{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T15:51:38Z","timestamp":1786117898711,"version":"build-2736575974"},"reference-count":28,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100004737","name":"National Health Research Institutes","doi-asserted-by":"publisher","award":["IV-113-GP-11"],"award-info":[{"award-number":["IV-113-GP-11"]}],"id":[{"id":"10.13039\/501100004737","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004737","name":"National Health Research Institutes","doi-asserted-by":"publisher","award":["IV-113-PP-12"],"award-info":[{"award-number":["IV-113-PP-12"]}],"id":[{"id":"10.13039\/501100004737","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004737","name":"National Health Research Institutes","doi-asserted-by":"publisher","award":["IV-114-GP-11"],"award-info":[{"award-number":["IV-114-GP-11"]}],"id":[{"id":"10.13039\/501100004737","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004737","name":"National Health Research Institutes","doi-asserted-by":"publisher","award":["IV-114-PP-12"],"award-info":[{"award-number":["IV-114-PP-12"]}],"id":[{"id":"10.13039\/501100004737","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100020950","name":"National Science and Technology Council","doi-asserted-by":"publisher","award":["MOST 108-2314-B-400-031-MY3"],"award-info":[{"award-number":["MOST 108-2314-B-400-031-MY3"]}],"id":[{"id":"10.13039\/501100020950","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100020950","name":"National Science and Technology Council","doi-asserted-by":"publisher","award":["NSTC 114-2314-B-400-020-MY3"],"award-info":[{"award-number":["NSTC 114-2314-B-400-020-MY3"]}],"id":[{"id":"10.13039\/501100020950","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,8,4]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Cunninghamella bertholletiae<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Rhizopus microsporus<\/jats:italic>\n                      are clinically important Mucorales that frequently exhibit mixed peaks in chromatograms from conventional Sanger sequencing of the internal transcribed spacer (ITS) region. Intragenomic ITS heterogeneity has been reported in\n                      <jats:italic toggle=\"yes\">R. microsporus<\/jats:italic>\n                      but has not yet been characterized in\n                      <jats:italic toggle=\"yes\">Cunninghamella<\/jats:italic>\n                      species. This study investigated ITS patterns in nine\n                      <jats:italic toggle=\"yes\">C. bertholletiae<\/jats:italic>\n                      , three\n                      <jats:italic toggle=\"yes\">Cunninghamella echinulata<\/jats:italic>\n                      , and two\n                      <jats:italic toggle=\"yes\">R. microsporus<\/jats:italic>\n                      isolates collected in Taiwan. The ITS-D1\/D2 regions were examined by PCR, cloning, and Sanger sequencing; one\n                      <jats:italic toggle=\"yes\">C. bertholletiae<\/jats:italic>\n                      isolate was further studied using PacBio HiFi sequencing. In\n                      <jats:italic toggle=\"yes\">C. bertholletiae<\/jats:italic>\n                      , cloning and Sanger sequencing revealed length variation at six homopolymeric tracts \u2265 8 bp within the ITS region, consistent with PacBio findings.\n                      <jats:italic toggle=\"yes\">C. echinulata<\/jats:italic>\n                      exhibited none or minimal intragenomic ITS variation. Both\n                      <jats:italic toggle=\"yes\">R. microsporus<\/jats:italic>\n                      isolates contained divergent ITS and D1\/D2 sequence patterns; one strain showed substantial indels, resulting in intragenomic divergence of 27.4% in ITS and 3.10% in D1\/D2. These hybrid ITS sequence patterns were highly similar to those observed in strains from Hong Kong, suggesting that ancient hybridization has been stably inherited. Overall, intragenomic divergence ranged 0.14%\u20130.55% in\n                      <jats:italic toggle=\"yes\">C. bertholletiae<\/jats:italic>\n                      , 0%\u20130.23% in\n                      <jats:italic toggle=\"yes\">C. echinulata<\/jats:italic>\n                      , and 1.19%\u201327.37% in\n                      <jats:italic toggle=\"yes\">R. microsporus<\/jats:italic>\n                      for ITS, and 0%\u20130.41%, 0%\u20130.41 %, and 1.43%\u20133.10% for D1\/D2, respectively. In conclusion, our study revealed the patterns of ITS heterogeneity, including length variation in long homopolymeric tracts in\n                      <jats:italic toggle=\"yes\">C. bertholletiae<\/jats:italic>\n                      and hybrid ITS patterns in\n                      <jats:italic toggle=\"yes\">R. microsporus<\/jats:italic>\n                      , which contribute to Sanger chromatogram ambiguity and pose challenges for ITS-based identification. Although D1\/D2 sequencing offers an alternative, its utility may also be affected by intragenomic divergence in\n                      <jats:italic toggle=\"yes\">R. microsporus<\/jats:italic>\n                      , highlighting the need for single-copy genes to improve species identification.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        C\n                        <jats:italic toggle=\"yes\">unninghamella bertholletiae<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">Rhizopus microsporus<\/jats:italic>\n                        are clinically important Mucorales that often yield ambiguous internal transcribed spacer (ITS) chromatograms in Sanger sequencing. We showed that in\n                        <jats:italic toggle=\"yes\">C. bertholletiae<\/jats:italic>\n                        , chromatogram ambiguity arises from length variation in six long homopolymeric tracts (\u22658 bp), while in\n                        <jats:italic toggle=\"yes\">R. microsporus,<\/jats:italic>\n                        it results from hybrid ITS types, as observed in isolates from Hong Kong. Such intragenomic ITS sequence heterogeneity poses challenges to ITS-based identification. Although D1\/D2 sequencing offers an alternative, its utility may also be affected by intragenomic divergence in\n                        <jats:italic toggle=\"yes\">R. microsporus<\/jats:italic>\n                        , highlighting the need for single-copy genes to improve species identification.