IDENTIFICATION OF VARIOUS MEDICALLY IMPORTANT CANDIDA SPECIES IN CLINICAL SPECIMENS BY PCR-RESTRICTION ENZYME ANALYSIS

A single primer pair amplifying a cytochrome P-450 lanosterol-14 alpha -demethylase (L1A1) gene fragment that encodes a highly conserved regi on was used to detect yeast DNA in clinical specimens, Positive PCR pr oducts were obtained from genomic DNAs of Candida albicans, C. parapsi losis, C. tropicalis, C. guilliermondii, C. krusei, C. (Torulopsis) gl abrata, and C. kefyr, No human, bacterial, or parasitic DNA was amplif ied, The sensitivity was evaluated for C. albicans genomic DNA by usin g various DNA concentrations (200 pg to 2 fg), The amplified DNAs of C andida species with unknown P-450 L1A1 gene sequences were subcloned a nd sequenced, Identification at the species level was achieved by dige stion of the PCR products with different restriction enzymes, A specif ic restriction enzyme analysis pattern was determined for each species investigated, Subsequently, we used PCR to detect specific yeast DNA directly with clinical specimens such as blood and bronchoalveolar lav age specimens. After appropriate treatment, the specimens were process ed by PCR and the results were compared with those obtained by traditi onal diagnostic procedures such as cultures and serology, Although pre liminary, the PCR results seem to correlate well, at least for blood, with those of antigen detection assays and traditional blood cultures, with a better and earlier detection of candidemia.