Evaluation of pathogen disinfection efficiency of electrochemical advanced oxidation to become a sustainable technology for water reuse.

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|RuO2-based plate and Si|BDD thin-film were comparatively employed as the anode, which was combined with a stainless-steel cathode in an undivided cell operating at 12 V. Despite the low resulting current density (<15 mA/cm2), both anodes demonstrated the production of oxidants in wetland effluent water. The disinfection efficiency was high for the bacteriophage MS2 (T99 in less than 7.1 min) and bacteria (T99 in about 30 min as maximum), but limited for CBV5 and TuV, spores and amoebas (T99 in more than 300 min). MS2 presented a rapid exponential inactivation regardless of the anode and bacteria showed similar sigmoidal curves, whereas human viruses, spores and amoebas resulted in linear profiles. Due the different sensitivity of microorganisms, different models must be considered to predict their inactivation kinetics. On this basis, it can be concluded that evaluating the viral inactivation from inactivation profiles determined for bacteria or some bacteriophages may be misleading. Therefore, neither bacteria nor bacteriophages are suitable models for the disinfection of water containing enteric viruses. The electrochemical treatment added as a final disinfection step enhances the inactivation of microorganisms, which could contribute to safe water reuse for irrigation. Considering the calculated low energy consumption, decentralized water treatment units powered by photovoltaic modules might be a near reality.

A novel tool for specific detection and quantification of chicken/turkey parvoviruses to trace poultry fecal contamination in the environment.

Poultry farming may introduce pathogens into the environment and food chains. High concentrations of chicken/turkey parvoviruses were detected in chicken stools and slaughterhouse and downstream urban wastewaters by applying new PCR-based specific detection and quantification techniques. Our results confirm that chicken/turkey parvoviruses may be useful viral indicators of poultry fecal contamination.

The Catalan Surveillance Network of SARS-CoV-2 in Sewage: design, implementation, and performance.

Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.

Isolation of a novel monkey adenovirus reveals a new phylogenetic clade in the evolutionary history of simian adenoviruses.

Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses.

Occurrence of water-borne enteric viruses in two settlements based in Eastern Chad: analysis of hepatitis E virus, hepatitis A virus and human adenovirus in water sources.

Hepatitis E virus (HEV) is a common cause of water-borne acute hepatitis in areas with poor sanitation. In 2004 an outbreak of HEV infection affected around 2,000 people in Eastern Chad (Dar Sila). This paper describes the decrease in the incidence of acute jaundice syndrome (AJS) from 2004 until 2009 when a mean incidence of 0.48 cases/1,000 people/year was recorded in the region. Outbreaks of AJS were identified in some of the camps in 2007 and 2008. Moreover, water samples from drinking water sources were screened for human adenoviruses considered as viral indicators and for hepatitis A virus and HEV. Screening of faecal samples from donkeys for HEV gave negative results. Some of the samples were also analysed for faecal coliforms showing values before disinfection treatment between 3 and >50 colony forming units per 100 mL. All water samples tested were negative for HEV and HAV; however, the presence of low levels of human adenoviruses in 4 out of 16 samples analysed indicates possible human faecal contamination of groundwater. Consequently, breakdowns in the treatment of drinking water and/or increased excretion of hepatitis viruses, which could be related to the arrival of a new population, could spread future outbreaks through drinking water.

NGS Techniques Reveal a High Diversity of RNA Viral Pathogens and Papillomaviruses in Fresh Produce and Irrigation Water.

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.

Analysis of the evolution in the circulation of HAV and HEV in eastern Spain by testing urban sewage samples.

The aim of the study was to analyse the evolution of the prevalence of HAV and HEV in the population of eastern Spain by analysing the viruses excreted in urban sewage. Raw urban sewage samples were collected and analysed during several years using RT-PCR techniques and sequencing analysis. Two limiting regions were analysed, one of them having implemented HAV vaccination programs. Acute symptomatic HEV cases were also examined. Results were compared with those from previous studies in the area using identical methodology. The percentage of positive HAV samples in urban sewage fell from 57.4% to 3.1% in 5-10 years in the two studied areas in Spain. Around 30% of the urban sewage samples were positive for HEV in the absence of agricultural sources of contamination. HEV RNA was also detected in four clinical cases of acute hepatitis. The dramatic reduction in the presence of HAV in raw urban sewage observed in eastern Spain could be most likely related to the general improvement in sanitation. However, these improvements would not have an equivalent effect on the circulation of HEV and this observation could be explained by the presence of animal reservoirs for HEV, which act as external sources of infections.

High Prevalence of Rotavirus A in Raw Sewage Samples from Northeast Spain.

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 ×104 and 3.30 × 108 RT-PCR Units/L and 9.51 × 104 and 1.16 × 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.

Microbiological contamination of conventional and reclaimed irrigation water: Evaluation and management measures.

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 Log10 of viruses and 5 Log10 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.

Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa.

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.

UV disinfection and flocculation-chlorination sachets to reduce hepatitis E virus in drinking water.

