ObjectivesTo characterize 250 drug-resistant Mycobacterium tuberculosis (MTB) isolates in Hong Kong with respect to their drug susceptibility phenotypes to five common anti-tuberculosis drugs (ofloxacin, rifampicin, ethambutol, isoniazid and pyrazinamide) and the relationship between such phenotypes and the patterns of genetic mutations in the corresponding resistance genes (gyrA, rpoB, embB, katG, inhA, ahpC and pncA).MethodsThe MIC values of the aforementioned anti-tuberculosis drugs were determined for each of the 250 drug-resistant MTB clinical isolates by the absolute concentration method. Genetic mutations in the corresponding resistance genes in these MTB isolates were identified by PCR-single-stranded conformation polymorphism/multiplex PCR amplimer conformation analysis (SSCP/MPAC), followed by DNA sequencing of the purified PCR products.ResultsResistance to four or five drugs was commonly observed in these MTB isolates; such phenotypes accounted for over 34% of the 250 isolates. The most frequently observed phenotypes were those involving both rifampicin and isoniazid, with or without additional resistance to the other drugs. A total of 102 novel mutations, which accounted for 80% of all mutation types detected in the 7 resistance genes, were recovered. Correlation between phenotypic and mutational data showed that genetic changes in the gyrA, rpoB and katG genes were more consistently associated with a significant resistance phenotype. Despite this, however, a considerable proportion of resistant MTB isolates were found to harbour no detectable mutations in the corresponding gene loci.ConclusionsThese findings expand the spectrum of potential resistance-related mutations in MTB clinical isolates and help consolidate the framework for the development of molecular methods for delineating the drug susceptibility profiles of MTB isolates in clinical laboratories.

A novel PCR-restriction fragment length polymorphism analysis of the hsp65 gene was developed. The restriction patterns for Mycobacterium tuberculosis and Mycobacterium avium complex (MAC) species were designed to be highly distinct, and the overall number of restriction patterns was designed to be limited. Four hundred specimens (17 reference strains and 383 clinical isolates) were tested, of which 98 were M. tuberculosis and 132 were MAC species. The assay was virtually 100% sensitive and specific for M. tuberculosis and MAC species. Moreover, it gave highly concordant results for other mycobacterial species other than M. terrae complex species. This assay can be completed in one day and is user-friendly and robust. Therefore, it is highly suitable for large-scale use in a clinical laboratory.

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