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首页> 外文期刊>Journal of Clinical Microbiology >Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens
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Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

机译：焦磷酸测序技术可快速检测临床分离株和临床标本中广泛耐药的结核分枝杆菌

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Treating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization.
机译：广泛耐药性（XDR）肺结核（TB）的治疗是一个严峻的挑战。从样本收集到获得结果，基于培养物的药物敏感性测试（DST）可能需要4周或更长时间。我们开发了一种焦磷酸测序（PSQ）测定法，包括八个子测定法，用于快速鉴定结核分枝杆菌复合物（MTBC）并同时检测与对定义XDR TB的药物具有抗性相关的突变。从DNA提取到结果可得的整个过程均在6小时内完成。该测定方法已验证可用于测试临床分离株和临床标本，从而缩短了分子DST的周转时间，并最大程度地提高了使用分子测试的益处。共研究了130个临床分离株和129个临床标本。 PSQ结果与表型DST结果之间的相关性：异烟肼为94.3％，利福平为98.7％，喹诺酮类药物（氧氟沙星，左氧氟沙星或莫西沙星）为97.6％，阿米卡星为99.2％，阿霉素为99.2％，卡那霉素为96.4％ 。对于测试临床标本，PSQ分析产生的MTBC检测灵敏度为98.4％，从所有子分析中产生完整测序结果的灵敏度为95.8％。 PSQ分析能够利用所提供的序列信息快速准确地检测出耐药性突变，从而可以进一步研究耐药性或易感性与每种突变的相关性，并积累此类知识以供将来对结果进行解释。因此，可以防止对已知不具有抗药性的突变报道错误的抗药性，这是该测定方法优于世界卫生组织认可的现有分子诊断方法的重要优势。

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