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.03485-25","type":"journal-article","created":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T13:26:00Z","timestamp":1783344360000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Challenges in ITS-based identification of\n                    <i>Cunninghamella bertholletiae<\/i>\n                    and\n                    <i>Rhizopus microsporus<\/i>\n                    due to intragenomic ITS sequence heterogeneity"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4020-5812","authenticated-orcid":true,"given":"Wan-Lin","family":"Wu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02r6fpx29","id-type":"ROR","asserted-by":"publisher"}],"name":"National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes","place":["Tainan, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-4317-0048","authenticated-orcid":true,"given":"Pui-Ching","family":"Choi","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02r6fpx29","id-type":"ROR","asserted-by":"publisher"}],"name":"National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes","place":["Tainan, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hsuan-Chen","family":"Wang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02r6fpx29","id-type":"ROR","asserted-by":"publisher"}],"name":"National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes","place":["Tainan, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-4769-3938","authenticated-orcid":true,"given":"Ming-I","family":"Hsieh","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02r6fpx29","id-type":"ROR","asserted-by":"publisher"}],"name":"National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes","place":["Tainan, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9607-9088","authenticated-orcid":true,"given":"Chi-Jung","family":"Wu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02r6fpx29","id-type":"ROR","asserted-by":"publisher"}],"name":"National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes","place":["Tainan, Taiwan"]},{"id":[{"id":"https:\/\/ror.org\/01b8kcc49","id-type":"ROR","asserted-by":"publisher"}],"name":"Division of Infectious Diseases, Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University","place":["Tainan, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_5_2_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1469-0691.2009.02972.x"},{"key":"e_1_3_5_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmi.2019.11.021"},{"key":"e_1_3_5_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmi.2018.07.011"},{"key":"e_1_3_5_5_2","doi-asserted-by":"publisher","DOI":"10.3109\/13693786.2012.690107"},{"key":"e_1_3_5_6_2","doi-asserted-by":"publisher","DOI":"10.3390\/jof11040298"},{"key":"e_1_3_5_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01230-18"},{"key":"e_1_3_5_8_2","doi-asserted-by":"publisher","DOI":"10.1111\/mec.14995"},{"key":"e_1_3_5_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2023.107317"},{"key":"e_1_3_5_10_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1207508109"},{"key":"e_1_3_5_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(19)30312-3"},{"key":"e_1_3_5_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.01685-08"},{"key":"e_1_3_5_13_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.01750-09"},{"key":"e_1_3_5_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.simyco.2014.07.004"},{"key":"e_1_3_5_15_2","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.35.5.1216-1223.1997"},{"key":"e_1_3_5_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.fbr.2022.04.002"},{"key":"e_1_3_5_17_2","doi-asserted-by":"publisher","DOI":"10.1006\/mpev.1996.0376"},{"key":"e_1_3_5_18_2","doi-asserted-by":"publisher","DOI":"10.5941\/MYCO.2017.45.4.318"},{"key":"e_1_3_5_19_2","doi-asserted-by":"publisher","DOI":"10.1007\/s13225-013-0229-6"},{"key":"e_1_3_5_20_2","volume-title":"Atlas of clinical fungi","author":"Guarro J","year":"2020","unstructured":"Guarro J, Gen\u00e9 J, Figueras MJ. 2020. Atlas of clinical fungi. 3rd ed. CBS-KNAW Fungal Biodiversity Centre.","edition":"3"},{"key":"e_1_3_5_21_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.01230-21"},{"key":"e_1_3_5_22_2","doi-asserted-by":"publisher","DOI":"10.1093\/emboj\/20.10.2587"},{"key":"e_1_3_5_23_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkg178"},{"key":"e_1_3_5_24_2","unstructured":"Biosystems A. 2016 Troubleshooting Sanger sequencing data. User Bulletin. Publication Number MAN0014435 Revision A.0. Thermo Fisher Scientific. Waltham MA . https:\/\/assets.thermofisher.com\/TFS-Assets\/LSG\/manuals\/MAN0014435_Trbleshoot_Sanger_seq_data_UB.pdf."},{"key":"e_1_3_5_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-019-0217-9"},{"key":"e_1_3_5_26_2","doi-asserted-by":"publisher","DOI":"10.1099\/00221287-90-1-115"},{"key":"e_1_3_5_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.diagmicrobio.2013.03.009"},{"key":"e_1_3_5_28_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.00036-09"},{"key":"e_1_3_5_29_2","doi-asserted-by":"publisher","DOI":"10.1007\/s13225-013-0229-6"}],"container-title":["Microbiology Spectrum"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.03485-25","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.03485-25","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T14:45:37Z","timestamp":1786027537000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/spectrum.03485-25"}},"subtitle":[],"editor":[{"given":"Sudha","family":"Chaturvedi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,8,4]]},"references-count":28,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2026,8,4]]}},"alternative-id":["10.1128\/spectrum.03485-25"],"URL":"https:\/\/doi.org\/10.1128\/spectrum.03485-25","relation":{},"ISSN":["2165-0497"],"issn-type":[{"value":"2165-0497","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,4]]},"assertion":[{"value":"2025-10-27","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-05-31","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-06","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e03485-25"}}