Hepatitis E Virus (HEV) is a major cause of waterborne outbreaks in areas with poor sanitation. As safe water supplies are the keystone for preventing HEV outbreaks, data on the efficacy of disinfection treatments are urgently needed. Here, we evaluated the ability of UV radiation and flocculation-chlorination sachets (FCSs) to reduce HEV in water matrices. The HEV-p6-kernow strain was replicated in the HepG2/C3A cell line, and we quantified genome number using qRT-PCR and infectivity using an immunofluorescence assay (IFA). UV irradiation tests using low-pressure radiation showed inactivation kinetics for HEV of 99.99% with a UV fluence of 232J/m(2) (IC 95%, 195,02-269,18). Moreover, the FCSs preparations significantly reduced viral concentrations in both water matrices, although the inactivation results were under the baseline of reduction (4.5 LRV) proposed by WHO guidelines.

Genome Sequence of a Cynomolgus Macaque Adenovirus (CynAdV-1) Isolate from a Primate Colony in the United Kingdom.

The genome sequence of a simian adenovirus from a cynomolgus macaque, denoted CynAdV-1, is presented here. Phylogenetic analysis supports CynAdV-1 in an independent clade, comprising a new simian adenovirus (SAdV) species. These genome data are critical for understanding the evolution and relationships of primate adenoviruses, including zoonosis and emergent human pathogens.

Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage.

Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.

Adenovirus and Norovirus Contaminants in Commercially Distributed Shellfish.

Shellfish complying with European Regulations based on quantification of fecal bacterial indicators (FIB) are introduced into markets; however, information on viruses, more stable than FIB, is not available in the literature. To assess the presence of noroviruses (NoVs) GI and GII and human adenoviruses (HAdV) in domestic and imported mussels and clams (n = 151) their presence was analyzed during winter seasons (2004-2008) in north-west Spanish markets through a routine surveillance system. All samples tested negative for NoV GI and 13 % were positive for NoV GII. The role of HAdV as viral indicator was evaluated in 20 negative and 10 positive NoV GII samples showing an estimated sensitivity and specificity of HAdV to predict the presence of NoV GII of 100 and 74 % (cut-off 0.5). The levels of HAdV and NoVs and the efficiency of decontamination in shellfish depuration plants (SDP) were evaluated analyzing pre- and post-depurated mussels collected in May-June 2010 from three different SDP. There were no statistically significant differences in the prevalence and quantification of HAdV between pre- and post-depurated shellfish and between seawater entering and leaving the depuration systems. Moreover, infectious HAdV were detected in depurated mussels. These results confirm previous studies showing that current controls and depuration treatments limiting the number of FIB do not guarantee the absence of viruses in shellfish.

Application of human and animal viral microbial source tracking tools in fresh and marine waters from five different geographical areas.

Integrated river basin management planning to mitigate the impacts of economic, demographic and climate change is an important issue for the future protection of water resources. Identifying sources of microbial contamination via the emerging science of Microbial Source Tracking (MST) plays a key role in risk assessment and the design of remediation strategies. Following an 18-month surveillance program within the EU-FP7-funded VIROCLIME project, specific MST tools were used to assess human markers such as adenoviruses (HAdV) and JC polyomaviruses (JCPyV) and porcine and bovine markers such as porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) via quantification with real-time PCR to analyze surface water collected from five sites within different climatic zones: the Negro River (Brazil), Glafkos River (Greece), Tisza River (Hungary), Llobregat River (Spain) and Umeälven River (Sweden). The utility of the viral MST tools and the prevalence and abundance of specific human and animal viruses in the five river catchments and adjacent seawater, which is impacted by riverine contributions from the upstream catchments, were examined. In areas where no sanitation systems have been implemented, sewage can directly enter surface waters, and river water exhibited high viral loads; HAdV and JCPyV could be detected at mean concentrations of 10(5) and 10(4) Genome Copies/Liter (GC/L), respectively. In general, river water samples upstream of urban discharges presented lower human viral loads than downstream sampling sites, and those differences appeared to increase with urban populations but decrease in response to high river flow, as the elevated river water volume dilutes microbial loads. During dry seasons, river water flow decreases dramatically, and secondary effluents can represent the bulk of the riverine discharge. We also observed that ice cover that formed over the river during the winter in the studied areas in North Europe could preserve viral stability due to the low temperatures and/or the lack of solar inactivation. Porcine and bovine markers were detected where intensive livestock and agricultural activities were present; mean concentration values of 10(3) GC/L indicated that farms were sometimes unexpected and important sources of fecal contamination in water. During spring and summer, when livestock is outdoors and river flows are low, animal pollution increases due to diffuse contamination and direct voiding of feces onto the catchment surface. The field studies described here demonstrate the dynamics of fecal contamination in all catchments studied, and the data obtained is currently being used to develop dissemination models of fecal contamination in water with respect to future climate change scenarios. The results concerning human and animal targets presented in this study demonstrate the specificity and applicability of the viral quantitative parameters developed to widely divergent geographical areas and their high interest as new indicators of human and animal fecal contamination in water and as MST tools.

Quantification of human and animal viruses to differentiate the origin of the fecal contamination present in environmental samples.

