Detection of resistance of Mycobacterium tuberculosis to rifampin and isoniazid in clinical specimens by real-time PCR

The aim of this study was to evaluate the possibility of a rapid prediction of resistance of Mycobacterium tuberculosis to the frontline antitubercular drugs rifampin (RMP) and isoniazid (INH) by searching for specific mutations by real-time PCR using the LightCycler instrument (Roche Biochemicals) directly in clinical specimens. Three genes were studied: rpoB, which is associated with RMP resistance, and katG and inhA, which are associated to INH resistance. For RMP, results of real-time PCR analysis were concordant with phenotypic susceptibility tests in 52 (49 susceptible and 3 resistant) out of 54 specimens tested. The discordant results were from two phenotypically susceptible specimens containing heteroresistant populations. Compared with phenotypic susceptibility tests, sensitivity and specificity of the real-time PCR assay were 100% and 96.1%, respectively; the predictive values for RMP susceptibility and resistance were 100% and 60%, respectively. For INH, real-time PCR detected the wild-type codons in all the 43 specimems from which phenotypically susceptible strains were isolated; mutations katG315 and -15 inhA were found only in 2 and 4 of 11 phenotypically INH-resistant specimens, respectively. In conclusion, the real-time PCR assay for rpoB mutations in clinical specimens appears to be excellent for the rapid and reliable prediction of sensitivity to RMP and helpful for the prediction of probable phenotypic resistance to the drug. On the other hand, prediction of resistance to INH by analysis of katG and inhA may be inappropriate in areas with low prevalence of tuberculosis, such as Italy, as the genetic mechanisms of resistance remain unidentified for approximately one third of the isolates.