Indicators for Waterborne Pathogens (2004) / Chapter Skim
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5 New Biological Measurement Opportunities
5 New Biological Measurement Opportunities
Pages 196-242
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From page 196... ...
This chapter provides an overview and discusses various issues and methods for making biological measurements. It underscores some of the key issues in making measurements both generically and specifically for pathogens and indicators of waterborne pathogens.
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From page 197... ...
Small spatial scales but long temporal scales (area C) may be useful in understanding "typical" conditions at a water supply intake on a river system for the purposes of developing treatment configurations to meet drinking water standards for finished water.
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From page 198... ...
. The current choices of detection methods for indicator bacterial species or groups were motivated by the associated technical difficulties in culturing many types of waterborne pathogens.
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. Typical culture methods for pathogen and indicator bacteria in water and other environmental samples greatly underestimate the true concentrations of viable and potentially infectious cells -- sometimes by as much as a thousandfold (Colwell and Grimes, 2000; Ray, 1989)
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From page 200... ...
. Because of the lack of scientific agreement of the public health significance of VBNC bacteria and the objections of some authorities even to the use of this terminology, this report does not attempt to address the VBNC issue in the context of microbial indicators of pathogens and human health risks from waterborne pathogens.
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From page 201... ...
Cells contain both DNA and RNA, whereas viruses contain either DNA or RNA but never both. The choice of nucleic acids and the ways in which they are measured in microorganisms can provide different kinds of information with regard to microbial identification, viability, and infectivity or culturability.
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From page 202... ...
Such molecules on the surfaces of cells and viruses often have high specificity or uniqueness in identifying a microorganism. Immunoassays can be used to detect, identify, and quantify such polysaccharides or glycoproteins, again using specific polyclonal or monoclonal antibodies raised against the microbe or the specific target molecules.
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From page 203... ...
When they do infect host cells, viruses begin to produce new, virus-specific molecules that can be targeted for detection by molecular and other chemical methods as evidence of their presence, infectivity, and concentration. Virus-specific nucleic acids, such as mRNA and proteins, including both structural and nonstructural proteins, can be targeted for detection by chemical, immunochemical, and molecular methods.
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From page 204... ...
Cell culture methods are now being used and have been found to be statistically comparable to animal infectivity for the determination of infectious oocysts (Slifko et al., 2002)
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From page 205... ...
However, as discussed below, all four components of the process must be considered to ensure accurate analysis of microbial water quality. Collection and Sampling Issues The first step in performing a measurement is collecting a sufficiently representative sample, and this remains one of the most challenging problems in water quality monitoring.
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From page 206... ...
Recent evidence from food microbiology and foodborne disease outbreaks indicates that measures of central tendency and the use of logarithmic transformations of microbial concentration data for the purposes of calculating geometric means and corresponding logarithmic measures of dispersions may be inappropriate for extrapolating to higher exposures and estimating corresponding health risks (Paoli, 2002)
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From page 207... ...
The widespread use of logarithmic transformations and measures of central tendency and dispersion of log-transformed data to estimate exposures and health risks needs to be reconsidered in water microbiology, epidemiology, and health risk assessment. An important characteristic of sampling when there is the likelihood of only low-level detection is that, although the species (microorganism or chemical)
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From page 208... ...
Statistically based sampling methods must be used in conjunction with analyses of multiple samples in order to estimate how microbial levels and human exposures change with varying water quality conditions. Sampling plans must be able to identify when and where microbial concentrations in water are at their highest levels since this is when human health risks are greatest.
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From page 209... ...
This method is referred to as RT-PCR. Quantitative Versus Qualitative Measurements It is important to recognize that while methods aimed particularly at treated drinking water or groundwater have focused on detecting presence versus absence of a particular indicator in a given volume, in most ambient water applications, obtaining quantitative information has been the ultimate goal.
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Various methodologies widely employed for making measurements of microorganisms in water samples are summarized later in this chapter and in Appendix C Data Collection and Processing Once measurement data have been scored, they are collected (compiled)
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. Direct Analysis of Microorganisms by Infectivity, Culturability, or Viability On the basis of indicating public health risk of infection or disease from exposure to microbially contaminated water, the direct measurement of a pathogen or a reliable microbial indicator of pathogens by culture or infectivity is generally considered the "gold standard," and both should be the goal of any new measurement technique.
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From page 212... ...
