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，每年约有900万新发结核病病例，200万人死于这种疾病。基于培养的方法仍然是诊断耐药结核病的黄金标准，但结核分枝杆菌的时间翻了一番，需要15-20个小时，这意味着基于培养的诊断(更不用说药物敏感性测试)可能需要几周的时间。然而，耐多药结核病的遗传学对聚合酶链式反应、分子信标或线探针检测提出了一个问题，因为已知的耐药标记的程度远远超过了传统聚合酶链式反应检测的多重能力。与此同时，由于试剂和阵列费用、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社区。因此，对于耐多药结核病的诊断，特别是在低资源环境下，需要的是具有PCR灵敏度的高度多路复用的测试(例如，阵列)，其配置为完全闭合扩增测试(类似于TaqMan(Tm)PCR)，具有将基于阵列的分子诊断向疾病最流行的低资源环境移动的技术足迹和价格点。为满足这一需求，Akonni生物系统公司开发了一种MDR-TB聚合酶链式反应TruArray(Tm)原型和现场便携的低成本分析系统。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测试覆盖范围，完成最终试剂盒(S)的制造放大和试剂包装研究，并在部署到结核病最流行的地区(美国边境县和南非)之前，在瓦兹沃斯临床分枝杆菌学中心实验室对分离株和修改后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(21CFR 820)开发测试相关的行政和管理工作。成功开发耐多药结核病阵列并将其分发给合作的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全球结核病监测和控制计划中，了解耐多药结核病在代表性不足的人群中的真实频率，并使感染患者能够使用正确的一线药物方案进行迅速治疗。 公共卫生相关性：MDR和XDR-TB聚合酶链式反应阵列将是首个将聚合酶链式反应和微阵列技术结合在一起的简单、廉价的结核分枝杆菌疾病监测和诊断检测方法。本文开发的底层平台同样将在传染病诊断的许多领域得到广泛应用。

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.