Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in formalin fixed, paraffin-embedded tissue specimens

Abstract

Background: Identification 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.

Methods: We 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.

Results: PCR 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.

Conclusions: While 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.

Figure 1

Fluorescence microscopy of formalin fixed fungi (C. tropicalis: a-d, A. terreus: e-h, P. variotii: i-l, C. bertholletiae: m-p) with DAPI and the universal eukaryotic probe EUK 516 (labelled with Cy3). Images were collected in three wave-length channels: DAPI (first column), FITC (second column) representing non-specific fluorescence, Cy3 (third column). Last column represents a fusion image of the DAPI and Cy3-channels.

Figure 2

Results from PCR and FISH in 42 samples from patients with proven invasive fungal infection.

Figure 3

Fluorescence Microscopy on tissue sections from patients with proven invasive fungal infection using DAPI. Invasive Candidiasis showing fluorescence of nuclei of yeasts and host cells (a). Invasive mold infection showing limited integration of DAPI in a sample with septate-(b) and non-septate mold hyphae (c). In addition, intense fluorescence of fungal cell wall in parts of the septate mold hyphae (b).

Figure 4

Hybridisation of tissue sections with DAPI and a broad range fungal rRNA probe (D223 and D223IO labelled with Cy3: c-d and g-h) or eukaryotic universal probe (EUK 516 labeled with Cy3: k-l) with tissue sections from a patient with candidiasis (a-d), aspergillosis (e-h) and mucormycosis (i-l). DAPI-channel (first column), FITC-channel (second column), Cy-3 channel (third column), fusion picture of DAPI and Cy3-channels (last column).

Figure 5

Liver biopsy from a patient with hepatosplenic candidiasis without culture confirmation. Sequencing of PCR amplicons revealed C. tropicalis. Probe Candida 317-FITC (d-f) shows hybridisation, in contrast to probe Asp-F-FITC (a-c). DAPI channel (first column), FITC-channel (second column), Fusion picture DAPI and FITC channels (last column). Note that there is substantial autofluorescence in the FITC channel.

Figure 6

Difficulties in the evaluation of hybridisation signals in samples with mould infection. Autofluoresence of hyphae (a-d) or surrounding tissue (e-h) in two tissue specimens from patients with invasive aspergillosis. Hybridisation with DAPI and eukaryotic universal probe (univ-SSU labelled with FITC). DAPI channel (first column), FITC-channel (second column), Cy-3 channel (third column). Fusion picture DAPI and FITC channels (last column).