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.     

Incidence of adenoviruses in raw and treated water

Adenoviruses are of major public health importance and are associated with a variety of clinical manifestations, i.e. gastroenteritis, eye infections and respiratory infections. The importance of water in the epidemiology of adenoviruses and the potential health risks constituted by adenoviruses in water sources and supplies are widely recognised. This study was conducted to assess the incidence of human adenoviruses in raw and treated water systems. Various raw and treated water were routinely monitored for the presence of adenoviruses, over a 1-year period (July 2000-June 2001). The supplies were derived from acceptable quality surface water sources using treatment processes, which conform to international standards for the production of safe drinking water. Adenoviruses were detected by firstly amplifying the viruses in cell cultures and then amplifying the extracted nucleic acids of these viruses using molecular techniques (nested PCR). The results indicated human adenoviruses present in 13 (12.75%) of the raw and 9 (4.41%) of the treated water samples tested. The combination of cell culture and nested PCR has proved to be a quick and reliable method for the detection of adenoviruses in water environments.     

Comparison of molecular markers to detect fresh sewage in environmental waters

Human-specific Bacteroides HF183 (HS-HF183), human-specific Enterococci faecium esp (HS-esp), human-specific adenoviruses (HS-AVs) and human-specific polyomaviruses (HS-PVs) assays were evaluated in freshwater, seawater and distilled water to detect fresh sewage. The sewage spiked water samples were also tested for the concentrations of traditional fecal indicators (i.e., Escherichia coli, enterococci and Clostridium perfringens) and enteric viruses such as enteroviruses (EVs), sapoviruses (SVs), and torquetenoviruses (TVs). The overall host-specificity of the HS-HF183 marker to differentiate between humans and other animals was 98%. However, the HS-esp, HS-AVs and HS-PVs showed 100% host-specificity. All the human-specific markers showed >97% sensitivity to detect human fecal pollution. E. coli, enterococci and, C. perfringens were detected up to dilutions of sewage 10⁻⁵, 10⁻⁴ and 10⁻³ respectively. HS-esp, HS-AVs, HS-PVs, SVs and TVs were detected up to dilution of sewage 10⁻⁴ whilst EVs were detected up to dilution 10⁻⁵. The ability of the HS-HF183 marker to detect fresh sewage was 3-4 orders of magnitude higher than that of the HS-esp and viral markers. The ability to detect fresh sewage in freshwater, seawater and distilled water matrices was similar for human-specific bacterial and viral marker. Based on our data, it appears that human-specific molecular markers are sensitive measures of fresh sewage pollution, and the HS-HF183 marker appears to be the most sensitive among these markers in terms of detecting fresh sewage. However, the presence of the HS-HF183 marker in environmental waters may not necessarily indicate the presence of enteric viruses due to their high abundance in sewage compared to enteric viruses. More research is required on the persistency of these markers in environmental water samples in relation to traditional fecal indicators and enteric pathogens.     

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.     

Detection of somatic phages, infectious enteroviruses and enterovirus genomes as indicators of human enteric viral pollution in surface water

In the present study, we aimed to determine whether the concentrations of somatic coliphages, infectious enteroviruses or the detection of enterovirus genomes were associated with the detection of human pathogenic viruses in surface water. Four French rivers were sampled monthly or semimonthly for the quantitative detection of somatic coliphages, infectious enteroviruses and the qualitative RT-PCR detection of enterovirus, hepatitis A virus, Norwalk I viruses, Norwalk II viruses, astrovirus and rotavirus genomes over 12 months. All the 68 water samples tested were positive for the quantitative detection of somatic coliphages (range of concentrations: 4 x 10(2) to 1.6 x10(5) FUl(-1)). Infectious enteroviruses were isolated by a cell culture system in only two (3%) of the 68 concentrated water samples tested, whereas enterovirus genomes were detectable in 60 (88%) of the same samples. A positive RT-PCR detection of the genome of hepatitis A virus, Norwalk-like virus genogroup II, astrovirus, rotavirus and Norwalk-like virus genogroup I was demonstrated, respectively, in 1.5% (1/68), 1.5% (1/68), 3% (2/68), 0% and 0% of the 68 concentrated water samples tested. All of these four water samples were positive for the detection of enterovirus genomes, whereas only one of them was positive for the isolation of enteroviruses on cell culture. Moreover, the genomic detection of human pathogenic viruses appeared not to be statistically associated with the concentration levels of somatic coliphages in the 68 concentrated water samples tested (Wilcoxon rank test; P=0.14). Taken together, our findings indicate that the quantitative detection of somatic coliphages and the isolation of enteroviruses on cell culture are not suitable parameters for the control of the viral contamination in surface water, whereas the detection of enterovirus genomes may be useful for predicting the presence of waterborne viruses.     

