SBIR-STTR Award

Chronic lung disease due to non-tuberculous mycobacteria (NTM-LD) is a silent and emerging epidemic in the U.S. and many parts of the world.1-3 The incidence and prevalence of NTM-LD is increasing yearly and now surpasses that of tuberculosis (TB) in the U.S.4 Two of the greatest known risk factors for NTM-LD are chronic obstructive pulmonary disease (COPD) and pre-existing bronchiectasis.3 In the U.S., approximately 12 million individuals have COPD, and it is the third leading cause of death in the U.S.5 In addition, the co-occurrence of bronchiectasis in patients with known COPD is estimated to be up to ~70%.6 Like TB, the requirement for prolonged combination drug therapy is a central tenet of NTM treatment. Consequently, it is essential that several drugs be administered concurrently to maximize sterilizing conditions. Unlike TB for which there are now rapid molecular tests for detection of infection and antimicrobial resistance and recently approved new drugs, diseases caused by NTM are a major concern for multiple reasons, foremost being their intrinsic resistance to most existing antimicrobials in the absence of a rapid diagnostic test for antimicrobial susceptibility or even NTM infection.7,8 Diagnosis of NTM-LD is complicated by the fact that clinical manifestations and radiographic findings for TB and NTM-LD are virtually indistinguishable. Thus, it is important when diagnosing NTM-LD to "rule out" TB even in regions of lower prevalence, such as the U.S. because of its infectious and deadly nature. To differentiate TB from NTM-LD, clinicians must rely on a combination of mycobacteriology and molecular tests to identify the etiological agent of the disease, which for NTM includes 13 clinically-relevant species/sub-species that must be distinguished from approximately 180 other NTM species that are rarely pathogenic. In addition to the challenge of accurately speciating mycobacteria, there is also the need to detect antibiotic resistance. Hence, the algorithm for mycobacterial diagnostic testing is complex, requiring varied testing methodologies, which are either slow to respond (culture), insensitive (smears), or insufficiently comprehensive (molecular). Molecular detection of respiratory mycobacterial infections and resistance to antibiotics is challenging. The specimen type (sputum) is viscous and highly heterogeneous; the mycobacteria bacilli are difficult to lyse; the number of species is considerable; and the polymorphisms that confer drug resistance are numerous. To address these challenges, we propose to automate and integrate the following into a one user-step test: chaotic mixing of glass beads using a rotating magnetic disc to homogenize sputum and lyse bacilli, a porous disc in a pipette tip to purify and concentrate nucleic acid, and a Lab-on-a-Film test to speciate and detect polymorphisms that confer drug resistance. For Phase 1, we propose to develop a Lab-on-a-Film test that has the required sensitivity to rule in/out TB, diagnose NTM-LD, and detect drug resistance when Mycobacterium tuberculosis (MTB) and/or clinically important NTM are present.

Public Health Relevance Statement:
PROJECT NARRATIVE Mycobacteria that cause respiratory infections, such as tuberculosis and non-tuberculous mycobacteria lung disease, require many months and tens of thousands of dollars for treatment. The diagnostic algorithm to identify (or rule out) the organism that is the cause of the infection (and detect drug resistance), is complex because of a lack of a comprehensive, sensitive and rapid test. We propose to develop a one-step molecular test that identifies (or rules out) clinically-relevant mycobacteria species and sub-species that cause respiratory infections and detects genetic mutations that confer drug-resistance. PHS 398/2590 (Rev. 09/04) Page Continuation Format Page

Project Terms:
