TB- FISH Assay for Direct Detection of M. tuberculosis in unprocessed sputum

说明（申请人提供）：结核病是一种穷人的疾病。活动性肺结核（TB）的80-90%是由肺结核引起的。患有肺部疾病的人具有高度传染性。因此，为了控制结核病的传播，需要在资源贫乏的国家采用敏感、快速和负担得起的诊断方法。有些地区可能没有养殖设施。通常，鉴定结核分枝杆菌复合体（MTB）的唯一手段是通过显微镜检查抗酸染色未经处理的痰涂片。不幸的是，从未经处理的痰液中制备的抗酸涂片缺乏敏感性和特异性。在未处理的痰液中直接检测结核分枝杆菌的TB-FISH（荧光原位杂交）试验可能是一个答案。TB- FISH检测结核分枝杆菌核糖体RNA。该测试可在室温下进行，不需要离心或培养箱。该试验使用用荧光染料标记的MTB特异性探针。该检测方法简单、快速且价格低廉（约5.00美元/次）。该检测包括六个步骤：涂片制备和固定，用专有试剂预处理；杂交，洗涤，反染色，并在荧光显微镜下观察处理后的涂片。从吸痰开始，总时间小于1.5 h。具体目标：开发一种简单、快速且价格低廉的结核分枝杆菌fish检测试剂盒，用于直接检测未经处理的痰液中的结核分枝杆菌。试剂盒应包含所有试剂和控制涂片。第一阶段：(1)制定协议。(2)准备用于试验开发的载玻片。(3)室温条件下TB- FISH检测性能的优化。(4)制定质量控制程序，以确保正确读取载玻片，确定纯培养物和加标痰样的检测限和检测灵敏度和特异性。(5) TB-FISH检测在临床样品中的性能。(6)美国潜在临床地点的确定。(7)分析编写报告II期：在美国设立生产设施，在美国进行临床试验。第三阶段：获得FDA批准。美国及海外市场营销。

DESCRIPTION (provided by applicant): Tuberculosis is a disease of the poor. Between 80-90% of active tuberculosis (TB) is due to pulmonary tuberculosis. Individuals with pulmonary disease are highly contagious. Therefore, in order to control spread of TB, diagnostic methods that are sensitive, rapid and affordable in resource-poor countries are required. Some areas may not have culturing facilities. Often, the only means of identification of M. tuberculosis complex (MTB) is by microscopic examination of acid-fast stained unprocessed sputum smears. Unfortunately the acid- fast smear prepared form unprocessed sputum lacks both sensitivity and specificity. TB-FISH (Fluorescent In Situ Hybridization) assay for direct detection of MTB in unprocessed sputum, may be one answer. The TB- FISH test detects MTB ribosomal RNA. The test can be performed at room temperature, and does not require centrifugation or incubators. The assay uses MTB specific probes labeled with fluorescent dyes. The assay is simple, rapid and in-expensive (~$5.00/test). The assay consists of six steps: smear preparation and fixation, pretreatment with a proprietary reagent; hybridization, washing, counterstaining and viewing the processed smear under a fluorescent microscope. The total time is less than 1.5 h from receipt of sputum. Specific Aims: Develop a simple, rapid and in-expensive TB-FISH test kit for direct detection of MTB in unprocessed sputum. The kit shall contain all the reagents and control smears. Phase I: (1) Development of protocols. (2) Preparation of slides for development of test. (3) Optimization of TB- FISH Assay for performance at room temperature. (4) Develop Quality Control procedures to ensure correct reading of slides and determination of the limit of detection and assay sensitivity and specificity in pure cultures and spiked sputum samples. (5) Performance of TB-FISH assay in clinical samples. (6) Identification of potential Clinical Sites in the US. (7) Analyzing and compiling Report Phase II: Set up manufacturing facilities in the US and perform clinical trials in the US. Phase III: Get FDA approval. Marketing within US and Overseas. Tuberculosis caused by Mycobacterium tuberculosis complex (MTB), is the second leading killer of adults in the world, with 8.8 million new cases diagnosed every year. Culture is the gold standard. Unfortunately it can take anywhere from one week to 2 months. Amplified assays have reduced the time to detection and confirmation to less than 2 days. They are especially useful in individuals with pulmonary tuberculosis, since they are highly infectious and require immediate isolation and initiation of antituberculosis therapy. Unfortunately, most of the TB endemic world is resource poor countries. Because of the cost of the amplified assays, they are of little or no use in the TB endemic world. Some don't even have culturing facilities. The only means of diagnosis is based on acid-fast staining of direct sputum smears that lacks sensitivity and specificity. Therefore, a test like TB-FISH assay with the following attributes would be very useful. 1. Can be performed directly on unprocessed sputum at room temperature. 2. Has specificity of amplified assays and sensitivity superior to direct acid-fast staining. 3. Does not require expensive equipment other than a microscope. 4. Cost less than <$5.00 5. From time of receipt of sputum sample to result is less than 2 hours.