SBIR-STTR Award

Of all diseases, Tuberculosis (TB) represents one of, if not, the greatest health disparity between whites and minorities [1]. To be specific, for every TB-infected white person in the United States, there are an estimated 9 African-Americans, 8 Latinos, 6 Native Americans, 23 Asians, and 21 Native Hawaiian/Pacific Islanders with this disease [2]. Compounded with this disparity is the prevalence of drug-resistant mutations of TB, which have an associated 1000 polymorphisms that span 36 genes, two promoter regions, and one ribosomal RNA coding region [3]. Current methodologies, available primarily to affluent healthcare communities, utilize microbial cultures, which require sophisticated laboratories and weeks before a result can be determined. Difficulties for minorities in a low socioeconomic class to commute and/or follow up with their physicians can result in a lack of appropriate treatment. A low-cost simple and rapid point-of-care (POC) test could expand drug-resistant TB diagnostics to these minority communities. However, current technologies lack sensitivity, specificity, and/or multiplexing capacity. We, therefore, propose to develop a POC device that offers the sensitivity of culture methods, specificity of nucleic acid methods, and a broad coverage of mutations. To accomplish this, we will expand upon our existing MDR-TB PCR-Microarray Biochips. These biochips consist of printed gel-element microarrays that have been shown to amplify target with immobilized primers in the gel elements. Previous work showed that at least 60 independent reactions can simultaneously amplify 1000, and in some cases 100 genomic copies, without needing to split, and thus dilute, the sample. Our team includes the Laboratorios Medicos Especializados in Juarez, Mexico. Team members from this facility will initially evaluate our sample purification device for Mycobacterium tuberculosis (MTB), previously shown to be sucessful at the hands of the British Columbia Centre for Disease Control (BC-CDC). Additionally, the Juarez team will verify Akonni's MDR-TB PCR-Microarray Biochip. In parallel, Akonni will expand the multiplexing capacity of the drug-resistant TB arrays, develop a lysis method, and translate the MDR-TB assay to Akonni's POC prototype device. During Phase II, the genotyping capacity will be expanded further and the POC devices will be translated to the Juarez clinic. This proposed test is projected to be a $3 consumable, operated on a $5000 instrument. (PUBLIC HEALTH RELEVANCE STATEMENT): Of all diseases, Tuberculosis (TB) represents one of, if not, the greatest health disparity between whites and minorities. To be specific, for every TB-infected white person in the United States, there are an estimated 9 African-Americans, 8 Latinos, 6 Native Americans, 23 Asians, and 21 Native Hawaiian/Pacific Islanders with this disease. The proposed project is to develop a point-of-care device for identifying drug-resistant strains of Tuberculosis that can be widely disseminated to minority populations.

Thesaurus Terms:
Active Follow-Up; African American; Afro American; Afroamerican; Asians; Assay; Au Element; Bedside Testings; Bioassay; Biologic Assays; Biological Assay; Black Populations; Black Or African American; British Columbia; Cdc; Centers For Disease Control; Centers For Disease Control (U.S.); Centers For Disease Control And Prevention; Centers For Disease Control And Prevention (U.S.); Clinic; Code; Coding System; Communities; Community Healthcare; Commuting; Cytolysis; Dna; Deoxyribonucleic Acid; Devices; Diagnostic; Disease; Disorder; Drug Resistant Tuberculosis; Drug Resistance; Elements; Gel; Genes; Genetic Alteration; Genetic Change; Genetic Polymorphism; Genetic Defect; Genomics; Genotype; Goals; Gold; Hand; Hawaiian; Hawaiian Population; Health Care, Community; Healthcares, Community; Heating; Instrumentation, Other; Killings; Laboratories; Latino; Lysis; M. Tb; M. Tuberculosis; M.Tb; M.Tuberculosis; Method Loinc Axis 6; Methodology; Methods; Mexico; Minority; Multidrug-Resistant Tuberculosis; Mutation; Mycobacterium Tuberculosis; Native Americans; Nucleic Acids; Pacific Island Americans; Pacific Islander; Pacific Islander American; Persons; Phase; Physicians; Polymorphism (Genetics); Polymorphism, Genetic; Population; Preparation; Prevalence; Printing; Promoter Regions; Promoter Regions (Genetics); Promotor Regions; Promotor Regions (Genetics); Reaction; Ribosomal Rna; Sampling; Sensitivity And Specificity; Specificity; Sputum; System; System, Loinc Axis 4; Technology; Testing; Testings, Bedside; Translating; Translatings; Tuberculosis; Tuberculosis, Drug Resistance; Tuberculosis, Drug Resistant; Tuberculosis, Mdr; Tuberculosis, Multi-Drug Resistant; Tuberculosis, Multidrug Resistance; Tuberculosis, Multidrug-Resistant; United States; United States Centers For Disease Control; United States Centers For Disease Control And Prevention; Work; Base; Biochip; Black American; Cost; Disease/Disorder; Disseminated Tb; Disseminated Tuberculosis; Drug Resistant; Follow-Up; Genetic Promoter Element; Genome Mutation; Health Disparities; Health Disparity; Instrument; Instrumentation; Language Translation; Member; Microbial; Oriental; Point Of Care; Point Of Care Testing; Polymorphism; Prototype; Public Health Relevance; Rrna; Resistance To Drug; Resistant Strain; Resistant To Drug; Socioeconomic; Socioeconomically; Socioeconomics; Tuberculous Spondyloarthropathy

