DECLARATION 2 PUBLICATIONS
1. INTRODUCTION
1.7 Health risk assessment
1.7.3 Exposure assessment
used in this case with acceptable results. This tool does not take into account the reaction when more than one infectious agent is present (Soller, 2006). In order to compare the severity of different diseases, use is often made of disability adjusted life years (DALYS). These compare the severity of the diseases by taking into account the number of days lost through premature death or disability as compared to the normal life span free of disease. To make use of this system, it is necessary to have data on the life expectancy of a community, the average duration of a disease and its severity (WHO, 2006a). This is not always available.
means of detecting the organisms is necessary for assessment. There are various methods for the detection of the organisms used as indicators as well as for the detection of pathogens themselves. Each has its benefits and drawbacks and these are thoroughly covered in the standard works such as the Standard Methods published by the American Water Works Association (APHA/AWWA, 2005). It is rare that pathogens are examined directly in wastewaters and it is more usual for indicators to be used (Gerba, 2000; Rose et al., 2004;
Roesner et al., 2006).
The most studied microorganisms are the bacteria which have been used as predictors of potential health risk for decades. The most commonly used bacterial indicators are the coliforms, with the focus gradually narrowing firstly from total coliforms to the thermotolerant coliforms and then to Escherichia coli itself as being the coliform most closely linked to the presence of faecal matter (APHA/AWWA, 2005). Enterococcus sp are now also often used (Rose et al., 1986; Christova-Boal et al., 1996; Ottoson and Stenström , 2003; Rose et al., 2004). In classical studies, the ability of these microorganisms to grow on or in selective culture media such as agars or broths has been used. These media have become more specific and selective with time.
Originally the ability to ferment lactose in a defined period at a defined temperature formed the criterion of identification of E. coli (APHA/AWWA, 2005). Current cultural techniques are now often based on the expression of enzymes peculiar to the coliforms or E. coli itself such as β-galactosidase and β-glucuronidase respectively (APHA/AWWA, 2005) This has allowed the identification process to be accelerated from several days to less than 24 hours. Media utilising the expression of these enzymes for identification purposes are produced by several companies such as Merck and IDEXX. Such techniques are also available for enterococci and some other organisms of interest in health risk analysis, where other specific enzymes are targeted (APHA/AWWA, 2005). These methods have the advantage of being relatively inexpensive and simple enough to be performed routinely in most water microbiology laboratories. For many of the bacteriological pathogens themselves however, pre-enrichment followed by enrichment and then selective culture are still needed for the classical analyses (APHA/AWWA, 2005) and therefore results are qualitative rather than quantitative. Examples of this are Salmonella sp., Shigella sp. and Vibrio cholerae. This enrichment process is time consuming but many of these analyses are also performed routinely by water laboratories. For the development of the databases of exposure, this time factor is not of importance, but it could prove critical in the identification of an outbreak of illness in a community.
The culture-based techniques allow the examination of large volumes of sample when filtration, immune-capture or centrifugation is used to capture the organism of interest. The greatest drawback of the cultural techniques is that they are not available for all organisms of interest, particularly the helminths and protozoa, as well as many viruses. In some cases, the organism has not as yet been grown in culture (e.g. Norovirus) and in other cases the organism can go into a viable but non-culturable state in which it can still cause infection but is un-culturable by standard techniques, e.g. Vibrio cholerae (Hurst et al., 2002).
The tissue culture technique required for the identification of human enterovirus is exacting and requires specialized tissue culture facilities unavailable to most basic laboratories. For this reason, indicator virus such as somatic or F-specific coliphage or the phages of other faecally- linked bacteria are regularly used as indicators of potential entero-viral contamination (APHA/AWWA, 2005). Other techniques such as the polymerase chain reaction (PCR), immune-capture and fluorescent in situ hybridization (FISH) are available at more sophisticated research laboratories. As these techniques were not available for this study, they are not discussed further here.
Whilst each of the above techniques could be used in the assessment of exposure, it is likely that classical culture techniques will continue for some time as the backbone of analysis, perhaps supported by more sophisticated techniques where possible. As indicators are generally used instead of pathogens, a relationship between the two needs to be used to estimate health risk. Brown (2009), states that indicators overestimate the presence of pathogens by approximately 1 000 times, whilst according to Hamilton et al. (2007b), when wastewater was used as an aerosol to irrigate crops, noincreased health risk was attributed to waterswith 104 to 105 total coliforms L–1, implying a ratio of less than one pathogen per 104 indicator organisms. According to Peterson and Ashbolt (2002) bacterial and viral pathogens normally range between 105 and 1010 per gram of faeces and Mara and Horan (2003) state that E. coli is present at levels of 107 to 109 whilst pathogens are only intermittently present at similar levels. Ottoson (2005) presents levels of 10 to 104 for Salmonella in sewage along with levels of up to 105.4 for E. coli and 102.4 to 104.6 for Enterococcus in greywater. In this dissertation a conversion factor of 10-2 has been used for E. coli and Staphylococcus, a factor of 10-4 for total coliforms, and Enterococcus has been considered as being present at approximately equivalent levels to pathogens in order to give a very conservative conversion to potential pathogens.