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

摘要 非结核分枝杆菌肺病（NTM-LD）在美国是一种无声的新兴流行病，许多国家 1 -3 NTM-LD的发病率和患病率每年都在增加，现在远远超过了 在美国，由结核分枝杆菌复合体引起的结核病（TB）的两个最大已知风险 NTM-LD的因素是慢性阻塞性肺疾病（COPD）和既存支气管扩张症。3 美国，大约1200万人患有COPD，这是美国第三大死亡原因。此外， 支气管扩张在已知COPD患者中的合并发生率高达~ 70%。6与TB一样， 延长的联合药物治疗是NTM-LD治疗的中心原则。因此，必须 同时施用几种药物以使杀菌活性最大化。虽然结核病受益于 开发快速分子诊断测试，以同时检测感染和抗菌素耐药性 从最近批准的新药来看，NTM-LD的诊断和治疗还没有经历过类似的过程。 进展。7，8 NTM-LD的诊断很复杂，因为临床表现和放射学检查 肺结核和NTM-LD的结果可能几乎无法区分。因此，当 诊断NTM-LD以“排除”结核病，即使在疾病流行率较低的地区，如美国， 因为TB和NTM-LD的治疗方法有很大的不同。为了区分结核病和NTM-LD，临床医生 必须依靠表型测定和分子检测的组合来鉴定病原体， 检测对关键抗生素的耐药性。因此，用于当代NTM诊断测试的算法是复杂的， 需要不同的检测方法，这些方法或者不敏感（抗酸杆菌涂片）， 获得结果（培养;长达6周），或不够全面（缺乏分子测试）。 呼吸道样本中NTM及其耐药性的分子检测具有挑战性。的 标本类型（痰液）粘稠且高度异质;细菌负荷通常较低但显著; 分枝杆菌难以裂解;临床相关的NTM种类的数量相当可观; 赋予药物抗性的多态性有很多。为了应对这些挑战，我们建议 自动化并将以下内容集成到一个用户步骤的测试中：使用旋转搅拌器对玻璃珠进行混沌混合 磁性圆盘用于均质化芽孢杆菌和裂解杆菌，吸液管尖端中的多孔圆盘用于纯化和浓缩 核酸，以及用于物种形成和检测赋予耐药性的多态性的薄膜实验室测试。为 第二阶段，我们建议开发一种测试，可以排除在/出结核病，物种临床相关的NTM，并检测 NTM-LD耐药标志物。为了评价该试验，我们建议在马约诊所进行临床研究， 国家犹太人健康中心和沃兹沃斯中心与临床NTM-LD专家合作，其中包括 成员：临床微生物学杂志编辑委员会，临床实验室标准协会 工作组成员、美国微生物学会研究员和该学会理事会成员。

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.