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

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.