Molecular approach to differential identification of Mycobacterium species and detection of drug-resistant tuberculosis

The recent increase in infections caused by tuberculous, as well as non-tuberculous Mycobacterium, has intensified the requirement for a rapid and reliable molecular identification tool as an alternative to current conventional methods. The differentiation between Mycobacterium tuberculosis and Mycobacterium bovis, along with the rapid detection of the resistance to first-line antituberculosis drug, pyrazinamide, are of great importance for the control of tuberculosis. The hypothesis of this study was that sequence diversity at specific targets of the Mycobacterium DNA can provide for rapid and accurate differential identification of mycobacterial species and detection of antimicrobial resistance. A unique sequence-based identification algorithm called "MycoAlign" that utilized the hypervariable sequence of the internal transcribed spacer (ITS) region of the rDNA gene was created for identification of Mycobacterium species. The study proved the feasibility of the MycoAlign assay as an alternative tool to the conventional identification methods. Sequence analysis of tested isolates has also showed five previously uncharacterized Mycobacterium species from five different immunocompromised patients with a general clinical picture of pulmonary infection. An evaluation of the phenotypic properties, the mycolic acid-HPLC profile, and the 16S rDNA-dependant phylogenetic analysis of the organism has placed the new species within the slow-growing Mycobacterium species and supported the conclusion that the described organism represented a new slow-growing Mycobacterium species, for which the name Mycobacterium nebraskiae sp. nov. was proposed. A temperature mediated Heteroduplex analysis (TMHA) assay using the denaturing high performance liquid chromatography (DHPLC) was also developed using the pncA gene as genetic target for rapid detection of pyrazinamide resistance and differentiation between M. tuberculosis and M. bovis. Chromatographic patterns generated by the two reference probes provided for the rapid detection of PZA-R with the simultaneous ability to distinguish between M. tuberculosis and M. bovis . In conclusion, the two described assays represented an alternative molecular tool that provided for rapid and accurate differential identification of all clinically important mycobacterial species including M. tuberculosis and M. bovis, and the detection of the pyrazinamide resistance among tuberculosis causing agents.