{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T11:01:28Z","timestamp":1774004488441,"version":"3.50.1"},"reference-count":63,"publisher":"PeerJ","license":[{"start":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T00:00:00Z","timestamp":1773964800000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Faculty of Medicine, Khon Kaen University, Thailand","award":["IN69013"],"award-info":[{"award-number":["IN69013"]}]},{"name":"Young Researcher Development Project of Khon Kaen University Year 2025"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"abstract":"<jats:p>\n                    Although fungal eye infections are a major cause of visual impairment worldwide, standard clinical laboratory methods remain slow, insensitive, and limited in their taxonomic resolution. Sequencing of the full ribosomal RNA (rRNA) operon provides a comprehensive marker for fungal identification. In this study, twenty fungal isolates associated with ocular infections were obtained from Srinagarind Hospital, Thailand, and characterized using four identification approaches. Initial hospital-based routine identification relied on conventional morphological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). To enhance resolution and to develop a comprehensive analytical pipeline, we further employed full rRNA operon sequencing using Oxford Nanopore Technologies (ONT), analyzed through three bioinformatic pipelines: EPI2ME\/Minimap2, NGSpeciesID with BLASTn, and internal transcribed spacer (ITS)-based phylogenetic analysis coupled with phylogenetic analysis. All isolates yielded complete operon sequences, thus ensuring comprehensive coverage of the target regions. NGSpeciesID produced high-confidence consensus sequences and species-level classifications for nearly all isolates (except one\n                    <jats:italic>Candida<\/jats:italic>\n                    specimen). Of these, 15 of the 20 isolates showed exhibited concordance with hospital identifications at the genus level (\u226597% identity). This approach successfully resolved closely related\n                    <jats:italic>Aspergillus<\/jats:italic>\n                    taxa (\n                    <jats:italic>i.e.<\/jats:italic>\n                    ,\n                    <jats:italic>A. terreus<\/jats:italic>\n                    ,\n                    <jats:italic>A.\u00a0luchuensis<\/jats:italic>\n                    ,\n                    <jats:italic>A.\u00a0oryzae<\/jats:italic>\n                    ), reclassified\n                    <jats:italic>Curvularia<\/jats:italic>\n                    isolates as\n                    <jats:italic>Bipolaris maydis<\/jats:italic>\n                    , and confirmed species-level assignments for\n                    <jats:italic>Fusarium<\/jats:italic>\n                    and\n                    <jats:italic>Rhodotorula<\/jats:italic>\n                    . By contrast, the EPI2ME workflow produced more variable classifications, providing species-level assignments for\n                    <jats:italic>Aspergillus<\/jats:italic>\n                    and\n                    <jats:italic>Rhodotorula<\/jats:italic>\n                    but mixed genus\/species profiles for several isolates, including seven isolate assignments unique to this method. ITS-based phylogenetic reconstruction recovered all expected clades, with\n                    <jats:italic>Curvularia<\/jats:italic>\n                    isolates clustering within their genus. However, node support varied substantially, highlighting the limited discriminatory power of ITS alone, which constrains taxonomic resolution to the species-complex level rather than consistently achieving the species-level identification of\n                    <jats:italic>Aspergillus<\/jats:italic>\n                    isolates. Overall, ONT-based full-operon sequencing demonstrates strong potential for fungal diagnostics, its performance depends on bioinformatic pipelines, database quality, and sequencing errors. Species-level resolution is particularly limited in\n                    <jats:italic>Aspergillus<\/jats:italic>\n                    , while incomplete reference datasets hinder the classification of isolates such as\n                    <jats:italic>Curvularia<\/jats:italic>\n                    . To improve reliability and clinical application, it will be essential to expand curated full-length rRNA references, integrate complementary loci, and refine analytical strategies.\n                  <\/jats:p>","DOI":"10.7717\/peerj.20997","type":"journal-article","created":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T08:49:11Z","timestamp":1773996551000},"page":"e20997","source":"Crossref","is-referenced-by-count":0,"title":["Identification of pathogenic fungi causing ocular infections using full rRNA operon sequencing with Oxford Nanopore Technologies"],"prefix":"10.7717","volume":"14","author":[{"given":"Thitima","family":"Suwannasaeng","sequence":"first","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pratsanee","family":"Hiengrach","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine, Khon Kaen 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Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sunchai","family":"Payungporn","sequence":"additional","affiliation":[{"name":"Center of Excellence in Systems Microbiology, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pasinee","family":"Sangsiwarit","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Parama","family":"Budmala","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Suthida","family":"Visedthorn","sequence":"additional","affiliation":[{"name":"Center of Excellence in Systems Microbiology, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pakorn","family":"Ruengket","sequence":"additional","affiliation":[{"name":"Center of Excellence in Systems Microbiology, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kanuengnit","family":"Srisak","sequence":"additional","affiliation":[{"name":"Clinical Microbiology Unit, Clinical Laboratory Section, Srinagarind Hospital Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Suwalak","family":"Chitcharoen","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand"},{"name":"Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen,  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