Rapid detection of isoniazid resistance in Mycobacterium tuberculosis isolates by use of real-time-PCR-based melting curve analysis.
نویسندگان
چکیده
The MeltPro TB/INH assay, recently approved by the Chinese Food and Drug Administration, is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test specially designed to detect 30 isoniazid (INH) resistance mutations in katG position 315 (katG 315), the inhA promoter (positions -17 to -8), inhA position 94, and the ahpC promoter (positions -44 to -30 and -15 to 3) of Mycobacterium tuberculosis. Here we evaluated both the analytical performance and clinical performance of this assay. Analytical studies with corresponding panels demonstrated that the accuracy for detection of different mutation types (10 wild-type samples and 12 mutant type samples), the limit of detection (2×10(3) to 2×10(4) bacilli/ml), reproducibility (standard deviation [SD], <0.4°C), and the lowest heteroresistance level (40%) all met the parameters preset by the kit. The assay could be run on five types of real-time PCR machines, with the shortest running time (105 min) obtained with the LightCycler 480 II. Clinical studies enrolled 1,096 clinical isolates collected from three geographically different tuberculosis centers, including 437 INH-resistant isolates and 659 INH-susceptible isolates characterized by traditional drug susceptibility testing on Löwenstein-Jensen solid medium. The clinical sensitivity and specificity of the MeltPro TB/INH assay were 90.8% and 96.4%, respectively. DNA sequencing analysis showed that, except for the 5 mutants outside the detection range of the MeltPro assay, a concordance rate between the two methods of 99.1% (457/461) was obtained. Among the 26 mutation types detected, katG S315T (AGC→ACC), inhA -15C→T, katG S315N (AGC→AAC), and ahpC promoter -10C→T accounted for more than 90%. Overall, the MeltPro TB/INH assay represents a reliable and rapid tool for the detection of INH resistance in clinical isolates.
منابع مشابه
تشخیص موتاسیون در کدون 315 ژن katG، مارکر مقاومت به ایزونیازید در سوش‌های مایکوباکتریوم توبرکولوزیس جدا شده از بیماران اصفهان و تهران با روش PCR-RFLP
Background and Objective: Drug resistance to tuberculosis is continuously increasing and is a significant threat to tuberculosis control programs because afew effective drugs are present against Mycobacterium tuberculosis. Although isoniazid (INH) is the most effective drug against tuberculosis, resistance to this drug also develops readily. Mutations in katG, specially the Ser315Thr substituti...
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In this study, Substitution at codon Ser315 of katG gene, a reliable marker for isoniazid (INH) resistance was analyzed and compared by three molecular methods such as DNA sequencing, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and PCR-single strand conformation polymorphism (PCR-SSCP) in 105 phenotypically resistant isolates obtained from various parts of Ind...
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متن کامل
Isoniazid MIC and KatG Gene Mutations among Mycobacterium tuberculosis Isolates in Northwest of Iran
Objective(s) Isoniazid (INH) is one of the main first line drugs used in treatment of tuberculosis and development of resistance against this compound can result in serious problems in treatment procedures. Resistance to INH is mediated mainly by mutation in KatG gene that is coded for the catalase enzyme. The proportional method for detection of INH-resistance is time consuming due to the slo...
متن کاملRapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis Using Real-Time PCR
BACKGROUND Accurate and rapid detection of drug-resistant Mycobacterium tuberculosis is fundamental for the successful treatment of tuberculosis (TB). OBJECTIVES The aim of this study was to determine the frequency of common mutations leading to isoniazid (INH) and rifampicin (RMP) resistance. PATIENTS AND METHODS In a cross-sectional study carried out in 2014, 90 patients with M. tuberculo...
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ورودعنوان ژورنال:
- Journal of clinical microbiology
دوره 52 5 شماره
صفحات -
تاریخ انتشار 2014