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

摘要 非结核分枝杆菌引起的慢性肺病（NTM-LD）是一种无声的新兴流行病， 美国1 -3 NTM-LD的发病率和患病率逐年增加， 在美国，NTM-LD的发病率超过了结核病（TB）。4已知的NTM-LD的两个最大风险因素是慢性 阻塞性肺病（COPD）和预先存在的支气管扩张症。3在美国，约1200万 个人患有COPD，并且它是美国的第三大死亡原因。 据估计，已知COPD患者的支气管扩张率高达约70%。6与TB一样， 延长联合药物治疗是NTM治疗的中心原则。因此，必须 同时施用几种药物以使灭菌条件最大化。与结核病不同， 现在，用于检测感染和抗菌素耐药性的快速分子检测和最近批准的新的 由于多种原因，NTM引起的疾病是一个主要问题，最重要的是它们的内在原因， 在缺乏抗菌药物快速诊断测试的情况下， 敏感性甚至NTM感染。7，8 NTM-LD的诊断由于临床 TB和NTM-LD的临床表现和放射学检查结果几乎无法区分。照经上所 在诊断NTM-LD以“排除”结核病时，即使在患病率较低的地区，如美国， 因为它的传染性和致命性。为了区分结核病和NTM-LD，临床医生必须依靠 结合分枝杆菌学和分子检测来鉴定疾病的病原体， NTM包括13个临床相关种属/亚种，必须与大约180个种属/亚种区分开 其他很少致病的NTM物种。除了准确识别物种之外， 此外，还需要检测抗生素耐药性。因此，分枝杆菌的算法 诊断测试是复杂的，需要各种各样的测试方法，这些方法要么反应缓慢， （培养），不敏感（涂片），或不够全面（分子）。 呼吸道分枝杆菌感染和抗生素耐药性的分子检测具有挑战性。的 标本类型（痰液）是粘稠的，高度异质性;分枝杆菌很难溶解; 物种的数量是相当可观的;并且赋予耐药性的多态性是众多的。到 为了应对这些挑战，我们建议将以下内容自动化并集成到一个用户步骤测试中： 使用旋转磁盘将玻璃珠混沌混合，以使痰液均质化并溶解杆菌， 在移液器吸头中的圆盘用于纯化和浓缩核酸，以及薄膜实验室测试用于物种形成和检测 基因多态性导致抗药性。对于第一阶段，我们建议开发一种薄膜实验室测试， 排除结核病、诊断NTM-LD和检测耐药性所需的灵敏度， 结核病（MTB）和/或临床上重要的NTM存在。

ABSTRACT 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.