Algorithms; Aminoglycosides; Bacillus; Biological Assay; Assay; Bioassay; Biologic Assays; Bronchiectasis; Cause of Death; Diagnosis; Disease; Disorder; DNA; Deoxyribonucleic Acid; Drug resistance; drug resistant; resistance to Drug; resistant to Drug; Combination Drug Therapy; Polychemotherapy; combination chemotherapy; combination pharmacotherapy; combined drug therapy; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Epidemic; Glass; Incidence; Infection; Laboratories; Lung diseases; Pulmonary Diseases; Pulmonary Disorder; Respiratory Disease; Respiratory System Disease; Respiratory System Disorder; disease of the lung; disorder of the lung; lung disorder; Chronic Obstructive Airway Disease; COPD; Chronic Obstruction Pulmonary Disease; Chronic Obstructive Lung Disease; Chronic Obstructive Pulmonary Disease; Methodology; Genus Mycobacterium; Mycobacterium; Mycobacterium Infections; Mycobacterial Infection; Mycobacterium tuberculosis; M tb; M tuberculosis; M. tb; M. tuberculosis; mtb; Nucleic Acids; Organism; living system; Patients; Genetic Polymorphism; polymorphism; Respiratory Tract Infections; Respiratory Infections; Risk Factors; Sputum; Testing; Time; Translating; Tuberculosis; M tuberculosis infection; M. tb infection; M. tuberculosis infection; M.tb infection; M.tuberculosis infection; MTB infection; Mycobacterium tuberculosis (MTB) infection; Mycobacterium tuberculosis infection; TB infection; disseminated TB; disseminated tuberculosis; infection due to Mycobacterium tuberculosis; tuberculosis infection; tuberculous spondyloarthropathy; Diagnostic radiologic examination; Conventional X-Ray; Diagnostic Radiology; Diagnostic X-Ray; Diagnostic X-Ray Radiology; Radiography; Roentgenography; X-Ray Imaging; X-Ray Medical Imaging; Xray imaging; Xray medical imaging; conventional Xray; diagnostic Xray; diagnostic Xray radiology; Antibiotic Resistance; Resistance to antibiotics; Resistant to antibiotics; antibiotic drug resistance; antibiotic resistant; Diagnostic tests; Film; Step Tests; Clinical; Phase; Individual; infectious organism; Infectious Agent; Diagnostic; Nature; Research Specimen; Specimen; anti-microbial susceptibility; Antimicrobial susceptibility; Complex; Protocol; Protocols documentation; infection resistance; Resistance to infection; mycobacterial; respiratory; magnetic; Magnetism; microbial; Chronic lung disease; Sampling; Genomics; Pathogenicity; Causality; causation; disease causation; Etiology; Address; Antimicrobial resistant; Resistance to antimicrobial; anti-microbial resistance; anti-microbial resistant; resistance to anti-microbial; resistant to anti-microbial; resistant to antimicrobial; Antimicrobial Resistance; DNA Alteration; DNA mutation; Genetic mutation; Sequence Alteration; genomic alteration; DNA Sequence Alteration; Detection; Low Prevalence; Molecular; Process; virtual; design; designing; Drug resistance in tuberculosis; Drug Resistance Tuberculosis; Drug Resistant TB; Drug Resistant Tuberculosis; Drug resistance in Mtb; Drug resistance in Mycobacterium Tuberculosis; Drug resistant M Tuberculosis; Drug resistant Mtb; Drug resistant Mycobacteria Tuberculosis; Mtb drug resistance; TB drug resistance; drug resistance M Tuberculosis; drug resistance Mycobacteria Tuberculosis; drug resistant M.tb; drug resistant in tuberculosis; Macrolide-resistance; Macrolide antibiotic-resistance; macrolide resistant; resistance to macrolide; resistant to macrolide; Prevalence; Resistance; resistant; antimicrobial; anti-microbial; clinically relevant; clinical relevance; novel therapeutics; new drug treatments; new drugs; new therapeutics; new therapy; next generation therapeutics; novel drug treatments; novel drugs; novel therapy; prototype; non-tuberculosis mycobacteria; non-tuberculosis mycobacterial; non-tuberculous mycobacteria; non-tuberculous mycobacterial; nontuberculosis mycobacterial; nontuberculous mycobacteria; nontuberculous mycobacterial; Lung infections; pulmonary infections; Rapid diagnostics; rapid test; rapid assay; rapid tests; detection test; detection tests; multiplex assay

