9
Methods to Identify and Detect Microbial Contaminants in Drinking Water

Mark D. Sobsey

The transmission of infectious diseases via contaminated water continues to be a risk to public health in the United States and throughout the rest of the. world. Source and finished drinking waters are vulnerable to microbial pathogen contamination from a variety of sources of human and animal fecal wastes and from the introduction and proliferation of nonfecal pathogenic microbes. Throughout most of the modem history of drinking water supply, concerns about pathogenic microbes have focused on enteric bacteria of human fecal origin. 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., Shigella spp., and Fibrio cholerae. The infectious disease risks in drinking water supplies from enteric viruses (such as hepatitis A virus), enteric parasites (such as Entamoeba histolytica and Giardia lamblia), and nonfecal bacterial pathogens (such as Legionella spp.) were not recognized until more recent times. These risks were recognized initially by the occurrence of waterborne outbreaks of disease mused by these pathogens.

Until recently there were no formal, legally required processes to identify or consider new or emerging water. me pathogens in the United States. It was only with reauthorization of the Safe Drinking Water Act in 1996 that the U.S. Environmental Protection Agency (EPA) was required to identify through a structured process candidate microbial pathogens for possible regulation in drinking water supplies. Prior to this the agency used an informal and largely reactive process to recognize, identify, and prioritize microbial pathogens for possible regulation. The new requirement for a proactive rational process to identify and consider microbial pathogens for possible regulation in drinking water is the essential motivation for this paper. As part of this process it is necessary to detect and quantify microbial pathogens in drinking water and its sources; to establish dose-response relationships as an essential step in health effects characterization for waterborne pathogens; and to identify, characterize, and quantify the virulence properties of these pathogens that influence their human health effects.



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--> 9 Methods to Identify and Detect Microbial Contaminants in Drinking Water Mark D. Sobsey The transmission of infectious diseases via contaminated water continues to be a risk to public health in the United States and throughout the rest of the. world. Source and finished drinking waters are vulnerable to microbial pathogen contamination from a variety of sources of human and animal fecal wastes and from the introduction and proliferation of nonfecal pathogenic microbes. Throughout most of the modem history of drinking water supply, concerns about pathogenic microbes have focused on enteric bacteria of human fecal origin. 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., Shigella spp., and Fibrio cholerae. The infectious disease risks in drinking water supplies from enteric viruses (such as hepatitis A virus), enteric parasites (such as Entamoeba histolytica and Giardia lamblia), and nonfecal bacterial pathogens (such as Legionella spp.) were not recognized until more recent times. These risks were recognized initially by the occurrence of waterborne outbreaks of disease mused by these pathogens. Until recently there were no formal, legally required processes to identify or consider new or emerging water. me pathogens in the United States. It was only with reauthorization of the Safe Drinking Water Act in 1996 that the U.S. Environmental Protection Agency (EPA) was required to identify through a structured process candidate microbial pathogens for possible regulation in drinking water supplies. Prior to this the agency used an informal and largely reactive process to recognize, identify, and prioritize microbial pathogens for possible regulation. The new requirement for a proactive rational process to identify and consider microbial pathogens for possible regulation in drinking water is the essential motivation for this paper. As part of this process it is necessary to detect and quantify microbial pathogens in drinking water and its sources; to establish dose-response relationships as an essential step in health effects characterization for waterborne pathogens; and to identify, characterize, and quantify the virulence properties of these pathogens that influence their human health effects.

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--> Purpose The purpose of this report is to consider and address the following questions: How should microbial contaminants for possible regulation in drinking water be identified, characterized, and quantified with respect to their risks to public health? What should be the essential elements of the process for waterborne microbial pathogen identification and characterization? What should be the basis for prioritizing, ranking, or choosing among the many potential drinking water pathogens for possible regulation? How should the microbial pathogen identification and selection process be integrated into the overall process of improving drinking water quality and reducing health risks through drinking water regulations? 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? Analysis Need for a Risk-Based Approach The recognition, identification, prioritization, and characterization of microbial pathogens in drinking water should be risk based and should consider the relationships and interactions of the microbes, their hosts, and the environment. The microbial world consists of a wide variety of different types or classes of microbial agents potentially present in water. Viruses, bacteria, protozoans, fungi, and algae are widespread in soil, sediments, water, air, and food and on objects and surfaces with which humans have contact ("fomites"). Most of these microbes are not pathogenic (harmless) and are incapable of infecting or colonizing immunocompetent persons unless they somehow gain access to sterile internal sites in the body (such as the bloodstream and various organs) through trauma, surgery, or other such means. However, persons with immunodeficiencies are at risk of infection, colonization, and illness from microbes considered nonpathogenic for immunocompetent persons. Therefore, recognition and identification of a possible waterborne pathogen depends in part on the susceptibilities of the population to infection, colonization, and illness from a microorganism. Some pathogens are always potentially pathogenic and are often referred to as "frank" pathogens. Other pathogens are never or rarely pathogenic for immunocompetent and otherwise "healthy" people. However, these microbes can sometimes cause infection, colonization, and illness in persons who have an immune deficiency, have other conditions that make them susceptible, or because they encounter the microbe in an unusual or atypical way. Such microbes are sometimes referred to as "conditional" or "opportunistic" pathogens. As previously noted, there are nonpathogenic microbes in the environment that are capable of infecting, colonizing, and causing illness in humans only if they are able to dramatically breach the body's natural barriers

