Identifizierung von Candida spp. mittels Fluoreszenz in situ Hybridisierung

Abstract

There is strong correlation between the rising number of immuncompromised patients and fungal infections. Candida spp. is the fourth common pathogen isolated from blood cultures. Fast differentiation is necessary because primary resistances against antimycotic agents such as Fluconazol and Amphotericin occur in certain Candida species. In this study fluorescence in situ hybridization (FISH) for rapid differentiation of Candida spp. was evaluated. FISH uses fluorescently labelled oligonucleotide probes of 15-20 base pairs that bind to complementary and specific target sequences on the fungal ribosomal RNA. FISH is a very useful method for fast, inexpensive and easy identification and is already successfully used in the field of medical microbiology for the differentiation of bacteria, for example in blood cultures. A set of 18 probes including species specific probes for C. albicans, C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis and Cryptococcus neoformans was designed and evaluated using 14 reference strains. The reliability of the probes was further tested on 162 well characterized clinical isolates. All reference strains and 134 of 162 clinical isolates strains were correctly identified by FISH. Eighteen strains were not identifiable to species level because the corresponding species was not included in the probe set. In 10 cases the malfunctioning of single probes resulted in an atypical staining pattern, so that the isolates were not identifiable. Finally the oligonucleotide probes were used for the direct identification and differentiation of Candida spp. in positive blood cultures, which showed yeast in the Gram-stain. Forty six samples were tested and in 43 cases the identification was correct. The three remaining isolates were not identifiable by FISH. In conclusion FISH proofed to be a very sensitive and specific method for identification of Candida spp. in clinical samples especially blood cultures.