Fluorescent staining using Blankophor for the diagnosis of sporotrichosis on fresh biopsies

Fluorescent staining using Blankophor for the diagnosis of sporotrichosis on fresh biopsies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Fluorescent staining using Blankophor for the diagnosis of sporotrichosis on fresh biopsies Regielly Cognialli, Marisol Dominguez Muro, Vânia Aparecida Vicente, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6991836/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Mycopathologia → Version 1 posted 5 You are reading this latest preprint version Abstract Sporotrichosis is an implantation mycosis with a high incidence in Brazil. Diagnosing human sporotrichosis poses significant challenges, which can lead to increased morbidity and prolonged treatment duration. Direct examination of fresh biopsies using Blankophor represents a valuable tool for rapid diagnosis, offering high sensitivity. Figures Figure 1 Short communication Sporotrichosis is caused by thermodimorphic fungi of the Sporothrix genus 1 . In recent decades, a new species, S. brasiliensis , has emerged in Brazil, leading to a large outbreak that has spread to other countries 2 – 5 . The case definition of sporotrichosis classifies cases as possible, probable, or proven, based on epidemiological, clinical, and laboratory diagnostic criteria 6 . Most cases are classified as probable due to challenges in performing fungal cultures and awaiting results, prompting the initiation of empirical treatment 6 . Additionally, atypical manifestations are often poorly recognized by clinicians, necessitating thorough differential diagnosis 7 . Fungal culture remains the gold standard for diagnosis of sporotrichosis; however, its sensitivity varies depending on factors such as fungal burden, prior treatment, potential bacterial contamination, and the type of specimen collected 8 , 9 . For biopsies, the average growth time in culture is 13 days, which is shorter for pus specimens (6 days) 6 . Conventional direct examination using 10–40% potassium hydroxide (KOH) has a sensitivity of only 1 to 2% 8 . Histopathological studies show sensitivity ranging from 18–35%, depending on the technique used 9 . Microscopic analysis has low sensitivity, mainly due to the scarcity of yeast structures in the specimen 8 , 9 . Serological methods have been validated and are promising, particularly ELISA and lateral flow assays; however, commercial kits are not yet available 10 . Molecular tests to detect Sporothrix DNA directly in clinical samples have also been studied, but commercial kits for these tests are not yet available either 8 . Nevertheless, for species identification from fungal isolates, some molecular techniques are employed, such as calmodulin gene sequencing and genotyping 11 , 12 . Fluorescent staining of fungi is a useful technique with high sensitivity for clinical specimens in histopathological and direct examinations, but it is not routinely used in clinical laboratories 8 , 13 , 14 . Various solutions can be used, such as optical brighteners like Calcofluor White and Blankophor which bind to chitin and cellulose 13 . Blankophor {4,4′-bis[(4-anilino-subst.1,3,5-triazin-2-yl)amino]stilben-2,2′-disulfonic acid} is diluted in 15–20% potassium hydroxide, which facilitates the digestion of clinical specimens 14 . After preparing slides with fresh specimens, the examination is conducted using a fluorescent microscope with an excitation wavelength below 400 nm and a barrier filter at 420 nm 14 . The fluorescence intensity can increase over time; it is often stronger after 24 hours, but fungal structures can usually be visualized within 15 minutes of preparation 14 . We conducted direct examinations using 40% KOH and Blankophor on eight fresh tissue biopsies—seven from humans and one from a dog. All patients presented clinical suspicion of probable zoonotic sporotrichosis, in accordance with the Brazilian Health Surveillance Guidelines, exhibiting compatible lesions and a history of epidemiological contact with infected cats 6 . All skin biopsies were obtained via punch technique and immediately stored in sterile containers with saline solution. Among the human cases (n = 7), three exhibited the fixed