Assessment of Partial Sequencing of the 65-Kilodalton Heat Shock Protein Gene ( hsp65 ) for Routine Identification of Mycobacterium Species Isolated from Clinical Sources

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Abstract
We assessed the ability of an in-house database, consisting of 111 hsp65 sequences from putative and valid Mycobacterium species or described groups, to identify 689 mycobacterial clinical isolates from 35 species or groups. A preliminary assessment indicated that hsp65 sequencing confirmed the identification of 79.4% of the isolates from the 32 species examined, including all Mycobacterium tuberculosis complex isolates, all isolates from 13 other species, and 95.6% of all M. avium - M. intracellulare complex isolates. Identification discrepancies were most frequently encountered with isolates submitted as M. chelonae , M. fortuitum , M. gordonae , M. scrofulaceum , and M. terrae . Reexamination of isolates with discrepant identifications confirmed that hsp65 identifications were correct in a further 40 isolates. This brought the overall agreement between hsp65 sequencing and the other identification methods to 85.2%. The remaining 102 isolates had sequence matches below our acceptance criterion, had nondifferential sequence matches between two or more species, were identified by 16S rRNA sequencing as a putative taxonomic group not contained in our database, or were identified by hsp65 and 16S rRNA gene sequencing as a species not in our biochemical test database or had conflicting identifications. Therefore, to incorporate the unconfirmed isolates it was necessary to create 29 additional entries in our hsp65 identification database: 18 associated with valid species, 7 indicating unique sequences not associated with valid or putative species or groups, and 4 associated with unique, but currently described taxonomic groups. Confidence in the hsp65 sequence identification of a clinical isolate is best when sequence matches of 100% occur, but our data indicate that correct identifications can be confidently made when unambiguous matches exceeding 97% occur, but are dependent on the completeness of the database. Our study indicates that for hsp65 sequencing to be an effective means for identifying mycobacteria a comprehensive database must be constructed. hsp65 sequencing has the advantage of being more rapid and less expensive than biochemical test panels, uses a single set of reagents to identify both rapid- and slow-growing mycobacteria, and can provide a more definitive identification.

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