Metagenomics for the study of viruses in urban sewage as a tool for public health surveillance.

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 10L 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.

Standard and new faecal indicators and pathogens in sewage treatment plants, microbiological parameters for improving the control of reclaimed water.

This study involved collaboration between three centres with expertise in viruses, bacteria and protozoa. The focus of the research was the study of the dissemination and removal of pathogens and faecal indicators in two sewage treatment plants (STP1 and STP2) using tertiary treatments. Samples were collected over a period of five months through the sewage treatment processes. Analysis of the samples revealed that the plants were not efficient at removing the faecal indicators and pathogens tested during the study. From entry point (raw sewage) to effluent level (tertiary treatment effluent water), the experimental results showed that the reduction ratios of human adenoviruses were 1.2 log10 in STP1 and 1.9 log10 in STP2. Whereas for Giardia spp. and Cryptosporidium spp. the reduction ratios were 2.3 log10 for both pathogens in STP1, and 3.0 and 1.7 log10 in STP2, respectively. Furthermore, the presence of faecal indicators and pathogens at different sampling points was evaluated revealing that the tested pathogens were present in reclaimed water. Human adenovirus and Arcobacter spp. showed positive results in infectivity assays for most of the tertiary effluent water samples that comply with current legislation in Spain. The pathogens detected must be evaluated using a risk assessment model, which will be essential for the development of improved guidelines for the re-use of reclaimed water.

HIV, HEV and cirrhosis: evidence of a possible link from eastern Spain.

OBJECTIVES: The aim of the study was to assess the seroprevalence of hepatitis E virus (HEV) infection in an HIV-infected population, as determined by HEV immunoglobulin G (IgG) antibodies (anti-HEV). METHODS: The design of the study was cross-sectional. Serum anti-HEV IgG was determined by enzyme immunoassay in 238 HIV-infected patients consecutively attending our out-patient clinic between April and May 2011. In HEV-seropositive patients, HEV RNA was analysed by nested reverse transcriptase-polymerase chain reaction (RT-PCR). Associations between anti-HEV and liver cirrhosis, route of HIV infection, hepatitis B virus (HBV) and hepatitis C virus (HCV) serological markers, age, sex and alanine aminotransferase (ALT) levels were examined by univariate and multivariate analysis. RESULTS: One hundred and forty patients (59%) had chronic liver disease (99% were HBV- and/or HCV-coinfected). Liver cirrhosis was detected in 44 individuals (19%). Two hundred and twelve patients (89%) were on antiretroviral treatment; the median CD4 T-cell count was 483 cells/µL [interquartile range (IQR) 313-662 cells/µL] and the HIV viral load was <25 HIV-1 RNA copies/mL. Overall, 22 patients (9%) were anti-HEV positive. Liver cirrhosis was the only factor independently associated with the presence of anti-HEV, which was documented in 23% of patients with cirrhosis and 6% of patients without cirrhosis (P=0.002; odds ratio 5.77). HEV RNA was detected in three seropositive patients (14%), two of whom had liver cirrhosis. CONCLUSIONS: Our findings show a high prevalence of anti-HEV in HIV-infected patients, strongly associated with liver cirrhosis. Chronic HEV infection was detected in a significant number of HEV-seropositive patients. Further research is needed to ascertain whether cirrhosis is a predisposing factor for HEV infection and to assess the role of chronic HEV infection in the pathogeneses of cirrhosis in this population.

Development of a qPCR assay for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment.

The Adenoviridae family comprises a wide diversity of viruses that may be excreted for long periods in feces or urine. Previous studies have suggested that the detection of human and animal adenoviruses as well as human and animal polyomaviruses by PCR could be used as an index of fecal contamination of human and animal origin. In this study, quantitative PCR assays targeting specifically porcine adenoviruses have been developed and applied to fecal and environmental samples, including pig slurries, urban sewage, slaughterhouse sewage and river water samples. To develop real-time quantitative PCR for the detection and quantitation of porcine adenoviruses, primers and a TaqMan probe targeting a 68-bp region of the porcine adenovirus hexon gene were designed to amplify specifically porcine adenovirus, and the conditions of the reaction were optimized. The assay detected 1-10 genome copies per test tube and was specific in showing no positive results when samples containing human or bovine adenoviruses were analyzed. Fecal samples contained mean concentrations of porcine adenoviruses of 10(5) GC/g while slaughterhouse wastewater samples showed mean values of 10(3) GC/ml. The assay detected porcine fecal pollution in samples that were highly diluted and had been collected at a considerable distance from the input source, such as river water. In general, the data presented here provide a quantitative tool for the analysis of porcine adenoviruses as indicators of the presence of porcine contamination in the environment, and support the detection of porcine adenoviruses by real-time quantitative PCR as a promising and valuable tool for source-tracking studies.