Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues
Joseph Jillwin1,2, Shivaprakash M Rudramurthy2, Shreya Singh2
1Present address: Lecturer of Microbiology, Xavier University School of Medicine, Oranjestad, Aruba.
Abstract:
Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species levelGap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (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-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 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 Mucorales-specific semi-nested 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 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues.Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.
Insights
This study developed a sequence-based protocol for identifying fungi in formalin-fixed and paraffin-embedded (FFPE) tissues. The optimized ITS1-PCR method, using conventional DNA extraction, effectively identifies fungal pathogens in FFPE samples.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Histopathology
Background:
- Histopathological examination (HPE) aids in diagnosing invasive fungal infections (IFIs) but lacks species-level identification.
- Existing molecular methods for fungal identification in FFPE tissues have limitations in DNA extraction, target selection, and standardization.
Purpose of the Study:
- To develop and optimize a sequence-based protocol for fungal identification from FFPE tissues.
- To evaluate different DNA extraction methods and PCR targets for accurate fungal DNA detection.
Main Methods:
- Standardized DNA extraction using phenol-chloroform-isoamyl alcohol (PCI) and commercial kits from 63 FFPE tissues.
- Evaluated PCR targeting various ribosomal DNA (rDNA) regions (ITS1-5.8S-ITS2, ITS1, ITS2, 18S, D1/D2).
- Developed a Mucorales-specific semi-nested PCR for aseptate hyphae and optimized ITS1-PCR for septate fungi and yeasts.
Main Results:
- Conventional PCI method yielded significantly higher DNA than commercial kits.
- ITS1 region showed the highest test accuracy (61.9%) for fungal identification.
- Optimized ITS1-PCR achieved 75.5% accuracy for septate fungi and 100% for yeasts; Mucorales-specific PCR achieved 68.3% accuracy.
Conclusions:
- Conventional PCI-based DNA extraction from thick tissue sections (50 µm) is a viable option for fungal identification in FFPE tissues.
- Recommended protocol: ITS1-PCR for septate fungi/yeasts and 18S rDNA semi-nested PCR for Mucorales.
