Lab-on-a-Film Multiplexed Test for Respiratory Mycobacterial Infections

呼吸道分枝杆菌感染的胶片实验室多重检测

• 批准号: 10689261
• 负责人: Christopher Gerard Cooney
• 金额: $ 99.94万
• 依托单位: AKONNI BIOSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-21 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10689261
• 关键词: Academy; Acid Fast Bacillae Staining Method; Address; Algorithms; American; Aminoglycoside resistance; Antibiotics; Antimicrobial Resistance; Antimicrobial susceptibility; Azithromycin; Bacillus; Bacteria; Biological Assay; Bronchiectasis; Cause of Death; Chronic; Chronic Obstructive Pulmonary Disease; Clarithromycin; Clinic; Clinical; Clinical Microbiology; Clinical Research; Collaborations; Combination Drug Therapy; Companions; Complex; Computer software; Consumption; DNA; DNA Sequence Alteration; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Drug resistance; Elements; Erythromycin; Etiology; Film; Freeze Drying; General Population; Genes; Genetic Polymorphism; Genomics; Genus Mycobacterium; Glass; Goals; Health; Incidence; Individual; Infection; Journals; Laboratories; Laboratory Diagnosis; Laboratory Study; Life; Liquid substance; Low Prevalence; Lung diseases; Lung infections; Macrolide-resistance; Magnetism; Methodology; Methyltransferase; Microbe; Microbial Biofilms; Microbiology; Microfluidics; Molecular; Molecular Diagnostic Testing; Mycobacterium Infections; Mycobacterium abscessus; Mycobacterium avium Complex; Mycobacterium tuberculosis complex; Nucleic Acids; Organism; Patients; Performance; Pharmaceutical Preparations; Phase; Phenotype; Point Mutation; Polymorphism Analysis; Porosity; Positioning Attribute; Prevalence; Process; Protocols documentation; Pulmonary Emphysema; Pulmonary Tuberculosis; Reagent; Regimen; Relapse; Resistance; Resistance to infection; Respiratory Tract Infections; Ribosomes; Risk Factors; Robotics; Rotation; Sampling; Sensitivity and Specificity; Soil; Specificity; Specimen; Sputum; Step Tests; Testing; Time; Tuberculosis; Veterans; Viscosity; Water; automated analysis; clinical diagnosis; clinically relevant; diagnostic algorithm; drug testing; editorial; follower of religion Jewish; imager; instrument; member; mycobacterial; new epidemic; non-tuberculosis mycobacteria; novel therapeutics; optimal treatments; portability; rRNA Genes; radiological imaging; rapid test; respiratory; test strip; tuberculosis treatment; virtual; working group

ABSTRACT 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.

摘要 非结核分枝杆菌肺病(NTM-LD)在美国和许多国家是一种默默无闻的新兴流行病。 世界部分地区1-3非霍奇金淋巴瘤的发病率和流行率每年都在增加，现在远远超过 由结核分枝杆菌复合体引起的结核病在美国是已知风险最大的两个 慢性阻塞性肺疾病(COPD)和既往存在的支气管扩张是NTM-LD的因素。 在美国，大约有1200万人患有慢性阻塞性肺病，这是美国第三大死因。 在已知的COPD患者中，支气管扩张的并存比例高达~70%。6与结核病一样，要求 长期联合用药是NTM-LD治疗的核心原则。因此，至关重要的是 同时给药几种药物，以最大限度地发挥杀菌作用。虽然结核病受益于 同时检测感染和耐药性的快速分子诊断试验的发展 从最近批准的新药来看，NTM-LD的诊断和治疗没有经历过类似的经历 NTM-LD的诊断因其临床表现和X线表现而变得复杂。 肺结核和NTM-LD的诊断结果可能几乎无法区分。因此，重要的是什么时候 即使在疾病发病率较低的地区，如美国，诊断NTM-LD时也要“排除”结核病， 因为结核病和NTM-LD的治疗有很大的不同。为了区分结核病和NTM-LD，临床医生 必须依靠表型分析和分子测试相结合的方法来确定病原体和 检测对关键抗生素的耐药性。因此，当代NTM诊断测试的算法是复杂的， 需要不同的测试方法，这些方法要么不敏感(抗酸杆菌涂片)，固有地缓慢 获得结果(培养；长达6周)，或不够全面(缺乏分子测试)。 从呼吸道样本中检测NTM及其耐药性的分子检测是具有挑战性的。这个 标本类型(痰)是粘性和高度异质性的；细菌负荷通常很低，但很明显； 分支杆菌很难分解；临床上相关的NTM种类数量相当多；以及 导致耐药性的基因多态有很多。为应付这些挑战，我们建议 自动化以下内容并将其集成到一个用户步骤测试中：使用旋转的 用于均质痰和裂解杆菌的磁盘，用于提纯和浓缩吸管尖端的多孔盘 核酸，以及一种膜上实验室测试，以确定和检测导致耐药性的多态。为 第二阶段，我们建议开发一种测试，可以排除/排除结核病，指定与临床相关的NTM，并检测 NTM-LD耐药标志物。为了评估这项测试，我们建议在梅奥诊所进行临床研究， 国家犹太人健康和沃兹沃斯中心与NTM-LD临床专家合作，其中包括 成员：临床微生物学杂志编辑委员会，临床实验室标准研究所 工作组、美国微生物学会院士和该学会理事会成员。