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

描述(申请人提供)：耐药结核病(TB)的诊断受到资金限制、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。在全球确诊的结核病病例中，只有一小部分对药物敏感性进行了筛查，因此，数十万结核病病例得到的抗结核药物治疗方案不足。其后果是灾难性的，包括高水平的发病率和死亡率、耐药性的放大以及疾病传播水平的增加。广泛耐药结核病XDR-TB的出现促使国际上呼吁更好地利用现有诊断方法和开发新的诊断方法。因此，世界卫生组织批准使用Line探针检测结核分枝杆菌复合体以及异烟肼和/或利福平。这些新的检测方法建立了一种新的诊断范式，使用快速分子方法在DNA水平识别突变，而不是使用速度慢得多的细胞培养方法。然而，直线探针法的技术要求很高，需要使用先进的实验室来降低交叉污染的风险。为了绕过这个问题，世界卫生组织最近批准了一种“受控聚合酶链式反应测试”，它可以在2小时内检测结核分枝杆菌复合体和利福平耐药性的存在。该系统的另一个优点是，它可以在一些涂片阴性病例中检测出结核分枝杆菌复合体。但是，这种新的测试只测量利福平的耐药性，因此目前只能通过传统的细胞培养方法来促进后续的药物敏感性测试，再次推迟了诊断。这里描述的研究旨在通过将一项开创性技术转移到南非来克服这些深刻的障碍，在南非，将使用StellenBosch大学储存库中保存的具有良好特征的结核分枝杆菌样本以及国家卫生实验室服务机构的常规临床样本对该技术进行评估。这项技术由马萨诸塞州布兰代斯大学的Wangh教授发明，被称为Lights-on-on/Lights-Off Probe and PrimeSafe“技术，允许同步、闭管检测与异烟肼、利福平、乙胺丁醇、氟喹诺酮和氨基葡萄糖苷的耐药性有关的突变，以及导致疾病的分枝杆菌种类。这项技术通过快速和经济实惠地定义突变谱，可以在初始诊断时用于优化药物治疗，从而有可能彻底改变许多传染病的药物敏感性的分子诊断领域。因此，这项技术具有降低发病率和死亡率的潜力，并有助于遏制全球结核病大流行。 公共卫生相关性：目前，用经批准的方法诊断耐药结核病(TB)可能需要数月时间，导致高发病率和死亡率、耐药性放大和进一步传播。这项研究旨在通过验证开创性技术来克服这些障碍，该技术允许以具有成本效益的单一封闭管形式同时识别对五种抗结核病药物的耐药性。这项技术的未来临床应用有可能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. PUBLIC HEALTH RELEVANCE: Currently diagnosis of drug resistant tuberculosis (TB) by approved methods may take months, leading to high morbidity and mortality, amplification of resistance, and further transmission. This study aims to overcome these barriers by validating pioneering technology that permits simultaneous identification of resistance to five anti-TB drugs in a cost-effective single closed- tube format. Future clinical application of this technology has the potential to stem the global TB pandemic.