cutaneous form and four displayed the lymphocutaneous form of infection. The anatomical distribution of human biopsy sites included: three from lower limbs (42.9%), three from upper limbs (42.9%), and one from the abdomen (14.2%). Human biopsy specimens measured 2–3 mm in diameter. The canine case (n = 1) involved a 2 mm diameter biopsy collected from a nasal bridge lesion. Following our standard protocol, biopsies were aseptically transferred to a sterile Petri dish in a Biological Safety Cabinet, immersed in sterile water containing gentamicin solution, and excess fluid was removed. The tissue was vertically minced into small fragments using a sterile scalpel and cultured on Sabouraud dextrose agar (HiMedia, Mumbai, India), Mycosel agar (Becton Dickinson, Ontario, Canada), and Brain Heart Infusion agar (HiMedia, Mumbai, India) at 30°C. For microscopic analysis, two slides were prepared per biopsy: one with a drop of 40% KOH and another with a drop of Blankophor, both covered with a coverslip. After two hours, slides were examined by an experienced mycologist (> 10 years of experience); KOH slides were observed under an Olympus CX31 microscope (Japan), while Blankophor slides were evaluated using a Zeiss Axioskop 2 mot plus microscope (Germany) equipped with a DAPI (4',6-diamidino-2-phenylindole) filter (excitation: 358 nm; emission: 461 nm). Figure 1 summarizes the findings observed in direct examination for each case using Blankophor and 40% KOH (Fig. 1 ). Fungal structures were not visualized in any of these cases using conventional direct examination with 40% KOH, likely due to the low fungal burden and interference from artifacts and tissue fibers.However, when Blankophor was employed, scarce yeast cells were observed in six cases. In one case (Case #5), a higher number of yeast cells was observed, likely due to the patient’s advanced HIV infection. The yeast cells were oval to round, with some elongated and "cigar-shaped”, often with “pipe stem” budding, measuring approximately 4–6 µm. The only case negative in the direct examination with Blankophor was from a human patient presenting with the fixed cutaneous form (Case #3). Sporothrix was successfully isolated in culture from all eight specimens in all culture media employed. The fungal isolates were deposited at Micobiological Collections of the Paraná Network – Taxonline (CMRP/Taxonline: https://www.cmrp-taxonline.com ). Fungal isolates were identified by partial calmodulin gene sequencing in seven cases as S. brasiliensis and in one case as S. schenckii . The generated sequences were deposited in GenBank. Table 1 summarizes the clinical features, along with the results from direct examination, culture, and the corresponding accession numbers from CMRP and GenBank. The sensitivity of Blankophor for detecting sporotrichosis in fresh biopsies was 87%. Table 1 Clinical characteristics, mycological findings (KOH and Blankophor direct examination), and strain accession numbers (CMRP/GenBank) of sporotrichosis cases included in the study. Case ID Source Clinical form Biopsy site Direct examination with 40% KOH Direct examination with Blankophor Culture CMRP / GenBank accession numbers #1 Human LC Left upper limb Negative Positive S. brasiliensis CMRP6648 / PV740341 #2 Canine FC Nasal bridge Negative Positive S. brasiliensis CMRP6863 / PV740333 #3 Human FC Abdomen Negative Negative S. brasiliensis CMRP6647 / PV740348 #4 Human LC Left upper limb Negative Positive S. brasiliensis CMRP6624 / PV740339 #5 Human FC Left lower limb Negative Positive S. brasiliensis CMRP6191 / PV40346 #6 Human LC Right lower limb Negative Positive S. schenckii CMRP7272 / PX000723 #7 Human FC Left lower limb Negative Positive S. brasiliensis CMRP7274 / PX000724 #8 Human LC Left upper limb Negative Positive S. brasiliensis CMRP7275 / PX000725 Legend: FC – fixed cutaneous; LC – lymphocutaneous The fluorescent brightener Blankophor, which binds specifically to fungal cell wall components, is a fast and reliable staining technique to detect fungi in clinical specimens as early as 1951 15,16 . Fluorescent staining for the diagnosis of sporotrichosis has previously been described for histopathological examination, demonstrating a high sensitivity of 65% 17 . However, the use of Blankophor in fresh tissue biopsies offers a rapid turnaround time (less than 24 hours), is cost-effective, involves a simple technique, and represents a valuable alternative for endemic regions, improving laboratory diagnosis. Waiting for culture results can lead to delayed diagnosis, increasing morbidity and prolonging treatment duration. The primary limitation of our study is the small sample size, comprising only eight skin biopsies. Additionally, the study was conducted in a specialized mycology laboratory located in an endemic region for sporotrichosis in Brazil, which may have influenced the high sensitivity observed in our results. While Blankophor fluorescent staining shows promise for diagnosing sporotrichosis in fresh biopsies, this method requires experienced personnel due to the typically low fungal burden and small yeast cell diameter. Although direct examination is not always conclusive for diagnosing sporotrichosis, it can guide the diagnostic process, particularly in cases with clinical suspicion and the need for differential diagnosis. Future studies with larger sample sizes should validate these preliminary findings and include comparative evaluations with other fluorescent stains, such as Calcofluor white, to better establish the method's diagnostic performance characteristics. Declarations Acknowledgements We want to thank Lili Volochen Lopuch for her technical assistance in biopsy processing. Author contributions RCRC and MDM participated to the conceptualization, data curation, formal analysis, investigation, methodology and to the writing of the manuscript. VAV, JFM, EFJM and FQT contributed to the writing of the manuscript. Authors approved the final version before submission. Funding The research also receives support from CNPq, project INCT (nº406645/2022-1) and project Universal (nº409422/2021-5). Conflict of interest EFJM received research grants from Mundipharma and Scynexis, is on the scientific advisory board for Pfizer, and has received speaker fees from Gilead Sciences. All other authors declare no conflict of interest. References Queiroz-Telles F, Bonifaz A, Rossow J, Chindamporn A. Sporothrix and Sporotrichosis. In: Rezaei N, ed. Encyclopedia of Infection and Immunity . Elsevier; 2022:376-396. doi:10.1016/B978-0-12-818731-9.00046-X Gremião IDF, Oliveira MME, Monteiro de Miranda LH, Saraiva Freitas DF, Pereira SA. Geographic Expansion of Sporotrichosis, Brazil. Emerg Infect Dis . 2020;26(3):621-624. doi:10.3201/eid2603.190803 do Prado CM, Razzolini E, Santacruz G, et al. First Cases of Feline Sporotrichosis Caused by Sporothrix brasiliensis in Paraguay. J Fungi (Basel) . 2023;9(10):972. doi:10.3390/jof9100972 Fernandez NB, Spruijtenburg B, Tiraboschi IN, et al. Genotyping and clonal origin of Sporothrix brasiliensis in human sporotrichosis cases in Argentina. Med Mycol Case Rep . 2024;43:100633. doi:10.1016/j.mmcr.2024.100633 Thomson P, González C, Blank O, et al. Sporotrichosis Outbreak Due to Sporothrix brasiliensis in Domestic Cats in Magallanes, Chile: A One-Health-Approach Study. J Fungi (Basel) . 2023;9(2):226. doi:10.3390/jof9020226 Cognialli RCR, Cáceres DH, Bastos F de AGD, et al. Rising Incidence of Sporothrix brasiliensis Infections, Curitiba, Brazil, 2011-2022. Emerg Infect Dis . 2023;29(7):1330-1339. doi:10.3201/eid2907.230155 Poester VR, Xavier MO, Munhoz LS, et al. Sporothrix brasiliensis Causing Atypical Sporotrichosis in Brazil: A Systematic Review. J Fungi (Basel) . 2024;10(4):287. doi:10.3390/jof10040287 Arenas R, Sánchez-Cardenas CD, Ramirez-Hobak L, Ruíz Arriaga LF, Vega Memije ME. Sporotrichosis: From KOH to Molecular Biology. J Fungi (Basel) . 2018;4(2):62. doi:10.3390/jof4020062 Orofino-Costa R, Macedo PM de, Rodrigues AM, Bernardes-Engemann AR. Sporotrichosis: an update on epidemiology, etiopathogenesis, laboratory and clinical therapeutics. An Bras Dermatol . 2017;92(5):606-620. doi:10.1590/abd1806-4841.2017279 Cognialli R, Bloss K, Weiss I, Caceres DH, Davis R, Queiroz-Telles F. A lateral flow assay for the immunodiagnosis of human cat-transmitted sporotrichosis. Mycoses . 