Mycolic Acid Analysis for Identification of Mycobacterium spp. using High-Performance Liquid Chromatography

Lee, Hyo Won; Cho, Byung Won; Kang, Moon Soo; Kim, Jong Soo; Lee, Jong Wook; Nahm, Chung Hyun; Choi, Jong Weon; Kim, Jin Ju; Pai, Soo Hwan

Abstract

BACKGROUND: Mycobacteria are traditionally identified with biochemical reactions. Since it takes 2 to 6 weeks, more rapid method is needed for timely treatment of mycobacterial infection. Mycolic acid analysis by high-performance liquid chromatography (HPLC) was recently introduced, which showed species-specificity with more than 95% sensitivity and 100% specificity for identifing Mycobacterium spp. within 2-4 hours. In this study, We performed mycolic acid analyses of standard strains of Mycobacterium spp. and two clinical isolates of known M. tuberculosis for demonstrating their species-specific nature and evaluated its reproduciblity.
METHODS
: 8 standard strains of Mycobacterium spp. (M. tuberculosis H37Rv, M. intracellurae, M. avium, M. fortuitum, M. chelonae subsp. chelonae, M. scrofulaceum, M. kansasii, M. gordonae) and 2 clinical isolates of known M. tuberculosis were analyzed. The extracted mycolic acids which were prepared by 3 steps were analyzed by HPLC with rC18 column.
RESULTS
Mean retention time (MRT) of low and high molecular weight internal standards were 3.757min+/-0.017 (C.V. <0.455%) and 9.829min+/-0.015 (C.V. <0.015%), respectively (n=30). The C.V. of MRT for M. intracellurae for positive control showing double cluster pattern was less than 0.3% from 4 injection. The C.V. of MRT for M. tuberculosis H37Rv and 2 clinical isolates of M. tuberculosis with single cluster pattern were less than 0.4%, and 0.9%, respectively. The chromatographic patterns of M. kansasii and M. gordonae showed a single cluster pattern, and M. avium, M. fortuitum, M. chelonae subsp. chelonae, and M. scrofulaceum showed a double cluster pattern which were species-specific nature.
CONCLUSIONS
We demonstrated HPLC method was rapid and highly reproducible.