Evaluation of a highly-informative LATE-PCR single-tube assay for M(X)DR-TB

描述（由申请人提供）：耐药结核病（TB）的诊断受到财政限制、基础设施不足、结核分枝杆菌生长速度缓慢以及抗生素耐药性遗传复杂性的阻碍。在全球诊断的结核病病例中，只有一小部分接受了药物敏感性筛查，因此数十万结核病病例的治疗方案不充分。其后果是灾难性的，包括发病率和死亡率高，抵抗力增强，疾病传播水平提高。广泛耐药结核病（XDR-TB）的出现促使国际社会呼吁更好地利用现有诊断方法并开发新的诊断方法。因此，WHO批准使用线探针检测M。结核复合症和异烟肼和/或利福平。这些新的检测方法建立了一个新的诊断模式，通过使用快速分子方法，在DNA水平上识别突变，而不是慢得多的细胞培养方法。然而，线探针检测在技术上要求很高，需要使用复杂的实验室来降低交叉污染的风险。为了避免这个问题，世界卫生组织最近批准了一种“包含PCR测试”，可以检测出M。结核复合菌和利福平耐药。该系统的另一个优点是它可以检测M。在一些涂片阴性病例中存在结核综合征。但是，这种新的检测方法只能测量利福平耐药性，因此目前只能通过常规的细胞培养方法来促进后续的药物敏感性检测，再次延迟诊断。本文所述的研究旨在通过向南非转让一项开创性技术来克服这些深刻的障碍，在南非将使用具有良好特征的M。保存在斯泰伦博斯大学储存库中的结核病样本，以及来自国家卫生实验室服务的常规临床样本。该技术被称为LATE-PCR with Lights-On/Lights-Off Probes and PrimeSafe”，由Brandeis大学（马萨诸塞州）的Wangh教授发明，允许同时封闭管检测对异烟肼、利福平、乙胺丁醇、氟喹诺酮和氨基葡萄糖苷以及引起疾病的分枝杆菌物种产生耐药性的突变。该技术有可能通过快速且经济实惠地定义突变谱来彻底改变许多传染病药物敏感性的分子诊断领域，这些突变谱可用于在初步诊断时优化药物治疗。因此，这项技术有可能降低发病率和死亡率，并有助于遏制全球结核病大流行。

DESCRIPTION (provided by applicant): The diagnosis of drug resistant tuberculosis (TB) is hampered by financial constraints, inadequate infrastructure, the slow growth rate of Mycobacterium tuberculosis, and the genetic complexity of antibiotic resistance. Only a fraction of tuberculosis cases diagnosed globally is screened for drug susceptibility, thus hundreds of thousands of TB cases are treated with inadequate regimens of anti-TB drugs. The consequences are catastrophic and include high levels of morbidity and mortality, amplification of resistance, and increasing levels of disease transmission. The emergence of the extensively drug resistant TB, XDR-TB, has prompted an international call for better use of current diagnostics and the development of novel diagnostics. Accordingly, the WHO approved the use of Line Probe assays for the detection of M. tuberculosis complex and isoniazid and/or rifampin. These novel assays established a new diagnostic paradigm by using rapid molecular methods that identified mutations at the DNA level, rather than far slower cell culture methods. However, Line Probe assays are technically demanding and require use of sophisticated laboratories to reduce the risk of cross contamination. To circumvent this problem the WHO recently approved a "contained PCR test" that detects the presence of M. tuberculosis complex and rifampin resistance within 2 hours. An added advantage of this system is that it can detect M. tuberculosis complex in some smear negative cases. But, this new test only measures rifampin resistance and thus currently only serves to prompt follow-up drug susceptibility testing by conventional cell culture methods, again delaying diagnosis. The study described here is aimed at overcoming these profound barriers by transferring a pioneering technology to South Africa, where it will be evaluated using well characterized M. tuberculosis samples held in the repository at Stellenbosch University, as well as, routine clinical specimens from the National Health Laboratory service. The technology, known as LATE-PCR with Lights-On/Lights-Off Probes and PrimeSafe", invented by Professor Wangh at Brandeis University (Massachusetts), allows for the simultaneous, closed-tube detection of mutations conferring resistance to isoniazid, rifampin, ethambutol, fluorquinolones and aminoglucosides, and mycobacterial species causing disease. This technology has the potential of revolutionizing the field of molecular diagnostics for drug susceptibility in many infectious diseases by rapidly and affordably defining a profile of mutations which can be used to optimize drug treatment at initial diagnosis. Thus this technology has the potential to reduce morbidity and mortality and to help stem the global TB pandemic.