RAPID DETECTION AND IDENTIFICATION OF CANDIDA-ALBICANS AND TORULOPSIS(CANDIDA) GLABRATA IN CLINICAL SPECIMENS BY SPECIES-SPECIFIC NESTED PCR AMPLIFICATION OF A CYTOCHROME-P-450 LANOSTEROL-ALPHA-DEMETHYLASE (L1A1) GENE FRAGMENT

PCR of a Candida albicans cytochrome P-450 lanosterol-alpha-demethylas e (P450-L1A1) gene segment is a rapid and sensitive method of detectio n in clinical specimens. This enzyme is a target for azole antifungal action. In order to directly detect and identify the clinically most i mportant species of Candida, me cloned and sequenced 1.3-kbp fragments of the cytochrome P450-L1A1 genes from Torulopsis (Candida) glabrata and from Candida krusei. These segments were compared with the publish ed sequences from C. albicans and Candida tropicalis. Amplimers for ge ne sequences highly conserved throughout the fungal kingdom were first used; positive PCR results were obtained for C. albicans, T. glabrata , C. krusei, Candida parapsilosis, C. tropicalis, Cryptococcus neoform ans, and Trichosporon beigelii DNA extracts. Primers were then selecte d for a highly variable region of the gene, allowing the species-speci fic detection from purified DNA of C. albicans, T. glabrata, C. krusei , and C. tropicalis. The assay sensitivity as tested for C. albicans i n seeded clinical specimens such as blood, peritoneal fluid, or urine was 10 to 20 cells per 0.1 ml. Compared with results obtained by cultu re, the sensitivity, specificity, and efficiency of the species-specif ic nested PCR tested with 80 clinical specimens were 71, 95, and 83% f or C. albicans and 100, 97, and 98% for T. glabrata, respectively.