APPLICATION OF MOLECULAR-BIOLOGICAL TECHN IQUES FOR DIAGNOSIS AND EPIDEMIOLOGY OF FUNGAL-INFECTIONS IN HUMANS

For mycological diagnosis molecular methods can be applied to detect t he pathogen directly without prior cultivation and to identify species and subspecies. For the detection of infecting agents specific DNA pr obes and/or the polymerase chain reaction (PCR) are widely used, where as normally only PCR can provide sufficient sensitivity for the direct detection of pathogens in clinical material. Prospects and Limitation s of PCR approaches for the detection of pathogenic fungi reported in the literature will be discussed. DNA polymorphisms which are useful f or species identification and epidemiological strain typing of medical ly relevant fungi can be detected by such methods as the analysis of r estriction fragment length polymorphisms (RFLP), and Southern hybridiz ation with appropriate DNA probes, and as karyotyping by pulsed field gel electrophoresis (PFGE). These techniques which could be applied su ccessfully to different epidemiological studies are, however, laboriou s and time-consuming. By using a PCR-fingerprinting method which can b e performed much simpler polymorphic DNA regions are amplified with di fferent non-specific primers. Distinctive and reproducible sets of amp lification products were observed for 26 different Candida and 8 other fungal species. The number and size of the amplification products obt ained were characteristic for each species. By comparing species-speci fic PCR-fingerprints of clinical isolates with those of reference stra ins, clinical isolates could be identified to the species level even i f they could not be identified by conventional tpying methods. With al l primers, PCR-fingerprints also displayed intraspecies variability. T herefore, PCR-fingerprinting can also be applied for epidemiological s train characterization among medically relevant fungi.