Molecular Diagnosis of Invasive Fungal Infections: Species Identification and Quantitative Analysis by Broad-Spectrum PCR Assays.

Invasive fungal infections (IFI) play an increasingly important role as life-threatening complications in immunocompromised patients. Early application of antimycotic agents is an essential prerequisite for successful therapy. However, standardized diagnostic techniques permitting rapid, sensitive and, no less importantly, economic screening for the clinically relevant fungi have been lacking. We have developed two different real-time PCR systems for quantitative analysis of pathogenic fungi. The Pan-AC assay* permits in a single reaction the detection of all important Aspergillus and Candida species, which are responsible for the great majority of IFI in immunosuppressed individuals. In view of the increasing incidence of invasive infections caused by hitherto uncommon fungal species, new diagnostic tests with very broad specificity are required. We have therefore developed an additional two-reaction Pan-fungus assay*, which facilitates quantitative detection of a wide spectrum of fungal species (n>50), including also the newly emerging pathogenic fungi. The assays display high sensitivity and show no cross-reactivity with non-fungal pathogens or human DNA sequences. We have established an additional rapid molecular assay based on PCR fragment length analysis of a variable region in the fungal genome permitting rapid identification of the fungal species present, in order to facilitate selection of the most appropriate antifungal treatment. Correct identification of specific fungal pathogens including different Aspergillus, Candida and Fusarium species detected by the above assays in patients with febrile neutropenia has been confirmed by sequence analysis. The new assays are readily applicable to routine clinical diagnosis and provide a rapid and economic approach to the screening and monitoring of invasive fungal infections.