2022;65(10):926-934. doi:10.1111/myc.13516 Spruijtenburg B, Meis JF, Verweij PE, de Groot T, Meijer EFJ. Short Tandem Repeat Genotyping of Medically Important Fungi: A Comprehensive Review of a Powerful Tool with Extensive Future Potential. Mycopathol . 2024;189(5):72. doi:10.1007/s11046-024-00877-8 Zhang Y, Hagen F, Stielow B, et al. Phylogeography and evolutionary patterns in Sporothrix spanning more than 14 000 human and animal case reports. Persoonia . 2015;35:1-20. doi:10.3767/003158515X687416 Hamer EC, Moore CB, Denning DW. Comparison of two fluorescent whiteners, Calcofluor and Blankophor, for the detection of fungal elements in clinical specimens in the diagnostic laboratory. Clin Microbiol Infect . 2006;12(2):181-184. doi:10.1111/j.1469-0691.2005.01321.x Rüchel R, Schaffrinski M. Versatile fluorescent staining of fungi in clinical specimens by using the optical brightener Blankophor. J Clin Microbiol . 1999;37(8):2694-2696. doi:10.1128/JCM.37.8.2694-2696.1999 Janke D. Fluorescence microscopy of fungi in the human corneum. Klin Wochenschr . 1951;29(17-18):326-327. doi:10.1007/BF01651187 Holländer H, Keilig W, Bauer J, Rothemund E. A reliable fluorescent stain for fungi in tissue sections and clinical specimens. Mycopathol . 1984;88(2-3):131-134. doi:10.1007/BF00436443 Li PP, Zhao XH, Yang JX. Development of a Rapid Diagnostic Method for Sporotrichosis. Int Arch Allergy Immunol . Published online October 25, 2024:1-8. doi:10.1159/000541465 Cite Share Download PDF Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Mycopathologia → Version 1 posted Reviewers agreed at journal 14 Aug, 2025 Reviewers invited by journal 14 Aug, 2025 Editor invited by journal 02 Aug, 2025 Editor assigned by journal 01 Aug, 2025 First submitted to journal 31 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6991836","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":500294950,"identity":"c8f48b31-ac12-4e56-969b-e031904d5471","order_by":0,"name":"Regielly Cognialli","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3895-1982","institution":"Universidade Federal do Parana Hospital de Clinicas","correspondingAuthor":true,"prefix":"","firstName":"Regielly","middleName":"","lastName":"Cognialli","suffix":""},{"id":500294951,"identity":"84b831fa-f579-484c-b665-bad9fca86c40","order_by":1,"name":"Marisol Dominguez Muro","email":"","orcid":"","institution":"Universidade Federal do Paraná: Universidade Federal do Parana","correspondingAuthor":false,"prefix":"","firstName":"Marisol","middleName":"Dominguez","lastName":"Muro","suffix":""},{"id":500294952,"identity":"da14b86e-d0fb-4187-bc5f-46eb3109c50f","order_by":2,"name":"Vânia Aparecida Vicente","email":"","orcid":"","institution":"Universidade Federal do Paraná: Universidade Federal do Parana","correspondingAuthor":false,"prefix":"","firstName":"Vânia","middleName":"Aparecida","lastName":"Vicente","suffix":""},{"id":500294953,"identity":"1fb25393-fde5-44e8-8bfb-bf9b6de7ff33","order_by":3,"name":"Jacques F. Meis","email":"","orcid":"","institution":"Cologne University of Applied Sciences: Technische Hochschule Koln","correspondingAuthor":false,"prefix":"","firstName":"Jacques","middleName":"F.","lastName":"Meis","suffix":""},{"id":500294954,"identity":"1ceb3081-ac8e-4c88-a26d-e9cf974d64f3","order_by":4,"name":"Eelco F.J. Meijer","email":"","orcid":"","institution":"Radboud University: Radboud Universiteit","correspondingAuthor":false,"prefix":"","firstName":"Eelco","middleName":"F.J.","lastName":"Meijer","suffix":""},{"id":500294955,"identity":"4d9320f6-ccdf-4f42-87d0-50cea626b657","order_by":5,"name":"Flávio Queiroz-Telles","email":"","orcid":"","institution":"Universidade Federal do Paraná: Universidade Federal do Parana","correspondingAuthor":false,"prefix":"","firstName":"Flávio","middleName":"","lastName":"Queiroz-Telles","suffix":""}],"badges":[],"createdAt":"2025-06-27 12:59:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6991836/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6991836/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11046-025-01041-6","type":"published","date":"2026-01-06T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89574372,"identity":"0e02d5e9-c19b-42e5-870b-ab256e5b3dcd","added_by":"auto","created_at":"2025-08-21 12:53:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32227125,"visible":true,"origin":"","legend":"\u003cp\u003eComparative direct microscopic examination of fresh biopsy specimens using Blankophor and 40% KOH across all cases.