Identification of nontuberculous mycobacteria using commercial DNA probes and gene sequencing

Diagnosis of NonTuberculous Mycobacteria (NMT) infection frequently runs into difficulties regarding a precise definition of the strains.The use of molecular assays is the technique of choice for the identification of species, but commercial methods recognize only a limitednumber of species.Aim of this study was molecular identification of NonTuberculous Mycobacteria (NTM) in clinical specimens using commercial methodsand automated sequencing.We analyzed 6192 clinical specimens for the isolation of Mycobacteria. One hundred and twelve strains of NTM were previously analyzedwith GenoType Mycobacteria CM/AS kit and then with entire 16S rDNA and partial hsp65 sequencing.100/112 NMT strains were identified with GenoType Mycobacteria CM/AS kit as: M. gordonae (n°25), M. xenopi (n°24), M. fortuitum (n°15),M. avium (n°12), M. intracellulare (n°7), M. chelonae (n°6), M. malmoense (n°4), M. peregrinum (n°3), M. mucogenicum (n°1), M. kansasii (n°1), M.abscessus (n°1), M. heckeshornense (n°1).Twelve unidentified strains were subjected to the entire 16S rDNA gene sequencing and nine wereidentified as M. arupense (n°7), M. avium complex (n°1), M. kumamotonense (n°1).Three unidentified strains were subjected to partial hsp65gene sequencing and one was identified as M. arupense.Conclusions. Direct sequencing of entire 16SrDNA gene and of partial hsp65 gene appears to be a useful tool for the study of strains that arenot identifiable by commercial methods.This new approach, applied to clinical diagnostic, allows also recognition of unusual strains or new species.