Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis

Abstract

A single primer pair amplifying a cytochrome P-450 lanosterol-14 alpha-demethylase (L1A1) gene fragment that encodes a highly conserved region was used to detect yeast DNA in clinical specimens. Positive PCR products were obtained from genomic DNAs of Candida albicans, C. parapsilosis, C. tropicalis, C. guilliermondii, C. krusei, C. (Torulopsis) glabrata, and C. kefyr. No human, bacterial, or parasitic DNA was amplified. The sensitivity was evaluated for C. albicans genomic DNA by using various DNA concentrations (200 pg to 2 fg). The amplified DNAs of Candida species with unknown P-450 L1A1 gene sequences were subcloned and sequenced. Identification at the species level was achieved by digestion of the PCR products with different restriction enzymes. A specific 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 lavage specimens. After appropriate treatment, the specimens were processed by PCR and the results were compared with those obtained by traditional diagnostic procedures such as cultures and serology. Although preliminary, 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.