Identifying Future Drinking Water Contaminants (1999) / Chapter Skim
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9 Methods to Identify and Detect Microbial Contaminants in Drinking Water
9 Methods to Identify and Detect Microbial Contaminants in Drinking Water
Pages 173-205
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From page 173... ...
These concerns led to the development of criteria and standards for bacteriological quality intended to protect against excessive risks from enteric bacterial pathogens such as Salmonella typhi and other nontyphoid Salmonella spp., Shigelia spp., and Vibrio cholferae. The infectious disease risks in drinking water supplies from enteric viruses (such as hepatitis A virus)
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From page 174... ...
How should analytical methods for detection, characterization, and quantification of microbial contaminants be applied to the process of identifying, characterizing, and quantifying the risks from waterborne pathogens being considered for regulation? What analytical methods are available for use in identifying, quantifying, and characterizing microbial pathogens in drinking water for possible regulation?
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From page 175... ...
This effort resulted in a modified quantitative risk assessment system that specifies the criteria, information needs, and analytical approaches for quantitative risk assessment for waterborne microbes (See Figure 9-1~. Presently, the EPA/ILST system for quantitative risk assessment of microbes in water is being applied by several research groups to determine its utility, and other researchers will peer review the QRA products for utility, Methods to Identify and Detect Microbial Contaminants in Drinking Water ~ 75
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From page 176... ...
Considering that the EPA and many of the nation's scientists in the areas of water microbiology, infectious diseases, water treatment, epidemiology, and risk assessment invested much effort and time in the development of this system, it seems appropriate to interface it with the process for microbial contaminant selection. Furthermore, it can be said that the elements of the EPA/ILST QRA system for microbes rely on the same types and sources of data and at least some of the same analytical methods that would be used in any structured process to identify, select, characterize, and prioritize candidate microbial contaminants for possible regulation in `drinking water (See Figure 9-2~.
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Methods to Identify and Detect Microbial Contaminants in Drinking Water ~ 77
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From page 178... ...
Another important feature of some of these viruses, as well as many of the bacterial and parasitic pathogens of concern in drinking water, is that they have known or suspected animal hosts and therefore are transmissible directly or indirectly from other animals to humans. The potential for animal-to-human transmission creates concerns about contamination of drinking water supplies with animal wastes containing these pathogens.
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From page 179... ...
in the case of some of these waterborne bacteria their risks to human health from ingestion or inhalation of water or contact with water are uncertain because they have not been conclusively documented by outbreaks or other epidemiological evidence of waterborne disease. However, their presence in drinking water and the uncertainty of their risks to human health from drinking water exposure suggest the need for further investigation and analyses.
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From page 180... ...
The role of the aquatic environment as a reservoir for and source of emergence of new virulent strains of bacteria is becoming increasingly recognized in the case of some bacteria. For example, the genes coding for the cholera toxin of Vibrio cholerae are borne on and can be infectiously transmitted 180 Identifying Future Drinking Water Contaminarlts
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From page 181... ...
Many of the others, including Giardia lamblia, Cryptosporiclium parrum, Toxoplasma gondii, Balantidium coli, as well as the microsporidia have nonhuman animal reservoirs that contribute to their presence in drinking water supplies and sources. The microsporidia are among the most ubiquitous protozoan parasites of animals and Methods to Identify and Detect Microbial Contaminants in Drinking Water 181
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From page 182... ...
A particular challenge to the detection of protozoans of public health concern in drinking water is that many of the currently available and widely used analytical methods, especially the various microscopic techniques (such as brightfield, immunofluorescent, phase contrast, and differential interference contrast microscopy) cannot always distinguish the human pathogenic genera, species, and strains from the many others that are noninfectious and therefore harmless to humans (EPA, 1996, 1998)
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From page 183... ...
A variety of assay and characterization procedures can be applied to the detection and quantitation of target pathogens in drinking water. These include enumeration or quantal assays of total, viable, active, or infectious target microbes and their distinction from nontarget microbes based on identification or characterization of genus, species, type, strain, and virulence or other relevant properties.
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From page 184... ...
Alternative filtration methods have been used to recover and detect bacteria and parasites, including microporous filters composed of nylon, 184 Identifying Future Drinking Water Corltamirlants
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Furthermore, recoveries from the filters are highly variable, resulting in large coefficients of variation. Additionally, because the target sample volumes are 100 L or more, there are Methods to Identify and Detect Microbial Contaminants in Drinking Water 185
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From page 186... ...
The current EPA-approved ICR method to detect culturable enteric viruses in drinking water supplies specifies use of commercially available, electropositive filter (EPA, 19961. Viruses adsorbed to both electronegative or electropositive filters are subsequently eluted and recovered by passing a relatively small volume of aqueous elusion medium through the filter.
