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ABSTRACTUsing INNO-LiPA-MYCOBACTERIA (Lipav1; Innogenetics) and the AccuProbe (Gen-Probe Inc./bioMe´rieux) techniques, 35 Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum(MAC/MAIS) complex strains were identified between January 2000 and December 2002. Thirty-four of 35 isolates were positive only for the MAIS complex probe by Lipav1 and were further analyzed by INNO-LiPA-MYCOBACTERIA version 2 (Lipav2), hsp65PCR restriction pattern analysis (PRA), and ribosomal internal transcribed spacer (ITS), hsp65, and 16S rRNA sequences. Lipav2 identified 14 of 34 strains at the species level, including 11 isolates positive for the newly specific MAC sequevar Mac-A probe (MIN-2 probe). Ten of these 11 isolates corresponded to sequevar Mac-A, which was recently defined as Mycobacterium chimeraesp. nov. Among the last 20 of the 34 MAIS isolates, 17 (by hsp65PRA) and 18 (by hsp65sequence) were characterized as M. avium. Ten of the 20 were identified as Mac-U sequevar. All these 20 isolates were identified as M. intracellulareby 16S rRNA sequence except one isolate identified as Mycobacterium paraffinicumby 16S rRNA and ITS sequencing. One isolate out of 35 isolates that was positive for M. aviumby AccuProbe and that was Mycobacteriumgenus probe positive and MAIS probe negative by Lipav1 and Lipav2 might be considered a new species. In conclusion, the new INNO-LiPA-MYCOBACTERIA allowed the identification of 40% of the previously unidentified MAIS isolates at the species level. The results of the Lipav2 assay on the MAIS isolates confirm the great heterogeneity of this group and suggest the use of hsp65or ITS sequencing for precise identification of such isolates.