens were detected in groundwater in December 2013 but contamination of human origin was observed in approximately 50% of the points sampled in July 2014 reaching concentrations up to 99 GC/100 mL for HAdV. Generally, microbial concentrations in river water were higher than those detected in groundwater. This observation indicates that pathogens are naturally attenuated when river water infiltrates and flows through the aquifer, however HAdV were detected at a sampling point located at 380 m from the river in the absence of FIB. The presence of human viral contamination may represent a risk for the use of groundwater as a drinking water source. Further research is needed to understand the dynamics of pathogens in river–groundwater interface over long time periods and a wide range of flow conditions (wet and dry periods) since the urban groundwater of this aquifer might be a valuable drinking water resource in Barcelona especially during drought periods. The methodology followed in this research can be applied to other urban aquifers with similar purposes since the scarcity and contamination of freshwater resources are worldwide issues. © 2019 Elsevier B.V., A. J gratefully acknowledges the financial support from the Excellence Initiative of the German Federal and State Governments through the “Open Topic Postdoc Position Fellow” at the Technische Universität Dresden. The authors would like to thank the European Commission, the Spanish Foundation for Science and Technology (FECYT) and Spanish State Research Agency (AEI) for funding in the frame of the collaborative international consortium (URBANWAT) financed under the 2018 Joint call of the WaterWorks2017 ERA-NET Cofund. This ERA-NET is an integral part of the activities developed by the Water JPI. Additionally authors would also thank the Ministry of Science, Innovation and Universities, for funding the project UNBIASED (Ref: RTI2018-097346-B-I00) under the 2018 call of the “Proyectos de I+D Retos Investigación.

Metagenomic analysis of virus, bacteria and protozoa in irrigation water.

Wastewater is an important resource in water-scarce regions of the world, and its use in agriculture requires the guarantee of acceptable public health risks. The use of fecal indicator bacteria to evaluate safety does not represent viruses, the main potential health hazards. Viral pathogens could complement the use of fecal indicator bacteria in the evaluation of water quality. In this study, we characterized the concentration and removal of human adenovirus (HAdV) and norovirus genogroup II (NoV GII), highly abundant and important viral pathogens found in wastewater, in two wastewater treatment plants (WWTPs) that use different tertiary treatments (constructed wetland vs conventional UV, chlorination and Actiflo® treatments) for a year in Catalonia. The main objective of this study was to develop a Quantitative Microbial Risk Assessment for viral gastroenteritis caused by norovirus GII and adenovirus, associated with the ingestion of lettuce irrigated with tertiary effluents from these WWTPs. The results show that the disease burden of NoV GII and HAdV for the consumption of lettuce irrigated with tertiary effluent from either WWTP was higher than the WHO recommendation of 10−6 DALYs for both viruses. The WWTP with constructed wetland showed a higher viral reduction on average (3.9 and 2.8 logs for NoV GII and HAdV, respectively) than conventional treatment (1.9 and 2.5 logs) but a higher variability than the conventional WWTP. Sensitivity analysis demonstrated that the input parameters used to estimate the viral reduction by treatment and viral concentrations accounted for much of the model output variability. The estimated reductions required to reach the WHO recommended levels in tertiary effluent are influenced by the characteristics of the treatments developed in the WWTPs, and additional average reductions are necessary (in WWTP with a constructed wetland: A total of 6.7 and 5.1 logs for NoV GII and HAdV, respectively; and in the more conventional treatment: 7 and 5.6 logs). This recommendation would be achieved with an average quantification of 0.5 genome copies per 100 mL in reclaimed water for both viruses. The results suggest that the analyzed reclaimed water would require additional treatments to achieve acceptable risk in the irrigation of vegetables with reclaimed water.

Conventional wastewater treatment does not completely remove and/or inactive viruses; consequently, viruses excreted by the population can be detected in the environment. This study was undertaken to investigate the distribution and seasonality of human viruses and faecal indicator bacteria (FIB) in a river catchment located in a typical Mediterranean climate region and to discuss future trends in relation to climate change. Sample matrices included river water, untreated and treated wastewater from a wastewater treatment plant within the catchment area, and seawater from potentially impacted bathing water. Five viruses were analysed in the study. Human adenovirus (HAdV) and JC polyomavirus (JCPyV) were analysed as indicators of human faecal contamination of human pathogens; both were reported in urban wastewater (mean values of 10 6 and 10 5 GC/L, respectively), river water (10 3 and 10 2 GC/L) and seawater (10 2 and 10 1 GC/L). Human Merkel Cell polyomavirus (MCPyV), which is associated with Merkel Cell carcinoma, was detected in 75% of the raw wastewater samples (31/37) and quantified by a newly developed quantitative polymerase chain reaction (qPCR) assay with mean concentrations of 10 4 GC/L. This virus is related to skin cancer in susceptible individuals and was found in 29% and 18% of river water and seawater samples, respectively. Seasonality was only observed for norovirus genogroup II (NoV GGII), which was more abundant in cold months with levels up to 10 4 GC/L in river water. Human hepatitis E virus (HEV) was detected in 13.5% of the wastewater samples when analysed by nested PCR (nPCR). Secondary biological treatment (i.e., activated sludge) and tertiary sewage disinfection including chlorination, flocculation and UV radiation removed between 2.22 and 4.52 log 10 of the viral concentrations. Climate projections for the Mediterranean climate areas and the selected river catchment estimate general warming and changes in precipitation distribution. Persistent decreases in precipitation during summer can lead to a higher presence of human viruses because river and sea water present the highest viral concentrations during warmer months. In a global context, wastewater management will be the key to preventing environmental dispersion of human faecal pathogens in future climate change scenarios.

Wastewater is a valuable resource for water-scarce regions, and is becoming increasingly important due to the rising frequency of droughts as a result of climate change. The health risks derived from ingestion of lettuce that has been irrigated with effluent from a wastewater treatment plant (WWTP) in Catalonia (Spain) were estimated following a quantitative microbial risk assessment (QMRA) approach using site-specific data. Norovirus (NoV) was selected for this analysis, since it is the most common cause of acute gastroenteritis outbreaks in Catalonia. Two scenarios, irrigation with secondary and with tertiary effluent, were analysed. An uncertainty analysis was conducted to determine the impact of possible internalization of NoV into edible parts of the lettuce. The mean disease burden for ingestion of lettuce irrigated with secondary and tertiary effluent was 7.8 × 10− 4 Disability Adjusted Life Years (DALYs) per person per year (pppy) and 3.9 × 10− 4 DALYs pppy, respectively. A sensitivity analysis revealed that the model parameters with higher influence on the probability of disease are the concentration of NoV in the effluent and the consumption of lettuce. In order to decrease the disease burden to the guidance level of 10− 6 DALYs pppy, the tertiary treatment should be able to achieve a 4.3 log reduction of the concentration of NoV. If internalization of NoV into lettuces occurs, this would require a reduction of 7.6 log. This is the first time that site specific data and virus internalization in crops are incorporated in a QMRA of irrigation of lettuce and its impact is quantified.