Multiplex TB-M.avium complex Fluorescent in Situ hybridization Assay, direct from

多重 TB-M.avium 复合物荧光原位杂交检测，直接来自

• 批准号: 7749251
• 负责人: JYOTSNA S SHAH
• 金额: $ 33.11万
• 依托单位: ID FISH TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749251
• 关键词: Acid Fast Bacillae Staining Method; Antitubercular Agents; Area; Biochemical; Biological Assay; Clinical; Clinical Trials; Complex; Detection; Developing Countries; Development; Disease; Ensure; Equipment; Fluorescent in Situ Hybridization; Genus Mycobacterium; HIV; Home environment; Hour; Housing; Label; Lung diseases; Marketing; Methods; Microscope; Mycobacterium avium Complex; Mycobacterium tuberculosis; Patients; Performance; Phase; Preparation; Procedures; Process; Quality Control; Reading; Reagent; Reporting; Ribosomal RNA; Sampling; Sensitivity and Specificity; Slide; Specificity; Sputum; Staining method; Stains; Temperature; Testing; Time; clinical research site; cost; fluorescence microscope; manufacturing facility; protocol development; public health relevance; sample fixation

DESCRIPTION (provided by applicant): TB-MAC FISH assay is a fluorescent in-situ hybridization assay that detects and differentiates Mycobacterium tuberculosis complex (MTB) and Mycobacterium avium complex (MAC) in culture and sputum. This assay uses fluorescent labeled MTB and MAC specific probes targeted to MTB and MAC ribosomal RNA (rRNA) respectively. 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 2 hours from receipt of sputum. In the US, non-M. tuberculosis mycobacteria (NTMs) are important cause of disease in HIV patients, with the number of NTM isolates exceeding those of MTB in some areas. Pulmonary disease, the most commonly reported localized manifestation of NTMs, is often associated with the MAC. Patients with pulmonary disease are highly infectious and require immediate isolation and initiation of antituberculosis therapy. Therefore, all smear-positive patients are kept in isolation until confirmed. This is very costly. Currently there are no tests to detect MAC directly from sputum. The standard method of detecting MAC is by culturing Nalc/NaOH processed sputum followed by culture confirmation, using Gen-Probe AccuProbe assay, home-brew amplified assays or biochemical methods. This can take anywhere from 3 days to 2 weeks. Thus, if there was a way of determining which of the smear-positive patients were MTB and/or MAC positive on the same day, this would reduce the patient management cost. The TB-MAC FISH test may be an answer. Specific Aims: Develop a simple, rapid and in-expensive TB-MAC FISH test kit for direct detection of MTB and MAC in culture and 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 TBMAC 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, assay sensitivity and specificity in pure cultures and spiked sputum samples. (5) Performance of TB-MAC 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. PUBLIC HEALTH RELEVANCE: In the US, Mycobacteria other than M. tuberculosis (NTMs) are important cause of disease, with the number of NTMs isolates exceeding those of M. tuberculosis in some areas. Pulmonary disease, the most commonly reported localized manifestation of NTMs, is often associated with the M. avium complex (MAC). Patients with pulmonary disease are highly infectious and require immediate isolation and initiation of antituberculosis therapy. Therefore, all smear-positive patients are kept in isolation until confirmed. This is very costly. Currently there are no tests to detect MAC directly from sputum. The standard method of detecting MAC is by culturing Nalc/NaoH processed sputum followed by culture confirmation, using Gen-Probe AccuProbe assay, home-brew amplified assays or biochemical methods. This can take anywhere from 3 days to 2 weeks. Thus, if there was a way of determining which of the smear-positive patients were MTB and/or NTMs positive on the same day, this would reduce the patient management cost. Therefore, a test like TB-MAC FISH assay with the following attributes would be very useful. 1. Can be performed directly on sputum or culture. 2. Has specificity of amplified assays and sensitivity equivalent or better than acid-fast staining. 3. Does not require expensive equipment other than a microscope. 4. From time of receipt of sputum sample to result is about 2 hours.

描述（由申请人提供）：TB-MAC FISH试验是一种荧光原位杂交试验，用于检测和区分培养物和痰液中的结核分枝杆菌复合体（MTB）和鸟分枝杆菌复合体（MAC）。该实验使用荧光标记的MTB和MAC特异性探针分别针对MTB和MAC核糖体RNA （rRNA）。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。从吸痰算起总时间不超过2小时。在美国，非m。结核分枝杆菌（NTM）是HIV患者发病的重要原因，在某些地区NTM分离株数超过MTB分离株数。肺部疾病是ntm最常见的局部表现，通常与MAC相关。肺部疾病患者具有高度传染性，需要立即隔离并开始抗结核治疗。因此，在确诊之前，所有痰检呈阳性的患者都将被隔离。这非常昂贵。目前还没有直接从痰液中检测MAC的方法。检测MAC的标准方法是通过培养Nalc/NaOH处理过的痰液，然后使用Gen-Probe AccuProbe法、自酿扩增法或生化法进行培养确认。这可能需要3天到2周的时间。因此，如果有一种方法可以确定哪些痰检阳性患者在同一天是MTB和/或MAC阳性，这将降低患者管理成本。TB-MAC FISH测试可能是一个答案。具体目的：开发一种简单、快速且价格低廉的结核分枝杆菌MAC FISH检测试剂盒，用于直接检测培养物和痰液中的结核分枝杆菌和MAC。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) TBMAC FISH法室温性能的优化。(4)制定质量控制程序，以确保正确读取载玻片并确定纯培养物和加标痰样的检测限、检测灵敏度和特异性。(5) TB-MAC FISH法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。公共卫生相关性：在美国，结核分枝杆菌以外的分枝杆菌（ntm）是重要的致病原因，在某些地区，ntm分离株的数量超过了结核分枝杆菌。肺部疾病是ntm最常见的局部表现，通常与鸟分枝杆菌复合体（MAC）有关。肺部疾病患者具有高度传染性，需要立即隔离并开始抗结核治疗。因此，在确诊之前，所有痰检呈阳性的患者都将被隔离。这非常昂贵。目前还没有直接从痰液中检测MAC的方法。检测MAC的标准方法是通过培养Nalc/NaoH处理过的痰液，然后使用Gen-Probe AccuProbe法、自酿扩增法或生化法进行培养确认。这可能需要3天到2周的时间。因此，如果有一种方法可以确定哪些痰检阳性患者在同一天是MTB和/或ntm阳性，这将降低患者管理成本。因此，像TB-MAC FISH这样具有以下属性的测试将非常有用。1. 可直接在痰液或培养物上进行。2. 具有扩增试验的特异性和相当于或优于抗酸染色的敏感性。3. 不需要昂贵的设备，除了显微镜。4. 从收到痰样到出结果约2小时。