Evaluation of conventional and molecular strategies for the rapid diagnosis and molecular characterisation of strains of Mycobacterium tuberculosis
Laboratory diagnosis of tuberculosis is often difficult and time consuming. This study has evaluated some new strategies for improved isolation and detection of Mycobacterium tuberculosis in clinical specimens. This work was conducted over several years examining samples from the a high tuberculosis prevalence population in Jeddah, Saudi Arabia and in the low tuberculosis prevalence setting in Queensland, Australia. Commercial nucleic acid amplification technologies were evaluated and compared with in-house real-time quantitative PCR strategies for both pulmonary and extrapulmonary specimens and for paraffin embedded tissue samples. The study examined strategies for the detection of multidrug resistance strains through the use of Lipa assay to detect mutations in the rpoB gene. Variable numbers of tandem DNA repeat (VNTR) typing was applied to samples from Saudi Arabia and Queensland, Australia to assess their discriminatory power and to demonstrate the diversity and uniqueness of strains of M. tuberculosis in distinct geographical regions. A combination of VNTR typing targeting six ETR loci and an additional three polymorphic MIRU loci was applied to a strains of MTB to enhance discrimination of strains. The results demonstrated that culture remains the "gold standard" for diagnosis and that a liquid culture system is essential for timely isolation of mycobacteria. Direct nucleic acid techniques are valuable diagnostic tools in samples where AFBs can be demonstrated but have markedly reduced sensitivity in AFB smear negative MTB culture positive samples. A combination of VNTR and MIRU typing provides excellent discrimination of strains of Mycobacterium tuberculosis. This stable typing strategy relies on PCR which allows for real-time epidemiology of transmission to be monitored.