Page 7: Guidelines for Canadian Drinking Water Quality: Guideline Technical Document – Enteric Viruses
6.0 Analytical methods
6.1 Detection of enteric viruses
Pathogen detection still suffers from methodological and interpretation limitations (Payment and Pintar, 2006). These limitations include the necessity to concentrate large volumes of water, the need for specialized laboratory equipment and highly trained personnel and the cost of analysis, as well as determining which pathogens to test for, given the multitude of pathogens that may be present, which can vary over time and space. Therefore, routine monitoring of drinking water for enteric viruses is currently not practical.
Although not used for routine monitoring purposes, detection of enteric viruses in source water samples can be used as a tool to evaluate risks that may be associated with using a specific raw water source and to ensure that appropriate treatment is in place. As well, during outbreak investigations where epidemiological evidence indicates that drinking water could be the source of infection, testing for enteric viruses can provide invaluable data to researchers and public health authorities.
Standard methods for enteric virus recovery and detection have been published (U.S. EPA, 1996, 2001c; APHA et al., 1998; ASTM, 2004). These methods have been validated and can be used by laboratories with the capacity to monitor for enteric viruses. The following sections provide an overview of these methodologies along with information on recent advancements in virus detection that have been used in research settings.
6.1.1 Sample concentration
Enteric viruses are generally present in small numbers in faecally contaminated water; as such, 10-1000 L of water may need to be filtered to concentrate the pathogens to a detectable level.
Two methods of filtration have traditionally been used for initial virus concentration: filtration by adsorption and filtration by size exclusion (ultrafiltration). Adsorption filtration can employ electropositive filters, such as those prescribed by the U.S. Environmental Protection Agency's (EPA) Information Collection Rule for the recovery of viruses from water (U.S. EPA, 1996), negatively charged filters (Beuret, 2003; Fuhrman et al., 2005; Villar et al., 2006) or nitrocellulose membranes (Hsu et al., 2006). At ambient pH, most enteric viruses are negatively charged; therefore, they are captured by electropositive filter media. To adsorb viruses using negatively charged filter media, a cation such as magnesium chloride needs to be added to the sample, and the pH of the sample may need to be adjusted to an acidic pH. Since the viruses adsorb to the filter media, they must subsequently be eluted from the filter using an alkaline solution that alters the surface charge of the viral particles so that they will elute back into solution. Eluents commonly incorporate beef extract, glycine, tryptose phosphate buffer and/or sodium hydroxide into the solutions (Katayama et al., 2002; Hörman et al., 2004; Brassard et al., 2005; Villar et al., 2006). Size exclusion methods, such as ultrafiltration, are independent of pH and have the advantage of not requiring an elution step (Olszewski et al., 2005). Ultrafiltration does have some disadvantages. Because of the small size of viruses, the filter pore size must be extremely small and can become clogged. Typically, only approximately 20 L of water can be filtered at one time (Griffin et al., 2003), although volumes up to 100 L are being used in some laboratories (Linquist et al., 2007). Ultrafiltration is also less cost- and time-effective than adsorption filtration (Fong and Lipp, 2005). There is some work ongoing investigating the use of ultrafiltration for the simultaneous recovery of protozoa, bacteria and viruses, which could be advantageous from an economical, and potentially time-saving, perspective (Morales-Morales et al., 2003; Hill et al., 2005).
The initial concentration of the water sample is usually followed by a secondary concentration step, reducing the sample volume to 1-2 mL, to produce a concentrate sufficient for detection of viruses. Secondary concentration methods include organic flocculation, polyethylene glycol precipitation and ultracentrifugation.
6.1.2 Detection methods
Following concentration of the sample, detection methods for the enteric viruses are used. In general, virus detection methods have recovery efficiencies around 50% (Payment et al., 2000). The most commonly used detection methods include cell culture methods and polymerase chain reaction (PCR) methods, or a combination of both methods.
Historically, cell culture was the most widely used technique for the detection of viruses, and it is still the best method for determining the occurrence of infectious viruses in water. The ability to detect infectious viruses in environmental samples is important for predicting health risks to the public. However, not all enteric viruses can be readily detected by cell culture. Some enteric viruses do not produce a clear cytopathogenic effect, which is necessary for visual detection. This can underestimate the concentration of viruses in a sample. For other enteric viruses, such as some noroviruses, successful use of cell culture has only recently been accomplished using new three-dimensional cell culture techniques (Straub et al., 2007). While some viruses grow rapidly in a few days, most cell culture assays require several weeks to confirm negative results and to detect slow-growing viruses. In addition, plaque assays may underestimate virus concentration since, as mentioned previously, not all viruses produce a clear cytopathogenic effect. Other reasons for underestimation include aggregation of viruses in a sample, resulting in an individual plaque being infected with more than one virus (Teunis et al., 2005); the inability to maintain the cell monolayer for sufficiently long periods for some slow-growing viruses to produce a visible plaque; and the presence of fast-growing enteric viruses, which can lead to an underestimate of the concentration of slow-growing viruses (Irving and Smith, 1981; Fong and Lipp, 2005).
