Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing.

The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

The clinical relevance of Mycobacterial pharmacogenetics.

Current anti-tuberculosis (anti-TB) drug sensitivity testing methods provide a dichotomous readout: isolates are reported as either drug susceptible or drug resistant. This report demonstrates that rapid molecular methods may provide information concerning both the level of resistance and cross-resistance to other anti-TB drugs that is important for optimal clinical management. Specific mutations detected by the Hain GenoType MTBDRplus test, recently approved by the World Health Organization (WHO) for rapid TB diagnosis and drug resistance testing, could inform the decision of whether to include high dose isoniazid (INH) when treating patients with INH mono-resistant TB, MDR-TB or XDR-TB. The presence of mutations in the inhA gene or promoter region generally confers low level INH resistance that can be overcome by high dose INH. The same mutations also confer resistance to ethionamide indicating little benefit from its inclusion in second line treatment regimens in such cases. This information has high clinical relevance since inhA mutations account for a large proportion of INH resistance, and optimized therapy regimens are crucial to improve patient outcomes and reduce the spread of drug resistant TB. This hypothesis needs to be tested in well controlled clinical and pharmacokinetic studies.