Use of denaturing high-performance liquid chromatography for rapid detection and identification of seven Candida species

Abstract

A novel denaturing high-performance liquid chromatography (DHPLC)-based technique allows rapid high-resolution analysis of PCR products. We used this technique for unequivocal molecular identification of seven Candida species. We show the application of this PCR/DHPLC approach for direct detection and identification of yeast species from blood cultures and for detection of Candida colonization in the gastrointestinal tract of allogeneic transplant patients.

FIG. 1.

DHPLC peak profile of mixed fungal amplicons. Shown are mixtures of PCR-amplified ITS2 DNA fragments from C. membranaefaciens (peak 1), C. tropicalis (peak 2), C. parapsilosis (peak 3), C. magnoliae (peak 4), C. lusitaniae (peak 5), C. dubliniensis (peak 6), C. albicans (peak 7), C. inconspicua (peak 8), C. krusei (peak 9), C. neoformans (peak 10), C. kefyr (peak 11), and C. glabrata (peak 12).

FIG. 2.

Peak profiles obtained from Candida variants. Profiles consisting of only one major peak were designated type I. Dashed line, marker profile containing type I reference peaks and the type II variant of C. tropicalis. (A) Besides type I, six additional Candida albicans variants (types II to VII) were identified by analyzing clinical isolates and blood culture samples. (B) Candida glabrata variants (types I to VI). (C) Three variants were observed for Candida tropicalis (types I to III). Note that the marker profile contains types I and II of C. tropicalis.