Surveillance of adenoviruses and noroviruses in European recreational waters

Exposure to human pathogenic viruses in recreational waters has been shown to cause disease outbreaks. In the context of Article 14 of the revised European Bathing Waters Directive 2006/7/EC (rBWD, CEU, 2006) a Europe-wide surveillance study was carried out to determine the frequency of occurrence of two human enteric viruses in recreational waters. Adenoviruses were selected based on their near-universal shedding and environmental survival, and noroviruses (NoV) selected as being the most prevalent gastroenteritis agent worldwide. Concentration of marine and freshwater samples was done by adsorption/elution followed by molecular detection by (RT)-PCR. Out of 1410 samples, 553 (39.2%) were positive for one or more of the target viruses. Adenoviruses, detected in 36.4% of samples, were more prevalent than noroviruses (9.4%), with 3.5% GI and 6.2% GII, some samples being positive for both GI and GII. Of 513 human adenovirus-positive samples, 63 (12.3%) were also norovirus-positive, whereas 69 (7.7%) norovirus-positive samples were adenovirus-negative. More freshwater samples than marine water samples were virus-positive. Out of a small selection of samples tested for adenovirus infectivity, approximately one-quarter were positive. Sixty percent of 132 nested-PCR adenovirus-positive samples analysed by quantitative PCR gave a mean value of over 3000 genome copies per L of water. The simultaneous detection of infectious adenovirus and of adenovirus and NoV by (RT)PCR suggests that the presence of infectious viruses in recreational waters may constitute a public health risk upon exposure. These studies support the case for considering adenoviruses as an indicator of bathing water quality.     

Detection of enteroviruses in untreated and treated drinking water supplies in South Africa

Enteric viruses have been detected in many drinking water supplies all over the world. A meaningful number of these supplies were treated and disinfected according to internationally acceptable methods. In addition, counts of bacterial indicators (coliform bacteria and heterotrophic plate count organisms) in these water supplies were within limits generally recommended for treated drinking water and these findings have been supported by epidemiological data on infections associated with drinking water. The shortcomings of conventional treatment methods and indicator organisms to confirm the absence of enteric viruses from drinking water, was generally ascribed to the exceptional resistance of these viruses. In this study, the prevalence of enteroviruses detected from July 2000 to June 2002 in sewage, river-, borehole-, spring- and dam water as well as drinking water supplies treated and disinfected according to international specifications for the production of safe drinking water was analysed. A glass wool adsorption-elution technique was used to recover viruses from 10--20 l of sewage as well as environmental water samples, in the case of drinking water from more than 100 l. Recovered enteroviruses were inoculated onto two cell culture types (BGM and PLC/PRF/5 cells) for amplification of viral RNA with nested-PCR being used to detect the amplified viral RNA. Results from the study demonstrated the presence of enteroviruses in 42.5% of sewage and in 18.7% of treated drinking water samples. Furthermore, enteroviruses were detected in 28.5% of river water, in 26.7% of dam/spring water and in 25.3% of borehole water samples. The high prevalence of coxsackie B viruses found in this study suggested, that a potential health risk and a burden of disease constituted by these viruses might be meaningful. These findings indicated that strategies, other than end-point analysis of treated and disinfected drinking water supplies, may be required to ensure the production of drinking water that does not exceed acceptable health risks. More reliable approaches to ensure acceptable safety of drinking water supplies may be based on control by multiple-barrier principles from catchment to tap using hazard assessment and critical control point (HACCP) principles.     

Detection of enteroviruses in treated drinking water

This study deals with the routine monitoring of drinking water for the presence of enteroviruses, over a period of 1 year. A rapid and simple method was employed for the simultaneous detection and typing of enteroviruses in large-volume water samples. This included an integrated cell culture/nested PCR approach, followed by restriction enzyme analysis. The two drinking water supplies studied were derived from acceptable quality surface water sources using treatment processes, which conform to international specifications for the production of safe drinking water. Enteroviruses (predominantly coxsackie B viruses) were detected in 11% and 16% of the drinking water samples from two treatment plants, respectively. This study confirms that acceptable water quality indicators do not necessarily reflect the virus content of drinking water.     

