Real-time PCR for single-nucleotide polymorphism detection in the 16S rRNA gene as an indicator for extensive drug resistance in Mycobacterium tuberculosis.

OBJECTIVES: A real-time PCR screening system was established for rapid detection of single-nucleotide polymorphisms (SNPs) at positions 1401, 1402 and 1484 of the 16S rRNA gene of Mycobacterium tuberculosis leading to resistance to amikacin, kanamycin and capreomycin. Resistances to the respective drugs may indicate the presence of an extensively drug-resistant (XDR) strain of M. tuberculosis. METHODS: Fifty-seven M. tuberculosis isolates that tested phenotypically susceptible or resistant to amikacin, capreomycin or both were subjected to 1401-2/1484 real-time PCR to screen for SNPs in the respective rrs region. RESULTS: 1401-2 and 1484 wild-type and mutant M. tuberculosis strains displayed distinct melting peaks. Of the cross-resistant strains, 86.7% displayed A1401G SNPs, 76.9% of amikacin-resistant strains did not display rrs SNPs and one capreomycin-resistant strain showed a C1402T SNP. CONCLUSIONS: Phenotypic drug susceptibility testing takes several weeks, but with the 1401-2/1484 real-time PCR a preliminary diagnosis can be made within a few hours. SNPs in the rrs region are not exclusively involved in the development of resistances to amikacin and capreomycin. However, 80.0% of XDR-tuberculosis samples tested were detected with the real-time PCR screening assay of the present study.

Rapid identification of multidrug-resistant Mycobacterium tuberculosis isolates by rpoB gene scanning using high-resolution melting curve PCR analysis.

BACKGROUND: Multidrug-resistant (MDR) Mycobacterium tuberculosis poses a serious threat to the control of tuberculosis (TB) and constitutes an increasing public health problem. The availability of rapid in vitro susceptibility tests is a prerequisite for optimal patient treatment. Rifampicin resistance caused by diverse mutations in the rpoB gene is an established and widely used surrogate marker for MDR-TB. We used a high-resolution melting (HRM) curve analysis approach to scan for mutations in the rpoB gene. METHODS: A total of 49 MDR-TB and 19 fully susceptible non-MDR-TB isolates, as determined by conventional drug susceptibility testing using the BACTEC-MGIT960 system, were used to evaluate the suitability of HRM curve analysis as a rapid and accurate screening system for rifampicin resistance. RESULTS: HRM analysis of the rpoB cluster I site allowed the correct allocation of 44 of the 49 MDR-TB isolates and all non-MDR-TB isolates. Three of the five MDR-TB isolates (60%) falsely identified as non-MDR-TB harboured the V176F mutation that could be specifically detected by an additional HRM assay. The combined HRM analysis of all strains and isolates exhibited 95.9% sensitivity and 100% specificity. CONCLUSIONS: With a positive predictive value of 100% and a negative predictive value of at least 99.9%, this combined HRM curve analysis is an ideal screening method for the TB laboratory, with minimal requirements of cost and time. The method is a closed-tube assay that can be performed in an interchangeable 96- or 384-well microplate format enabling a rapid, reliable, simple and cost-effective handling of even large sample numbers.