Bodmer Thomas, Ströhle Angelika
Institute for Infectious Diseases, University of Bern.
J Vis Exp. 2012 Apr 9(62):e3547. doi: 10.3791/3547.
Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population(1), and almost two million people are killed by TB each year. Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy. The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities. Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in > 95% of the MDR-TB isolates. The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community. Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene. It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results. It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated. Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.
由结核分枝杆菌（MTB）引起的结核病（TB）仍然是一个重大的公共卫生问题：该感染影响着全球多达三分之一的人口（1），每年有近200万人死于结核病。世界卫生组织的终止结核病战略强调要为所有结核病患者提供普遍可及的高质量、以患者为中心的治疗。在呼吸道标本中快速检测MTB并根据可靠的耐药性检测结果进行药物治疗是成功实施该战略的前提条件。然而，在世界许多地区，结核病诊断仍依赖于灵敏度低、标准化程度差的痰涂片显微镜检查方法。无效的结核病检测以及耐药MTB菌株的出现和传播日益危及全球结核病控制活动。有效的肺结核诊断需要在全球范围内提供标准化、易于使用且可靠的诊断工具，以便能够直接检测MTB复合群以及对关键抗生素（如利福平（RIF））的耐药性。后者的结果可作为耐多药MTB（MDR-TB）的标志物，在>95%的MDR-TB分离株中均有报道。快速获得可靠的检测结果可能会直接转化为合理的患者管理决策，最终治愈个体患者并打破社区内结核病传播的链条。赛沛公司（美国加利福尼亚州桑尼维尔）的Xpert MTB/RIF检测以显著的方式满足了上述要求。它是一种核酸扩增检测方法，用于1）检测痰液或浓缩痰液沉淀物中的MTB复合群DNA；以及2）检测rpoB基因的利福平耐药相关突变。它设计用于赛沛公司的GeneXpert Dx系统，该系统集成并自动化样本处理、核酸扩增以及使用实时PCR和逆转录PCR检测靶序列。该系统由一台仪器、个人计算机、条形码扫描仪以及用于运行检测和查看结果的预加载软件组成。它采用一次性使用的Xpert MTB/RIF检测卡，其中装有PCR试剂并进行PCR过程。由于检测卡是独立的，可消除样本之间的交叉污染。目前用于检测MTB的核酸扩增方法复杂、劳动强度大且技术要求高。Xpert MTB/RIF检测有潜力为结核病和耐药性带来标准化、灵敏且非常特异的诊断检测，前提是普及型即时检测点能够负担得起。为了便于获取，创新诊断基金会（FIND）已协商大幅降价。目前FIND协商的价格以及有资格享受折扣的国家名单可在网上查询。
