Development and Clinical Evaluation of a New Multiplex PCR Assay for a Simultaneous Diagnosis of Tuberculous and Nontuberculous Mycobacteria

Abstract

Background: The prevalence of non-tuberculous mycobacteria (NTM) has been increasing worldwide in both developed and developing countries. NTM infection is clinically indistinguishable from tuberculosis and therefore poses significant challenges in patient management, especially in patients chronically treated for pulmonary TB. In this study, we evaluated a new highly sensitive Multiplex MTB/NTM assay that can differentiate M. tuberculosis complex (MTBC) from all NTM, including the treatable and most common NTM, M. avium complex (MAC). Methods: We developed and optimized a new open- Multiplex MTB/NTM assay with two gene-targets for MTBC (IS6110/senX3-regX3) and two targets for MAC (IS1311/DT1) with samples spiked with stored strains and testing 20 replicates. Patients with presumptive TB and NTM were enrolled at the Pneumology Department of The University Teaching Hospital of Point G, in Mali. Finding: In the development stage, the new assay showed a high analytic performance with 100% detections of MTBC and MAC at only 5 colony forming units (CFUs). Overall, without the treatment failure cases, the Multiplex assay and the Xpert showed a sensitivity and specificity of 83·3% [66·4-92·6] and 96·6% [88·6-99·0], and 96·7% [83·3-99·4] and 80% [55·5-82·3], respectively. The Multiplex assay successfully detected all (5/5) the MAC cases. Interpretation: Our new Multiplex assay demonstrates better specificity than Xpert for all group studies, in addition to detecting potential NTM cases. The assay could therefore complement the widely used Xpert assay and enhance discrimination of TB and NTM infections. Funding Statement: This work was supported by the National Institutes of Health (R03AI137674, U54EB027049, D43TW010350 and UM1AI069471). Declaration of Interests: There are no conflicts of interest. Ethics Approval Statement: The study was approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Odonto-stomatology of the University of Sciences, Technics and Technologies of Bamako (USTTB) and the Institutional Review Board of Northwestern University.