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This study evaluated the performance of droplet digital PCR (ddPCR), internal transcribed spacer (ITS) sequencing, and metagenomic next-generation sequencing (mNGS) using 111 formalin-fixed, paraffin-embedded tissue samples with histologically confirmed fungal infections. All three methods showed comparable detection rates for filamentous fungi (84.2%\u201394.7%; P = .2). For yeast-like fungi, however, mNGS demonstrated significantly higher detection (66.7%) than ddPCR (46.3%) and ITS sequencing (35.2%) (P &amp;lt; .01). mNGS also achieved superior genus- and species-level identification (81.1% for both) compared to ddPCR (65.8% and 64.9%) and ITS sequencing (61.3% and 50.5%) (P &amp;lt; .01). Additionally, mNGS identified two unusual fungi (Scedosporium apiospermum and Schizophyllum commune) previously misdiagnosed as Aspergillus. These findings support the integration of mNGS into clinical diagnostic workflows for the accurate identification of yeast-like and rare fungal pathogens, thereby enabling targeted antifungal therapy.<\/jats:p>","DOI":"10.1093\/mmy\/myag049","type":"journal-article","created":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T12:05:13Z","timestamp":1778760313000},"source":"Crossref","is-referenced-by-count":1,"title":["Fungal species identification in FFPE tissues: A comparative evaluation of droplet digital PCR, ITS sequencing, and metagenomic next-generation sequencing"],"prefix":"10.1093","volume":"64","author":[{"given":"Jialu","family":"Che","sequence":"first","affiliation":[{"name":"Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute Department of pathology, , 101149 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Du","sequence":"additional","affiliation":[{"name":"Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute Department of pathology, , 101149 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingshi","family":"Piao","sequence":"additional","affiliation":[{"name":"Beijing TongRen Hospital, Capital Medical University Department of Pathology, , 100730 Beijing ,","place":["China"]},{"name":"Beijing Key Laboratory of Head and Neck Molecular Diagnostic Pathology , 100730 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujie","family":"Dong","sequence":"additional","affiliation":[{"name":"Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute Department of pathology, , 101149 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Zhang","sequence":"additional","affiliation":[{"name":"Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute Department of pathology, , 101149 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dan","family":"Su","sequence":"additional","affiliation":[{"name":"Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute Department of pathology, , 101149 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chen","family":"Zhang","sequence":"additional","affiliation":[{"name":"Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute Department of pathology, , 101149 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingli","family":"Zhao","sequence":"additional","affiliation":[{"name":"Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute Department of pathology, , 101149 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weili","family":"Du","sequence":"additional","affiliation":[{"name":"Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute Department of pathology, , 101149 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4179-1737","authenticated-orcid":false,"given":"Nanying","family":"Che","sequence":"additional","affiliation":[{"name":"Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis & Thoracic Tumor Research Institute Department of pathology, , 101149 Beijing ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,5,14]]},"reference":[{"key":"2026060205011359800_bib1","doi-asserted-by":"publisher","first-page":"e428","DOI":"10.1016\/S1473-3099(23)00692-8","article-title":"Global incidence and mortality of severe fungal disease","volume":"24","author":"Denning","year":"2024","journal-title":"Lancet Infect Dis"},{"key":"2026060205011359800_bib2","article-title":"Geneva: World Health Organization","author":"WHO Fungal Priority Pathogens List to Guide Research, Development and Public Health Action","year":"2022"},{"key":"2026060205011359800_bib3","doi-asserted-by":"publisher","first-page":"787","DOI":"10.1080\/14787210.2023.2227790","article-title":"Will invasive fungal infections be the last of us? 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