Abstract
BackgroundIdentification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients.MethodsWe designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach.ResultsPCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background.ConclusionsWhile PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.
Highlights
Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy
The probe Cand 317 was designed for the detection of causative agents of invasive candidiasis such as C. albicans, and C. glabrata
We developed and evaluated four rRNA-fluorescence in situ hybridisation (FISH) probes targeting fungal pathogens frequently found in human infections
Summary
Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients. The identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy because of different in vitro susceptibility. Targeting the fungal ribosomal RNA by fluorescence in situ hybridisation (FISH) is an alternative molecular diagnostic approach that is not well studied in FFPE samples. The results of FM were compared with quantitative PCR targeting the fungal ribosomal RNA gene in order to assess the advantages and limitations of these two molecular approaches for the identification of the etiology of invasive fungal infections
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