\u003c/p\u003e\n\u003cp\u003eA) Blankophor fluorescent stain from Case #1 showing yeast structure morphologically consistent with \u003cem\u003eSporothrix\u003c/em\u003e spp. (B) Corresponding 40% KOH preparation from Case#1 shows no detectable fungal elements. (C) Blankophor fluorescent stain from Case #2 showing yeast structure morphologically consistent with \u003cem\u003eSporothrix\u003c/em\u003e spp. (D) Corresponding 40% KOH preparation from Case#2 shows no detectable fungal elements. (E) Blankophor fluorescent stain from Case #3 shows absence of fungal structures. (F) Corresponding 40% KOH preparation from Case#3 shows no detectable fungal elements. (G) Blankophor fluorescent stain from Case #4 showing yeast structure morphologically consistent with \u003cem\u003eSporothrix\u003c/em\u003e spp. (H) Corresponding 40% KOH preparation from Case#4 shows no detectable fungal elements. (I) Blankophor fluorescent stain from Case #5 showing multiple yeast structures morphologically consistent with \u003cem\u003eSporothrix\u003c/em\u003e spp. (J) Corresponding 40% KOH preparation from Case#5 shows no detectable fungal elements. (K) Blankophor fluorescent stain from Case #6 showing yeast structure morphologically consistent with \u003cem\u003eSporothrix\u003c/em\u003e spp. (L) Corresponding 40% KOH preparation from Case#6 shows no detectable fungal elements. (M) Blankophor fluorescent stain from Case #7 showing yeast structure morphologically consistent with \u003cem\u003eSporothrix\u003c/em\u003espp. (N) Corresponding 40% KOH preparation from Case#7 shows no detectable fungal elements. (O) Blankophor fluorescent stain from Case #8 showing yeast structure morphologically consistent with \u003cem\u003eSporothrix\u003c/em\u003e spp. (P) Corresponding 40% KOH preparation from Case#8 shows no detectable fungal elements. \u0026nbsp;Scale bar = 5 μm (consistent across all panels).\u003c/p\u003e","description":"","filename":"BLANKPHORprancha300dpi.png","url":"https://assets-eu.researchsquare.com/files/rs-6991836/v1/2f413b6c26b27a3cd5b78e5e.png"},{"id":100069229,"identity":"ef4b9a6f-dcff-404b-b672-b6c402bbc70a","added_by":"auto","created_at":"2026-01-12 16:11:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":47179828,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6991836/v1/cda7f89a-39af-49c4-b25b-845d58076f35.pdf"}],"financialInterests":"","formattedTitle":"Fluorescent staining using Blankophor for the diagnosis of sporotrichosis on fresh biopsies","fulltext":[{"header":"Short communication","content":"\u003cp\u003eSporotrichosis is caused by thermodimorphic fungi of the \u003cem\u003eSporothrix\u003c/em\u003e genus\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In recent decades, a new species, \u003cem\u003eS. brasiliensis\u003c/em\u003e, has emerged in Brazil, leading to a large outbreak that has spread to other countries\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The case definition of sporotrichosis classifies cases as possible, probable, or proven, based on epidemiological, clinical, and laboratory diagnostic criteria\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Most cases are classified as probable due to challenges in performing fungal cultures and awaiting results, prompting the initiation of empirical treatment\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Additionally, atypical manifestations are often poorly recognized by clinicians, necessitating thorough differential diagnosis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFungal culture remains the gold standard for diagnosis of sporotrichosis; however, its sensitivity varies depending on factors such as fungal burden, prior treatment, potential bacterial contamination, and the type of specimen collected\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. For biopsies, the average growth time in culture is 13 days, which is shorter for pus specimens (6 days)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Conventional direct examination using 10\u0026ndash;40% potassium hydroxide (KOH) has a sensitivity of only 1 to 