Quantification, identification and genotyping of fungi

Due to the increasing number of patients at risk, fungal infections have become omnipresent. The treatment of these infections is often empirical, as rapid and accurate methods for the diagnosis of these infections are lacking. Therefore, I have focussed on the development of methods for the quantification, identification and genotyping of fungi in order to develop novel approaches allowing accurate prevention and rapid diagnosis of fungal infections. Methods based on solid-phase cytometry (SPC) were developed for the quantification of bacteria and fungi as well as A. fumigatus in air samples by using viability staining and immunofluorescence labelling, respectively. Comparison of the results obtained with SPC and those obtained with plating showed that SPC is more rapid and has a lower detection limit. Moreover, SPC was found suitable to specifically detect airborne A. fumigatus in the presence of other fungi. Next, we used SPC to investigate whether A. fumigatus strains resistant to itraconazole are present in air. Because 1.8% of airborne A. fumigatus strains were found to be resistant to itraconazole, we can conclude that the environment possibly serves as a reservoir for infections with itraconazole-resistant strains. In order to improve the diagnosis of candidemia, we developed a method based on immunomagnetic separation and SPC to detect Candida cells in blood. Application of this method to clinical samples demonstrated that it was diagnostically more sensitive and more rapid than traditional culture-based techniques. In a third part we compared different genotyping methods for A. fumigatus and concluded that VNTR typing was the optimal tool for epidemiological studies on A. fumigatus. Subsequently, this method was used to analyse isolates retrieved from patients with cystic fibrosis (CF) and invasive aspergillosis (IA). We found that the respiratory tract of persons with CF is usually colonised with multiple A. fumigatus strains and that a limited number of them is able to cause persistent colonisation. In contrast, the same A. fumigatus strain was isolated from various deep organs of patients with IA. Both studies suggested that the impact of patient-to-patient transmission is limited. In a final study, we used SPC and VNTR typing for the quality control of 15 probiotic products containing S. boulardii. Using VNTR typing, we confirmed that the identity of all Saccharomyces strains is S. boulardii. Additionally, SPC counts were higher than plate counts as all viable cells were enumerated instead of culturable cells only. Initially, limited differences were observed between the numbers of cells present in the various products. When exposed to simulated gastric conditions, less than 1% of the S. boulardii cells in all products tested was able to survive. However, the probiotic product Enterol proved more resistant to acidic conditions than the other products