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Appendix C: Pathogen Discovery, Detection, and Diagnostics
Pages 313-330

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From page 313...
... Departments of Microbiology and Immunology and of Medicine Stanford University, Stanford, California VA Palo Alto Health Care System, Palo Alto, California PATHOGEN DISCOVERY1 Microbial Diversity and the Limitations of Cultivation Methods Beginning in the late 1970s and early 1980s, explorations of the natural microbial world focused on extreme environments and exploited the use of newly described molecular approaches for phylogenetic analysis and classification. Recovery of sequence-based signatures of life directly from these environments confirmed revolutionary proposals for three aboriginal lines of descent (Woese and Fox, 1977)
From page 314...
... Because the internal environmental conditions of the human body are viewed as more hospitable to life and are more easily replicated in the laboratory than are many external environmental conditions, we often assume that microbial cultivation efforts have been relatively successful. To be sure, there is no dearth of known, cultivated microbial pathogens.
From page 315...
... For example, high-clensity microarrays of oligonucleoticles or amplifieci DNA products can be clesigneci to screen complex pools of microbial nucleic acid for specific agents in a massively parallel and efficient manner. This technical platform obviates the need to clone and sequence large numbers of variant microbial molecules, which is particularly relevant to the analysis of clinical specimens with a significant burden of "background" microorganisms (see the further discussion below)
From page 316...
... Although the clinical significance of the newly discovered community membership has not yet been established, it is clear that the human endogenous microflora play an im
From page 317...
... Alternatively, anatomic sites of interest can be targeted specifically for signature detection using laser capture microdissection (Emmert-Buck et al., 1996; Becich, 2000~. Recognition and Classification of Microbial Disease Based on Host Gene Expression Patterns The limitations of methods for analysis of microbial signatures and the emergence of technology platforms for rapid, highly parallel gene expression measurements have facilitated a potentially important, independent approach for identification of microbial disease.
From page 318...
... The transition to an analysis of humans with and without known infectious diseases ex viva is accompanied by a number of interesting but complex questions. What is the most useful and practical type of clinical specimen from which to record genome-wide expression patterns and discern meaningful information about infection?
From page 319...
... Because of the time demands and resource constraints found in today's clinical workplace, as well as recent laboratory downsizing, recovery of fastidious microbes from clinical specimens has almost certainly suffered (Bartlett et al., 2000~. For example, increasing delays from the time of sputum specimen collection to the inoculation of appropriate culture media have probably contributed to the decreasing recovery rates of S
From page 320...
... Solid-phase immunological assays, such as dipsticks and optical immunoassays, have established a niche in the clinical workplace, but their utility has been demonstrated in only a limited number of infectious diseases settings (Needham et al., 1998~. PCR is the most widely used method for microbial nucleic acid detection; other signal and target amplification techniques for nucleic acid detection, such as ligase chain reaction, have generated more limited commercial markets (Tang et al., 1997; Fredricks and Relman, 1999~.
From page 321...
... Sensitive and specific diagnostic tests are vitally important adjuncts to clinical diagnosis; however, screening and diagnostic tests cannot replace the crucial need for careful studies that define likelihood of exposure or examine correlations between detection and disease. One issue of particular importance concerns the complexity and widespread distribution of microbial sequence "background" or "noise" observed in the analysis of human clinical specimens (both experimental and biological)
From page 322...
... Few efforts are invested in improving methods for the detection of less commonly found pathogens. In addition, important lessons have been learned in recent years about the use and limitations of molecular methods for microbial pathogen discovery (see the earlier discussion)
From page 323...
... And fourth, in the real world of clinical medicine, specimen handling and storage may introduce exogenous contamination, spurious signals, or target degradation. Standardization of Procedures The preceding discussion has suggested that current diagnostic and detection procedures lack sufficient standardization and validation.
From page 324...
... These resources would assist with standardization and validation of methods and help minimize reliance on an overly narrow set of detection reagents. ANTICIPATING MICROBIAL THREATS Intelligence Gathering: Human Intention Deliberate release of a biological agent as a weapon poses a number of additional challenges in detection and diagnosis, including a much-expanded spectrum of agents and greater difficulty in calculating pretest probabilities and positive predictive values.
From page 325...
... These needs may ultimately require the development and use of high-density DNA microarrays designed for broad-range microbial surveys. A Second Human Genome Project: Microbial Community Genomics As one broad approach to the problem of poorly understood endogenous flora, one might consider a second human genome project (Relman and Falkow,2001~.
From page 326...
... As mentioned earlier, endogenous microbial flora might be the source of informative patterns that would indicate exposure to or incipient development of infectious disease, as well as prior behavior of the host. It is already clear that automated and miniaturized technology platforms, such as microfluidics chips and cartridges, will speed the development of point-of-care, rapid, hands-off diagnostic tests (Beigrader et al., 2000; Pourahamadi et al., 2000~.
From page 327...
... Proc Natl Acad Sci U S
From page 328...
... Proc Natl Acad Sci U S
From page 329...
... Proc Natl Acad Sci U S
From page 330...
... 330 MICROBIAL THREATS TO HEALTH for calicivirus (Mostashari, New York City Department of Health, Personal Communication, 20011. Investigators generally expect that syndromic surveillance will be able to detect a wide variety of diseases and conditions, not only acts of bioterrorism or emerging infections.


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