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--> and proliferate in normally sterile sites within the body. These nonpathogenic or "saprophytic" microbes are common in aquatic and other environments. A waterborne pathogen may emerge or acquire increased public health importance because of changes in host susceptibility to infection. Factors influencing host susceptibility in the population include increases in the number of immunocompromised persons, increased use of immunosuppressive agents (among persons receiving cancer chemotherapy or undergoing organ transplants), increases in the elderly segment of the population, and poor nutrition. In identifying and prioritizing emerging waterborne pathogens the susceptibilities of these higher-risk population subgroups to specific infectious diseases is an important consideration (Morris and Potter, 1997). The relationships between waterborne microbes and their human hosts are complex and are influenced by a variety of factors involving the characteristics and conditions of the microbe, the human and in some cases animal hosts, and the environment. Therefore, it seems necessary to identify, characterize, and quantify these relationships in order to determine if a potentially waterborne microbe should be considered or classified as a drinking water contaminant for possible regulation. Furthermore, the need to prioritize or otherwise determine the importance of a microbe for possible regulation in water suggests that a structured and quantitative approach must be used for such an evaluation or assessment. Adapting Quantitative Risk Assessment to Recognize, Identify, Prioritize, and Characterize Drinking Water Pathogens Over the past two decades considerable progress has been made in quantitative risk assessment (QRA) for making management decisions about waterborne pathogens. Initially, the National Academy of Sciences/National Research Council risk assessment strategy was adapted to assess microbial risks (Regli et al., 1991; Rose and Gerba, 1991; Rose et al., 1991; Sobsey et al., 1993). This process consists of hazard identification, exposure assessment, effects assessment, and risk characterization. Using this approach, quantitative risk assessments were done initially for several recognized waterborne pathogens, such as Giardia lamblia and rotaviruses. More recently, the process of quantitative risk assessment for pathogens in water was reconsidered and revised through a consensus-building International Life Sciences Institute (ILSI) workshop process (ILSI Risk Science Institute Pathogen Risk Assessment Working Group, 1996). 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/ILSI 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,

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--> Figure 9-1 Risk assessment for waterborne microbial pathogens:  EPA/ILSI paradigm. clarity, and transparency. The EPA/ILSI QRA approach was not intended for the purpose of selecting microbial contaminants for possible regulation in drinking water. Furthermore, it certainly is not the only way to identify, prioritize, and assess the risks from microbes in drinking water. However, this microbial QRA system does specify information needs and analytical methods that can be readily adapted to the recognition, identification, prioritization, and initial characterization of risks from a possible waterborne microbe. 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/ILSI 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). Human Pathogens in Water: Classification, Sources, and Properties Known and potential human pathogens in water include the spectrum of agents ranging, in order of increasing complexity, from prions, to viruses, to bacteria, and other prokaryotes, and the microbial eukaryotes (the protists), including protozoans, fungi, and algae (Moe, 1997). Prions have not been implicated in waterborne disease, but recent evidence for human spongiform encephalopathies from ingestion of beef contaminated with bovine spongiform agents suggests that vehicles such as food and possibly water contaminated with prions pose a risk of exposure (Ironside, 1998; Knight and Stewart, 1998). Furthermore, these agents are very small compared to other microbes, which makes them difficult to remove by physical-chemical processes, and they are extremely resistant to virtually all physical and chemical agents, which makes them persistent in the environment and resistant to virtually all drinking water disinfectants.

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--> A. ELEMENTS CONSIDERED IN PATHOGEN OCCURRENCE   Spatial distribution (clumping, particle-association, clustering)   Concentrations in environmental vehicles and foods   Seasonality and climatic effects   Temporal distribution, duration, and frequency   Niche (potential to multiply or survive in specific media)   Amplification, die-off, persistence   Indicators/surrogates predictive of pathogen B. ELEMENTS CONSIDERED IN EXPOSURE ANALYSIS   Identification of water and other media   Unit of exposure   Temporal nature of exposure (single or multiple; intervals)   Route of exposure and transmission potential   Demographics of exposed population   Size of exposed population   Behavior of exposed population C. ELEMENTS CONSIDERED IN PATHOGEN CHARACTERIZATION   Virulence and pathogenicity of the microorganism   Pathological characteristics and diseases caused   Survival and multiplication of the microorganism   Resistance to environmental control measures   Host specificity   Infection mechanism and route; portal of entry   Potential for secondary spread   Taxonomy and strain variation D. ELEMENTS CONSIDERED IN HOST CHARACTERIZATION   Demographics of the exposed population (age, density, etc.)   Immune status   Pregnancy   Concurrent illness or infirmity   Nutritional status   Genetic background   Behavioral and social factors E. ELEMENTS CONSIDERED IN HEALTH EFFECTS   Morbidity, mortality, sequelae of illness   Severity of illness   Duration of illness   Chronicity or recurrence   Potential for secondary spread   Quality of life FIGURE 9-2 Microbial contaminant identification, selection and characterization using elements of the EPA/ILSI risk-based approach: Description of the information needs.

