Curvilinear-gradient high-performance liquid chromatography for identification of mycobacteria

Abstract

Over a 1-year period, 502 mycobacterial cultures submitted to the Microbial Diseases Laboratory were identified by high-performance liquid chromatography (HPLC) in parallel with standard biochemical methods. Identification by HPLC using a curvilinear gradient was achieved by comparing the chromatograms of the unknown cultures to chromatograms for known reference strains, together with calculation of peak height or peak area ratios, as necessary. The overall agreement between HPLC and biochemical identification was 97.2%. In addition, 7 of 12 cultures of Mycobacterium bovis were identified by HPLC as the BCG strain. Of 111 cultures biochemically identified as members of the M. avium complex (MAC), 108 were confirmed as MAC by DNA probe and 106 were confirmed by HPLC. Of the latter 106, 58 probe-positive strains were identified as M. avium, 38 were identified as M. intracellulare, and 10 were identified as Mycobacterium sp. strain "X" by HPLC. Of the remaining five nonchromogenic cultures, four had MAC-like chromatograms that did not match any in our library sufficiently to permit definitive identification. Of the latter four, two were confirmed as MAC strains by DNA probe and two were not. The last of the cultures biochemically identified as MAC (1 of 111) was a mixture of MAC and non-MAC strains. Overall, only 2 of 502 cultures yielded results by HPLC that differed from those obtained by standard biochemical methods. The HPLC result was confirmed in both cases by an independent national reference laboratory. In the 12 instances in which HPLC did not provide identification, the chromatograms were either uninterpretable or did not match available reference chromatograms. These findings show that the identification obtained by HPLC concurs well with that obtained by both the standard biochemical methods and the DNA probes. Thus, identification by HPLC provides mycobacteriology laboratories with a reproducible and specific method for accurate and timely identification of most medically important mycobacteria.