{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:52:09Z","timestamp":1787028729132,"version":"build-2736575974"},"reference-count":33,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2023,9,26]],"date-time":"2023-09-26T00:00:00Z","timestamp":1695686400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>Fungal keratitis (FK) is an invasive infection of the cornea primarily associated with Aspergillus and Fusarium species. FK is treated empirically with a limited selection of topical antifungals with varying levels of success. Though clinical infections are typically characterized by a dense network of mature mycelium, traditional models used to test antifungal susceptibility of FK isolates exclusively evaluate susceptibility in fungal cultures derived from asexual spores known as conidia. The purpose of this study was to characterize differences in fungal response when topical antifungal treatment is initiated at progressive phases of fungal development. We compared the efficacy of voriconazole and luliconazole against in vitro cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. A porcine cadaver corneal model was used to compare antifungal efficacy of voriconazole and luliconazole in ex vivo tissue cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. Our results demonstrate phase-dependent susceptibility of both A. flavus and F. keratoplasticum to both azoles in vitro as well as ex vivo. We conclude that traditional antifungal susceptibility testing with conidial suspensions does not correlate with fungal susceptibility in cultures of a more advanced developmental phase. A revised method of antifungal susceptibility testing that evaluates hyphal susceptibility may better predict fungal response in the clinical setting where treatment is often delayed until days after the initial insult.<\/jats:p>","DOI":"10.3390\/jof9100966","type":"journal-article","created":{"date-parts":[[2023,9,26]],"date-time":"2023-09-26T08:58:17Z","timestamp":1695718697000},"page":"966","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Phase-Dependent Differential In Vitro and Ex Vivo Susceptibility of Aspergillus flavus and Fusarium keratoplasticum to Azole Antifungals"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3358-5485","authenticated-orcid":false,"given":"Darby","family":"Roberts","sequence":"first","affiliation":[{"name":"Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacklyn","family":"Salmon","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc A.","family":"Cubeta","sequence":"additional","affiliation":[{"name":"Department of Entomology and Plant Pathology, Center for Integrated Fungal Research, North Carolina State University, Raleigh, NC 27606, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7771-9166","authenticated-orcid":false,"given":"Brian C.","family":"Gilger","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1084","DOI":"10.1038\/s41433-020-01339-3","article-title":"Infectious keratitis: An update on epidemiology, causative microorganisms, risk factors, and antimicrobial resistance","volume":"35","author":"Ting","year":"2021","journal-title":"Eye"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Hoffman, J.J., Burton, M.J., and Leck, A. (2021). Mycotic Keratitis\u2014A Global Threat from the Filamentous Fungi. J. Fungi, 7.","DOI":"10.3390\/jof7040273"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Brown, L., Kamwiziku, G., Oladele, R.O., Burton, M.J., Prajna, N.V., Leitman, T.M., and Denning, D.W. (2022). The Case for Fungal Keratitis to Be Accepted as a Neglected Tropical Disease. J. Fungi, 8.","DOI":"10.3390\/jof8101047"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Ahmadikia, K., Gharehbolagh, S.A., Fallah, B., Eshkaleti, M.N., Malekifar, P., Rahsepar, S., Getso, M.I., Sharma, S., and Mahmoudi, S. (2021). Distribution, Prevalence, and Causative Agents of Fungal Keratitis: A Systematic Review and Meta-Analysis (1990 to 2020). Front. Cell. Infect. Microbiol., 11.","DOI":"10.3389\/fcimb.2021.698780"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e489","DOI":"10.1016\/S2214-109X(20)30488-5","article-title":"The Lancet Global Health Commission on Global Eye Health: Vision beyond 2020","volume":"9","author":"Burton","year":"2021","journal-title":"Lancet Glob. Health"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1016\/j.survophthal.2018.12.003","article-title":"The persistent dilemma of microbial keratitis: Global burden, diagnosis, and antimicrobial resistance","volume":"64","author":"Ung","year":"2019","journal-title":"Surv. Ophthalmol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Cullen, M., Jacob, M.E., Cornish, V., VanderSchel, I.Q., Cotter, H.V.T., Cubeta, M.A., Carbone, I., and Gilger, B.C. (2019). Multi-locus DNA sequence analysis, antifungal agent susceptibility, and fungal keratitis outcome in horses from Southeastern United States. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0214214"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"321","DOI":"10.2746\/095777309X400270","article-title":"Clinical features and outcomes of severe ulcerative keratitis with medical and surgical management in 41 horses (2000-2006)","volume":"21","author":"Utter","year":"2009","journal-title":"Equine Veter- Educ."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1111\/j.2042-3306.2010.tb05630.x","article-title":"Therapy of equine infectious keratitis: A review","volume":"42","author":"Clode","year":"2010","journal-title":"Equine Veter. J."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"243","DOI":"10.2746\/095777309X409929","article-title":"Equine keratomycosis: An international problem","volume":"21","author":"Brooks","year":"2009","journal-title":"Equine Veter. Educ."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e00069-19","DOI":"10.1128\/CMR.00069-19","article-title":"Antifungal Susceptibility Testing: Current Approaches","volume":"33","author":"Berkow","year":"2020","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1016\/j.cveq.2017.08.001","article-title":"Antifungal Therapy in Equine Ocular Mycotic Infections","volume":"33","author":"Ledbetter","year":"2017","journal-title":"Veter. Clin. N. Am. Equine Pract."