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--> Viruses A variety of enteric and respiratory viruses of humans and in some cases other animals as well are potential agents of waterborne disease. For many of these viruses the role of water has been clearly established because of documented waterborne outbreaks, or it is strongly suspected because the viruses have been detected in drinking water or its sources. Some of these viruses are shown in Table 9-1, but other viruses and virus groups may also pose risks from exposure via drinking water. A notable feature of all of these viruses except the coronaviruses and picobirnaviruses is that they are nonenveloped (consisting only of a nucleic acid surrounded by an outer protein coat or capsid). Nonenveloped viruses tend to be more resistant to various physical and chemical agents and more stable in the environment than the enveloped viruses, which probably contributes to their potential to cause waterborne disease. 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. As previously noted, similar concerns also apply to many bacterial and parasitic pathogens. Bacteria Many enteric and respiratory bacteria infect and cause morbidity and mortality in humans via the water route. Some of these bacteria also infect other TABLE 9-1 Important Viral Contaminants of Drinking Water Virus or Virus Group Outbreak or Detected in Water Animal Sources Enteroviruses: (polios, echos2, coxsackies2, etc.) Yes No Hepatitis A virus2 Yes No Hepatitis E virus Yes Pigs? Reoviruses Yes Yes Rotaviruses Yes Yesb Adenoviruses2 Yes Yesb Caliciviruses2: Norwalk, Snow Mountain, etc. Yes No for some; maybe for others Astroviruses Yes No Parvoviruses; picornaviruses Maybe Uncertain Coronaviruses No Uncertain Picobirnaviruses and picotrirnaviruses Unknown Maybe 2 On EPA's microbial contaminants priority list. b Humans and animals are usually infected by different ones but not always.

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--> animals, often asymptomatically, and some, such as the Legionella spp., the Mycobacteria spp., and the heterotrophic plate count bacteria, have abiotic environmental reservoirs. For many of these bacteria the role of waterborne transmission has been documented by waterborne outbreaks, or it is strongly suspected because the bacteria have been detected in drinking water and its sources (see Table 9-2). 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. The risks posed by various bacteria potentially present in drinking water differ among the various genera and species as well as within the same genus and species of a bacterium. These differences in risks to human health pose considerable challenges to the detection and identification of these bacteria in water. Similar concerns apply to the protozoan parasites, algae, and fungi. Strains or variants of the same genus and species of bacterium can differ dramatically in their ability to cause disease because this ability is largely dependent on the presence of virulence factors or properties. In some cases the virulence factors or properties of the bacterium responsible for disease are essential constituents of the cell. This appears to be the case for Salmonella typhi, the causative agent of typhoid fever, whose essential virulence properties are the O antigen (the lipopolysaccharide outer membrane of the cell wall; an endotoxin) and the Vi antigen (a capsule polysaccharide) (Salyers and Whitt, 1994; Levine, 1998). For many other bacteria, such as strains of Escherichia coli, Aeromonas hydrophila, and Yersinia enterocolitica, the ability to be a pathogen and cause disease is clearly associated with the presence of specific virulence properties that may or may not be present in specific strains or types. These virulence factors are often transmissible from one cell to another via transmissible plasmids or bacterial viruses (bacteriophages). Plasmids are extrachromosomal, small, circular DNA molecules that replicate separately from the bacterial chromosome and can move from one cell to another by a process called conjugation. Bacteriophages also can transmit virulence factors from one host cell to another, especially if the infecting bacteriophages do not kill the cell and instead integrate their DNA into the bacterial chromosome. Strains of a species bacterium possessing no virulence factors generally are not pathogenic and do not produce disease. Strains of the same species of bacterium possessing one or more specific virulence factors are pathogenic and capable of producing disease. Furthermore, the pathology and clinical features of the disease depend on the properties and activities of these virulence factors. For example, strains of E. coli lacking virulence properties generally are not pathogenic, and most humans and other mammals harbor about a billion E. coli cells per gram in their lower intestinal

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--> TABLE 9-2 Some Important Bacterial Contaminants of Drinking Water Bacterium/Group Outbreak or Detected in Water Animal Feces Non-Fecal Sources Salmonella spp. (except S. typhi) Yes Yes No Salmonella typhi Yes No No Campylobacter spp. Yes Yes No Escherichia cell Yes Yes Maybe Helicobacter pyloria Yes Unknown Unknown Heromonas hydrophilaa Yes Yes Yes Yersinia enterocolitica Yes Yes Yes Fibrio cholerae and some other Fibrio sp. Yes Yes Yes Shigella spp. Yes No No Legionella sppb. Yes No Yes Mycobacterium avium-intracellularea and other Mycobacterium spp. Yes Yes Yes Leptospira spp. Yes Yes Yes Heterotrophic plate Yes Yes Yes Count bacteria Yes Yes Yes a On EPA's microbial contaminants priority list. b On EPA's microbial contaminants priority list in groundwater. tracts with no adverse effects. However, E. cell strains possessing one or more virulence properties are capable of causing different diseases. For example, enterohemorrhagic strains of E. cell strains possess the Shiga-like toxin (SLT). They are infectious at relatively low doses (< one thousand cells) and cause a bloody diarrhea without fever that can advance to life-threatening hemolytic-uremic syndrome, a form of hemolytic anemia and kidney failure, especially in young children. Other strains of E. coli possess a heat-stable toxin (ST) and/or heat labile toxin (LT; similar to the cholera toxin of Fibrio cholerae). These enterotoxigenic strains of E. coli are infectious at higher doses (> one million cells) and cause disease by increasing adenylate cyclase (LT) and guanylate cyclase (ST) activities in the small intestine that lead to watery diarrhea, electrolyte imbalance, and dehydration. The roles of human and animal hosts as well as the environment in the selection for and emergence of new strains of virulent bacteria are becoming increasingly appreciated. For example, there is growing evidence that cattle and other agricultural (livestock) animals are major reservoirs of such waterborne and foodborne bacterial pathogens as enterohemorrhagic E. coli, Salmonella spp., and Yersinia enterocolitica (Tauxe, 1997). 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 Fibrio cholerae are borne on and can be infectiously transmitted

