High prevalence of rotavirus a in raw sewage samples from Northeast Spain

Rotavirus A (RVA) is the most common virus associated with infantile gastroenteritis worldwide, being a public health threat, as it is excreted in large amounts in stool and can persist in the environment for extended periods. In this study, we performed the detection of RVA and human adenovirus (HAdV) by TaqMan qPCR and assessed the circulation of RVA genotypes in three wastewater treatment plants (WWTPs) between 2015 and 2016 in Catalonia, Spain. RVA was detected 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. RVA VP7 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, commonly associated with human, bovine and porcine strains. It is important to mention that the RVA strains isolated from the WWTPs were different from those recovered from piglets and calves living in the same area of single sampling in 2016. These data highlight the importance of monitoring water matrices for RVA epidemiology and may be a useful tool to evaluate and predict possible emergence/reemergence of uncommon strains in a region

Development of improved low-cost ceramic water filters for viral removal in the Haitian context

Household-based water treatment (HWT) is increasingly being promoted to improve water quality and, therefore, health status in low-income countries. Ceramic water filters (CWFs) are used in many regions as sustainable HWT and have been proven to meet World Health Organization (WHO) microbiological performance targets for bacterial removal (24 log); however, the described viral removal efficiencies are insufficient to significantly reduce the associated risk of viral infection. With the objective of improving the viral removal efficiencies of ceramic water filters, new prototypes with different oxide compositions and firing atmospheres have been developed and evaluated. For removal efficiencies human adenoviruses, MS2 bacteriophage and Escherichia coli were quantified in all prototypes. A new model of CWF that was fired in a reductive atmosphere presented virus and bacteria removal efficiencies greater than 3.0 log and 2.5 log, respectively, which would fulfill the viral targets that are recommended by the WHO. Ceramic characterization of the selected filters, which were fired in a reductive atmosphere, showed that a larger specific surface area than those of control filters and higher fraction of a positive Z-potential fraction are the most likely explanations for this increase in virus removal

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

Raw Sewage Harbors Diverse Viral Populations

At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity

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

[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.Gonzales-Gustavson, E.; Cárdenas-Youngs, Y.; Calvo, M.; Figueira, M.; Hundesa, A.; Amoros, I.; Moreno Trigos, MY.... (2017). Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa. Journal of Microbiological Methods. 134:46-53. https://doi.org/10.1016/j.mimet.2017.01.006S465313

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

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.The authors wish to thank the staff from all the WWTPs monitored for their help and technical support during the sampling campaigns. The authors acknowledge the funding received from the ACA and the ASPCAT from the Catalan Government (Generalitat de Catalunya). ICRA authors acknowledge the funding provided by the Generalitat de Catalunya through the Consolidated Research Group grants ICRA-ENV 2017 SGR 1124 and ICRA-TiA 2017 SGR 1318. ICRA researchers also thank the funding from the CERCA program of the Catalan Government.Peer reviewe

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

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

Rotavirus A (RVA) is the most common virus associated with infantile gastroenteritis worldwide, being a public health threat, as it is excreted in large amounts in stool and can persist in the environment for extended periods. In this study, we performed the detection of RVA and human adenovirus (HAdV) by TaqMan qPCR and assessed the circulation of RVA genotypes in three wastewater treatment plants (WWTPs) between 2015 and 2016 in Catalonia, Spain. RVA was detected 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. RVA VP7 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, commonly associated with human, bovine and porcine strains. It is important to mention that the RVA strains isolated from the WWTPs were different from those recovered from piglets and calves living in the same area of single sampling in 2016. These data highlight the importance of monitoring water matrices for RVA epidemiology and may be a useful tool to evaluate and predict possible emergence/reemergence of uncommon strains in a region