{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T14:52:50Z","timestamp":1787496770859,"version":"build-2736575974"},"reference-count":23,"publisher":"Microbiology Society","issue":"2","funder":[{"DOI":"10.13039\/501100001411","name":"Indian Council of Medical Research","doi-asserted-by":"crossref","award":["AMR\/106\/2018-ECD-II"],"award-info":[{"award-number":["AMR\/106\/2018-ECD-II"]}],"id":[{"id":"10.13039\/501100001411","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001411","name":"Indian Council of Medical Research","doi-asserted-by":"crossref","award":["3\/1\/3\/JRF-2012\/HRD-28 (10821)"],"award-info":[{"award-number":["3\/1\/3\/JRF-2012\/HRD-28 (10821)"]}],"id":[{"id":"10.13039\/501100001411","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,2,1]]},"abstract":"<jats:p>\n                    <jats:bold>Introduction.<\/jats:bold>\n                    Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus\/species level\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Gap Statement<\/jats:bold>\n                    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.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Aim.<\/jats:bold>\n                    Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Methodology.<\/jats:bold>\n                    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.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Results.<\/jats:bold>\n                    The DNA yield by conventional PCI method was significantly higher (\n                    <jats:italic>P<\/jats:italic>\n                    &lt;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\u200a%) as the target compared to 7.9, 29.9 and 22.2\u200a% 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\u200a%, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50\u2009\u00b5m) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8\u200a%) tissues and Mucorales-specific semi-nested PCR in 86 (68.3\u200a%) tissues.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Conclusion.<\/jats:bold>\n                    Conventional PCI-based DNA extraction from thick tissue (50\u2009\u00b5m) 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.\n                  <\/jats:p>","DOI":"10.1099\/jmm.0.001282","type":"journal-article","created":{"date-parts":[[2020,11,30]],"date-time":"2020-11-30T10:27:05Z","timestamp":1606732025000},"source":"Crossref","is-referenced-by-count":34,"title":["Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues"],"prefix":"10.1099","volume":"70","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2592-6761","authenticated-orcid":false,"given":"Joseph","family":"Jillwin","sequence":"first","affiliation":[{"name":"Present address: Lecturer of Microbiology, Xavier University School of Medicine, Oranjestad, Aruba"},{"name":"Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, 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