SPECIFIC DETECTION OF CANDIDA-ALBICANS AND CANDIDA-TROPICALIS BY FLUORESCENT IN-SITU HYBRIDIZATION WITH AN 18S RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBE

In situ hybridization of whole cells with rRNA-targeted, fluorescently labelled oligonucleotide probes is a powerful method to specifically detect microorganisms in their natural habitat without cultivation and subsequent identification by phenotypic characterization. To examine the use of this method for the specific detection of pathogenic Candid a species, we have designed an oligonucleotide probe which binds to th e 18S rRNA of C. albicans and C. tropicalis, the two most important pa thogenic Candida species, and differentiates them from other clinicall y relevant species. After establishing suitable hybridization conditio ns, we confirmed the specificity of our probe 020 in RNA dot blot hybr idizations with a series of reference strains and clinical isolates of medically important Candida species. All C. albicans and C tropicalis strains hybridized with the probe, whereas all strains of C. parapsil osis, C. glabrata, C. krusei, C. guilliermondii, C. kefyr and C. lusit aniae did not. When we used the fluorescently labelled probe 020 to sp ecifically detect single cells of the two target species by in situ hy bridization, both C. albicans and C. tropicalis reacted strongly with the probe and could be clearly differentiated from C. krusei and C. pa rapsilosis, although the latter organism contains only two nucleotide mismatches in the probe target region. This discrimination capacity wa s also seen when mixed suspensions of C. albicans and C. parapsilosis were hybridized with the probe. After infection of a human endothelial cell line with C. albicans and C. krusei, C. albicans cells adhering to the endothelial cells were easily distinguishable from the C. kruse i cells by fluorescent in situ hybridization with probe 020. In additi on, germ tubes and hyphae of C. albicans were also efficiently labelle d. The application of fluorescently labelled rRNA-targeted oligonucleo tide probes therefore appears to be a valuable tool for the specific d etection and identification of different members of the genus Candida, which does not require any cultivation.