SBIR-STTR Award

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

TB-FISH is Dual Probe Fluorescent In-Situ Hybridization assay for detecting and differentiating Mycobacterium tuberculosis complex (MTB) ribosomal RNA (rRNA) and Mycobacterium species (rRNA) on a SINGLE methanol fixed smear, prepared either from a culture or directly from a specimen. The assay uses MTB and Mycobacterium genus (M-Genus) specific probes labeled with different fluorescent dyes. The assay is simple, rapid and in-expensive (< $5.00/test and a one time expense of ~$1200 for filters). The assay consists of six steps - Smear preparation and fixation, pretreatment, hybridization, and washing, counterstaining and viewing the processed smear under a fluorescent microscope. MTB and Mycobacterium shall fluoresce with different colors under specific dual pass filters. The total assay time is about 2 hours. Specific Aims: Develop a simple, rapid and in-expensive TB-FISH test kit for culture confirmation and direct detection of MTB in sputum samples. The kit shall contain all the reagents and control smears. Tuberculosis is the disease of the poor. Over one-third of the world's population (mostly in Africa and Asia) is at risk. The only means of identification of MTB is by microscopic examination of acid-fast stained smears. Unfortunately the acid-fast smear lacks both sensitivity and specificity. We demonstrated in Phase I that the MTB-FISH assay sensitivity in pure culture is between 1000 to 10,000 cells/ml for all the MTB complex bacteria. Based on a clinical study performed on over 100 patient smears, the assay sensitivity was better than acid-fast stained smears. As compared to culture, the FISH assay sensitivity in smear positive samples is >95% and between 40-67% in smear negative samples, with overall sensitivity > 80%, The acid-fast smear sensitivity is around 60%. As compared to culture, the MTB assay has 100% specificity in cultures and sputum. Based on the Phase I studies, the FISH assay may provide the specificity equivalent to amplified DNA probe assays, and sensitivity better than acid-fast smears. In the industrialized nations, this test may be useful for individuals with pulmonary tuberculosis since they are highly infectious and require immediate isolation and initiation of antituberculosis therapy. Specific Aims for Phase II (1) Purchase microscopes and filters for Clinical Sites and MTb manufacturing department (2) Manufacture TB-FISH Kits and determine shelf-life of reagents. (3) Evaluate Analytical Performance - Sensitivity and Specificity (4) Perform Interference Study - Endogenous and Exogenous (5) Evaluate Assay Reproducibility (6) Evaluate Clinical Performance of the TB-FISH Assays at 4-6 sites. (7) Develop inexpensive Microscope for FISH assays. Proceeding to Phase III will be based on completing the clinical trials and demonstrating specificity of at least 95% and sensitivity equivalent to or better than acid-fast stained smear as compared to culture. Specific Aims for Phase III (1) Filing PMA. (2) Marketing. (3) Work with WHOM to obtain their stamp of approval. This will avoid long delays in approval of the tests in many countries. (4) Develop M. avium complex FISH assay for detecting and differentiating MAI complex from other mycobacteria on a SINGLE smear.

Public Health Relevance:
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. Most of the TB endemic world is also 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. Based on the feasibility study performed in Phase I, we would like get a FDA approval for the TB-FISH assay. Therefore, in Phase II we shall perform all the studies required by FDA for a PMA filing.

Public Health Relevance Statement:
Project Narrative Project Title: TB-FISH (M. tuberculosis Fluorescent In -Situ Hybridization) Assy for MTB Detection in Unprocessed Sputum Shah Tuberculosis caused by Mycobacterium tuberculosis complex (MTB), is the second leading killer of adults in the world, with 9.2 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. Most of the TB endemic world is also 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. Based on the feasibility study performed in Phase I, we would like get a FDA approval for the TB-FISH assay. Therefore, in Phase II we shall perform all the studies required by FDA for a PMA filing.

Project Terms:
21+ years old; AFB; Acid Fast; Acid Fast Bacillae; Acid Fast Bacillae Staining Method; Adult; Africa; Africa South of the Sahara; Alcohol, Methyl; Antitubercular Agents; Antitubercular Drugs; Asia; Assay; Au element; Bacteria; Bioassay; Biologic Assays; Biological Assay; Carbinol; Cells; Clinical; Clinical Research; Clinical Sensitivity; Clinical Study; Clinical Trials; Clinical Trials, Phase I; Clinical Trials, Phase II; Clinical Trials, Unspecified; Color; Comment; Comment (PT); Comment [Publication Type]; Commentary; Commentary (PT); Complex; Country; DNA Hybridization Probes; DNA Probes; Detection; Developed Countries; Developed Nations; Development; Diagnosis; Disease; Disorder; Early-Stage Clinical Trials; Editorial Comment; Editorial Comment (PT); Equipment; Experimental Designs; FISH Technic; FISH Technique; FISH analysis; Feasibility Studies; Fixation; Fluorescence Agents; Fluorescent Agents; Fluorescent Dyes; Fluorescent in Situ Hybridization; Genus Mycobacterium; Goals; Gold; Guidelines; Hour; Human, Adult; In Situ Hybridization, Fluorescence; Individual; Industrialized Countries; Industrialized Nations; Label; Life; Low income; Lung TB; Lung Tuberculosis; M. tb; M. tuberculosis; M.tb; M.tuberculosis; Marketing; Medical Device; Methanol; Methods; Microscope; Microscopic; Mycobacterium; Mycobacterium tuberculosis; Patients; Performance; Phase; Phase 1 Clinical Trials; Phase 2 Clinical Trials; Phase I Clinical Trials; Phase I Study; Phase II Clinical Trials; Population; Preparation; Process; Published Comment; Pulmonary TB; Pulmonary Tuberculosis; Reagent; Reproducibility; Research Resources; Research Specimen; Resources; Ribosomal RNA; Risk; Sampling; Sensitivity and Specificity; Site; Specificity; Specimen; Sputum; Staining method; Stainings; Stains; Sub-Saharan Africa; Subsaharan Africa; Suggestion; Temperature; Testing; Time; Tube; Tuberculosis; Tuberculosis, Pulmonary; Tuberculostatic Agents; UV laboratory microscope; Ultraviolet Microscopes; Viewpoint; Viewpoint (PT); Wood Alcohol; Work; adult human (21+); anti-tuberculosis; antituberculosis; base; clinical investigation; clinical research site; clinical site; cost; disease/disorder; disseminated TB; disseminated tuberculosis; fluorescence microscope; fluorescence/UV microscope; fluorescent dye/probe; fluorescent microscope; laboratory fluorescence light microscope; phase 1 study; phase 1 trial; phase 2 study; phase 2 trial; phase I trial; phase II trial; protocol, phase I; protocol, phase II; public health relevance; rRNA; sample fixation; study, phase II; tuberculous spondyloarthropathy