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--> by a filamentous bacteriophage (Faruque et al., 1995). The natural history of V. cholerae, including the mechanisms and evolution of virulence and pathogenicity, as well as its taxonomy, appear to be intimately linked to the aquatic environment and the interactions of humans with this environment. Molecular epidemiological studies reveal clonal diversity among toxigenic V. cholerae strains. The continual emergence of new epidemic clones may be taking place in aquatic ecosystems through interaction of the phages bearing the cholera toxin with different strains or antigenic types of V. cholerae . These new strains may then be selected for during epidemics in human populations. This appears to be an example of the evolution of new toxigenic strains of a human pathogen in natural aquatic ecosystems systems and its selection during outbreaks in human hosts. Within the aquatic ecosystem, interactions of the genetic elements of the microbes and their host reservoirs mediate the transfer of virulence genes, thereby resulting in the creation and the subsequent selection in humans of these new pathogenic strains. The extent to which such evolution and selection occurs for other human pathogens in aquatic ecosystems is unknown and deserves further investigation. Protozoan Parasites In the past three decades, protozoan parasites have emerged as important waterborne pathogens (Marshall, 1998). Some of the important protozoan parasites infecting humans and found in water are listed in Table 9-3. The ameba Entamoeba histolytica, the cause of amebic dysentery, has long been recognized as a waterborne pathogen. However, outbreaks of waterborne amebic dysentery have not been reported for decades in the United States and there are no major nonhuman reservoirs of this parasite. It was only with the recognition in the 1960s and 1970s of Giardia lamblia as a waterborne pathogen having important animal reservoirs and considerable resistance to chlorination and other drinking water disinfection practices that serious attention began to focus on this agent and other human pathogenic protozoans in drinking water. Since then, Cryptosporidium parvum has become a high-priority pathogen for regulation in drinking water because of documented waterborne disease, many animal reservoirs, ubiquitous presence in drinking water sources, relatively small size, and resistance to chlorine and other drinking water disinfectants. Other protozoan parasites, including the free-living amebas (e.g., Acanthamoeba spp), the coccidians Cyclospora cayatenensis and Toxoplasma gondii, the microsporidia, and the ciliate Balantidium coli are now recognized as human pathogens that deserve consideration in drinking water. Some of these agents, such as the free-living amebas, have a natural aquatic habitat. Many of the others, including Giardia lamblia, Cryptosporidium parvum, 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

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--> TABLE 9-3 Important Protozoan Parasite Contaminants of Drinking Water Parasite Outbreak or Detected in Water Animal Feces Non-fecal Sources Acanthcanoeba spp.a Yes No Yes Cryptosporidiurn parvum Yes Yes No Cyclospora cayatenensisa Yes Unknown Unknown Giardia lamblia Yes Yes No Entarnoeba histolytica Yes Rare No Balantidium cell Yes Yes (pigs) No Microsporidiaa (Enterocytozoon and Septata) Yes? Yes Maybe Toxoplasma gondiia Yes Yes No a On EPA's microbial contaminants priority list. infect hosts ranging from insects and other invertebrates to fish, birds, and mammals. Only a few species of human microsporidia have been recognized, and the Significance to human health of the many microsporidia of other animals is unknown at this time. 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). Furthermore, these microscopic methods cannot distinguish the infectious parasites posing a human health risk from the noninfectious inactivated ones no longer posing risks to human health. Even the latest of sensitive and specific molecular genetic methods, such as polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism (RFLP) analyses, may not distinguish human pathogenic from nonhuman nonpathogenic strains of a parasite such as Cryptosporidium (Champliaud et al., 1998). The limitations of currently available detection methods in identifying species and strains of protozoans capable of causing human infection and disease also apply to the methods of detecting other classes of microbes in drinking water that pose risks to human health. Methods for Detecting Pathogenic Microbes in Water Introduction The detection of pathogenic microbes in water typically involves three main steps: (1) recovery and concentration, (2) purification and separation, and (3) assay and characterization. In most cases the concentrations of pathogenic microbes in drinking water are so low that practical detection requires an initial

