Diagnostic MDR- and XDR-TB PCR TruArray

描述（由申请人提供）：结核分枝杆菌（TB）感染了世界上大约三分之一的人口，每年大约有900万新的结核病病例发生，200万人死于该疾病。基于培养的方法仍然是诊断耐药结核病的金标准，但是结核分枝杆菌的时间翻倍，为15 - 20小时，这意味着基于培养的诊断（更不用说药物敏感性测试）可能需要数周时间。然而，耐多药结核病的遗传学给PCR、分子信标或线探针检测带来了问题，因为已知的耐药特征的范围大大超过了传统PCR检测的多路处理能力。与此同时，由于试剂和阵列的费用，大型和昂贵的分析仪器，以及通常需要高级学术学位才能操作，使用或解释的劳动和时间密集型协议，微阵列仍然局限于研究界。因此，耐多药结核病诊断所需要的，特别是在资源匮乏的环境中，是一种具有PCR灵敏度的高度多路测试（例如阵列），配置为完全封闭的扩增子测试（类似于TaqMan(tm) PCR），具有技术覆盖范围和价格点，可将基于阵列的分子诊断推向疾病最普遍的资源匮乏环境。Akonni Biosystems开发了一种耐多药结核PCR TruArray（tm）原型和现场便携式低成本分析系统来满足这一需求。该项目的目标是适度扩大耐多药和广泛耐药结核病基因分型的检测覆盖率，完成对所得试剂盒的生产规模和试剂包装研究，并在部署到结核病最流行的地区（美国边境县；南非）之前，在Wadsworth中心临床分枝杆菌学实验室对分离物和改良的痰样本进行临床前验证实验。技术目标的基础是与根据FDA质量体系法规（21 CFR 820）开发测试相关的行政和管理工作。成功开发和分发耐多药结核病系列以开展合作，国际参考中心将提供必要的临床前和实际使用数据，以支持将检测试剂盒分发到结核病监测和控制的全球规划中，了解代表性不足的人群中耐多药结核病的真实频率，并使感染者能够及时接受正确的一线药物治疗方案。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (TB) infects approximately one-third of the world's population, with approximately nine million new cases of TB occurring each year and two million deaths attributable to the disease. Culture based methods remain the gold standard for diagnosing drug resistant TB, but M. tuberculosis has a doubling time of 15 - 20 hours, which means that culture-based diagnosis (let alone drug-susceptibility testing) can require several weeks. The genetics of MDR-TB, however, poses a problem for PCR, molecular beacon, or line-probe tests, because the extent of known drug-resistance signatures greatly exceeds the multiplexing capacity of conventional PCR assays. At the same time, microarrays are still limited to the research community due to reagent and array expense, large and expensive analysis instruments, and labor and time intensive protocols that typically require an advanced academic degree to operate, use or interpret. What is therefore needed for MDR-TB diagnostics, especially in low-resource settings, is a highly multiplexed test (e.g. an array) with the sensitivity of PCR, configured as an entirely closed-amplicon test (akin to TaqMan(tm) PCR), with a technology footprint and price point that moves array-based molecular diagnostics towards low-resource settings where the disease is most prevalent. Akonni Biosystems has developed a prototype MDR-TB PCR TruArray(tm) and field-portable, low-cost analysis system to address this need. The objective of this project is to modestly expand test coverage for MDR- and XDR-TB genotyping, complete the manufacturing scale-up and reagent packaging studies for the resulting kit(s), and perform pre-clinical verification experiments on isolates and amended sputum samples at the Wadsworth Center Laboratory of Clinical Mycobacteriology prior to deployment in regions (U.S. border counties; South Africa) where TB is most prevalent. Underlying the technical aims is the administrative and management effort associated with developing the tests according to the FDA Quality System Regulation (21 CFR 820). Successful development and distribution of the MDR-TB array to collaborating, international reference centers will provide the necessary pre-clinical and real-world use data to support distribution of the test kit into global programs in tuberculosis surveillance and control, understand the true frequency of MDR-TB in under-represented populations, and enable prompt treatment of infected individuals with a correct regimen of front-line drugs. PUBLIC HEALTH RELEVANCE: The MDR- and XDR-TB PCR array will be the first-of-its-kind combination of PCR and microarray technology in a simple, inexpensive test for Mycobacterium tuberculosis disease surveillance and diagnosis. The underlying platform developed herein will likewise find broad application in many areas of infectious disease diagnostics.