Other novel analytical methods have addressed the rapid detection of biological agents in drinking water. A bioluminescence method for water samples was developed as a simple method for E. coli analysis (Lee and Deininger, 2004). The process uses antibody binding separation steps before analysis to produce a bioluminescence response within an hour. A great deal of effort is also being put into mass spectrometric analysis of biological species, including water-borne pathogens (Magnuson et al., 2000; Fenselau and Demirev, 2001). Such analyses can target specific protein or toxins, even from intact organisms and can be used as an in situ test. A number of remote and/or in situ sampling systems are also being developed, which enable active monitoring of many basic water quality parameters, including an estimate of biological activity based on residual chlorine, dissolved oxygen, pH, and algal chlorophyll. Sampling systems allow dynamic collection in any water environment, so the autosampler can shift to regions of interest, essentially tracking water supply contamination with real-time measurements. This type of analysis could soon be implemented directly in the water main pipe, serving as a first level of warning. Such systems will likely be combined with nucleic acid and/or immunoassays for rapid, in situ pathogen testing of our water supplies.
Limitations. The schemes summarized above are increasingly used for biosurveillance, as costs for their implementation and maintenance decrease. These methods offer sensitive, rapid assay formats with the ability to be tailored to any pathogen with a known biomarker. As our knowledge of these organisms advances and the relevant sequence information is garnered, more thorough biothreat analysis will become routine. Because these assays are based on known genetic or other biological information, they have limited utility for identification of emerging pathogens or for pathogens for which we have limited information. Also, although a few techniques have demonstrated the capability to ascertain viability, the majority of assays provide only a preliminary positive result, and viability must be confirmed by growth in culture. One of the biggest limitations to biomonitoring is cost, and no inexpensive, reliable portable systems are yet commercially available. The cost of implementation, coupled with the enormous scale of the water supply infrastructure, makes the widespread incorporation of this type of system impractical. Also, as many of these formats are tied to specific pathogens, a single comprehensive platform is difficult to envision. For example, although microarrays have the ability to address thousands of gene targets, organisms such as Salmonella have hundreds to thousands of subtypes, including recent multidrug-resistant strains (Mulvey et al., 2004), making them more difficult to identify at the subtype level.
Approach to Biosurveillance. The EPA determines biosurveillance standards for treated water. The surveillance process involves three levels of action. If the routine level of screening identifies a potential problem, the second (confirmatory) level of testing is initiated, and more detailed testing is performed to validate the initial positive test. The detailed testing includes more frequent sampling, sampling of other parts of the supply and distribution chain, and an expanded regimen of agents tested. The types of tests performed are commensurate with the situation, that is, rapid, high-throughput, organism-specific tests are used when an incident is identified. The purpose of the expanded testing is to assess the public health risk and determine the need for further actions. The final stage of action seeks to determine the source of contamination. This phase of analysis guides remediation, mitigation, and policy to prevent recurrences.
Routine Monitoring. Routine monitoring most commonly entails surface water analysis for the presence of total col-iform by the water suppliers. There are no federal guidelines mandating routine testing of many commonly occurring pathogens. Testing is, in practice, limited to the minimal regulatory requirements plus a few voluntary monitoring tests, such as for Cryptosporidium and Giardia species, performed with low frequency. Such monitoring does not serve in a detect-to-prevent mode and is unlikely to be useful as an early warning indicator. Testing largely focuses on the watershed and its supply chain. Utilities will maintain records on multiple indicators and baseline concentrations as a means to identify atypical levels of contaminants. As noted above, utilities will also record data from which they can infer the presence of unwanted organisms. Test results are available for real-time analysis and are often included in microbiological surveillance strategies.
Confirmation Analysis. The second level of surface drinking water source assessment requires assays for specific pathogens. Utilities perform tests most often to confirm the presence of natural pathogens, such as protozoa and enteric viruses, or common environmental contaminants, such as bacterial organisms (E. coli O157:H7, Shigella, Salmonella). Although many laboratories can perform screening assays for the above agents, the water utilities usually assign confirmatory testing to specialized laboratories under strict protocols (see "Laboratory Networks''), which involve advanced methods and an expedited response. Methods such as real-time PCR can enumerate pathogen concentrations and be used to better assess the threat level, but these methods cannot assess organism viability, which is determined via organism growth, for example, cell culturing.
Source Contamination Determination. The third level of water surveillance involves trace-back analysis, with the purpose of identifying the source(s) of microbial contamination. Although this testing has practically no early warning value, it is critical for preemptive purposes to avoid or minimize future outbreaks. This analysis provides a framework within which to establish regulations, contain the pathogen so as to limit the extent of public illness, and define the requirements for remediation. These responses are influenced by the identity of the causative pathogen, its quantity, source, and dispersal range.
