MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM-AVIUM COMPLEX ISOLATES GIVING DISCORDANT RESULTS IN ACCUPROBE TESTS BY PCR-RESTRICTION ENZYME ANALYSIS, 16S RIBOSOMAL-RNA GENE SEQUENCING, AND DT1-DT6 PCR

Based on cultural and biochemical tests, a total of 84 strains (72 cli nical and 12 environmental isolates from the Caribbean Isles, Europe, and the Indian subcontinent) were identified as members of the Mycobac terium avium complex (MAC), They were further characterized with MAC, M. avium, and M. intracellulare probes of the AccuProbe system, and th is was followed by selective amplification of DT6 and DT1 sequences, S eventy isolates gave concordant results; 63 were identified as M. aviu m, 5 were identified as M. intracellulare, and 24 remained untypeable by both methods, Fourteen isolates gave discrepant results, as they we re DT1 positive but gave negative results by the M. intracellulare Acc uProbe test, Consequently, a detailed molecular analysis of all DT1-po sitive isolates (14 discrepant strains plus 5 M. intracellulare strain s) was performed by PCR-restriction analysis (PRA) of the hsp65 gene a nd 16S rRNA gene sequencing, The results confirmed the reported hetero geneity of M. intracellulare, as only 6 of 19 isolates (32%) gave PRA results compatible with published M. intracellulare profiles while the rest of the isolates were grouped in four previously unpublished prof iles, 16S rRNA gene sequencing showed that only 8 of 19 isolates (42%) were related to M. intracellulare IWGMT 90247 (EMBL accession no. X88 917), the rest being related to MCRO19 (EMBL accession no, X93030) and MIWGTMR10 (EMBL accession no. X88915). In conclusion, we have charact erized a significant number of MAC isolates which were not identified by the AccuProbe test, PRA, or 16S rRNA sequencing, However, all of th em were identifiable by DT1-DT6 PCR (they were DT6 negative and DT1 po sitive) and could be tentatively identified as M. intracellulare based on previously published observations. It is noteworthy that the major ity of such isolates (14 of 19) were from the Indian subcontinent, wit h 12 of 14 being environmental isolates, Our study confirms the marked heterogeneity of M. intracellulare isolates and shows the utility of in-house DT1 PCR to detect this group of isolates, which would otherwi se have been missed by the AccuProbe system in a routine clinical micr obiology laboratory.