Molecular identification of pathogenic fungi in formalin- fixed and paraffin-embedded tissues

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species level Gap Statement Available protocols for the molecular identification of fungi from formalin- fixed and paraffinembedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation. Aim. Development of sequence based fungal identification protocol after extraction of DNA from formalin- fixed and paraffin embedded (FFPE) tissues. Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit based extraction and conventional phenol chloroformisoamyl alcohol [PCI] method) and sequence based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8SITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Seminested PCR targeting Mucoralesspecific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1- PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucoralesspecific seminested PCR in 126 FFPE tissues containing aseptate hyphae. Results. The DNA yield by conventional PCI method was significantly higher (P<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S- ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1- PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 mu m) used for DNA extraction. ITS1- PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucoralesspecific seminested PCR in 86 (68.3 %) tissues. Conclusion. Conventional PCI- based DNA extraction from thick tissue (50 mu m) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1- PCR for septate fungi and yeast, and seminested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.

Automated summary

This study developed a new protocol for fungal identification from formalin-fixed and paraffin-embedded (FFPE) tissues, using DNA extraction from 63 FFPE tissues with histopathologically confirmed invasive fungal infections (IFIs). The results showed that DNA yield was significantly higher with conventional PCI method compared to commercial kit, and PCR targeting ITS1 region had the best test accuracy among the different rDNA regions tested.