Nontuberculous mycobacteria lung disease (NTM-LD) is a silent and emerging epidemic in the U.S. and many parts of the world.1-3 The incidence and prevalence of NTM-LD is increasing yearly and now far exceeds that of tuberculosis (TB) caused by Mycobacterium tuberculosis complex in the U.S.4 Two of the greatest known risk factors for NTM-LD are chronic obstructive pulmonary disease (COPD) and pre-existing bronchiectasis.3 In the U.S., approximately 12 million individuals have COPD, the third leading cause of death in the U.S.5 In addition, the co-occurrence of bronchiectasis in patients with known COPD is up to ~70%.6 Like TB, the requirement for prolonged combination drug therapy is a central tenet of NTM-LD treatment. Consequently, it is essential that several drugs be administered concurrently to maximize sterilizing activity. While TB has benefited from the development of rapid molecular diagnostic tests to simultaneously detect infection and antimicrobial resistance and from recently approved new drugs, the diagnosis and treatment of NTM-LD have not experienced similar advances.7,8 The diagnosis of NTM-LD is complicated by the fact that clinical manifestations and radiographic findings for pulmonary TB and NTM-LD may be virtually indistinguishable. Thus, it is important when diagnosing NTM-LD to "rule out" TB even in regions of lower prevalence of the disease, such as the U.S., because treatment for TB and NTM-LD are substantially different. To differentiate TB from NTM-LD, clinicians must rely on a combination of phenotypic assays and molecular tests to identify the etiological agent and to detect resistance to key antibiotics. Hence, the algorithm for contemporary NTM diagnostic testing is complex, requiring varied testing methodologies, which are either insensitive (acid-fast bacilli smear), inherently slow to obtain the results (culture; up to 6 weeks), or insufficiently comprehensive (lack of molecular tests). Molecular detection of NTM and its antimicrobial resistance from respiratory samples is challenging. The specimen type (sputum) is viscous and highly heterogeneous; bacterial burden is often low but significant; mycobacteria are difficult to lyse; the number of clinically relevant NTM species is considerable; and the polymorphisms that confer drug resistance are numerous. To address these challenges, we propose to automate and integrate the following into a one user-step test: chaotic mixing of glass beads using a rotating magnetic disc to homogenize sputa and lyse bacilli, a porous disc in a pipette tip to purify and concentrate nucleic acid, and a Lab-on-a-Film test to speciate and detect polymorphisms that confer drug resistance. For Phase 2, we propose to develop a test that can rule in/out TB, speciate clinically-relevant NTM, and detect NTM-LD drug resistance markers. To evaluate this test, we propose to perform clinical studies at Mayo Clinic, National Jewish Health, and Wadsworth Center in collaboration with clinical NTM-LD experts, which includes members of: the Journal of Clinical Microbiology Editorial Board, Clinical Laboratory Standards Institute Working Group, fellows of the American Academy of Microbiology and Board of Governors of the Academy.

Public Health Relevance Statement:
PROJECT NARRATIVE Mycobacteria that cause respiratory infections, such as nontuberculous mycobacteria lung disease, require many months and tens of thousands of dollars for treatment. The diagnostic algorithm to identify (or rule out) the organism that is the cause of the infection (and detect drug resistance), is complex because of a lack of a comprehensive, sensitive and rapid test. We propose to develop a molecular test that identifies clinically- relevant mycobacteria species and sub-species that cause respiratory infections and detects genetic mutations that confer drug-resistance.

Project Terms:
Academy; Algorithms; Antibiotics; Antibiotic Agents; Antibiotic Drugs; Miscellaneous Antibiotic; Bacillus; Bacteria; Biological Assay; Assay; Bioassay; Biologic Assays; Bronchiectasis; Cause of Death; Clinical Research; Clinical Study; Diagnosis; Laboratory Diagnosis; Disease; Disorder; DNA; Deoxyribonucleic Acid; Drug resistance; drug resistant; resistance to Drug; resistant to Drug; Combination Drug Therapy; Polychemotherapy; combination chemotherapy; combination pharmacotherapy; combined drug therapy; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Elements; Epidemic; Erythromycin; E-Mycin; Ery-Tab; Eryc; Eryderm; Erythrocin; Erythromycin A; Ilotycin; Pediamycin; RP-Mycin; Robimycin; Freeze Drying; Freeze Dryings; Lyophilization; Genes; Glass; Goals; Health; Incidence; Infection; Institutes; Laboratories; Lung