Sahebi Leyla, Ansarin Khalil, Monfaredan Amir, Farajnia Safar, Nili Seiran, Khalili Majid
PhD of Molecular Epidemiology, Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran.
Department of Hematology, Faculty of Medicine, Tabriz Branch, Islamic Azad University, Tabriz, IR Iran.
Jundishapur J Microbiol. 2016 Apr 16;9(10):e29147. doi: 10.5812/jjm.29147. eCollection 2016 Oct.
Accurate and rapid detection of drug-resistant 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 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' and 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 315 P1 (511 - 519 sequence) and P2 (524-533 sequence) were responsible for seven, nine and seven cases, respectively. The mean (standard deviation (SD)) of melting temperature (Tm) in 315 codon, P1 and P2 sequences in susceptible and mutant isolates was as follows: 85.4°C (0.18) and 87.54°C (0.62); P1 84.6°C (0.61) and 82.9°C (0.38); 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 315 mutations and 80% of 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 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.
准确快速地检测耐药性是成功治疗结核病（TB）的基础。
本研究旨在确定导致异烟肼（INH）和利福平（RMP）耐药的常见突变频率。
在2014年开展的一项横断面研究中，从伊朗五个边境省份选取了90例患者。在进行全面的临床病史和体格评估后，采用实时聚合酶链反应（PCR）技术检测患者的katG和rpoB基因中的突变。将结果与标准比例法以及多重等位基因特异性PCR（MAS-PCR）的结果进行比较。
在分离株中总共发现了23种突变，其中，密码子315 P1（511 - 519序列）和P2（524 - 533序列）分别导致了7例、9例和7例突变。敏感和突变分离株中katG 315密码子、P1和P2序列的解链温度（Tm）平均值（标准差（SD））如下：katG 85.4°C（0.18）和87.54°C（0.62）；P1 84.6°C（0.61）和82.9°C（0.38）；P2 83.4°C（0.18）和85.3°C（0.19）。与标准比例试验相比，实时PCR检测INH和RMP耐药突变的敏感性分别为75%和83.3%。与MAS-PCR试验相比，确定了100%的katG 315突变和80%的rpoB突变。总体而言，10%的患者被诊断为结核病复发。年龄和既往结核病治疗史增加了rpoB序列中的突变几率（分别为P = 0.046，P = 0.036）。
通过熔解分析技术的实时PCR检测与突变相关的耐药性显示出较高的鉴别能力。该技术与标准比例试验和MAS-PCR结果具有高度一致性。
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