Anwendung molekularbiologischer Methoden für Diagnostik und Epidemiologie humaner Pilzinfektionen

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Abstract
For mycological diagnosis molecular methods can be applied to detect the pathogen directly without prior cultivation and to identify species and subspecies. For the detection of infecting agents specific DNA probes and/or the polymerase chain reaction (PCR) are widely used, whereas normally only PCR can provide sufficient sensitivity for the direct detection of pathogens in clinical material. Prospects and limitations of PCR approaches for the detection of pathogenic fungi reported in the literature will be discussed. DNA polymorphisms which are useful for species identification and epidemiological strain typing of medically relevant fungi can be detected by such methods as the analysis of restriction fragment length polymorphisms (RFLP), and Southern hybridization with appropriate DNA probes, and as karyotyping by pulsed field gel electrophoresis (PFGE). These techniques which could be applied successfully to different epidemiological studies are, however, laborious and time consuming. By using a PCR-fingerprinting method which can be performed much simpler polymorphic DNA regions are amplified with different non-specific primers. Distinctive and reproducible sets of amplification products were observed for 26 different Candida and 8 other fungal species. The number and size of the amplification products obtained were characteristic for each species. By comparing species-specific PCR-fingerprints of clinical isolates with those of reference strains, clinical isolates could be identified to the species level even if they could not be identified by conventional typing methods. With all primers, PCR-fingerprints also displayed intraspecies variability. Therefore, PCR-fingerprinting can also be applied for epidemiological strain characterization among medically relevant fungi.