diseases; Pulmonary Diseases; Pulmonary Disorder; disease of the lung; disorder of the lung; lung disorder; Methodology; Methyltransferase; EC 2.1.1; methylase; transmethylase; Microbiology; Genus Mycobacterium; Mycobacterium; Mycobacterium avium Complex; M avium Complex; M. avium Complex; M. avium intracellulare; MAIC; Mycobacterium avium-intracellulare; Mycobacterium avium-intracellulare Complex; Mycobacterium Infections; Mycobacterial Infection; Mycobacterium tuberculosis; M tb; M tuberculosis; M. tb; M. tuberculosis; mtb; Nucleic Acids; living system; Organism; Patients; Phenotype; polymorphism; Genetic Polymorphism; Emphysema; emphysematous; Pulmonary Emphysema; Reagent; Relapse; Airway infections; Respiratory Infections; Respiratory Tract Infections; Ribosomes; Risk Factors; rRNA; Ribosomal RNA; Robotics; Sensitivity and Specificity; Software; Computer software; Soil; Specificity; Sputum; Testing; Time; Tuberculosis; M tuberculosis infection; M. tb infection; M. tuberculosis infection; M.tb infection; M.tuberculosis infection; MTB infection; Mycobacterium tuberculosis (MTB) infection; Mycobacterium tuberculosis infection; TB infection; disseminated TB; disseminated tuberculosis; infection due to Mycobacterium tuberculosis; tuberculosis infection; tuberculous spondyloarthropathy; Pulmonary Tuberculosis; Lung TB; Lung Tuberculosis; Pulmonary TB; Veterans; Water; Hydrogen Oxide; Azithromycin; Azadose; Azitrocin; Azythromycin; Ultreon; Zithromax; Zitromax; Clarithromycin; 6-O-Methylerythromycin; 6-O-methyl-erythromycin; Biaxin; Microbial Biofilms; biofilm; Diagnostic tests; Film; Step Tests; Journals; Magazine; Point Mutation; follower of religion Jewish; Jewish; base; Chronic; Clinical; Phase; Individual; Collaborations; editorial; fluid; liquid; Liquid substance; clinical diagnosis; Companions; instrument; Diagnostic; Research Specimen; Specimen; Life; anti-microbial susceptibility; Antimicrobial susceptibility; Complex; Clinic; Protocol; Protocols documentation; infection resistance; Resistance to infection; mycobacterial; respiratory; magnetic; Magnetism; American; Performance; member; Laboratory Study; General Public; General Population; Position; Positioning Attribute; molecular diagnostic assays; Molecular Diagnostic Testing; Sampling; portability; Genomics; µfluidic; Microfluidics; Causality; causation; disease causation; Etiology; Clinical Microbiology; Address; Antimicrobial resistant; Resistance to antimicrobial; anti-microbial resistance; anti-microbial resistant; resistance to anti-microbial; resistant to anti-microbial; resistant to antimicrobial; Antimicrobial Resistance; DNA Alteration; DNA mutation; Genetic mutation; Sequence Alteration; genomic alteration; DNA Sequence Alteration; Detection; Low Prevalence; Molecular; Process; Acid Fast Bacillae Staining Method; Acid Fast; Acid Fast Bacillae; Development; developmental; working group; work group; virtual; Macrolide-resistance; Macrolide antibiotic-resistance; macrolide resistant; resistance to macrolide; resistant to macrolide; Aminoglycoside resistance; Aminoglycoside resistant; resistance to aminoglycoside; resistant to aminoglycoside; Prevalence; Consumption; Resistance; resistant; Microbe; clinically relevant; clinical relevance; novel therapeutics; new drug treatments; new drugs; new therapeutics; new therapy; next generation therapeutics; novel drug treatments; novel drugs; novel therapy; tuberculosis treatment; TB therapy; TB treatment; tuberculosis therapy; non-tuberculosis mycobacteria; non-tuberculosis mycobacterial; non-tuberculous mycobacteria; non-tuberculous mycobacterial; nontuberculosis mycobacterial; nontuberculous mycobacteria; nontuberculous mycobacterial; Regimen; radiological imaging; Radiography; Roentgenography; radiologic imaging; Mycobacterium abscessus; M abscessus; M. abscessus; optimal treatments; optimal therapies; Lung infections; pulmonary infections; imager; automated analysis; rapid test; rapid assay; rapid tests; test strip; diagnostic algorithm; Chronic Obstructive Pulmonary Disease; COPD; Chronic Obstruction Pulmonary Disease; Chronic Obstructive Lung Disease; Mycobacterium tuberculosis complex; M. tuberculosis complex; M.tb complex; Mtb complex