Tuberculosis ranks in the top ten worldwide cause of death and now ranks ahead of HIV, making it the number one infectious disease killer. Perhaps, most alarming is the global prevalence of multidrug-resistant TB (MDR- TB) and extensively drug-resistant TB (XDR-TB): more than 5% of new TB cases are MDR-TB and 10% of these have XDR-TB. And, the number of enrolled XDR-TB cases has grown from 58 countries in 2010 to 105 in 2015. The concern to prevent the spread of TB has fueled a TB diagnostics market that is expected to reach $3.1 billion by 2024. The predominant method for diagnosing XDR-TB is still culture. And, culture for XDR-TB detection requires 6 to 16 weeks. The only commercially-available alternative is a test that requires a skilled technician to perform 20 manual steps, which often include an upfront culture step and PCR to improve sensitivity. One of these steps is to transfer amplified DNA from a PCR tube to a line probe strip that requires visual interpretation. The transfer step is susceptible to PCR contamination; the visual interpretation is susceptible to incorrect readings; and the complex workflow is susceptible to mistakes. Developing molecular tests for XDR-TB are inherently complex for primarily three reasons: (1) the sample type (typically sputum) is highly viscous and heterogenous, and thus extracting and purifying microbial DNA presents significant challenges, (2) there are hundreds of genetic mutations that confer TB-drug resistance, and (3) the market is cost sensitive because those infected are often impoverished. This year, Akonni received a 2017 North American New Product Innovation Award from Frost and Sullivan. The test that we are developing includes an automated method of extracting DNA from sputum and a Lab-on- a-Film diagnostic, which consists of hundreds of molecular gel drops printed on a conventional film. This film costs 350X less than standard microarray glass. We laminate this film to additional layers of flexible films and pressure-sensitive adhesives that form chambers and channels – without need for pumps, valves, or special coatings. The resulting Lab-on-a-Film assembly is then used for integrated PCR/hybridization and includes a waste chamber absorbent for eventual confinement of all liquids in order to prevent PCR contamination. The promise of this assembly strategy is that manufacturing throughput can increase from one part per minute using robotics to one part per second using the newspaper printing production method: reel-to-reel manufacturing. During Phases 1 and 2 of this SBIR project, we developed and delivered a fully-automated sample-to-answer system, which included the Lab-on-a-Film consumable, to our collaborator in Mexico and demonstrated sample-to-answer genotyping of both isoniazid and rifampin resistant strains of Mycobacterium tuberculosis. Under a separate R01 project with University of California, San Diego Medical School, we developed a modular (non-automated) system and an XDR-TB assay using our gel drop arrays on glass. For Phase 2B, we propose to: (1) further the development of the Lab-on-a-Film assembly so that it can be produced with reel-to- reel manufacturing, (2) transfer the XDR-TB assay to our Lab-on-a-Film device, and (3) couple it with our sample-to-answer instrument for a fully-automated workflow. We propose to evaluate this system with retrospective XDR-TB sputa specimens collected under the R01 project.

Public Health Relevance Statement:
NARRATIVE Diagnosis and treatment of drug resistant tuberculosis is currently limited by its reliance on slow-growing, culture-based drug susceptibility testing. The goal of this project is to advance a Lab-on-a-Film (low cost) consumable test with a companion sample-to-answer instrument in order to detect extensively drug-resistant tuberculosis (XDR-TB) in sputum.

NIH Spending Category:
Antimicrobial Resistance; Biodefense; Bioengineering; Emerging Infectious Diseases; Infectious Diseases; Orphan Drug; Rare Diseases; Tuberculosis

Project Terms:
Adhesives; American; Award; Bacillus (bacterium); base; Biological Assay; California; Cause of Death; Clinical; Clinical Research; Communicable Diseases; Companions; Complex; cost; Country; design; Detection; Development; Devices; Diagnosis; Diagnostic; DNA; DNA Sequence Alteration; Drops; Drug resistance in tuberculosis; Enrollment; extensive drug resistance; Extreme drug resistant tuberculosis; Film; flexibility; Gel; Genotype; Glass; Goals; HIV; improved; innovation; instrument; Isoniazid resistance; laboratory development; Life; Liquid substance; Manuals; medical schools; Methods; Metric System; Mexico; microbial; Molecular; Multidrug-Resistant Tuberculosis; Mycobacterium tuberculosis; Newspapers; One-Step dentin bonding system; Performance; Pharmaceutical Preparations; Phase; point of care; pre-clinical; Predisposition; Preparation; pressure; Prevalence; prevent; Printing; Process; Production; Protocols documentation; Pump; Readiness; Reading; Reagent; Regulation; resistant strain; Rifampicin resistance; Robotics; Sampling; Sensitivity and Specificity; Shipping; Small Business Innovation Research Grant; Specificity; Specimen; Sputum; System; Testing; Tube; Tuberculosis; tuberculosis diagnostics; Universities; Visual; wasting