Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting

Abstract

Introduction: Nontuberculous mycobacteria (NTM) species, as human pathogens, are increasing in the world, as is the difficulty of accurately identifying them. Differential diagnosis, especially between the M. tuberculosis complex and NTM species, and the characterization of NTM species is important. This study aimed to evaluate the performance of a molecular system based on multiplex real-time PCR with high-resolution melting (HRM) for the identification and differentiation of NTM species of clinical importance of an endemic area for tuberculosis in northeastern Brazil. Methods: The technical protocol of the molecular system was based on multiplex real-time PCR-HRM, and evaluated the sensitivity and specificity of the detection of NTM species in mycobacterial clinical isolates from the studied region. The gold standard method was specific gene sequencing. Results: The sensitivity and specificity of multiplex real-time PCR-HRM modified for differentiation between NTM and M. tuberculosis were 90% and 100%, respectively. The PCR-HRM sensitivities for the characterization of NTM species (M. kansasii, M. abscesses, M. avium, and M. fortuitum) were 94.59%, 80%, 57.14%, and 54%, respectively. Conclusions: The multiplex real-time PCR-HRM modified assay has the potential to rapidly and efficiently identify nontuberculous mycobacteria of clinical importance, which is crucial for immediate implementation of the appropriate therapy and thus avoiding complications and sequelae in patients.

Detection of non-tuberculosus mycobacteria (NTMs) in lung samples using 16S rRNA

“…Similar analytical sensitivity analyses were used in the study by Peixoto and his coworkers. (25) The standardised qPCR technique showed an almost perfect agreement with the culture (Kappa 0.93), which was confirmed by sequencing. Only one sample was positive for culture, but negative for qPCR and sequencing.…”

“…(35,36) The hsp65 and rpoB genes have been described for this purpose. (8,25,34) The most frequently identified NTM species in this study was M. gordonae (33%), being a common mycobacterial species. (37) M. gordonae is considered to be a low virulence strain that is also very associated with laboratory contamination; hence the importance of oth- er factors to make the differential diagnosis of disease caused by NTM, thus reinforcing the need for careful procedures in order to differentiate contamination from infection, (4,38) since M. gordonae hardly causes disease.…”

Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in nontuberculous mycobacteria isolates from Kenya

“…Non-tuberculous Mycobacteria (NTM) are a group of about 170 Mycobacteria species that do not include Mycobacterium tuberculosis and Mycobacterium leprae (Falkinham, 2017;Koh et al, 2006;Peixoto et al, 2020;Sam et al, 2020;Simons et al, 2011). In mycobacterial culture, NTM varies in their growth characteristics, with rapid growing NTM (RGM) forming visible colonies within seven days of incubation and slow-growing NTM (SGM) taking up to fourty days.…”

TB or NTM: Can a new multiplex PCR assay be the answer?

“…In contrast, real-time PCR is an alternative and cheaper technique. A few recent studies have evaluated real-time PCR for the detection of NTM [7] , [8] , [9] . Peixoto et al.…”

“…Peixoto et al. [7] evaluated a multiplex real-time PCR with high-resolution melting for the identification and differentiation between Mtb and NTM, as well as among NTM species of clinical importance. However, this assay involved three different stages of PCR, one to differentiate Mtb and NTM and two more to differentiate the NTM species.…”