Diagnostic MDR- and XDR-TB PCR TruArray

描述（申请人提供）：结核分枝杆菌（TB）感染了大约三分之一的世界人口，每年大约有900万新发TB病例，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M。结核病的倍增时间为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，因为已知耐药特征的范围大大超过了传统PCR检测的多重检测能力。与此同时，由于试剂和阵列费用、大型且昂贵的分析仪器以及通常需要高级学位才能操作、使用或解释的劳动和时间密集型方案，微阵列仍然限于研究界。因此，特别是在低资源环境中，MDR-TB诊断所需要的是具有PCR灵敏度的高度多重测试（例如阵列），其被配置为完全封闭的扩增子测试（类似于TaqMan（tm）PCR），具有将基于阵列的分子诊断推向疾病最流行的低资源环境的技术足迹和价格点。Akonni生物系统公司开发了一种原型MDR-TB PCR TruArray（TM）和现场便携式低成本分析系统，以满足这一需求。本项目的目的是适度扩大MDR和XDR-TB基因分型的检测覆盖范围，完成所得试剂盒的生产规模扩大和试剂包装研究，并在结核病最流行的地区（美国边境县;南非）部署之前，在沃兹沃斯临床分枝杆菌中心实验室对分离株和修正痰液样本进行临床前验证实验。技术目标的基础是根据FDA质量体系法规（21 CFR 820）开发检测相关的行政和管理工作。MDR-TB阵列的成功开发和向合作的国际参考中心的分发将提供必要的临床前和真实世界使用数据，以支持将检测试剂盒分发到结核病监测和控制的全球计划中，了解代表性不足的人群中MDR-TB的真实频率，并使感染者能够使用正确的一线药物方案进行及时治疗。 公共卫生相关性：MDR-和XDR-TB PCR阵列将是PCR和微阵列技术在结核分枝杆菌疾病监测和诊断中的第一个简单，廉价的测试组合。本文开发的基础平台同样将在传染病诊断的许多领域中得到广泛应用。

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.