Mycobacterium avium complex DNA Detection

Detection of Mycobacterium avium complex DNA.

Background

Rapid and accurate identification of pathogenic Mycobacteria is important for correct therapeutic intervention against an infectious disease. Biochemical or phenotypic methods for identification of a Mycobacteria first requires growth of the microorganism in a suitable media. Furthermore, complete identification of organisms can be time consuming in cases where a biochemical reaction needs extended incubation or a phenotype takes a long time to appear on a plate requiring long time viability in the culture media. Identification of pathogenic Mycobacteria by PCR amplification of microbial DNA followed by DNA sequencing or probe can by-pass the requirement for growth of microorganism on a media for slow growing or fastidious organisms and the requirement for extended incubation for scoring biochemical or phenotypic results for many other organisms. Therefore, sequence and probe-based identification of microorganisms offer a faster turn-around time and more accurate result compared to conventional methods based on biochemical and phenotypic tests.

Principle

• Fluorescence Resonance Energy Transfer (FRET) methodology, using hybridization probes targeted to species-specific hsp65 sequences, can be used to specifically identify Mycobacterium avium complex and closely related Mycobacterium species DNA by a characteristic melting temperature.

Clinical Indication

• Clinical suspicion of infection of blood, body fluids (e.g., CSF), or tissues.
• Pathological evidence or clinical suspicion of infectious agents in fixed paraffin embedded tissue specimens.
• Epidemiological or clinical suspicion of contaminated instruments or environmental surfaces.

Clinical Utility

• The rapid identification of bacteria, including cells with fastidious growth properties, dead cells, or an organism not otherwise speciated by morphological/biochemical means.
• The identification of the infectious agent may influence the choice of antibacterial therapy and/or monitoring of effectiveness.
• The confirmation of a diagnosis of bacterial infection vs. other pathological process (e.g., malignancy).
• The identification of the infectious agent may provide prognostic information important for patient management.
• The detection and monitoring of nosocomial infections

Sensitivity

Diagnostic sensitivity is the ability of the assay to detect a disease. If a disease-causing organism(s) is cultured from the specimen and subsequently sequenced, the diagnostic sensitivity approaches 100%. Compared to the requirement for bacteria to grow in culture, direct PCR from specimens including body fluids, fresh tissues, and/or paraffin-embedded formalin fixed tissues greatly increases the sensitivity of identifying the disease-causing organism. However, the precise diagnostic sensitivity cannot be measured. Even reconstitution experiments are inadequate due to the degree of infection, sampling bias, etc.

• Analytical sensitivities or minimal detection limits are expressed in copies of bacterial or genomic DNA in a single amplification reaction performed on purified DNA. For this assay, analytical sensitivity was estimated to be 5 genome equivalent per PCR reaction with hsp65 amplified probe.
• The sensitivity of detecting microbial DNA from a tissue/fluid sample will vary depending on the organism load in the sample submitted to our lab for testing, pretreatment such as formaldehyde fixation or staining, and any process that introduces exogenous micro-organisms or microbial DNA into the sample submitted for testing.

Specificity

Diagnostic specificity is the degree to which an assay is negative when disease is absent. In sequence-based organism identification, diagnostic specificity is difficult to determine due to the possible presence of an environmental contaminant in a specimen, which may grow in culture or amplify directly from a specimen. Results must be interpreted in conjunction with clinical history to judge whether an identified organism is a likely pathogen.

• Every measure is taken to maintain a high diagnostic specificity by rigorous protocols to avoid laboratory contamination.
• Analytic specificity is the degree to which interfering substances are not detected by an assay. For direct PCR amplification of body fluids or tissues, the presence of known and unknown interfering substances is monitored. Typically, PCR of DNA purified from fresh tissues stored in formalin (up to days at a time) is difficult to amplify.
• NOTE: Analytical & clinical interpretation of positive results must take into account the robustness of PCR amplification, the possibility that the organism identified is an environmental contaminant, possible sampling bias (e.g., 5 year old paraffin tissue block vs. fresh tissue), the consistency of identification with anatomic site and/or morphological and biochemical data, and DNA amplification.

For interesting cases employing this test methodology, please see REFERENCES