Nucleic acid hybridization and target or probe amplification methods are the molecular techniques most commonly used for direct organism detection in clinical specimens.

Advantages and Disadvantages

 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 organism. Therefore, a positive assay indicates the presence of an organism but also pro vides 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 associated 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 com ponent 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 sp.) or for which reliable cultivation methods are not widely available (e.g., Ehrlichia sp.).

The high specificity 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 deter mine the clinical relevance of finding a particular organ ism 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 are not completely reliable in directly detecting organisms. The quantity of target nucleic acid may be insufficient, or the patient specimen may contain substances that inter fere with or cross-react in the hybridization and signal generating reactions. One approach developed by Gen-Probe (San Diego, California) 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 was discussed with direct hybridization methods, patient specimens 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 an invaluable means of 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:

• Ability to detect nonviable organisms that are not retrievable by cultivation-based methods

• Ability to detect and identify organisms that cannot be grown in culture or are extremely difficult to grow (e.g., hepatitis B virus and the agent of Whipple’s disease)

• More rapid detection and identification of slow growing organisms (e.g., mycobacteria, certain fungi)

 • Ability to detect previously unknown agents directly in clinical specimens by using broad-range primers (e.g., use of primers that anneal to a region of target DNA conserved among all bacteria)

• Ability to quantitate infectious agent burden in patient specimens, an application that has particular importance for managing HIV, cytomegalovirus (CMV), and hepatitis B and hepatitis C infections.

Despite these significant advantages, limitations still exist, notably the ability to find only 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 to screen specimens for the presence of any organism (e.g., bacteria, fungi, parasite). Specimens 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 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 com plication 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

 Given their inherent advantages and disadvantages, molecular direct detection methods are most useful when:

• One or two pathogens cause the majority of infections (e.g., Chlamydia trachomatis and Neisseria gonorrhoeae as common agents of genitourinary tract infections)

• Further organism characterization, such as antimicrobial susceptibility testing, is not required for management of the infection (e.g., various viral agents)

• Either no reliable diagnostic methods exist or they are notably suboptimal (e.g., various bacterial, parasitic, viral, and fungal agents)

• Reliable diagnostic methods exist but are slow (e.g., Mycobacterium tuberculosis) • Quantitation of infectious agent burden that influences patient management (e.g., AIDS) is desired

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. 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 aca demic 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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مواضيع ذات صلة

Identification of Microorganisms Grown in Culture

Laboratory Manifestations of Iron Overload

Differential Diagnosis of Iron Deficiency

Laboratory Manifestations of Iron Deficiency

Staining Techniques for Light Microscopy

Selection of Culture Media

Sterilization and Disinfection

Biochemical Assays for Metabolic Diseases

Laboratory Diagnosis of Urinary Tract Infections

Identification of Mycoplasma Infections

Laboratory Diagnosis of Infections of the Eyes

Laboratory Diagnosis of Central Nervous System Infections