Nucleic acid hybridization and target or probe amplification methods are the molecular techniques most commonly used for direct organism detection in clinical specimens.
When considering the advantages and disadvantages of molecular approaches to direct organism detection, comparison with the most commonly used conventional method (i.e., direct smears, culture, and microscopy) is helpful.
Specificity. Both hybridization and amplification methods are driven by the specificity of a nucleotide sequence for a particular oiganism. Therefore, a positive assay not only indicates the presence of an organism but also provides the organism's identity, potentially precluding the need for follow-up culture. Although molecular methods may not be faster than microscopic smear examinations, the opportunity to avoid delays assodated with culture can be a substantial advantage.
However, for many infectious agents, detection and identification are only part of the diagnostic requirement. Determination of certain characteristics, such as strain relatedness or resistance to antimicrobial agents, is often an important diagnostic or epidemiologic component that is not possible without the availability of culture. For this reason, most molecular direct detection methods target organisms for which antimicrobial susceptibility testing is not routinely needed (e.g., chlamydia) or for which reliable cultivation methods are not widely available (e.g., ehrlichia).
The high spedfiaty of molecular techniques also presents a limitation in what can be detected with any one assay, that is, most molecular assays focus on detecting the presence of only one or two potential pathogens. Even if tests for those organisms are positive, the possibility of a mixed infection involving other organisms has not been ruled out. If the tests are negative, other procedures may be needed to determine whether additional pathogens are present. In contrast, smear examination and cultivation procedures can detect and identify a broader selection of possible infectious etiologies. Of importance, Gram-stained smear results are often needed to determine the clinical relevance of finding a particular organism upon culture or detection using molecular assays. However, given the rapid development of molecular technology, protocols that widen the spectrum of detectable organisms in any particular specimen are becoming available. ASRs for real-time PCR that can detect as many as six to seven organisms are commercially available. Finally, a concern always associated with any amplification-based assay is the possibility for cross contamination between samples and/or by amplified byproduct. Thus, it is of utmost importance for any laboratory performing these assays to employ measures to prevent false-positive results.
Sensitivity. Hybridization-based methods can have difficulty directly detecting organisms. The quantity of target nucleic acid may be insufficient, or the patient specimen may contain substances that interfere with or cross-react in the hybridization and signal-generating reactions. One approach developed by Gen-Probe (San Diego, Calif) to enhance sensitivity has been to use DNA probes targeted for bacterial ribosomal RNA, of which there are up to 10,000 copies per cell. Essentially, amplification is accomplished by the choice of a target that exists within the cell as multiple copies rather than as a single copy.
Amplification Techniques Enhance Sensitivity. As discussed with direct hybridization methods, patient specimens also may contain substances that interfere with or inhibit amplification reactions such as PCR. Nonetheless, the ability to amplify target or probe nucleic acid to readily detectable levels has provided a tremendous means for overcoming the lack of sensitivity characteristic of most direct hybridization methods.
Besides the potential for providing more reliable test results than direct hybridization (i.e., fewer false-negative results), amplification methods have other advantages that include:
Despite these significant advantages, limitations still exist, notably the ability to only find the organisms toward which the primers have been targeted. Additionally, no cultured organism is available if subsequent characterization beyond identification is necessary. As with hybridization, the first limitation may eventually be addressed using broad-range amplification methods that would be used to screen specimens for the presence of any organism (e.g., bacteria, fungi, parasite). Specimens that are positive by this test would then be processed further for a more specific diagnosis. The second limitation is more difficult to overcome and is one reason why culture methods will remain a major part of diagnostic microbiology for some time to come.
An interesting consequence of using highly sensitive amplification methods is the effect on clinical interpretation of results. For example, if a microbiologist detects organisms that are no longer viable, can he or she assume the organisms are or were involved in the infectious process being diagnosed? Also, amplification may detect microorganisms present in insignificant quantities as part of the patient's normal or transient flora, or as an established latent infection, that have nothing to do with the current disease state of the patient
Finally, as previously mentioned, an underlying complication in the development and application of any direct detection method is that various substances in patient specimens can interfere with the reagents and conditions required for optimum hybridization or amplification. Specimen interference is one of the major issues that must be addressed in the design of any useful direct method for molecular diagnosis of infectious diseases.
Applications for Direct Molecular Detection of Microorganisms
A large number and variety of commercially available molecular systems and products for the detection and identification of infectious organisms are now available. These include automated or semiautomated systems such as Roche Diagnostic System COBAS instrument which is capable of processing and assaying 22 specimens in 3'/2 hours. Many of these systems and products are mentioned throughout this textbook. Additionally, many direct detection assays have been developed by diagnostic manufacturers and research laboratories associated with academic medical centers. Therefore, direct molecular diagnostic methods based on amplification will continue to expand and enhance our understanding and diagnosis of infectious diseases. However, as with any laboratory method, their ultimate utility and application will depend on their accuracy, potential impact on patient care, advantages over currently available methods, and resources required to establish and maintain their use in the diagnostic setting.
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