Direct detection in clinical samples of multiple gene mutations causing resistance of Mycobacterium tuberculosis to isoniazid and rifampicin using fluorogenic probes

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Abstract
Background: This study evaluates a method based on real-time PCR for direct detection in clinical samples of the common mutations responsible for isoniazid and rifampicin resistance of Mycobacterium tuberculosis. Methods: Six pairs of fluorogenic 5′ exonuclease probes (Taqman®), mutated and wild-type, were designed for six targets: codon 315 of katG, substitution C209T in the regulatory region of inhA, and codons 513, 516, 526 and 531 of rpoB. Results: A total of 98 clinical samples harbouring resistant bacilli from 55 patients and 126 samples harbouring susceptible bacilli from 126 patients were processed. The isolates from samples were tested for drug susceptibility with the radiometric method and sequenced for the same genetic targets. Among the samples, 93 harboured isoniazid-resistant bacilli. According to the sequencing results, 30 had mutations in katG, 30 in inhA and 33 (35.4%) had no mutations in these targets. All 27 clinical specimens harbouring rifampicin-resistant bacilli showed mutations in rpoB. The detection threshold of this method in detecting target genes in serial dilutions of artificial samples was 1.5 × 103 cfu/mL. In clinical samples, the sensitivity ranged from 30.4 to 35.3% for smear-negative samples and from 95.1 to 99.2% for smear-positive samples, with a specificity of 100%. In this study, the overall sensitivity in detecting patients having the target mutations was 74.3%. Conclusions: The main advantage of the described method is the possibility of detecting rifampicin and isoniazid resistance within 48–72 h after sample collection, with a sensitivity of nearly 100% in smear-positive samples if the chosen target is responsible for the resistance.

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