Detection of infectious enteroviruses and adenoviruses in tap water in urban areas in Korea.

We investigated the viral contamination of tap water at 11 urban sites in Korea between 1997 and 1998 over a period of 11 months. A total of 23 tap water samples were examined for infectious enteroviruses and adenoviruses by a cell culture technique followed by polymerase chain reaction (PCR) amplification. To identify the recovered viruses, sequence analysis of PCR products was performed. Infectious enteroviruses and adenoviruses were detected in 11 (47.8%) and 9 (39.1%) of the samples, respectively. Both enteroviruses and adenoviruses were detected in five samples (21.7%). The level of viral contamination was quite high, ranging from 2 x 10(-3) to 2.9 x 10(-2) Most Probable Number of Infectious Unit L(-1), far above the recommended virus level in drinking water set by the US EPA. Poliovirus type I derived from vaccine was frequently detected and the remainder comprised coxsackievirus B type or echovirus type 6, which were causative agents of aseptic meningitis in Korea in 1997 and 1998, respectively. Several types of adenovirus were detected in tap water samples and some water samples were found to contain adenoviruses which were closely related to enteric adenovirus types 40 and 41.     

Comparison of cytopathogenicity, immunofluorescence and In situ DNA hybridization as methods for the detection of adenoviruses

Three different methods were compared for their efficiency at detection of adenoviruses. The samples examined for viral analysis consisted of concentrates prepared from raw sewage, chosen as providing a representation of the spectrum of viruses being intestinally shed from a large population at any given time. When using one single cell line, HEp-2, the overall numbers of adenoviruses detected using cytopathogenicity and immunofluorescence were roughly equal. In situ hybridization was approx. 40% more sensitive than either of these other methods as determined by average virus titers for the different samples, and also proved to be better by means of a nonparametric comparison. The 293 cell line was approx. 5 times more sensitive for detecting adenoviruses by cytopathogenicity as compared with the HEp-2 cell line, but proved unsuitable in our hands for quantitatively detecting indigenous adenoviruses by immunofluorescence. The relative number of indigenous adenoviruses present in the sewage concentrates we examined was, on average, 94-fold greater than that of enteroviruses. Assay of enteroviruses was performed by plaque assay in the BGM cell line.     

Use of eukaryotic mitochondrial DNA to differentiate human, bovine, porcine and ovine sources in fecally contaminated surface water

A molecular method based on the detection of mitochondrial DNA from various animal species was developed to track the origin of surface water pollutions, and to differentiate human and animal sources. Mitochondrial DNA sequences were used to design PCR primers specific for human, bovine, ovine and porcine DNA using single, multiplex and nested PCR protocols. The primers were tested with DNA extracted from untreated domestic sewage, agricultural soils run-off, swine farm effluents and water from two rivers with known pollution sources. At least one of the four species was detected in most of these samples. The limit of detection in wastewater was 10(3)-10(4) cells L(-1) with a multiplex PCR protocol. This is the first report of a method using eukaryotic genetic DNA to detect and differentiate animal DNA from fecal sources in water. This innovative method is simple and could be used to quickly differentiate sources of pollution in a watershed.     

Effect of pathogen concentrations on removal of Cryptosporidium and Giardia by conventional drinking water treatment

The presence of waterborne enteric pathogens in municipal water supplies contributes risk to public health. To evaluate the removal of these pathogens in drinking water treatment processes, previous researchers have spiked raw waters with up to 10(6) pathogens/L in order to reliably detect the pathogens in treated water. These spike doses are 6-8 orders of magnitude higher than pathogen concentrations routinely observed in practice. In the present study, experiments were conducted with different sampling methods (i.e., grab versus continuous sampling) and initial pathogen concentrations ranging from 10(1) to 10(6) pathogens/L. Results showed that Cryptosporidium oocyst and Giardia cyst removal across conventional treatment were dependent on initial pathogen concentrations, with lower pathogen removals observed when lower initial pathogen spike doses were used. In addition, higher raw water turbidity appeared to result in higher log removal for both Cryptosporidium oocysts and Giardia cysts.     

Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR)

We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.     

The Use of Bone Charcoal in the Treatment of Rural Water Supplies

As a result of the Water Act of 1989 on the quality of water intended for human consumption, a number of small spring sources in Yorkshire were the subject of legal undertakings for remedial action to reduce the concentration of trihalomethanes in the distribution system. The trihalomethanes are formed when the colour in these waters, which is made up of predominantly fulvic and humic acids, is chlorinated. Therefore, in order to solve the problem of trihalomethanes in treated water, colour removal was necessary.
The paper describes the use of bone charcoal in slow sand filters to remove colour from rural water supplies. It covers six months pilot-plant work which was undertaken at Marsett water-treatment works (near Richmond), and the implementation at other plants within Yorkshire. low-rate filtration through bone charcoal ensured that the colour and trihalomethane concentrations in filtered water complied with EC standards, and the material coped well with rapid changes in raw water quality.     

Removal of bacterial fecal indicators, coliphages and enteric adenoviruses from waters with high fecal pollution by slow sand filtration

The aim of the present study was to estimate the performance of slow sand filtration (SSF) facilities, including the time needed for reaching stabilization (maturation), operated with surface water bearing high fecal contamination, representing realistic conditions of rivers in many emerging countries. Surface water spiked with wastewater was infiltrated at different pore water velocities (PWV) and samples were collected at different migration distances. The samples were analyzed for phages and to a lesser extent for fecal bacteria and enteric adenoviruses. At the PWV of 50 cm/d, at which somatic phages showed highest removal, their mean log₁₀ removal after 90 cm migration was 3.2. No substantial differences of removal rates were observed at PWVs between 100 and 900 cm/d (2.3 log₁₀ mean removal). The log₁₀ mean removal of somatic phages was less than the observed for fecal bacteria and tended more towards that of enteric adenoviruses This makes somatic phages a potentially better process indicator than Escherichia coli for the removal of viruses in SSF. We conclude that SSF, and by inference in larger scale river bank filtration (RBF), is an excellent option as a component in multi-barrier systems for drinking water treatment also in areas where the sources of raw water are considerably fecally polluted, as often found in many emerging countries.     

Estimating the impact on health of poor reliability of drinking water interventions in developing countries

Recent evidence suggests that many improved drinking water supplies suffer from poor reliability. This study investigates what impact poor reliability may have on achieving health improvement targets. A Quantitative Microbiological Risk Assessment was conducted of the impact of interruptions in water supplies that forced people to revert to drinking raw water. Data from the literature were used to construct models on three waterborne pathogens common in Africa: Rotavirus, Cryptosporidium and Enterotoxigenic E. coli. Risk of infection by the target pathogens is substantially greater on days that people revert to raw water consumption. Over the course of a few days raw water consumption, the annual health benefits attributed to consumption of water from an improved supply will be almost all lost. Furthermore, risk of illness on days drinking raw water will fall substantially on very young children who have the highest risk of death following infection. Agencies responsible for implementing improved drinking water provision will not make meaningful contributions to public health targets if those systems are subject to poor reliability. Funders of water quality interventions in developing countries should put more effort into auditing whether interventions are sustainable and whether the health benefits are being achieved.     

