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

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.