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

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.