Many different viruses are excreted by humans and animals and are frequently detected in fecal contaminated waters causing public health concerns. Classical bacterial indicator such as E. coli and enterococci could fail to predict the risk for waterborne pathogens such as viruses. Moreover, the presence and levels of bacterial indicators do not always correlate with the presence and concentration of viruses, especially when these indicators are present in low concentrations. Our research group has proposed new viral indicators and methodologies for determining the presence of fecal pollution in environmental samples as well as for tracing the origin of this fecal contamination (microbial source tracking). In this paper, we examine to what extent have these indicators been applied by the scientific community. Recently, quantitative assays for quantification of poultry and ovine viruses have also been described. Overall, quantification by qPCR of human adenoviruses and human polyomavirus JC, porcine adenoviruses, bovine polyomaviruses, chicken/turkey parvoviruses, and ovine polyomaviruses is suggested as a toolbox for the identification of human, porcine, bovine, poultry, and ovine fecal pollution in environmental samples.

Effect of temperature and sunlight on the stability of human adenoviruses and MS2 as fecal contaminants on fresh produce surfaces.

Determining the stability, or persistence in an infectious state, of foodborne viral pathogens attached to surfaces of soft fruits and salad vegetables is essential to underpin risk assessment studies in food safety. Here, we evaluate the effect of temperature and sunlight on the stability of infectious human adenoviruses type 2 and MS2 bacteriophages on lettuce and strawberry surfaces as representative fresh products. Human adenoviruses have been selected because of their double role as viral pathogens and viral indicators of human fecal contamination. Stability assays were performed with artificially contaminated fresh samples kept in the dark or under sunlight exposure at 4 and 30°C over 24h. The results indicate that temperature is the major factor affecting HAdV stability in fresh produce surfaces, effecting decay between 3 and 4 log after 24h at 30°C. The inactivation times to achieve a reduction between 1 and 4-log are calculated for each experimental condition. This work provides useful information to be considered for improving food safety regarding the transmission of foodborne viruses through supply chains.

New methods for the concentration of viruses from urban sewage using quantitative PCR.

Viruses are among the most important pathogens present in water contaminated with feces or urine and represent a serious risk to human health. Four procedures for concentrating viruses from sewage have been compared in this work, three of which were developed in the present study. Viruses were quantified using PCR techniques. According to statistical analysis and the sensitivity to detect human adenoviruses (HAdV), JC polyomaviruses (JCPyV) and noroviruses genogroup II (NoV GGII): (i) a new procedure (elution and skimmed-milk flocculation procedure (ESMP)) based on the elution of the viruses with glycine-alkaline buffer followed by organic flocculation with skimmed-milk was found to be the most efficient method when compared to (ii) ultrafiltration and glycine-alkaline elution, (iii) a lyophilization-based method and (iv) ultracentrifugation and glycine-alkaline elution. Through the analysis of replicate sewage samples, ESMP showed reproducible results with a coefficient of variation (CV) of 16% for HAdV, 12% for JCPyV and 17% for NoV GGII. Using spiked samples, the viral recoveries were estimated at 30-95% for HAdV, 55-90% for JCPyV and 45-50% for NoV GGII. ESMP was validated in a field study using twelve 24-h composite sewage samples collected in an urban sewage treatment plant in the North of Spain that reported 100% positive samples with mean values of HAdV, JCPyV and NoV GGII similar to those observed in other studies. Although all of the methods compared in this work yield consistently high values of virus detection and recovery in urban sewage, some require expensive laboratory equipment. ESMP is an effective low-cost procedure which allows a large number of samples to be processed simultaneously and is easily standardizable for its performance in a routine laboratory working in water monitoring. Moreover, in the present study, a CV was applied and proposed as a parameter to evaluate and compare the methods for detecting viruses in sewage samples.

Description of a novel viral tool to identify and quantify ovine faecal pollution in the environment.

Farmed animals such as sheep, cattle, swine and poultry play an important role in microbial contamination of water, crops and food, and introduce large quantities of pathogens into the environment. The ability to determine the origin of faecal pollution in water resources is essential when establishing a robust and efficient water management system. Animal-specific viruses have previously been suggested as microbial source tracking tools, but specific ovine viral markers have not been reported before now. Previous studies have shown that polyomaviruses are host-specific, highly prevalent and are commonly excreted in urine. Furthermore, they have been reported to infect several vertebrate species but not sheep. That situation encouraged the study of a new putative ovine polyomavirus (OPyV) and its use to determine whether faecal pollution originates from ovine faecal/urine contamination. Putative OPyV DNA was amplified from ovine urine and faecal samples using a broad-spectrum nested PCR (nPCR). Specific nested PCR and quantitative PCR assays were developed and applied to faecal and environmental samples, including sheep slurries, slaughterhouse wastewater effluents, urban sewage and river water samples. Successful amplification by PCR was achieved in sheep urine samples, sheep slaughterhouse wastewater and downstream sewage effluents. The assay was specific and was negative in samples of human, bovine, goat, swine and chicken origin. Ovine faecal pollution was detected in river water samples by applying the designed methods. These results provide a quantitative tool for the analysis of OPyV as a suitable viral indicator of sheep faecal contamination that may be present in the environment.