Nontuberculous Mycobacteria DNA Detection

Detection and identification of Nontuberculous Mycobacteria DNA

Background

While Mycobacterium tuberculosis complex members are the most commonly known human pathogens, Non-tubercolosis Mycobacteria (also known as environmental mycobacteria, atypical mycobacteria and mycobacteria other than tuberculosis (MOTT)) are also implicated in tuberculosis-like disease, localized lymphadenitis, gastrointestinal disease, and disseminated infections. Rapid and accurate identification of pathogenic Mycobacteria important for correct therapeutic intervention against an infectious disease. Acid fast bacilli can be very difficult to grow due to their nutritional requirements. Some specimens may never reveal the presence of a pathogen because of low abundance and/or lack of viability. The use of PCR to detect Mycobacterial DNA extracted directly from clinical specimens facilitates the identification of these pathogens.

For an accurate detection of the presence of Mycobacteria in clinical specimens, the UWMC Molecular Diagnosis Section utilizes a combination of several techniques. The detection of Mycobacterium avium complex, for example, relies on fluorescence resonance energy transfer (FRET) probes in a nested PCR protocol that targets the heat shock protein 65 gene (hsp65). Utilization of MAI complex specific fluorescent probes on a real-time PCR platform facilitates rapid and highly sensitive detection of MAI complex DNA in clinical specimens.

For other Non-tuberculous Mycobacteria (including rapidly growing mycobacteria (RGM)) and Mycobacterium leprae, detection and speciation involves a multi-locus PCR strategy. Identifications are performed by DNA sequencing, which provides direct, unambiguous data and can distinguish medically relevant subspecific phylogenetic lineages.

Principle

• Sequencing of specific gene(s) provides a rapid and accurate identification of clinically significant organism. Judicious primer selection allows amplification of sequences at the species level or at the phylogenetic level. Raw sequences are assembled then classified by comparison to public sequence databases and the UWMC Clinical Microbiology Sequence Database in order to provide organism identification.
• With >20000 sequences available in public databases, 16S rRNA gene sequencing is considered by current taxonomists to be the gold standard in bacterial identification and classification. It contains conserved regions useful for the design of universal primers that amplify the gene from all pathogenic and nonpathogenic bacteria in addition to hypervariable regions that contain species-specific signature sequences useful for bacterial identification to species level.
• Fluorescence Resonance Energy Transfer (FRET) methodology, using hybridization probes targeted to species-specific hsp65 sequences, can be used to distinguish between the most frequently isolated Mycobacteria species (M. abscessus, M. avium, M. chelonae, M. fortuitum, M. gordonae, M. haemophilum, M. intracellulare, M. kansasii, M. marinum and M. tuberculosis). Each of these species have their own characteristic melting points. They are distinguished from other Mycobacteria isolates and closely related species that are commonly isolated in the clinical laboratory. The “MAC” probe sets were designed specifically to detect and identify Mycobacterium avium complex.

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 of blood, 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 genomic DNA in a single amplification reaction performed on purified DNA. Analytical sensitivity was estimated to be 100 genome equivalent per PCR reaction with alternate 16s gene primer set, 5 genome equivalent per PCR reaction with hsp65 amplified probe, and 5 genome equivalent per PCR reaction with rpoB gene primer set.
• 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 are 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