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--> step of concentration or enrichment from the water. Because many concentration and recovery procedures for pathogens also recover and concentrate other microbes and other constituents in the water sample, subsequent purification procedures are needed to separate the target pathogens from these other materials. Furthermore, the volumes of concentrated samples often are still too large for sensitive detection and analysis of the target pathogens, and therefore additional steps of concentration as well as purification are needed. The physical and chemical properties of the microbes have an important influence on their ability to be recovered by the various physical and chemical separation methods available. The size, shape, and density of the microbes influence various physical methods of recovery, such as filtration, sedimentation, and flotation. The surface properties of the microbes, such as their hydrophilicity, surface charge, isoelectric point, hydrophobicity, permeability, and chemical reactivity, will influence chemical and physical-chemical methods of recovery and separation. Most microbes are hydrophilic and negatively charged near neutral pH, but most are also somewhat hydrophobic and their surface has both hydrophobic and hydrophilic domains. The environmentally stable stages or forms of some microbes, including bacterial spores and protozoan cysts, oocysts, and spores, have thick outer "walls." Some bacteria have other surface features influencing physical-chemical behavior, such as outer polysaccharide layers (capsules), and appendages such as flagella and pili (fimbrae). These many and varied physical and chemical properties must be considered in the development and application of methods to recover and concentrate microbes from water. 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. Recently, nucleic acid amplification methods such as PCR and nucleic acid identification and characterization methods, such as hybridization (gene probes), RFLP analysis, and nucleotide sequencing, have been applied to the detection of microbes in water. Despite the potential sensitivity and specificity of these methods, they are not always capable of reliably detecting and quantifying infectious or viable organisms because they often detect the nucleic acid of noninfectious inactivated microbes (Sobsey et al., 1998). There are similar concerns about the various immunochemical methods to detect and quantify microbes in water; these methods detect and quantify antigens that may still be present and reactive in noninfectious or inactivated microbes.

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--> facilitates the removal of sample-related interfering components. Furthermore, because the pathogens have the opportunity to multiply, it becomes possible to relate gene probe detection to pathogen infectivity. Despite these advantages, this approach still relies on culturing and so it cannot be applied to noncultivable pathogens. Nucleic Acid Amplification With the recent development of PCR and other in vitro enzymatic amplification techniques for target gene sequences, the direct detection of low levels of human pathogens in environmental samples becomes more plausible, practical, and economically feasible than ever before (Persing et al., 1993; Dieffenbach and Dveksler, 1995; Persing, 1996; Sobsey et al., 1996). 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). Despite these advances and successes, a variety of strategic issues and technical problems must be further addressed in order for PCR and related nucleic acid amplification and detection methods to become practical and reliable for the direct detection of emerging pathogens in environmental samples. There are several essential steps in the development and application of PCR, other related enzymatic amplification techniques, and nucleic acid hybridization techniques such as oligonucleotide probing for successful detection of human pathogens in environmental samples. These key steps are (1) identification and selection of oligonucleotide primers and hybridization probes for target genomic sequences; (2) testing of selected primers and probes for sensitivity, specificity and selectivity; (3) further purification and concentration of the pathogens in environmental sample concentrates to enable efficient and reliable enzymatic amplification of low numbers of target genomic sequences; (4) testing of the methods for their applicability to natural pathogen strains and actual field samples; and (5) verifying that PCR amplification and oligonucleotide probing detect human pathogens that are potentially infectious and therefore pose a risk to human health. Selection of oligonucleotide primers and probes for target pathogens requires that sequence data be available. Such data are now available for some waterborne pathogens, and the database is increasing for a variety of emerging waterborne pathogens. However, these data are not comprehensive for all pathogens of concern, and they are still limited for some of the epidemiologically most important pathogens, such as the human caliciviruses and Cryptosporidium parvum. Proper selection of PCR primers and oligonucleotide probes requires detailed knowledge of the genomic organization and function and the nucleotide sequences of the target pathogens. Of particular importance are the type and

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--> function of nucleic acid target (genome, chromosomal gene, extrachromosomal gene, rRNA, mRNA, etc.), its length (size), and location, and the extent to which its sequence is related to that of other, nontarget but genetically related microbes. It is essential to select oligonucleotide primers and probes having the following characteristics: (1) appropriate length, (2) desired sequence composition for specificity and selectivity, and (3) appropriate melting and annealing temperatures and other physical characteristics to prevent the formation of undesirable secondary structures, primer dimers, other primer interactions, and other artifacts that would interfere with RT, PCR, or nucleic acid hybridization. Because of the large number of different waterborne pathogens, efforts have been made to amplify as many as possible using a single primer pair for those belonging to a genetically related taxonomic group. For the human enteric viruses, pan-specific primers and oligonucleotide probes have been developed for the human enteroviruses, group A rotaviruses, human caliciviruses, and adenoviruses. However, additional studies are needed to verify that these primers and probes do not amplify or detect similar or identical nucleotide sequences in the genomes of nonhuman animal viruses belonging to the same taxonomic groups. If so, alternative genomic sequences having greater specificity for the human pathogenic strains of these taxonomic groups must be selected. If this is not possible, additional analytical methods, such as RFLP or nucleotide sequencing, must be applied to the amplicons in order to conclusively identify them as being from the strain or type target microbe that infects and poses a health risk to humans. PCR and RT-PCR detection of specific pathogens in a broader taxonomic group is achievable by several different methods. One method involves the use of additional primers internal to the group-specific primers that would amplify subsequently from the initially amplified cDNA ("nested amplification"). Another approach is hybridization using highly specific oligoprobes that would hybridize only with amplicons from a single pathogen type or strain. Selected oligonucleotide primers and probes must be tested for specificity and selectivity. It must be verified that primers have the ability to amplify a DNA of correct molecular weight and that the amplicon will hybridize with a specific nucleic acid probe (e.g., oligoprobe). Furthermore, it must be verified that the probes are nonreactive with the nucleic acid of nontarget microbes. The sensitivities or lower detection limits of PCR or other nucleic acid amplification methods for target pathogens can be tested by determining the greatest dilution of a known quantified pathogen suspension that can be successfully amplified. The same sample is also quantified by infectivity, microscopic enumeration of the numbers of microbes or other methods so that the endpoint PCR titer can be compared to these other titers. Other approaches to quantifying nucleic acid amplification by PCR and RT-PCR have been described by Freeman et al. (1999). The quantification methods should include determining if there are materials in the sample interfering with PCR amplification and should estimate the concentration of target microbes in the sample. 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