PCR-based detection methods have been developed for most of the key enteric viruses of concern for waterborne transmission. Recent improvements in technology have also made what is now known as real-time or quantitative PCR (q-PCR) the PCR method most often used for detection and quantitation of enteric viruses. It should be noted, however, that quantitation using q-PCR is not yet very precise and is reliant on materials that are currently not routinely available. Also, direct comparison of q-PCR results with cell culture results is not possible. PCR detection methods have some advantages over cell culture methods: they are rapid (results within 24 h), highly sensitive and, if properly designed, very specific, in comparison with cell culture. The main disadvantages of PCR-based methods are that they are unable to determine if the viruses are infectious and they are subject to inhibition by common environmental compounds, such as humic and fulvic acids, heavy metals and phenolic compounds (Fong and Lipp, 2005). Inhibitors can be removed from the samples, but this requires additional processing and results in loss of sensitivity. Knowing if a virus is infectious is important from a public health perspective to determine if there is a public health concern. For example, a recent study looking at adenoviruses in a water source was unable to find infectious virus using cell culture, but approximately 16% of the samples tested positive for the presence of adenoviruses using q-PCR (Choi and Jiang, 2005). These limitations need to be considered when interpreting PCR results.
Methods integrating cell culture and PCR make it possible to shorten the processing time (compared with cell culture alone) and to detect infectious viruses. Cell culture methods can also be combined with immunological methods to improve virus detection. Integrated methods have been reported to be both sensitive and specific, including for those viruses that are difficult to assay using conventional cell culture, such as adenoviruses and rotaviruses (Payment and Trudel, 1993; Jothikumar et al., 2000; Hurst et al., 2001; Payment, 2001, 2007; Reynolds et al., 2001; Greening et al., 2002; Ko et al., 2003). An additional advantage of combining cell culture with immunological or molecular methods is improvement in the sensitivity of the assay, as the infected cells amplify the quantity of virus, providing more target material for detection.
6.2 Detection of viral indicators
Methods for the detection of viruses in water are not practical for routine monitoring, and therefore various surrogate parameters (i.e., indicators) have been proposed to evaluate water treatment efficiency or to indicate the presence of enteric viruses in water (Deere et al., 2001; WHO, 2004). The indicators proposed to date include E. coli, total coliforms, enterococci, Clostridium perfringens spores and bacteriophages.
6.2.1 E. coli
Escherichia coli is the microbial indicator that is used most often for determining faecal contamination of water sources. Further information on detection methods for E. coli is provided in the Guideline Technical Document on E. coli (Health Canada, 2006a).
6.2.2 Total coliforms
Total coliforms, although not an indicator of faecal contamination, are useful as an indicator of overall water quality. Further information on detection methods for total coliforms is provided in the Guideline Technical Document on total coliforms (Health Canada, 2006b).
6.2.3 Enterococci
Enterococci can be used to indicate faecal contamination and indirectly indicate the presence of viruses (U.S. EPA, 2000; Ashbolt et al., 2001). Standardized methods for the detection of enterococci in water have been published (APHA et al., 1998; U.S. EPA, 2002a,b). Commercial kits for the detection of these indicators are also available.
6.2.4 Clostridium perfringens
Clostridium perfringens spores are indicators of both recent and past faecal contamination, but they are not as numerous as coliforms in faeces or contaminated water. Clostridium perfringens spores are also used as indicators of treatment efficiency. Standardized detection methods for C. perfringens have been published (ASTM, 2002; HPA, 2004).
6.2.5 Bacteriophages
Three types of bacteriophages are generally used as indicators: the somatic coliphages, male-specific F-RNA bacteriophages (also referred to as F-specific coliphage) and Bacteroides phages (i.e., phages infecting Bacteroides fragilis, B. thetaiotaomicron and Bacteroides strain GB-124). In the United States, standardized methods for the detection of somatic and male-specific coliphages have been developed (U.S. EPA, 2001a,b). The International Organization for Standardization (ISO) has also published standardized methods (ISO 10705 series) for the detection of bacteriophages (Mooijman et al., 2001, 2005).