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1111\/vop.12995","article-title":"In vitro antifungal susceptibility of Fusarium species and Aspergillus fumigatus cultured from eleven horses with fungal keratitis","volume":"25","author":"Martinez","year":"2022","journal-title":"Veter. Ophthalmol."},{"key":"ref_14","unstructured":"Castano, G., Elnahry, A.G., and Mada, P.K. (2023, June 17). \u201cFungal Keratitis,\u201d in StatPearls, Available online: http:\/\/www.ncbi.nlm.nih.gov\/books\/NBK493192\/."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Liu, S., Le Mauff, F., Sheppard, D.C., and Zhang, S. (2022). Filamentous fungal biofilms: Conserved and unique aspects of extracellular matrix composition, mechanisms of drug resistance and regulatory networks in Aspergillus fumigatus. npj Biofilms Microbiomes, 8.","DOI":"10.1038\/s41522-022-00347-3"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4450","DOI":"10.1167\/iovs.12-9848","article-title":"Characterization of Fusarium Keratitis Outbreak Isolates: Contribution of Biofilms to Antimicrobial Resistance and Pathogenesis","volume":"53","author":"Mukherjee","year":"2012","journal-title":"Investig. Opthalmology Vis. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1093\/jac\/dkn402","article-title":"Phase-dependent antifungal activity against Aspergillus fumigatus developing multicellular filamentous biofilms","volume":"62","author":"Mowat","year":"2008","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1007\/s42770-023-00964-w","article-title":"Candida species causing fungal keratitis: Molecular identification, antifungal susceptibility, biofilm formation, and clinical aspects","volume":"54","author":"Bezerra","year":"2023","journal-title":"Braz. J. Microbiol."},{"key":"ref_19","unstructured":"CLSI (2017). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamenttous Fungi, Clinical and Laboratory Standards Institute. [3rd ed.]. CLSI standard M38."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"404","DOI":"10.5694\/j.1326-5377.2007.tb01313.x","article-title":"Antifungal agents","volume":"187","author":"Chen","year":"2007","journal-title":"Med. J. Aust."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1007\/s00430-020-00672-4","article-title":"Luliconazole, a highly effective imidazole, against Fusarium species complexes","volume":"209","author":"Gharaghani","year":"2020","journal-title":"Med. Microbiol. Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"918","DOI":"10.1111\/vop.12774","article-title":"In vitro susceptibility of Aspergillus and Fusarium associated with equine keratitis to new antifungal drugs","volume":"23","author":"Roberts","year":"2020","journal-title":"Veter. Ophthalmol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"711","DOI":"10.1080\/13693780500129814","article-title":"Human albumin promotes germination, hyphal growth and antifungal resistance by Aspergillus fumigatus","volume":"43","author":"Rodrigues","year":"2005","journal-title":"Med. Mycol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"7774","DOI":"10.1167\/iovs.12-10810","article-title":"Keratitis-Associated Fungi Form Biofilms with Reduced Antifungal Drug Susceptibility","volume":"53","author":"Zhang","year":"2012","journal-title":"Investig. Opthalmology Vis. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Brand, A. (2011). Hyphal Growth in Human Fungal Pathogens and Its Role in Virulence. Int. J. Microbiol., 2012.","DOI":"10.1155\/2012\/517529"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2760","DOI":"10.1128\/AAC.41.12.2760","article-title":"Comparison of in vitro antifungal susceptibilities of conidia and hyphae of filamentous fungi","volume":"41","author":"Guarro","year":"1997","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2106","DOI":"10.1128\/AAC.40.9.2106","article-title":"Comparison study of broth macrodilution and microdilution antifungal susceptibility tests for the filamentous fungi","volume":"40","author":"Pujol","year":"1996","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"James, J.E., Lamping, E., Santhanam, J., and Cannon, R.D. (2021). PDR Transporter ABC1 Is Involved in the Innate Azole Resistance of the Human Fungal Pathogen Fusarium keratoplasticum. Front. Microbiol., 12, Available online: https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2021.673206.","DOI":"10.3389\/fmicb.2021.673206"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2092","DOI":"10.1128\/AAC.01189-10","article-title":"Azole Resistance of Aspergillus fumigatus Biofilms Is Partly Associated with Efflux Pump Activity","volume":"55","author":"Rajendran","year":"2011","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"48","DOI":"10.3109\/13693780802713497","article-title":"The effects of antifungal agents to conidial and hyphal forms of Aspergillus fumigatus","volume":"48","author":"Tavakol","year":"2010","journal-title":"Med. Mycol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Subroto, E., van Neer, J., Valdes, I., and de Cock, H. (2022). Growth of Aspergillus fumigatus in Biofilms in Comparison to Candida albicans. J. Fungi, 8.","DOI":"10.3390\/jof8010048"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1128\/EC.00287-12","article-title":"Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms","volume":"12","author":"Rajendran","year":"2013","journal-title":"Eukaryot. Cell"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1167\/tvst.10.9.31","article-title":"Genipin in an Ex Vivo Corneal Model of Bacterial and Fungal Keratitis","volume":"10","author":"Koudouna","year":"2021","journal-title":"Transl. Vis. Sci. Technol."}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/9\/10\/966\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:58:13Z","timestamp":1760129893000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/9\/10\/966"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,9,26]]},"references-count":33,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2023,10]]}},"alternative-id":["jof9100966"],"URL":"https:\/\/doi.org\/10.3390\/jof9100966","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,9,26]]}}}