Pre-clinical Development of an Advanced Genexpert Test for Drug Resistant Mtb

描述（由申请人提供）：对检测耐药结核病（TB）的快速和敏感方法的需求从未如此之大。耐多药（MDR）和广泛耐药（XDR）结核病的发病率正在以惊人的速度增长。即使在没有MDR和XDR TB的情况下，对于接受基于莫西沙星的治疗方案的患者，也越来越需要快速氟喹诺酮（FQ）耐药性测试，预计这些方案将在未来部分取代基于异烟肼（INH）的治疗。 我们的团队开发了Xpert MTB/RIF（Xpert）检测试剂盒，这是有史以来第一个独立的、接近患者的检测试剂盒，可以进行结核病检测，也可以检测对药物利福平（RIF）的耐药性。这种检测方法使结核病检测变得简单和快速，正在改变全球结核病检测。然而，在耐药性检测方面，Xpert检测试剂盒仅限于通过使用RIF耐药性作为替代物来识别可能患有MDR的患者。Xpert检测试剂盒无法判断RIF耐药患者是否仍对异烟肼敏感，因此仍可以接受含异烟肼方案治疗。它也不能确定哪些RIF耐药患者可以用FQ和/或氨基糖苷类药物治疗，而不是那些患有XDR TB的患者。因此，患者面临过度治疗或治疗不足的风险，可能会造成严重后果。此外，未确诊的XDR患者可以长时间保持感染性，进一步传播疾病。 在此，我们建议将联合收割机高度创新的10色实时PCR检测（使用新型大斯托克斯位移染料）、创新的三相PCR流控、松散的分子信标探针、现有的GeneXpert平台和基本的Xpert检测试剂盒相结合，以创建一种新的检测方法，用于鉴定对INH、FQs、阿米卡星和卡那霉素的耐药性，这些耐药性是Xpert检测方法未检测到的重要耐药性类型。这种新的“Xtend-XDR”检测试剂盒将补充目前的Xpert检测试剂盒，使其有可能为结核病患者选择适当的治疗（和感染控制）方法。Xtend-XDR检测试剂盒将使用与Xpert相同的检测程序和检测平台，并且很大程度上仅限于在主要Xpert检测中检测出TB呈阳性（且RIF耐药）的患者。这些特点将使其具有很高的成本效益。 我们已经开发了所有基本检测组件的主要证据。在当前提案中，我们将完成检测开发，并创建生产工艺、质量控制和检测软件。最后，我们将进行临床试验所需的分析和临床前研究。这些活动正是目前外地住房储备金伙伴关系所要求的类型。我们的工作将在以下具体目标进行：1.最终确定所有试验参数和湿格式的内部对照组分。2.调整检测试剂盒以进行机器人组装，并修改组装线以满足检测盒需求。3.为临床操作员开发分析软件和简单的界面。4.进行分析和临床前研究，为临床试验做准备。

DESCRIPTION (provided by applicant): The need for rapid and sensitive methods to detect drug-resistant tuberculosis (TB) has never been greater. The incidence of multi-drug-resistant (MDR) and extensively-drug resistant (XDR) TB is increasing at an alarming rate. Even in the absence of MDR and XDR TB, rapid Fluoroquinolone (FQ) resistance testing will be increasingly needed for patients who are treated with the Moxifloxacin-based regimes that are expected to partially replace Isoniazid (INH)-based treatment in the future. Our group developed the Xpert MTB/RIF (Xpert) assay, the first ever self-contained, near-patient assay that can perform TB detection and also detect resistance to the drug Rifampicin (RIF). This test is transforming TB detection worldwide by making TB detection simple and rapid. However in regards to drug resistance detection, the Xpert assay is limited to identifying patients who potentially have MDR by using RIF-resistance as a surrogate. The Xpert assay cannot tell whether a patient with RIF resistance remains INH-susceptible, and thus could still be treated with an INH-containing regime. Nor can it identify which RIF-resistant patients can be treated with a FQ and/or aminoglycoside as opposed to those who have XDR TB. Thus, patients are at risk for being either over treated or undertreated, with potentially serious consequences. Furthermore, patients with undiagnosed XDR can remain infectious for prolonged periods, further spreading their disease. Here, we propose to combine highly innovative 10-color real-time PCR detection (that makes use of novel large Stokes-shift dyes), innovative three phase PCR fluidics, sloppy molecular beacon probes, the existing GeneXpert platform and basic Xpert assay cartridge to create a new assay that identifies resistance to INH, the FQs, amikacin and kanamycin, important types of resistance not detected by the Xpert assay. This new "Xtend-XDR" assay will complement the current Xpert assay, making it possible to select appropriate treatment (and infection control) approaches in patients identified with TB. The Xtend-XDR assay will use the same testing procedures and testing platform as the Xpert, and largely be restricted to patients who already test TB positive (and RIF resistant) in a primary Xpert test. These features will make it highly cost effective. We have developed proof of principal for all essential assay components. In the current proposal, we will finalize assay development, and create manufacturing processes, quality control, and assay software. Finally, we will perform the analytic and pre-clinical studies needed to prepare for clinical trials. These activities are exactly the type requested in the current partnership RFA. Our work will be performed in the following specific aims: 1. Finalize all assay parameters and internal control components in wet format. 2. Adapt assay for robotic assembly and modify assembly line for cartridge needs. 3. Develop analytic software and a simple interface for clinical operators. 4. Perform analytic and pre-clinical studies in preparation for clinical trials.