The three levels of surveillance are complementary and shape the overall public health response. Surveillance is often a dynamic process, influenced by experience from past outbreaks, public policy, and ongoing data collection. Obviously, we can define the extent of an outbreak most clearly when comprehensive pre- and postexposure data are available.
Sampling and Actual Testing Methods. Sampling is a critical step in the identification of biological agents. For water sampling, a concentration step is performed because many laboratory techniques are concentration sensitive and have specific limits of detection that could compromise the result for a diluted sample. Filter sets can also serve as an initial means for unknown identification, because bacteria, viruses, or protozoa can be selectively filtered based on particle size. The order in which tests are performed is influenced by any clinical or physical evidence that suggests a particular pathogen. The EPA sets testing guidelines, such as culture protocols, for the common water-borne agents (EPA, n.d.). For bacteria, membrane filtration concentrates microorganisms from water samples, which are then spotted on selective media for culture growth, serological classification, and biochemical analysis. Concomitant with culturing, other established techniques may be performed, such as PCR and immunology-based analysis. Detection of protozoa and viruses requires analysis of much larger volumes of water than are used for bacterial analysis, because these organisms are typically present in lower concentrations. For viruses, the material from the collection filters undergoes microscopic examination for visual confirmation as well as exposure to a number of cell lines selective for particular viruses and, ultimately, PCR analyses. General parasitologi-cal analysis is via fluorescent immunologic-based separation and identification and, again, PCR assays. An important note is that although many clinical laboratories are equipped with PCR capabilities, the above methods, such as culturing, are the universally accepted measure for definitive classification. The time required to complete a real-time PCR assay is generally 2 hours for common rapid cycling instruments. The assay results depend on the sample concentration, that is, the amount of organism present. Because the assays can be interpreted in real time, an abundant biological agent can provide a positive real-time PCR result within 30 to 60 minutes of the 2-hour assay. Although the use of PCR instruments at every stage of the water testing process is still cost prohibitive, portable PCR instruments have been developed that can be deployed to problem sites as needed. Ideally, an in situ test would able to monitor the actual water mains, but currently this type of system is impractical, owing to both to the cost associated with such an instrument and to the dilution factor associated with water sampling. Because of this, PCR is considered the most rapid and effective measure of the presence of an organism, and organizations such as the Laboratory Response Network (LRN) have defined primer sequences and target genes that must be used for identification.
If the sample is an unknown and a select agent is suspected, a specialty laboratory receives the sample initially. The filtration/concentration process for unknowns uses ultracentrifuga-tion with selective filters. Because bacterial and viruses differ substantially in size, filters with varied pore sizes and polarities can provide a gross discrimination of the type of microbe before culture-based and molecular analyses (immunoassays, PCR) are performed. The "Molecular Methods'' section below describes the more common advanced assays for biological detection. Also, a number of specific and/or commercially available tests, including handheld assays and PCR formats are listed in a Department of Justice guide, "An Introduction to Biological Agent Detection Equipment for Emergency First Responders'' (Fatah et al., 2001).
Limitations to Surveillance. One of the biggest limitations facing comprehensive biosurveillance is cost, because of the sheer size of the water system and the multiple points at which it is vulnerable to intentional contamination. A number of concern areas in need of improvement are listed in Figure 9.4. Because of the scale of the physical infrastructure, the safety policies, testing proficiency, and communication network for the nation's water supply can be thought of as a massive collective effort, organized on the federal, state, and local levels. More than $140 million in federal funding (2002-2004) has been spent on physical security, training, and vulnerability assessments, yet little mandatory implementation has taken place (U.S. Government Accountability Office, 2004).The lack of federal guidelines requiring testing of the most commonly present organisms, and the limited implementation of advanced biosurveillance techniques are issues of concern. The limited extent of drinking water surveillance in terms of biological organisms, in and of itself, can be perceived as low prioritization. Although many agents, if present, would be sufficiently eliminated by the current treatment system, several pathogens have demonstrated resistance to the concentrations of disinfectants used in common practice (Burrows and Renner, 1999). Still, comprehensive testing of all pathogens is not considered practical, and general water quality tests only determine total coliform density. The lack of comprehensive guidelines will likely serve confusion and difficulties in future incidents.
Inherent limitations to sampling and analysis exist because many water supply sources are continually fed, resulting in
sample dilution. Because of this, filtration and analysis protocols were developed by the EPA, typically filtering 50-liter water samples for testing, to concentrate any biological pathogen. In addition, as mentioned above, distributed water can originate from multiple sources and can undergo varied treatment measures and limited testing along the distribution route. As the basic goal of a water supplier is to provide potable water, comprehensive surveillance would require multiple check points for testing, allowing trace-back to the source of contamination, verification of the efficacy of the water treatment procedures, and rapid signaling of a problem before it develops into a public health crisis. Eventually, the current monitoring of total coliform counts, and E. coli specifically, will not be perceived as sufficient to meet public health needs.
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