1887

Abstract

Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species level

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.

Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues.

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.

The DNA yield by conventional PCI method was significantly higher (<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.

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.

Funding
This study was supported by the:
  • Indian Council of Medical Research (Award 3/1/3/JRF-2012/HRD-28 (10821))
    • Principle Award Recipient: JosephJillwin
  • Indian Council of Medical Research (Award AMR/106/2018-ECD-II)
    • Principle Award Recipient: ArunalokeChakrabarti
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/content/journal/jmm/10.1099/jmm.0.001282
2020-11-30
2021-08-02
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