Molecular-based assays using either PCR or RT-PCR methodologies to amplify DNA or RNA targets extracted from infected cells or oligonucleotide probes that can detect nucleic acids in situ have also been developed for speciation and genotyping. In a study of surface waters and filter backwash waters, infectious oocysts of a variety of strains were detected in 4.9 and 7.4 percent of the samples, respectively, using cell culture methods (DiGiovanni et al., 1999)
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From page 213... ...
In the latter case, the individual components may not be unique to the particular microorganism of interest, but their concentration, co-occurrence, or sequence generates an overall response profile characteristic of the microorganism. Nucleic Acid Analysis DNA sequences can be present at only one copy per cell, which poses an extremely difficult detection challenge.
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From page 214... ...
Although these molecular biology methods directed at nucleic acids were developed primarily for the Human Genome Project1 with application in clinical medicine, these powerful techniques have direct applicability to waterborne pathogen or indicator detection is clearly feasible and has already been done (Cook, 2003; Griffin et al., 2003; Keer and Birch, 2003)
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From page 215... ...
It is important to recognize that the detection of waterborne pathogens or indicators whether viable and potentially infectious or not, may provide sufficient information to assess vulnerability to contamination or to make decisions about public health risk (see also Chapters 4 and 6)
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As the methods for recovery, concentration, and purification of target microbes and their nucleic acids are further improved for the application of various nucleic acid methods, including microarray technology, it is likely that these approaches will become more widely applicable to the detection, quantification, and identification of microbes in water and other environmental media. The committee concludes that the introduction of molecular techniques for nucleic acid analysis is a growth opportunity for the field of waterborne pathogen detection and recommends that U.S.
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From page 217... ...
Cryptosporidium parvum oocysts were found in the source water at the time of outbreak, and a sharply decreasing level of oocysts in sewage samples was observed over a three-month period following the outbreak. The use of immunocapture technologies in conjunction with electrochemical detectors is one of several approaches to rapid and improved immunological detection of waterborne pathogens and indicators.
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From page 218... ...
Significant advances will be required before mass spectrometry can be used in the field because the instruments are generally large, and require significant amounts of power. Attributes of Methods All of the methods described thus far have both positive and negative aspects associated with their use in the detection of waterborne pathogens or microbial indicators of pathogens.
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From page 219... ...
With new and emerging technologies, it is likely to become possible to simultaneously measure multiple analytes in a water sample, thus providing a better basis for judging the microbial quality of the water from which it is taken and any associated health risks. Although any of these methods can be used for pathogen detection and identification, the simultaneous use of multiple capture methods based on orthogonal detection principles (e.g., antibodies and nucleic acid probes)
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From page 220... ...
220 high high to to moderate moderate possible high high high high Component to applicable to applicable but to to to to Cell Moderate Low Moderate Not Low Not No, Low Low Low Low Attributes Desirable rapid high by to to Acid moderate possible to but Nucleic High High High High Moderate Low No, Moderate Moderate Moderate Low Methods Detection rapid high to to high moderate to to Microbial of Immunological High High Low Low Moderate Low No Moderate Moderate Moderate Low high Categories to moderate moderate high high high high to to to to to to Major Culture Low High Moderate Moderate Low Moderate Yes Low Low Low Low of or Comparison personnel desired 5-1 to results sensitivity viability Feasibility and field requirements Attributes of organism in applicability requirements target infectivity Training Utility Cost Volume TABLE Method Specificity Broad Precision Adequate Rapidity Quantifiable Measures Logistical
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From page 221... ...
Organizations Involved in Developing Standards Several organizations and associations are involved in the development of standard methods for evaluating water quality or microorganisms in water (see Box 5-2 and Table 5-2)
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. As noted throughout this report, EPA currently has published methods for various bacterial, protozoan, and viral indicators and pathogens in response to specific rules and programs under the Safe Drinking Water Act (SDWA)
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To date, the standardization of microbiological methods in the United States has generally followed one or more models, but most have in common consideration of the intended application, and one typical route to standardization is inclusion in the aforementioned Standard Methods for the Examination of Water and Wastewater. Another typical route to the standardization of a microbiological
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American Public Health Association (APHA; Not-for-profit health, drinking water, www.apha.org) ; American Water Works and wastewater associations Association (www.awwa.org)
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is approved for use by the EPA EPA's Office of Science and Technology is responsible for preparing standards to be used in support of government regulations. EPA publishes laboratory analytical methods that are used by industrial and municipal facilities in analyzing the chemical and biological components of wastewater, drinking water, and other environmental samples required by EPA regulations under the authority of the CWA and SDWA (see also Tables 1-1 and 1-2)
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From page 226... ...
