Routine use of molecular methods for identification of non-tuberculous mycobacteria species in the clinical laboratory

Abstract number: P1815

Moure R., Andreu A., Castelblanco E., Mick V., Santín M., Martín R., Alcaide F.

Objective: To date, many nontuberculous mycobacteria (NTM) species are isolated from clinical samples. The identification to the species level is frequently relevant for patient management, and molecular methods seem to be the best approach for that purpose. Nowadays, there is not a clear systematic procedure for molecular identification of these microorganisms. The objective of this study was to determine the usefulness and limitations of molecular techniques to identify NTM species and to find the best identification strategy.

Methods: A prospective study of all NTM isolates from infected patients in our geographical area (Costa de Ponent, Barcelona) was carried out (2004–2007; 41-months). Genetic characterisation to species level was determined as follows: a) two reverse-hybridisation commercial systems: INNO-LiPA (June 2004-March 2007) and GenoType assays (since April 2007); b) two in-house methods: PCR-RFLP analysis of hsp65 gene (PRA), and 16S rDNA sequencing (16Seq). In addition, conventional phenotypic methods (growth rate, pigmentation, and biochemical tests) were used in combination with in-house molecular methods.

Results: A total of 489 isolates of MNT were identified during the study period: Four hundred and fifty-one of them (92.2%) were identified by commercial systems: 316 of 347 (91.1%) by INNO-LiPA (12 different species) and 135 of 142 (95.1%) by GenoType (17 different species). The MNT species identified by both methods were as follows: M. gordonae (142), M. avium (60), M. chelonae (44), M. fortuitum (44), M. intracellulare (40), M. kansasii (39), M. xenopi (34), M. avium-M. intracellulare (17), M. scrofulaceum (10), M. mucogenicum (4), M. celatum (3), M. genavense (2), M. malmoense (2), M. marinum (2), M. peregrinum (2), M. simiae (2), M. abscessus (1), M. interjectum (1), M. smegmatis (1), and M. szulgai (1). The remaining 38 isolates that were not characterised by commercial techniques (7.8%) were identified by PRA (22; 4.5%) or/and 16Seq (16; 3.3%).

Conclusions: Although a high number of different NTM species were isolated in our geographical area, the greater part of them was identified by the available commercial methods. GenoType assay showed a higher level of species discrimination than INNO-LiPA system. Despite PRA and, specially, 16Seq are the finest molecular methods to identify the majority of NTM species, conventional phenotypic techniques should be added in many cases to do a precise and definitive identification.