Distribution of enteroviruses in sediments contiguous with a deep marine sewage outfall

The distribution of enteroviruses, coliforms, fecal coliforms and fecal streptococcus discharged from a raw sewage outfall pipe 3.6 km from shore at a depth of 44 m was studied. Enteroviruses and indicator bacteria were only isolated from water in an area within 200 m of the outfall. However, viruses were isolated from sediments as far as 3.6 km from the outfall at recreational bathing beaches. The concentration of enteroviruses in the sediment at the outfall boil ranged from 112 to 78 PFU l⁻¹ and 0 to 30 PFU l⁻¹ at the bathing beach. Indicator bacteria were not detected in the water or sediment at the bathing beach. Analysis of sediments for viruses may give a better idea of the long term distribution of sewage discharged from the outfall pipe.     

Use of bituminous coal for concentration of enteroviruses from sewage and effluent

A method has been developed for concentration of enteroviruses from untreated and treated domestic wastewater using bituminous coal bed as a virus adsorbent. A bed made from 1.5 g of 120 mesh coal powder was used for concentrating enteroviruses from 100 ml of clarified sewage at different pH values with and without addition of AlCl3. To enhance the adsorption of viruses, requisite quantities of aluminium chloride were added so that a final concentration of 0.0005 M could be achieved. At pH 3.0 maximum adsorption (82.8%) of poliovirus type 1 from artificially contaminated clarified sewage was observed without addition of AlCl3. However, at pH 5.0 maximum virus adsorption of 98.7% was achieved after addition of aluminium chloride. An average recovery of 86.9% of adsorbed viruses at pH 5.0 was achieved from coal bed with 3% flocculating beef extract at pH 9.5. This method for concentration of enteroviruses incorporating use of coal was compared with that of Millipore membrane filter method applied to raw sewage and clarified sewage. The results obtained from the methodology using coal as adsorbent was subjected to Student's "t" test and it was observed that its efficiency is confirmed for recovery of enteroviruses from raw and nonclarified sewage. These results are also comparable with that obtained with MF method. The results presented in this paper are indicative of the potential of this method for both treated and raw sewage.     

Survival of bacterial indicator organisms and enteric viruses in the Mediterranean coastal waters off Tel-Aviv

Field and laboratory studies were initiated on the die-away kinetics of coliforms and enteric viruses in coastal waters off Tel-Aviv. Samples were taken from the sewage boil located approx. 880 m out to sea and from three main Tel-Aviv beaches designated as sampling stations, ranging 3 km to the north and 2 km to the south. Routine bacteriological tests were made from these stations; in addition, approx. 50% of the samples were also examined for enteroviral content and occasionally, samples were taken from most of the other Tel-Aviv beaches for comparison of bacterial and viral content.The concentrations of total coliforms, fecal coliforms and fecal streptococci were correlated with the concentrations of enteroviruses. In general, the data suggests that the number of enteric bacteria in the sea was reduced relatively more rapidly than that of the enteroviruses, while fecal streptococci displayed a die-away rate similar to that of enteroviruses. The percentage of samples positive for enteroviruses was found to be lower during the summer months of the year than in the remaining months. The concentration of coliforms was also significantly lower during the summer months. This suggests that the decreased microbial level is associated with increased summertime daylight and solar radiation which affect the micro-organisms' die-away rate. About 76% of the positive enteroviral samples were found at beaches with a fecal coliform bacterial level considered safe for bathing beaches.     

Occurrence of rotaviruses and enteroviruses in recreational waters of Oak Creek, Arizona

Recent epidemiological studies have shown a relationship between swimming in recreational waters meeting bacteriological standards and gastroenteritis with a suggested viral etiology. No previous studies have been conducted in the United States on the occurrence of human pathogenic enteric viruses in freshwater recreational areas. The presence of enteroviruses and rotaviruses was investigated in Oak Creek, Arizona, a heavily used recreational area. Water samples were filtered through positively charged filters (168–1555 I.), eluted with beef extract, and assayed for human enteroviruses and rotaviruses. Eighteen of the 41 recreational water samples were positive for enterovirus or rotavirus. Of these, nine samples exceeded the Arizona State recommended limit of 1 PFU 40 l⁻¹ for full body contact in effluent dominated recreational waters. Several virus positive samples met the recommended fecal coliform standards (200 CFU 100 ml⁻¹) for recreational waters indicating the inadequacy of bacterial standards for monitoring viral water quality. The isolation of the pathogenic enteric viruses (i.e., poliovirus 1, echovirus 1, coxsackievirus B1 and B6 and rotavirus) from this popular recreational water demonstrates the potential for transmission of viral disease.     

