Rapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis Using Real-Time PCR.

Abstract

Accurate and rapid detection of drug-resistant Mycobacterium tuberculosis is fundamental for the successful treatment of tuberculosis (TB).The aim of this study was to determine the frequency of common mutations leading to isoniazid (INH) and rifampicin (RMP) resistance.In a cross-sectional study carried out in 2014, 90 patients with M. tuberculosis from five border provinces of Iran were selected. After a full clinical history and physical evaluation, real-time polymerase chain reaction (PCR) technique was performed for the detection of mutations in the patients' katG and rpoB genes. The results were compared with results of a standard proportion method as well as a multiplex allele-specific PCR (MAS-PCR).A total of 23 mutations were found in isolates among which, codon katG 315, rpoB P1 (511 - 519 sequence) and rpoB P2 (524-533 sequence) were responsible for seven, nine and seven cases, respectively. The mean (standard deviation (SD)) of melting temperature (Tm) in katG 315 codon, rpoB P1 and P2 sequences in susceptible and mutant isolates was as follows: katG 85.4°C (0.18) and 87.54°C (0.62); rpo? P1 84.6°C (0.61) and 82.9°C (0.38); rpo? P2 83.4°C (0.18) and 85.3°C (0.19), respectively. In comparison to the standard proportion test, the sensitivity of real-time PCR in detecting INH- and RMP-resistant mutations was 75% and 83.3%, respectively. In comparison to the MAS-PCR test, 100% of katG 315 mutations and 80% of rpoB mutations were determined. Overall, 10% of the patients were diagnosed with a recurrence of TB. Age and previous history of TB treatment increased mutation odds in rpoB sequences (P = 0.046, P = 0.036, respectively).Detection of drug resistance associated with mutations through real-time PCR by melting analysis technique showed a high differentiating power. This technique had high concordance with the standard proportion test and MAS-PCR results.