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--> incorporates into the amplicon), fluorescent signal decrease (by loss of fluorescent signal upon incorporation of the fluorescent primer into the amplicon), or other chemical methods. Also, the amount of target DNA amplified in the sample can be compared to the amount of target DNA amplified under the same conditions from a positive control sample containing a known amount of nucleic acid from the same target microbe suspended in a noninhibitory solution. Using this approach, however, it is not possible to determine if lack of or low amplification of the target is due to inhibition by sample constituents, to few target nucleic acids being present, or a combination of both. Another approach to quantifying PCR or RT-PCR and determining the effects of sample-related inhibitors is to add a known amount of an internal nucleic acid standard as a positive control into the sample. Adding specific amounts of positive control nucleic acid sequences in the reaction mixture and determining the extent of amplification of this target (which differs from the true target) makes it possible to quantify both sample inhibition and the amount of actual target nucleic acid in the sample. This approach has been applied to the detection of human caliciviruses by RT-PCR (Schwab et al., 1997, 1998). Sample Preparation for Pathogen Detection in Water by Nucleic Acid Methods Typical environmental sample concentrates for pathogens are too large in volume (10 to 50 ml) and too contaminated with extraneous interfering constituents for reliable and sensitive enzymatic amplification of target pathogen genome sequences, especially at the low levels (low target nucleic acid numbers) typically found in most water samples. Therefore, in some previous studies target pathogen nucleic acid has been isolated from environmental samples by standard techniques of nucleic acid extraction, purification, and concentration, such as proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. However, these methods are cumbersome, laborious, and often inefficient, thus leading to poor recoveries and inadequate detection limits. Furthermore, disruption of pathogens to liberate target nucleic acid at an early stage of sample concentration and purification makes it impossible to compare pathogen detection by nucleic acid amplification to pathogen detection by other methods, such as infectivity or particle count. Preferred sample cleanup and concentration strategies maintain pathogen integrity as long as possible prior to enzymatic amplification. The goal is to reduce the sample volume to <100 mL, remove interfering and inhibitory substances (e.g., particulates, salts, proteins, lipids, carbohydrates, and natural organic matter) and also place the pathogens in an aqueous medium compatible with enzymatic amplification as well as the other quantitation procedures, such as infectivity or particle count. 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

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--> by other, chemical extraction methods as a final or near-final treatment step. This makes it possible to compare PCR endpoint titers with infectivity assay or particle count titers without intervening or additional purification and concentration steps that could cause differential losses. Candidate chemical purification and concentration methods for subsequent nucleic acid amplification and some other pathogen detection methods include precipitation (e.g., polyethylene glycol for viruses); chromatography (e.g., Sephadex gels in spin-columns); chelation of metals (using EDTA, EGTA, "Chelex," etc.); ultrafiltration using high (e.g., 100,000 daltons) molecular weight cutoff filters; organic solvent (e.g., chloroform, fluorocarbon) extraction; detergent (e.g., Tweens and other nonionic detergents) treatment; enzymatic digestion (e.g., proteases, amylases, lipases, and nucleases); and antibody (immunoaffinity) capture on paramagnetic beads (immunomagnetic separation). Immunoaffinity capture and purification of pathogens as antigens is a cleanup and concentration option that has been successfully applied to some viruses, bacteria, and parasites. However, it is not applicable to some pathogens because of the lack of reagent quality antisera or the antigenic diversity of a large pathogen group lacking a common antigen (and hence requiring many antisera). Overall, nucleic acid amplification by PCR or RT-PCR and hybridization using oligonucleotide probes is a specific, selective, and sensitive approach to the detection of pathogens in environmental samples. Oligonucleotide primers and probes can be selected to detect broad pathogen groups, such as the enteroviruses and Salmonella or specific pathogens, such as hepatitis A virus. In particular these methods can be used to detect fastidious pathogens, such as human caliciviruses and Cyclospora cayatenensis. Under optimized conditions method sensitivity or detection limit is less than one infectious unit and in principle as little as one target gene sequence. Methods have been developed and evaluated to concentrate and purify target pathogens from environmental samples such as water for successful detection by nucleic acid amplification and oligoprobe hybridization. These methods have been successfully applied to the detection of viral, bacterial, and protozoan pathogens in field samples of water. However, the inability of most of these nucleic acid methods to conclusively detect only the infectious pathogens is a limitation that remains to be overcome. Microscopic and Analytical Imaging Methods to Detect Waterborne Pathogens Microscopic methods include ordinary (light) microscopy (bright-field and dark-field), phase contrast, differential contrast, fluorescence, laser scanning, video, and other forms of microscopic and image analysis (Lawrence et al., 1997). These methods are not effective for viruses or other very small (ultra-small) agents, and some are not effective for very transparent agents unless the microbes are stained. The detection of internal and external structural features (e.g., organelles and reproductive units) is improved by various colored stains, fluorescent stains, advanced optical systems (phase contrast, Nomarski interference contrast, differential interference, and laser scanning), and the use of imaging by sensitive charge-coupled device cameras. For example, phase contrast and differential interference contrast microscopy are used to visualize

