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.

Mycobacterium smegmatis Skin Infection Following Cosmetic Procedures: Report of Two Cases

“… 17 Many other laboratories developed multiplex real-time PCR assays to detect tuberculous and nontuberculous mycobacteria simultaneously. 18 , 19 Our university hospital molecular laboratory also used multiplex real-time PCR targeting the NTM HSP65 gene and 16S rRNA gene and panfungal PCR targeting the 28S rRNA gene on tissue biopsy specimens to detect the NTM and fungal species. In our experience, the combination of the expected clinical presentations, histological findings, and molecular testing results often leads to a definitive diagnosis of NTM infection with specific species information.…”

Nanopore-Based Metagenomic Sequencing in Respiratory Tract Infection: A Developing Diagnostic Platform

“…NTM-PD is driven by a multitude of mycobacterial species, and is again diagnosed by traditional mycobacterial culture, which often takes 4–6 weeks to process and obtain results [ 42 ]. PCR-based methods are less common here and are often targeted at Mycobacterium tuberculosis rather than mycobacterial species more broadly [ 43 ]. NTM-PD therapy may therefore be delayed until confirmatory culture results are received, with potential negative effects on patient outcomes.…”

Beyond antibiotics: recent developments in the diagnosis and management of nontuberculous mycobacterial infection

“…Multiplex PCR assay for detection of MAC An exciting new development in rapid detection of NTM came in 2021, when SARRO et al [36] evaluated the performance of a new highly sensitive quantitative PCR assay for simultaneous detection of Mtb and Mycobacterium avium complex (MAC), using primers for two separate genes present in MAC species. The Xpert MTB/RIF molecular assay for the rapid detection of Mtb in clinical samples prior to culture revolutionised diagnosis of tuberculosis worldwide.…”

“…However, due to the large number of NTM species with varying genetic sequences, often low bacillary count within clinical specimens resulting in scanty genetic material and challenges differentiating environmental contamination from pathological infection, rapid PCR methods to detect NTM directly from clinical specimens have not been previously available [37]. SARRO et al [36] report a sensitivity of 83.3% and a specificity of 96.6% for their new multiplex PCR assay in the detection of treatment-naïve Mtb infection, which is relatively comparable to the sensitivity of 96.7% and specificity of 80.0% they found with the traditional Xpert MTB/RIF assay. Significantly, however, five confirmed cases of MAC infection were all detected using this novel assay, giving both a sensitivity and specificity of 100% in this very small sample size.…”