title: [Use of different PCR-based techniques integrated into a non-tuberculous identification algorithm].
abstract: BACKGROUND
  The aim of the present work was to demonstrate the utility of a non-tuberculous mycobacteria (NTM) identification algorithm, which integrates different PCR-based techniques and basic phenotypic features. Moreover, the algorithm for pattern restriction analysis of hsp65 (hsp65 PRA) interpretation has been updated.
  
  
  METHODS
  The workflow chosen consisted of the identification by a DNA hybridization probe method, followed by PCR-restriction enzyme analysis of hsp65 (hsp65 PRA) in those isolates that cannot be identified by hybridization probes. If necessary, 16S rRNA gene and hsp65 gene sequencing were used for speciation.
  
  
  RESULTS
  A total of 236 NTM were collected, in which 102 (43.2%) isolates were identified by DNA specific probes and 76 (32.2%) isolates were identified with hsp65 PRA. Partial sequencing of the 16S rRNA gene was used for species identification of the remaining 58 (24.5%) isolates. Fifty-three (22.4%) were identified using this method. Five isolates (2.1%) were submitted for partial sequencing of hsp65 gene and one isolate was identified with this method. Four strains (1.7%) could not be identified at species level. Three new PRA patterns were found. Seven isolates tested positive with the AccuProbe Mycobacterium avium complex identification test but did not test positive with the M. avium or Mycobacterium intracellulare specific probes. Five and two of these isolates were identified as M. intracellulare and Mycobacterium colombiense, respectively.
  
  
  CONCLUSIONS
  This approach allowed us to identify almost all NTM isolates found in this study, including some recently described species.
authors.affiliation: Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau, Barcelona, España. oscar.esparcia@marinasalud.es
authors.forename: Óscar
authors.initials: Ó
authors.lastname: Esparcia
authors.forename: Montserrat
authors.initials: M
authors.lastname: Español
authors.forename: Montserrat
authors.initials: M
authors.lastname: Garrigó
authors.forename: Carmen
authors.initials: C
authors.lastname: Moreno
authors.forename: Michel
authors.initials: M
authors.lastname: Montemayor
authors.forename: Ferran
authors.initials: F
authors.lastname: Navarro
authors.forename: Pere
authors.initials: P
authors.lastname: Coll
mesh_terms: D000465:Algorithms; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D009161:Mycobacterium / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004269:DNA, Bacterial