Molecular Detection of Nontuberculous Mycobacteria: Advantages and Limits of a Broad-Range Sequencing Approach

Abstract

The isolation of nontuberculous mycobacteria (NTM) from clinical specimens has become very common in recent years. Such organisms are typically environmental and occasionally pathogenic for humans and animals. Standard diagnosis of mycobacterial infections relies on direct examination and culture. However, molecular tools are now available which allow quicker and more accurate diagnosis. Detection of NTM can be performed directly from clinical samples, although identification is mostly carried out after isolation. Sequencing of genomic targets (such as 16S rRNA, ITS, rpoB or hsp65) allows accurate and rapid identification, but has some technical limitations. A brief summary of the molecular methods available for NTM identification and a discussion of the problems associated with the use of sequencing analysis together with a description of available algorithms for NTM identification are the major objectives of this review.

Identification of Mycobacteria from Solid and Liquid Media by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in the Clinical Laboratory

“…Gene sequencing and chromatographic analysis of lipids using HPLC and gas-liquid chromatography methods are technically cumbersome, relatively expensive, and available in only a few specialized clinical laboratories (12). DNA sequencing using 16S rRNA alone cannot differentiate between closely related rapidly growing mycobacterial species, including M. chelonae and the M. abscessus group and between M. mucogenicum and M. phocaicum, and it requires the use of hsp65 or rpoB sequencing (15 (18,22). Further studies are warranted with additional mycobacterial strains, especially those belonging to the groups and complexes so as to improve the discrimination capabilities for these species.…”

“…Other targets for molecular technique-based species identification of mycobacteria include the heat shock protein (hsp65) gene, the intergenic gene between 16S and 23S, and the gene coding for the beta subunit of RNA polymerase (rpoB) (9-11). Molecular techniques have helped greatly to improve the turnaround time; however, these require a high level of expertise and a special laboratory setup (13)(14)(15). High-pressure liquid chromatography (HPLC) for the analysis of mycolic acids (␤-hydroxy-␣-fatty acids) is also used to identify mycobacteria from culture; however, this technique is available in only a few specialized laboratories (12).…”

Nontuberculous Mycobacteria in Noncystic Fibrosis Bronchiectasis

“…In specialized laboratories, molecular tests are available for rapid identifications of most common NTM species. Sequencing of genomic targets (such as 16S rRNA) allows accurate and rapid identification, even if some technical limitations exist, such as in case of samples with polymicrobial patterns and the deficiencies in public sequence databases [ 53 ].…”

Nontuberculous Mycobacteria – Clinical and Laboratory Diagnosis: Experiences from a TB Endemic Country

“…Roth et al demonstrated that in situations where 16S rRNA gene sequences were indistinguishable, such as for M. kansasii and M. gastri , or highly similar, such as for M. malmoense and M. szulgai , the 16S–23S rRNA gene ITS sequences were a helpful supplement for the differentiation of closely related species. As with the 16S rRNA gene sequence, M. marinum and M. ulcerans have identical ITS sequences, thus, cannot be differentiated utilizing this analysis [ 14 , 15 ].…”