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--> the internal structures of Giardia lamblia and Cryptosporidium parvum isolated from water. This helps distinguish them from algae and other particles of similar size and shape. Fluorescent (ultraviolet light) microscopy methods are useful for transparent cells or other cells or organelles that are difficult to detect. The cells are reacted with a fluorochrome that interacts with a cellular components or macromolecules, which are then detected by ultraviolet light microscopy. Capturing microscopic images by digital methods for image analysis using computers has greatly advanced the capabilities to detect, quantify, and characterize pathogens and other microbes in environmental samples. For example, computer-assisted laser scanning and video microscopy have been applied to the analysis of Cryptosporidium parvum oocysts in environmental samples (Anguish and Ghiorse, 1997). Another technology employing advanced image analyses to detect, quantify and characterize pathogens in water is laser-based flow cytometry plus fluorescent cell sorting, which are methods that have been previously described above. Immunoassays to Detect Pathogens in Water Immunofluorescent detection by microscopy or other methods is a specific and potentially powerful way to detect pathogens and other microbes in water if there are enough target pathogens in the sample for detection (McDermott, 1997). Antibodies directed against antigens of the target pathogen can be labeled (conjugated) with a fluorochrome or fluorescent dye (e.g., fluorescein isothiocyanate or another fluorochrome) for direct immunofluorescence. These fluorescent antibodies are reacted with the target microbe, and then the microbe preparation is washed to remove meted fluorescent antibody. The sample is then examined for the target microbe by ultraviolet light microscopy or another analytical method to detect the immunofluorescent signal (e.g., fluorometry). Alternatively, secondary fluorochrome-labeled antibodies directed against the primary antibodies (now serving as antigens) of the species of animal in which the antibodies against the microbe were raised can be used in an indirect immunofluorescence assay. The target microbial antigen is reacted initially with a specific antibody, and then the resulting antigen-antibody complex is reacted with fluorochrome-labeled antispecies antibody to provide the signal for immunofluorescent detection. Enzyme immunoassays employ enzyme-conjugated antibodies directed against target pathogens. The antigen-antibody complex is detected and quantified by the ability of the enzyme to react with a substrate that typically produces either a colored product for colorimetry or emits light for luminometry. 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

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--> ("sandwich" or immunocapture assay format). As with other immunoassays, enzyme immunoassays can be direct (enzyme-conjugated, primary antibody against antigens of the target microbe) or indirect (enzyme-conjugated, antispecies secondary antibody). Studies have repeatedly shown that solid-phase enzyme immunoassays generally are too insensitive for direct detection of microbial pathogens in water, as they require a minimum of 10,000 to 100,000 target microbes (or their antigens) for detection. In most situations drinking water and its sources rarely contain high enough levels of most target pathogens for direct immnoenzymatic detection. However, enzyme immunoassays also have been combined with methods to propagate target pathogens by various culture methods, thereby increasing their numbers for immnoenzymatic detection. For example, cell culture infectivity has been used to propagate noncytopathogenic viral pathogens and thereby enhance their detection (Payment, 1997). Agglutination methods are used to detect pathogens by combining dispersed cells, viruses, or other forms of pathogen antigens with antibodies (on a slide, for example) and allowing for antigen-antibody reactions to produce agglutination (clumping) that can be scored as negative or various degrees of positive (strong, medium, or weak). One modification is latex bead agglutination in which antibodies against a specific microbial antigen (especially nonparticulate or "soluble" antigen) are attached to latex beads. The beads are reacted with the sample. If the sample contains the specific antigen, agglutination occurs by the reaction of antigens with antibodies on the beads resulting in the beads clumping together (agglutinating). As with enzyme immunoassays, agglutination tests are too insensitive to directly detect and quantify most waterborne pathogens in drinking water and other aquatic samples. The target microbes must first be propagated in order to obtain a sufficient number of them or a sufficient amount of antigen to detect and antigenically characterize them by agglutination methods. Signature Biolipid and Other Biochemical Detection Methods Some pathogens are detectable became they contain distinctive macromolecules or biochemicals that aid in their identification and detection. These include cell wall component assays for lipopolysaccharides, muramic acids, and assays for signature biolipids (White, 1995). Signature lipid biomarker analysis is based on the use of techniques such as liquid extraction and thin layer chromatography to separate and purify the microbial lipids from the microbes in the environmental sample. This is followed by quantitative analysis using gas chromatography/mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Using these techniques it has been shown that Cryptosporidium parvum contains a characteristic phosphatidyl-ethanolamine, that may make it possible to detect this parasite in environmental samples (Schrum et al., 1997).