Accuracy Studies There are generally three approaches for determining the accuracy of a biological measurement method. First, a sample with a known concentration is tested; however, reference standards for microorganisms may often be difficult to obtain so this approach is not used very often.
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In addition, maintenance of instruments, stability of reagents, and types of controls to be used will have to be described in the validation of a microbiological method. The level of false positives and false negatives associated with the use of a method can be assigned through the validation process.
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It also focused on compliance monitoring and thus (for example) did not provide incentives for testing methods to address microorganisms on the 1998 Drinking Water Contaminant Candidate List (EPA, 1998; see also Chapter 6)
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. Recent and ongoing major developments in new methods in microbiology with applications for public health-related water quality have necessitated a new approach for their rapid assessment, standardization, and validation.
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Greater efforts are required to support methods development for new and emerging microbial detection technologies, for more pathogens, and for new and improved candidate indicators of waterborne pathogens. Approaches for the development and maintenance of an on-line database of new microbiological methods for the analysis of water should be investigated.
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CHALLENGES AND PITFALLS OF NEW TECHNOLOGY A variety of challenges and potential pitfalls will be associated with developing and using improved and rapid methods for microbiological water quality compared with continued use and reliance on time-tested, widely accepted, traditional microbiological methods. These are discussed below.
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From page 232... ...
For example, a laboratory that routinely conducts less than 100 Salmonella tests per week has little or no need for a sophisticated, automated, and very expensive instrument that can perform thousands of tests per week. The committee recognizes the lack of technical, infrastructure, and financial resources required to implement water monitoring in many parts of the United States and recommends that efforts be made to support the development of inexpensive and rapid fieldable methods for testing microbial water quality.
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From page 233... ...
Although the microbiological community is moving ever closer to that reality, it is not yet achievable. LOOK TO THE FUTURE Today's measurement techniques are aimed at detecting viable organisms or specific components present in the organism of interest and correlating their presence to human health risk assessment.
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From page 234... ...
Such systems can be built on chips in which a sensor is present for every analyte of potential interest. Alternatively, such arrays may be able to measure patterns of response in which signatures of various analytes signify the presence of various water quality conditions, organisms, or toxins.
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Consistent with its previous related recommendations, the committee recommends that epidemiologic studies should be designed and performed to both establish the correlation among indicator and pathogen concentrations and health risk, and reestablish the health risks associated with existing and new pathogen indicators for new (non-culture-based) detection methods.
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From page 236... ...
Furthermore, the widespread use of logarithmic transformations and measure of central tendency and dispersion of log-transformed data to estimate exposures and health risks needs to be reconsidered in water microbiology, epidemiology, and health risk assessment. At present, most water quality measurement methods are single-parameter based.
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From page 237... ...
With the prospect for such an enormous amount of data to be collected from the many sensors disposed on arrays, the potentially large numbers of sensor arrays deployed for water monitoring, and the continuous data streams coming from these sensor networks, greater attention must be paid to the fields of data analysis, intelligent decision making, and archiving. There is need for a database that compiles and serves as a clearinghouse for all microbiological methods that have been utilized and published for studying water quality.
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From page 238... ...
· There is a need to address the sensitivity of miniaturized detection methods and ensure that sample collection, preprocessing or processing, concentration, and purification are given adequate attention to achieve representativeness and have the ability to detect microbial concentrations posing unacceptable health risks. This represents one of the most important technological challenges to the analysis of pathogens and indicators in water and other environmental samples and will become more important with the introduction of micro- and nanotechnologies.
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· The committee recommends that epidemiologic studies should be designed and performed to both establish the correlation among indicator and pathogen concentrations and health risk, and reestablish the health risks associated with existing and new pathogen indicators for new (non-culture-based) detection methods.
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1999. Detection of infectious Cryptosporidium parvum oocysts in surface and filter backwash water samples by immunomagnetic separation and integrated cell culture-PCR.
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2001. Develop ment and application of a quantitative, specific assay for Cryptosporidium parvum oocysts de tection in high-turbidity environmental water samples.
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1999. Methods to identify and detect microbial contaminants in drinking water.
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