García de Viedma Darío, del Sol Díaz Infantes Maria, Lasala Fátima, Chaves Fernando, Alcalá Luis, Bouza Emilio
Servicio de Microbiología y Enfermedades Infecciosas, Hospital Gregorio Marañón, Madrid, Spain.
J Clin Microbiol. 2002 Mar;40(3):988-95. doi: 10.1128/JCM.40.3.988-995.2002.
The emergence of resistance to antituberculosis drugs is a relevant matter worldwide, but the retrieval of antibiograms for Mycobacterium tuberculosis is severely delayed when phenotypic methods are used. Genotypic methods allow earlier detection of resistance, although conventional approaches are cumbersome or lack sensitivity or specificity. We aimed to design a new real-time PCR method to detect rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains in a single reaction tube. First, we characterized the resistant isolates in our area of Spain by DNA sequencing. Some mutation was found within the rpoB core region in all the RIF-resistant (RIF(r)) strains. Forty-six percent of the INH-resistant (INH(r)) strains showed a mutation in katG codon 315, and most of these were associated with high MICs. Eighteen of the RIF(r), INH(r), and multidrug-resistant strains sequenced were tested by our real-time PCR assay; and full concordance of the results of the PCR with the sequencing data was obtained. In addition, a blind test was performed with a panel of 15 different susceptible and resistant strains from throughout Spain, and our results were also in 100% agreement with the sequencing data. Ours is the first assay based on rapid-cycle PCR able to simultaneously detect in a single reaction tube a large variety of mutations associated with RIF resistance (12 different mutations affecting 8 independent codons, including the most prevalent mutations at positions 526 and 531) and the most frequent INH resistance mutations. Our design could be a model for new, rapid genotypic methods able to simultaneously detect a wide variety of antibiotic resistance mutations.
耐抗结核药物的出现是一个全球性的重要问题，但是当使用表型方法时，结核分枝杆菌药敏谱的获取会严重延迟。基因型方法能够更早地检测出耐药性，尽管传统方法繁琐，或者缺乏敏感性或特异性。我们旨在设计一种新的实时PCR方法，以便在单个反应管中检测耐利福平（RIF）和耐异烟肼（INH）的结核分枝杆菌菌株。首先，我们通过DNA测序对西班牙我们所在地区的耐药分离株进行了特征分析。在所有耐利福平（RIF(r)）菌株的rpoB核心区域内发现了一些突变。46%的耐异烟肼（INH(r)）菌株在katG密码子315处出现突变，其中大多数与高最低抑菌浓度相关。对18株经测序的RIF(r)、INH(r)和耐多药菌株进行了我们的实时PCR检测；PCR结果与测序数据完全一致。此外，我们用来自西班牙各地的15种不同的敏感和耐药菌株进行了盲测，结果也与测序数据完全一致。我们的检测方法是首个基于快速循环PCR的检测方法，能够在单个反应管中同时检测与利福平耐药相关的多种突变（12种不同突变影响8个独立密码子，包括第526和531位最常见的突变）以及最常见的异烟肼耐药突变。我们的设计可以成为能够同时检测多种抗生素耐药突变的新型快速基因型方法的一个范例。