Radio-iodine in thyroid glands of swans, farm animals and humans, also in algae and river water from the Thames Valley, England

A highly sensitive counting system has been used to measure radio-iodine in environmental samples from the Thames Valley. Iodine-125 and occasionally iodine-131 have been found in the thyroid glands of most of the swans that have died on the River Thames, the River Wey and the Grand Union Canal, and in algae and water samples from the Thames and many of its tributaries. The presence of this activity is ascribed to the waste discarded into the drainage system by hospitals and research laboratories, reaching the rivers via the effluent from sewage treatment works. The Thames is used as a source of drinking water, particularly in London and its western approaches. Weed and water samples collected from river water abstraction points, reservoirs, tap water supplies, and animal water troughs fed from this supply all contained low levels of iodine-125. The drinking water route can account for the iodine-125 found in the thyroids of farm animals from west Surrey and in a few people living in London. The amounts found constitute a trivial radiation dose to man and animals as they are far below the acceptable limit of exposure for man.     

Occurrence of rota- and enteroviruses in drinking and environmental water in a developing nation

Rotaviruses have been implicated as a major cause of childhood and traveler's diarrhea in developing countries. Since water is known to be a vehicle of transmission of other enteric viruses, we sought to determine if water could play a role in the transmission of rotavirus infections in a developing nation by applying recently developed techniques for the concentration of viruses from tapwater and environmental (lake, river, ocean and aqueduct) water in Mexico. In an initial survey during the rainy season in August 1978, rotavirus was detected in 10 of 10 drinking water samples and coxsackie B4 or B6 virus in 5 of 10. In a larger survey during the dry season in December 1979, rotavirus was recovered from 3 and enteric viruses from 8 of 21 drinking water samples. Water quality data, available for the 1979 survey, indicated that while many tapwater samples did not meet U.S. coliform standards, some samples containing infectious virus did. Our data suggest that current bacteriological water quality standards for potable water do not reflect viral contamination.     

Microbiological surveillance of private water supplies in England - The impact of environmental and climate factors on water quality

A passive surveillance system captured information on 34,904 microbiological samples from 11,233 private drinking water supplies within England as well as the associated constructional, climatic and environmental variables. Escherichia coli was detected in 6588 (18.87%) of samples and at least one positive sample was detected from 3638 (32.39%) of sites. However, this estimate of supplies failing to meet the European drinking water E. coli standard was probably an underestimate as the more samples taken per supply, the more likely the supply was to fail. A multivariable model of private water supplies data showed a strong seasonal impact, with samples between January and May being significantly less contaminated with E. coli than samples between June and December. Samples from springs (OR 2.5, CI 2.0-3.1) or surface waters (OR 2.4, CI 0.8-7.0) were more likely to fail than groundwater sources, as were supplies with no effective treatment (OR1.8, CI 1.5-2.3). Commercial supplies were less likely to fail than domestic supplies (OR 0.63, CI 0.48-0.83) and the probability of failure was linearly associated with the density of sheep in the area and rainfall on the previous day. A Monte Carlo modelling approach was used to estimate that, had sufficient samples been taken, 54% (95% confidence intervals 49-59%) of all private water supplies in England were likely to be unsatisfactory. These findings will be able to inform risk assessments of private water supplies prior to microbiological results being available.     

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.     

Low- and medium-pressure UV inactivation of microsporidia Encephalitozoon intestinalis

Newly recognized waterborne pathogens such as microsporidia are being detected in the world's water supplies with increasing frequency. Many of these organisms have been shown to cause negative health impacts for both immunocompetent as well as immunocompromised individuals. It is imperative that these emerging pathogens be investigated for their ability to resist both traditional and novel disinfection technologies that are currently in use or under consideration for drinking water treatment. Low- and medium pressure UV light is at the cutting edge of disinfection technologies for the drinking water industry. While previous UV disinfection studies have focused on the inactivation of Cryptosporidium and Giardia as well as viruses and common bacteria, this research reports the ability of low- and medium pressure UV light to inactivate > 3.6 log10 of microsporidia Encephalitozoon intestinalis spores at a dose of 6 mJ/cm2 or higher as determined using a cell culture approach.     

Recovery of viruses from waste-water and effluent by the direct inoculation method

Chloroform or antibiotic treated sewage and effluent samples were inoculated directly onto MK₂ or Vero cell monolayers for enterovirus recovery. The results obtained showed a high enterovirus recovery. The average PFU (100 ml)⁻¹ from sewage samples was 4496 and from effluent samples was 5684. The conventional ratio of enteroviruses to coliforms in sewage is considered to be 1:65,000. By the direct inoculation method the ratio obtained was 1: 20,000 for sewage and 1: 2000 for effluent. It is believed the number of enteroviruses in sewage to be even higher than the ones obtained.