2%\u003csup\u003e8\u003c/sup\u003e. Histopathological studies show sensitivity ranging from 18\u0026ndash;35%, depending on the technique used\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Microscopic analysis has low sensitivity, mainly due to the scarcity of yeast structures in the specimen\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Serological methods have been validated and are promising, particularly ELISA and lateral flow assays; however, commercial kits are not yet available\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Molecular tests to detect \u003cem\u003eSporothrix\u003c/em\u003e DNA directly in clinical samples have also been studied, but commercial kits for these tests are not yet available either\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Nevertheless, for species identification from fungal isolates, some molecular techniques are employed, such as calmodulin gene sequencing and genotyping\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFluorescent staining of fungi is a useful technique with high sensitivity for clinical specimens in histopathological and direct examinations, but it is not routinely used in clinical laboratories\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Various solutions can be used, such as optical brighteners like Calcofluor White and Blankophor which bind to chitin and cellulose\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Blankophor {4,4\u0026prime;-bis[(4-anilino-subst.1,3,5-triazin-2-yl)amino]stilben-2,2\u0026prime;-disulfonic acid} is diluted in 15\u0026ndash;20% potassium hydroxide, which facilitates the digestion of clinical specimens\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. After preparing slides with fresh specimens, the examination is conducted using a fluorescent microscope with an excitation wavelength below 400 nm and a barrier filter at 420 nm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The fluorescence intensity can increase over time; it is often stronger after 24 hours, but fungal structures can usually be visualized within 15 minutes of preparation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe conducted direct examinations using 40% KOH and Blankophor on eight fresh tissue biopsies\u0026mdash;seven from humans and one from a dog. All patients presented clinical suspicion of probable zoonotic sporotrichosis, in accordance with the Brazilian Health Surveillance Guidelines, exhibiting compatible lesions and a history of epidemiological contact with infected cats\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. All skin biopsies were obtained via punch technique and immediately stored in sterile containers with saline solution. Among the human cases (n\u0026thinsp;=\u0026thinsp;7), three exhibited the fixed cutaneous form and four displayed the lymphocutaneous form of infection. The anatomical distribution of human biopsy sites included: three from lower limbs (42.9%), three from upper limbs (42.9%), and one from the abdomen (14.2%). Human biopsy specimens measured 2\u0026ndash;3 mm in diameter. The canine case (n\u0026thinsp;=\u0026thinsp;1) involved a 2 mm diameter biopsy collected from a nasal bridge lesion.\u003c/p\u003e\n\u003cp\u003eFollowing our standard protocol, biopsies were aseptically transferred to a sterile Petri dish in a Biological Safety Cabinet, immersed in sterile water containing gentamicin solution, and excess fluid was removed. The tissue was vertically minced into small fragments using a sterile scalpel and cultured on Sabouraud dextrose agar (HiMedia, Mumbai, India), Mycosel agar (Becton Dickinson, Ontario, Canada), and Brain Heart Infusion agar (HiMedia, Mumbai, India) at 30\u0026deg;C. For microscopic analysis, two slides were prepared per biopsy: one with a drop of 40% KOH and another with a drop of Blankophor, both covered with a coverslip. After two hours, slides were examined by an experienced mycologist (\u0026gt;\u0026thinsp;10 years of experience); KOH slides were observed under an Olympus CX31 microscope (Japan), while Blankophor slides were evaluated using a Zeiss Axioskop 2 mot plus microscope (Germany) equipped with a DAPI (4\u0026apos;,6-diamidino-2-phenylindole) filter (excitation: 358 nm; emission: 461 nm).