Accurate identification of mycobacterial isolates is an essential task of the clinical microbiology laboratory that enables initiation of proper antimicrobial therapy. For many organisms, traditional phenotypic identification may be difficult, laborious and time consuming. This issue is confounded by phenotypic variation within species, the many newly described pathogenic species and the limited battery of phenotypic tests available to distinguish among all established and potential bacterial pathogens. It is sometimes essential to identify bacterial isolates to species level in order to detect unsuspected pathogens, ascribe pathogenicity to species so far considered to be nonpathogenic and to identify new bacterial species.
With >20000 sequences available in public databases, 16S rRNA gene sequencing is considered by current taxonomists to be the gold standard in identification and classification of bacteria, including the slower growing mycobacteria. 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.
For rapidly growing mycobacteria (RGM), the 65-KDa heat shock protein gene is more variable than the 16S rRNA gene, making this target a better discriminator for closely related species such as M. Abscessus and M. chelonae. Among RGM, there are important differences in antimicrobial susceptibilities and virulence. The hsp65 gene sequencing generates direct, unambiguous data and can distinguish medically relevant subspecific phylogenetic lineages.
In many specimens acid fast bacilli can be seen microscopy of tissue sections but are very difficult to grow due to their fastidious nature, or are not viable as a result of antimicrobial therapy. 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 this DNA extracted directly from clinical specimens facilitates the identification of these pathogens.
For interesting cases emplyoying this test methodology, please see our Clinical Significance page.