Real-time PCR detection of adenoviruses, polyomaviruses, and torque teno viruses in river water in Japan

The prevalence of DNA viruses in water from the Tamagawa River, Japan was quantitatively surveyed for 6 months, from April to September 2003. A total of 18 river water samples were subjected to virus concentration method using an electronegative membrane, followed by DNA extraction and direct quantitative real-time polymerase chain reaction (qPCR) for DNA viruses. Adenoviruses of serotypes 40 and 41 were detected most frequently in the river water samples tested (61.1%), at a concentration ranging from 3.16 × 10³ to 1.38 × 10⁵ copies/l, followed by JC polyomaviruses (11.1%) and torque teno viruses (5.6%). No sample was positive for BK polyomaviruses. In addition, for selective detection of virus particles, adenoviruses 40 and 41 were tested with qPCR combined with an immunomagnetic separation technique; they were detected in only 16.7% of the samples, showing a concentration ranging from 7.42 × 10² to 4.24 × 10⁴ copies/l. This study is significant since it is the first study to demonstrate the prevalence of polyomaviruses in water samples in Japan and to use immunomagnetic separation qPCR to detect adenovirus particles in aquatic environments.     

The control of potential health Risks related to drinking water in the UK

In the United Kingdom, potable water put into supply is required to be‘wholesome’ The term ‘wholesome’ is interpreted as clear, palatable and safe to drink. About 99% of potable supplies are provided by Regional Water Authorities and Water Companies (for England and Wales), Regional Councils and Island Councils (for Scotland) and the Department of the Environment (NI) (for Northern Ireland). Those water authorities draw their raw water from upland surface waters, lowland surface waters (including lakes and rivers) and underground waters. Although each source provides approximately one-third, of supply, the proportion varies considerably in different parts of the UK. Consequently the control of potential health risks related to drinking water also varies according to the source of supply. The paper describes briefly the treatment practice for the various sources, including disinfection practice. More specifically the paper describes current UK practice or developments in the control or investigation of plumbosolvency, fluoridation, nitrate, trihalomethanes, other organic micropollutants, sodium, asbestos and tar linings in pipes. The possibilities for the surveillance of the 1% of private supplies are also discussed.     

Viruses and bacteriophages

Many of the enteric viruses which are transmitted from person to person by thefecal-oral route are found in raw and treated wastewater, and because of their persistence under adverse conditions may also be found in slightly polluted waters.There is no routine examination procedure of water and wastewater for entero-viruses,mainly because of the cumbersome isolation techniques, high cost and the need for highly skilled laboratory personnel.Phages are specific to single species of bacteria, are known for many entericbacteria, and are very often used for final identification of enteric pathogenic bacteria.Coliphages are provalent in raw and treated sewage as well as in polluted water, where enteric viruses may also be found.Coliphages were often mentioned as possible viral indicators in polluted water.To be a perfect indicator, they should comply with minimum criteria as follows: (a) they should be present wherever human enteric viruses are present: (b) the coliphage numbers recovered should be equal to or larger than those of enteric viruses recovered: (c) the coliphages should be at least as resistant as enteric viruses to adverse environmental conditions: (d) isolation and quantification of the coliphage should be faster and less expensive than isolation of the enteroviruses.Comparative studies show that the coliphage to enterovirus ratio in wastewater is about 10³:1. Levels of poliovirus 1 (attenuated) to coliphage f2 remained stable for a few months in oxidation pond effluents.f2 coliphage exhibited higher resistance to chlorination than poliovirus 1 (attenuated). When the two strains were kept in water of different quality, f2 survived longer. In addition, all coliphage counts were completed within 24 h. while those of enteroviruses required about a week. Results indicate very strongly that coliphages can be used as viral indicators and this is already the practice in a few European and other countries.     

Epidemoilogical Studies on cancer and organic compounds in U.S. drinking waters

Two national surveys of U. S. drinking waters have been conducted todetermine the presence and concentration levels of organic compounds, particularly trihalomethanes. Most drinking water supplies from surface water sources have been shown to contain numerous organic compounds at very low levels. Chloroform is present in virtually all drinking water supplies that are treated with chlorine as a disinfectant.The health effects of organic compounds have been investigated by animal and epidemiological studies.Numerous epidemiological studies have been conducted on various geographic sections of the United States. Fourteen epidemiological studies that were hypothesis generating studies were conducted to determine if there is a positive — association between drinking water and increased cancer mortality. These studies may indicate which organ(s) might potentially be the affected organ(s). Further epidemiological studies that tested various hypotheses were conducted A summary of these studies is presented, which indicates that there is potentially an increased risk of bladder, colon, and rectum cancer from drinking waters containing trihalomethanes.     