\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the findings observed in direct examination for each case using Blankophor and 40% KOH (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Fungal structures were not visualized in any of these cases using conventional direct examination with 40% KOH, likely due to the low fungal burden and interference from artifacts and tissue fibers.However, when Blankophor was employed, scarce yeast cells were observed in six cases. In one case (Case #5), a higher number of yeast cells was observed, likely due to the patient\u0026rsquo;s advanced HIV infection. The yeast cells were oval to round, with some elongated and \u0026quot;cigar-shaped\u0026rdquo;, often with \u0026ldquo;pipe stem\u0026rdquo; budding, measuring approximately 4\u0026ndash;6 \u0026micro;m. The only case negative in the direct examination with Blankophor was from a human patient presenting with the fixed cutaneous form (Case #3). \u003cem\u003eSporothrix\u003c/em\u003e was successfully isolated in culture from all eight specimens in all culture media employed. The fungal isolates were deposited at Micobiological Collections of the Paran\u0026aacute; Network \u0026ndash; Taxonline (CMRP/Taxonline: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cmrp-taxonline.com\u003c/span\u003e\u003c/span\u003e). Fungal isolates were identified by partial calmodulin gene sequencing in seven cases as \u003cem\u003eS. brasiliensis\u003c/em\u003e and in one case as \u003cem\u003eS. schenckii\u003c/em\u003e. The generated sequences were deposited in GenBank. Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the clinical features, along with the results from direct examination, culture, and the corresponding accession numbers from CMRP and GenBank. The sensitivity of Blankophor for detecting sporotrichosis in fresh biopsies was 87%.\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eClinical characteristics, mycological findings (KOH and Blankophor direct examination), and strain accession numbers (CMRP/GenBank) of sporotrichosis cases included in the study.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCase ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClinical form\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBiopsy site\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDirect examination with 40% KOH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDirect examination with Blankophor\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCulture\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCMRP / GenBank accession numbers\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeft upper limb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. brasiliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMRP6648 /\u003c/p\u003e\n \u003cp\u003ePV740341\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNasal bridge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. brasiliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMRP6863 /\u003c/p\u003e\n \u003cp\u003ePV740333\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbdomen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. brasiliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMRP6647 /\u003c/p\u003e\n \u003cp\u003ePV740348\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeft upper limb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. brasiliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMRP6624 /\u003c/p\u003e\n \u003cp\u003ePV740339\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeft lower limb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. brasiliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMRP6191 /\u003c/p\u003e\n \u003cp\u003ePV40346\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRight lower limb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. schenckii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMRP7272 / PX000723\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeft lower limb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. brasiliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMRP7274 / PX000724\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeft upper