Anna Carratalà

Molecular detection of pathogens in water - the pros and cons of molecular techniques.

Pollution of water by sewage and run-off from farms produces a serious public health problem in many countries. Viruses, along with bacteria and protozoa in the intestine or in urine are shed and transported through the sewer system. Even in highly industrialized countries, pathogens, including viruses, are prevalent throughout the environment. Molecular methods are used to monitor viral, bacterial, and protozoan pathogens, and to track pathogen- and source-specific markers in the environment. Molecular techniques, specifically polymerase chain reaction-based methods, provide sensitive, rapid, and quantitative analytical tools with which to study such pathogens, including new or emerging strains. These techniques are used to evaluate the microbiological quality of food and water, and to assess the efficiency of virus removal in drinking and wastewater treatment plants. The range of methods available for the application of molecular techniques has increased, and the costs involved have fallen. These developments have allowed the potential standardization and automation of certain techniques. In some cases they facilitate the identification, genotyping, enumeration, viability assessment, and source-tracking of human and animal contamination. Additionally, recent improvements in detection technologies have allowed the simultaneous detection of multiple targets in a single assay. However, the molecular techniques available today and those under development require further refinement in order to be standardized and applicable to a diversity of matrices. Water disinfection treatments may have an effect on the viability of pathogens and the numbers obtained by molecular techniques may overestimate the quantification of infectious microorganisms. The pros and cons of molecular techniques for the detection and quantification of pathogens in water are discussed.

Newly described human polyomaviruses Merkel Cell, KI and WU are present in urban sewage and may represent potential environmental contaminants

Recently, three new polyomaviruses (KI, WU and Merkel cell polyomavirus) have been reported to infect humans. It has also been suggested that lymphotropic polyomavirus, a virus of simian origin, infects humans. KI and WU polyomaviruses have been detected mainly in specimens from the respiratory tract while Merkel cell polyomavirus has been described in a very high percentage of Merkel cell carcinomas. The distribution, excretion level and transmission routes of these viruses remain unknown.Here we analyzed the presence and characteristics of newly described human polyomaviruses in urban sewage and river water in order to assess the excretion level and the potential role of water as a route of transmission of these viruses. Nested-PCR assays were designed for the sensitive detection of the viruses studied and the amplicons obtained were confirmed by sequencing analysis. The viruses were concentrated following a methodology previously developed for the detection of JC and BK human polyomaviruses in environmental samples. JC polyomavirus and human adenoviruses were used as markers of human contamination in the samples. Merkel cell polyomavirus was detected in 7/8 urban sewage samples collected and in 2/7 river water samples. Also one urine sample from a pregnant woman, out of 4 samples analyzed, was positive for this virus. KI and WU polyomaviruses were identified in 1/8 and 2/8 sewage samples respectively. The viral strains detected were highly homologous with other strains reported from several other geographical areas. Lymphotropic polyomavirus was not detected in any of the 13 sewage neither in 9 biosolid/sludge samples analyzed.This is the first description of a virus isolated from sewage and river water with a strong association with cancer. Our data indicate that the Merkel cell polyomavirus is prevalent in the population and that it may be disseminated through the fecal/urine contamination of water. The procedure developed may constitute a useful tool for studying the excreted strains, prevalence and transmission of these recently described polyomaviruses.

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.

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.

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.

Comparative inactivation of murine norovirus, human adenovirus, and human JC polyomavirus by chlorine in seawater.

ABSTRACT Viruses excreted by humans affect the commercial and recreational use of coastal water. Shellfish produced in contaminated waters have been linked to many episodes and outbreaks of viral gastroenteritis, as well as other food-borne diseases worldwide. The risk can be reduced by appropriate treatment following harvesting and by depuration. The kinetics of inactivation of murine norovirus 1 and human adenovirus 2 in natural and artificial seawater by free available chlorine was studied by quantifying genomic copies (GC) using quantitative PCR and infectious viral particles (PFU). Human JC polyomavirus Mad4 kinetics were evaluated by quantitative PCR. DNase or RNase were used to eliminate free genomes and assess potential viral infectivity when molecular detection was performed. At 30 min of assay, human adenovirus 2 showed 2.6- and 2.7-log10 GC reductions and a 2.3- and 2.4-log10 PFU reductions in natural and artificial seawater, respectively, and infectious viral particles were still observed at the end of the assay. When DNase was used prior to the nucleic acid extraction the kinetic of inactivation obtained by quantitative PCR was statistically equivalent to the one observed by infectivity assays. For murine norovirus 1, 2.5, and 3.5-log10 GC reductions were observed in natural and artificial seawater, respectively, while no viruses remained infectious after 30 min of contact with chlorine. Regarding JC polyomavirus Mad4, 1.5- and 1.1-log10 GC reductions were observed after 30 min of contact time. No infectivity assays were conducted for this virus. The results obtained provide data that might be applicable to seawater used in shellfish depuration.

Evidence of viral dissemination and seasonality in a Mediterranean river catchment: Implications for water pollution management.

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 log10 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.

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.

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.

Iron oxide-mediated semiconductor photocatalysis vs. heterogeneous photo-Fenton treatment of viruses in wastewater. Impact of the oxide particle size

The photo-Fenton process is recognized as a promising technique towards microorganism disinfection in wastewater, but its efficiency is hampered at near-neutral pH operating values. In this work, we overcome these obstacles by using the heterogeneous photo-Fenton process as the default disinfecting technique, targeting MS2 coliphage in wastewater. The use of low concentrations of iron oxides in wastewater without H2O2 (wüstite, maghemite, magnetite) has demonstrated limited semiconductor-mediated MS2 inactivation. Changing the operational pH and the size of the oxide particles indicated that the isoelectric point of the iron oxides and the active surface area are crucial in the success of the process, and the possible underlying mechanisms are investigated. Furthermore, the addition of low amounts of Fe-oxides (1mgL-1) and H2O2 in the system (1, 5 and 10mgL-1) greatly enhanced the inactivation process, leading to heterogeneous photo-Fenton processes on the surface of the magnetically separable oxides used. Additionally, photo-dissolution of iron in the bulk, lead to homogeneous photo-Fenton, further aided by the complexation by the dissolved organic matter in the solution. Finally, we assess the impact of the presence of the bacterial host and the difference caused by the different iron sources (salts, oxides) and the Fe-oxide size (normal, nano-sized).