Guidelines for Canadian Drinking Water Quality: Supporting Documentation - Enteric Viruses

Analytical Methods

Direct Detection of Viruses

Many of the enteric viruses cause unapparent infections and thereby remain unreported. Although viruses have been associated with waterborne outbreaks, their importance as waterborne pathogens has only recently been recognized. As detection methods improve, and with increased epidemiological surveillance, the role of viruses as waterborne pathogens is becoming increasingly recognized (U.S. EPA 1989; AWWA 1999a; LeChevallier 1999; Jaykus 2000; Lees 2000).

Routine monitoring for waterborne pathogens, including enteric viruses, remains difficult. Detection methods may be expensive and are often imprecise or lack critical quality control. Enteric viruses are present in small numbers in faecally contaminated water; as a result, water samples of 10-1000 L must be concentrated in order to detect these pathogens. Filtration is often the preferred method for virus concentration. Two methods of filtration have traditionally been used: filtration by adsorption and filtration by size exclusion (ultrafiltration). Chemical precipitation with polyethylene glycol or aluminium hydroxide has also been used for recovery and concentration of viruses from water.

Methods for detection of viruses in water are employed mainly by specialized laboratories that have developed the expertise and can maintain the required level of biosafety for handling pathogenic viruses. Current testing methods still rely on cell culture to detect the infective enteric viruses that can be grown on selected host cell lines. Cell culture methods for the detection of a wide variety of these viruses have been developed and are currently being improved through the use of molecular methods, such as reverse transcriptase-polymerase chain reaction (RT-PCR) and integrated cell culture (ICC)-PCR. These methods have been developed to detect culturable viruses and combine cell culture and molecular detection of viral genomic nucleic acid. The detection of viral mRNA by RT-PCR assay in innoculated cell cultures is a very sensitive, specific and rapid method for the detection of viruses. These methods have been successfully applied to the detection of viruses in water (Payment and Trudel 1993; Jothikumar et al. 2000; Hurst et al. 2001; Payment 2001; Reynolds et al. 2001; Ko et al. 2003).

The detection of viruses in water samples may serve as a tool to evaluate risks associated with water usages, water treatment efficiency or possible health risk. During outbreak investigations, testing for viruses in water can provide invaluable data to researchers and public health authorities. Therefore, apart from specific scientific objectives, the direct detection of enteric viruses in drinking water should be restricted to those incidents where epidemiological evidence indicates that drinking water could be the source of infection. This should also be done with the understanding that negative results (i.e., finding no viruses) do not necessarily indicate that viruses were absent at the time of sampling or at the time when the population was exposed (Payment and Franco 1993; Gerba et al. 1996; Payment et al. 1997, 2001; Hurst et al. 2001; Payment and Hunter 2001).

Viral Indicators

Since the direct detection of enteric viruses in water is difficult, interest has focused on surrogate parameters (i.e., indicators) to evaluate water treatment efficiency or to indicate the presence of viruses in drinking water (WHO 1996; Deere et al. 2001).

In Untreated Surface Water or Groundwater

Coliform bacteria, particularly Escherichia coli, along with other bacteria - i.e., enterococci (faecal streptococci) and Clostridium perfringens (clostridial) spores - and bacteriophages, are the main groups of organisms that have been considered as indices of faecal pollution and thereby the potential presence of enteric viruses. In surface water, these provide some level of indication of the presence of viruses when the pollution originates from human sources. This relationship may not exist when the source of faecal pollution is of animal origin (Payment et al. 2000; Deere et al. 2001).

In groundwater, microbial indicators are selectively removed during percolation of water through the soil. Viruses, because of their small size, can travel much farther and have been reported in groundwater apparently not contaminated by indicator bacteria (Abbaszadegan et al. 1998, 1999).

In Treated Drinking Water

In treated drinking water, viruses have also been found in the absence of coliform bacteria. This is not surprising, as viruses are much more resistant to filtration and disinfection than these bacteria. The presence of E. coli indicates faecal contamination and, therefore, the potential presence of viruses; however, the absence of E. coli does not indicate the absence of viruses. This observation has raised questions regarding the appropriateness of using coliform bacteria as indicators of viral contamination. The quest for surrogates of viruses in drinking water has been the focus of recent studies. Organisms other than coliforms have been proposed, including enterococci, C. perfringens spores and bacteriophages. Although no surrogate for enteric viruses has been found to meet all the criteria for an ideal indicator in all types of water, bacteriophages and C. perfringens have gained some acceptance. The natural resistance to treatment of the clostridial spores and the bacteriophages suggests that they could be helpful in determining treatment efficacy.

Bacteriophages are viruses that infect bacteria via receptor sites on the host cell surface. Several reviews have been published on the use of bacteriophages as indicators for the presence of enteric viruses in fresh water and faecally polluted treated or untreated drinking water. Bacteriophages have also been used as indicators of treatment efficiency; they are sometimes added to drinking water during research studies to estimate removal and/or inactivation of human enteric viruses during treatment, but they pose no threat to human health. In general, enteric viruses are more similar to bacteriophages than to faecal indicator bacteria in origin and ecology. For example, they share similar survival characteristics when discharged into the aquatic environment. Three types of bacteriophages have been proposed as indicators: the somatic coliphages, male-specific F-RNA bacteriophages (MS2 T-specific coliphage or F-specific coliphage) and Bacteroides phages (i.e., phages infecting Bacteroides fragilis). However, there is no agreement yet on which of the three is most appropriate for detection of enteric viruses, and there are still numerous issues relating to standardization and quality control (Payment and Armon 1989; IAWPRC Study Group on Health Related Microbiology 1991; Payment and Franco 1993; Havelaar 1993; Havelaar et al. 1993; Havelaar and Sobsey 1995; WHO 1996; AWWA 1999a; Payment et al. 2000; Grabow 2001; Hurst et al. 2001). In the United States, standardized methods for the detection of coliphages have just been proposed (Method 1601/1602; U.S. EPA 2001a, 2001b), and an International Organization for Standardization method is used in Europe (Mooijman et al. 2001). These methods could be used as the basis for comparison studies.

Clostridium perfringens spores have been proposed as indicators of the presence of viruses and treatment efficiency. They are indicators of both recent and past faecal contamination, but, because they are not as numerous as coliforms in faeces or contaminated water, larger sample volumes must be analysed. Testing for C. perfringens may not be relevant for utilities drawing water from protected watersheds, but could be useful for utilities drawing water from unprotected surface waters. Clostridium perfringens spores are as resistant as enteric viruses during drinking water treatment. Their absence suggests that there is a very low probability of finding viruses in water. A direct correlation was observed between viruses and C. perfringens in polluted source waters (Payment and Franco 1993; Payment et al. 1997; Ashbolt et al. 2001).

Enterococci are more resistant to environmental stresses and to disinfection than are coliform bacteria; however, they are present in faeces and domestic sewage in smaller numbers than coliform bacteria. There are no data correlating the presence of enterococci with viruses in drinking water, but they have been found in groundwater, where they can serve as indicators of faecal pollution and indirectly of the presence of viruses (U.S. EPA 2000b; Ashbolt et al. 2001).