Evaluation of public health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human-specific bacteriological marker

Each year the National Resource Defense Council addresses the quality of US beaches by routine bacterial indicators. In the Great Lakes region the indicator used is Escherichia coli and for 2007 more beaches were closed and impacted than ever before. In this study, water quality was addressed at two Lake Michigan Beaches over the 2004 swimming season by monitoring infectious enteric viruses by cell culture and integrated PCR and for a human sewage marker based on the Enterococcal Surface Protein (esp). Our goals for this study were to 1) examine the occurrence and variety of human enteric viruses present during peak usage of the beaches 2) determine key variables for development of predictive models for viruses; and 3) use quantitative risk assessment to estimate the potential health impact. Our results demonstrate that for both beaches predictive models of virus pollution were best described utilizing physical parameters like wind speed, wind direction and water temperature. The esp marker was not predictive of human viruses. The daily risk of acquiring a viral infection at either of the beaches ranged from 0.2 to 2.4/1000 swimmers using a quantitative microbial risk assessment model, with three swims during a day at the beach for children and over the season, the risk was 9-15/1000 swimmers using adenovirus as the model.Conclusions: Lake Michigan recreational beaches are being adversely impacted by human fecal pollution. Monitoring for the traditional indicators of water quality does not address viral risks and models can be developed and potentially used as real-time water quality forecasting tools.     

One-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan

Sewerage systems are important nodes to monitor human enteric pathogens transmitted via water. A quantitative virus survey was performed once a month for a year to understand the seasonal profiles of noroviruses genotype 1 and genotype 2, enteroviruses, and adenoviruses in sewerage systems. A total of 72 samples of influent, secondary-treated wastewater before chlorination and effluent were collected from six wastewater treatment plants in Japan. Viruses were successfully recovered from 100ml of influent and 1000ml of the secondary-treated wastewater and effluent using the acid rinse method. Viruses were determined by the RT-PCR or PCR method to obtain the most probable number for each sample. All the samples were also assayed for fecal coliforms (FCs) by a double-layer method. The seasonal profiles of noroviruses genotype 1 and genotype 2 in influent were very similar, i.e. they were abundant in winter (from November to March) at a geometric mean value of 190 and 200 RT-PCR units/ml, respectively, and less frequent in summer (from June to September), at 4.9 and 9.1 RT-PCR units/ml, respectively. The concentrations of enteroviruses and adenoviruses were mostly constant all the year round, 17 RT-PCR units/ml and 320 PCR units/ml in influent, and 0.044 RT-PCR units/ml and 7.0 PCR units/ml in effluent, respectively.     

Molecular quantitative analysis of human viruses in California stormwater

Many human pathogenic viruses are transmitted via the oral-fecal route and water is one possible vector, representing a risk for public health. Sixty-one large-volume water samples from storm drains in California were processed by a two-step hollow fiber ultrafiltration procedure followed by molecular analysis for human enterovirus and adenovirus types. Each sample was spiked with a surrogate, the benign bacteriophage PP7. Both surrogate and human viruses were quantified by newly designed TaqMan PCR assays. Equations were developed that account for the main variables in the procedure: recovery of the ultrafiltration, efficiency of nucleic acid extraction, and effect of inhibitors on the amplification of viral targets. Adenovirus 40/41 was detected in one sample at 230 genomes per liter, and no other adenovirus or enterovirus types were found. Samples that resulted in nondetects are reported together with the corresponding sample-specific limit of detection (S(LOD)), a useful tool when estimating the public health risk associated with the contact or ingestion of water. Virus concentrations did not correlate with traditional viable indicator concentrations or any of the physicochemical parameters measured. In contrast, coliform concentrations were correlated with total suspended solids. To our knowledge, this is the first study where all factors known to influence limits of detection have been investigated and integrated into equations that are widely applicable to the quantification of viruses or other microbial targets by PCR.