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From page 187... ...
A variety of physical, chemical, and immunochemical methods are used for this purpose. Sedimentation and flotation using density solutions or gradients are Methods to Identify arid Detect Microbial Contaminants in Drinking Water ]
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From page 188... ...
For example, particle size exclusion chromatography using Sephadex gel has been used to separate enteric viruses from solutes in the sample matrix and achieve a high degree of purification for subsequent detection by cell culture or nucleic acid amplification-nucleic acid hybridization methods (Sobsey et al., 1996~. Extraction methods using organic solvents, detergents, Iytic enzymes, and other chemicals have been used to partition target microbes from impurities in the sample (phase partitioning)
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From page 189... ...
Culture or Infectivity Assays for Bacteria Culture of bacterial pathogens is widely used in clinical diagnostic microbiology, and, for many waterborne bacterial pathogens, culture methods are adapted from those initially developed for medical diagnosis. Typical approaches are culturing the target microbes from specified volumes of water by preenrichment and enrichment methods using broth media or filtering the organisms from specified volumes of water and placing the filters in broth or agar culture media.
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All 190 Identifj~ir~g Future Drinking Water Contaminants
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From page 191... ...
Some enteric viruses are readily cultured in susceptible Methods to Identify and Detect Microbial Contaminants in Drinking Water 191
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From page 192... ...
Detection is also possible by PCR, immunoblotting, and electron microscopy. For other waterborne parasites, such as Giardia lamblia and Cyclospora 192 Identifying Future Drinking Water Contaminants
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From page 193... ...
Combined Cell Culture and Nucleic Acic! Detection and Amplif cation of Waterborne Pathogens For some enteric viruses and protozoan parasites, detection and quantitation of the infectious pathogens are improved by using the combined techniques of cell culture and then nucleic acid hybridization and/or amplification.
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From page 194... ...
Nucleic acid methods for pathogen detection, quantitation and characterization in water are described in the next section. Detection of Waterborne Pathogens by Nucleic Acid Methods Methods for nucleic acid cloning, synthesis, hybridization, sequencing, and other analyses now make it possible to detect pathogens in environmental samples.
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From page 195... ...
For example, methods to detect enteric viruses in water by nucleic acid amplification using PCR and RT-PCR have advanced in recent years to the point where they have been successfully applied to investigating waterborne outbreaks caused by nonculturable human caliciviruses viruses (Bellar et al., 1997) and surveying for enteric viruses in drinking water sources (Abbaszadegan et al., 1998~.
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From page 196... ...
This can be done by measuring or quantifying the amount of amplicon (DNA product) produced under defined PCR or RT-PCR conditions using electrochemoluminescence, immunoassays, fluorescence signal increase (using a fluorescent primer that 196 Identifying Future Drinking Water Corltaminants
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From page 197... ...
For PCR or RT-PCR, the target nucleic acid is then liberated from the purified and concentrated pathogens by one or more of several physical methods, such as heat, freeze-thaw, sonication and bead beating, and/or Methods to Identify and Detect Microbial Contaminants in Drinking Water 197
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From page 198... ...
For example, phase contrast and differential interference contrast microscopy are used to visualize 198 Identifying Future Drinking Water Coniaminants
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From page 199... ...
Enzyme immunoassays often are done on a solid phase to which the pathogen antigens have been applied, such as a membrane filter or the bottom of a microtiter plate well. To provide increased specificity and to facilitate separation of the target microbial antigen from other particles and solutes in the sample, the target antigen can be captured on the solid phase using a specific antibody Methods to identify and Detect Microbial Contaminants in Drinking Water 199
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From page 200... ...
. As with enzyme immunoassays, agglutination tests are too insensitive to directly detect and quantify most waterborne pathogens in drinking water and other aquatic samples.
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From page 201... ...
Waterborne pathogen detection and quantitation by nucleic acid amplification and hybridization methods has advanced greatly in the last decade, has been successfully applied to pathogen detection in field samples, and appears to be a most promising technology for the future. However, further Methods to Identify and Detect Microbial Contaminants ir' Drinking Water
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From page 202... ...
Viability and activity methods to detect and quantify waterborne pathogens also continue to improve, and they have been applied to field samples of water on a limited basis. However, there continues to be uncertainty about the ability of many of these methods to detect truly infectious waterborne pathogens, and many of the existing methods are cumbersome, tedious, and unable to detect low concentrations of target pathogens in water.
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From page 203... ...
and integrated cell culture-PCR (CCPCR)
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From page 204... ...
and viruses in drinking water. Journal of the American Water Works Association 83~11~:76-84.
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1993. Using a conceptual framework for assessing risk to health from microbes in drinking water.
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