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--> Summary and Conclusions The identification and detection of microbial contaminants in drinking water must continue to be a high priority for assessing the risks from and managing the microbial quality of drinking water supplies. The essential information needs for quantitative microbial risk assessment can be applied to detecting, identifying, and characterizing microbial contaminants in drinking water supplies. The recognition of new waterborne pathogens as well as re-emerging ones posing increased risks due to newly acquired virulence properties and other traits requires further improvement of waterborne pathogen detection methods and better use of the advanced analytical methods now available. There must be continued efforts to improve and apply the methods for waterborne pathogen recovery, concentration, purification, separation from interfering materials, detection, isolation, assay, and characterization. Methods for waterborne pathogen recovery and concentration by centrifugation, filtration and precipitation can be further improved and newly developed methods can be more widely applied. Purification and separation methods, such as immunomagnetic capture, chromatography (by ion exchange, adsorption, chelation, and size exclusion) and chemical treatment (with enzymes, detergents, and organic solvents) are advancing and becoming more widely used for waterborne pathogens. Methods to assay, quantify, and characterize waterborne pathogens are advancing, but greater efforts are needed to determine pathogen infectivity and viability. Culture methods for viruses, bacteria, and parasites are preferred because they detect and quantify infectivity, which is the most relevant unit of measure in terms of health risk. For bacterial pathogens in water, better methods to detect infectivity are needed and existing methods to detect infectious bacteria must be more widely used. The detection of stressed, injured, and viable but nonculturable bacteria in water continues to be a technological challenge, and available methods to detect such bacterial pathogens in water continue to be underutilized. Detection and quantitation of waterborne vital pathogens by cell culture infectivity is possible for some of them, but many of the important waterborne enteric viruses are still not culturable. The combined use of cell culture and nucleic acid amplification (PCR, RT-PCR, etc.) and hybridization has improved the detection of some of the more fastidious waterborne viral pathogens, but these methods must become more widely used. Recently, it has become possible to detect and quantify the infectivity of some of the important waterborne parasites (Cryptosporidium parvum and some microsporidia) by cell culture methods. However, these methods require further refinement and verification and they have yet to be extensively applied in occurrence studies or exposure assessments. 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

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--> improvements in these nucleic acid amplification and hybridization methods are needed to reliably distinguish infectious from non-infectious waterborne pathogens. The combination of these nucleic acid methods with culture methods appears to be the most promising approach for sensitive and specific detection and quantitation of infectious waterborne pathogens. The methods to purify and concentrate the target microbes and their nucleic acids from water by various physical and chemical means have advanced greatly in recent years. However, these methods need further improvement, consolidation, and simplification in order to achieve reliable, sensitive, and specific detection and quantitation of the target pathogens recovered and concentrated from water. 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. Similarly, microscopy, analytical imaging, immunoassays, and biochemical analyses continue to advance as methods to detect, identify, and characterize pathogens in water. However, these methods lack sensitivity for direct detection and may not distinguish infectious from non-infectious organisms. The usefulness of these methods is improved when they are applied to pathogens that have been concentrated and purified from water or have been enriched in numbers by culture methods. Advances continue to be made in immunological, biochemical, nucleic acid, and bioassay methods to characterize potentially waterborne pathogens. The bases for and mechanisms of virulence and pathogenicity of these pathogens are becoming better understood at the biochemical, immunological, and genetic level. Such advances provide the needed tools for improved detection and identification of these microbes in water, understanding the role of the aquatic environment in their persistence, evolution, and selection, and better characterization of the human health risks from these waterborne pathogens. References Abbaszadegan, M., P. W. Stewart, M. W. LeChevallier, and C. P. Gerba. 1998. Application of PCR Technologies for Virus Detection in Groundwater. Final Report. Denver, Colo.: American Water Works Association Research Foundation. Anguish, L. J., and W. C. Ghiorse. 1997. Computer-assisted laser scanning and video microscopy for analysis of Cryptosporidium parvum oocysts in soil, sediment and feces. Applied Environmental Microbiology 63(2):724-733. Arrowood, M. J., L.-T. Xie and M. R. Hurd. 1994. In vitro assays of maduramicin activity against Cryptosporidium parvum. Journal Eukaryotic Microbiology 41:23S. Borchardt, M. A., and S. K. Spencer. 1998. Concentration of waterborne pathogens using a continuous separation channel centrifuge. In Proceedings of the 1998 Water Quality Technology Conference, Denver, 1998. Brock, T. D. 1983. Membrane Filtration: A User's Guide and Reference Manual. Madison, Wis.: Science Tech, Inc.

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