Diagnostics and Management of Invasive Fungal Infections

The development and patenting (A 1934/2005) of different molecular techniques based on real-time PCR (RQ- PCR) permitting highly sensitive detection and quantitative analysis of a broad spectrum of human pathogenic fungi can be regarded as the most important achievement of the current project. The Pan-AC RQ-PCR assay facilitates the detection and quantitative assessment of all clinically relevant Aspergillus and Candida species, which are currently responsible for the great majority of invasive fungal infections in humans. The second important test developed in our laboratory, the Pan-fungus RQ-PCR assay, permits rapid and economic detection of more than 80 different fungal species and displays the broadest specificity of all molecular fungus detection assays available to date. This assay covers not only all Aspergillus and Candida species of clinical interest, but also detects virtually all newly emerging fungal species, whose incidence has recently been shown to rise in immunocompromised patients. The new fungus detection assays were employed to investigate prospectively collected peripheral blood specimens primarily from patients who underwent allogeneic stem cell transplantation in our hospital. More than 5.000 tests were performed in serial samples of pediatric patients and revealed the presence of several different, occasionally also rare types of pathogenic fungi. The incidence of invasive fungal infections detected by molecular screening in the cohort of patients investigated was approximately 20%. Species identification by PCR fragment length analysis, a technique established in our laboratory during the course of the project, was initially confirmed by DNA sequencing to assess the reliability of this technical approach. In the final phase of the project, we have tackled the development of an alternative technique for rapid identification of fungal species based on microbead hybridization and flow-cytometric detection. This promising technique is currently in an advanced stage of development and may be amenable to patenting. Owing to the high specificity, the introduction of this technique as a diagnostic test would be an important step forward in clinical fungus diagnosis. An important lesson that has emanated from this project was the extremely high risk of contamination of molecular fungus detection assays attributable to the ubiquitous presence of fungal spores and traces of fungal DNA. In order to be able to continue with the investigation of clinical specimens at a high level of safety, it is necessary to extend the laboratory and supply it with additional biohazard equipment. Based on the experience of the present study, we have recently initiated follow-up projects with the intention to further develop the new fungus detection techniques to establish and market validated diagnostic tests in collaboration with an industrial partner. We are convinced that the insights gained from the current study supported by the FWF will be instrumental in our ongoing effort to optimize clinical diagnosis of fungal infections, which can be expected to contribute to improved treatment outcome in immunocompromised patients.