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

描述(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和痰中的结核分枝杆菌复合体(MTB)和禽分枝杆菌复合体(MAC)。该方法使用荧光标记的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，用专有试剂进行预处理；杂交、洗涤、反染色和在荧光显微镜下查看处理后的涂片。从收到痰起总时间不到2小时。在美国，非M。结核分枝杆菌(NTM)是HIV患者的重要致病因素，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NTMS最常见的局部性表现，通常与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，所有涂阳患者都被隔离，直到确诊。这是非常昂贵的。目前还没有直接从痰中检测MAC的检测方法。检测MAC的标准方法是培养NALC/NaOH处理的痰，然后进行培养确认，使用基因探针AccuProbe试验、自制酿造扩增试验或生化方法。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涂阳患者在同一天是结核分枝杆菌和/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。具体目标：开发一种简单、快速和廉价的TB-MAC FISH检测试剂盒，用于直接检测培养和痰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)为试验的开展准备幻灯片。(3)室温条件下TBMAC FISH检测方法的优化。(4)制定质量控制程序，以确保在纯培养和添加痰样本中正确读取载玻片并确定检测下限、检测灵敏度和特异性。(5)TB-MAC FISH检测在临床标本中的应用。(6)在美国确定潜在的临床地点。(7)分析和编写报告第二阶段：在美国建立制造设施并在美国进行临床试验。第三阶段：获得FDA的批准。在美国和海外进行市场营销。公共卫生相关性：在美国，结核分枝杆菌(NTMS)以外的分枝杆菌(NTMS)是重要的致病原因，在某些地区，NTMS分离株的数量超过了结核分枝杆菌。肺部疾病是NTMS最常见的局部性表现，通常与鸟型支原体复合体(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，所有涂阳患者都被隔离，直到确诊。这是非常昂贵的。目前还没有直接从痰中检测MAC的检测方法。检测MAC的标准方法是培养NALC/NaOH处理的痰，然后进行培养确认，使用基因探针AccuProbe试验、自制酿造扩增试验或生化方法。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涂阳患者在同一天是结核分枝杆菌和/或非结核分枝杆菌阳性，这将减少患者管理成本。因此，像TB-MAC FISH这样具有以下属性的测试将是非常有用的。1.可直接在痰或培养上进行。2.具有扩增试验的特异性，灵敏度与抗酸染色相当或更好。3.除了显微镜外，不需要昂贵的设备。4.从收到痰标本到检测结果的时间约为2小时。

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.