limb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. brasiliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCMRP7275 / PX000725\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003eLegend: FC \u0026ndash; fixed cutaneous; LC \u0026ndash; lymphocutaneous\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThe fluorescent brightener Blankophor, which binds specifically to fungal cell wall components, is a fast and reliable staining technique to detect fungi in clinical specimens as early as 1951\u003csup\u003e15,16\u003c/sup\u003e. Fluorescent staining for the diagnosis of sporotrichosis has previously been described for histopathological examination, demonstrating a high sensitivity of 65%\u003csup\u003e17\u003c/sup\u003e. However, the use of Blankophor in fresh tissue biopsies offers a rapid turnaround time (less than 24 hours), is cost-effective, involves a simple technique, and represents a valuable alternative for endemic regions, improving laboratory diagnosis. Waiting for culture results can lead to delayed diagnosis, increasing morbidity and prolonging treatment duration.\u003c/p\u003e\n\u003cp\u003eThe primary limitation of our study is the small sample size, comprising only eight skin biopsies. Additionally, the study was conducted in a specialized mycology laboratory located in an endemic region for sporotrichosis in Brazil, which may have influenced the high sensitivity observed in our results. While Blankophor fluorescent staining shows promise for diagnosing sporotrichosis in fresh biopsies, this method requires experienced personnel due to the typically low fungal burden and small yeast cell diameter. Although direct examination is not always conclusive for diagnosing sporotrichosis, it can guide the diagnostic process, particularly in cases with clinical suspicion and the need for differential diagnosis. Future studies with larger sample sizes should validate these preliminary findings and include comparative evaluations with other fluorescent stains, such as Calcofluor white, to better establish the method\u0026apos;s diagnostic performance characteristics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to thank Lili Volochen Lopuch for her technical assistance in biopsy processing.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRCRC and MDM participated to the conceptualization, data curation, formal analysis, investigation, methodology and to the writing of the manuscript. VAV, JFM, EFJM and FQT contributed to the writing of the manuscript. Authors approved the final version before submission.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research also receives support from CNPq, project INCT (n\u0026ordm;406645/2022-1) and project Universal (n\u0026ordm;409422/2021-5).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEFJM received research grants from Mundipharma and Scynexis, is on the scientific advisory board for Pfizer, and has received speaker fees from Gilead Sciences. All other authors declare no conflict of interest.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQueiroz-Telles F, Bonifaz A, Rossow J, Chindamporn A. \u003cem\u003eSporothrix\u003c/em\u003e and Sporotrichosis. In: Rezaei N, ed. \u003cem\u003eEncyclopedia of Infection and Immunity\u003c/em\u003e. Elsevier; 2022:376-396. doi:10.1016/B978-0-12-818731-9.00046-X\u003c/li\u003e\n\u003cli\u003eGremi\u0026atilde;o IDF, Oliveira MME, Monteiro de Miranda LH, Saraiva Freitas DF, Pereira SA. Geographic Expansion of Sporotrichosis, Brazil. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e. 2020;26(3):621-624. doi:10.3201/eid2603.190803\u003c/li\u003e\n\u003cli\u003edo Prado CM, Razzolini E, Santacruz G, et al. First Cases of Feline Sporotrichosis Caused by \u003cem\u003eSporothrix brasiliensis\u003c/em\u003e in Paraguay. \u003cem\u003eJ Fungi (Basel)\u003c/em\u003e. 2023;9(10):972. doi:10.3390/jof9100972\u003c/li\u003e\n\u003cli\u003eFernandez NB, Spruijtenburg B, Tiraboschi IN, et al. Genotyping and clonal origin of \u003cem\u003eSporothrix brasiliensis\u003c/em\u003e in human sporotrichosis cases in Argentina. \u003cem\u003eMed Mycol Case Rep\u003c/em\u003e. 2024;43:100633. doi:10.1016/j.mmcr.2024.100633\u003c/li\u003e\n\u003cli\u003eThomson P, Gonz\u0026aacute;lez C, Blank O, et al. Sporotrichosis Outbreak Due to \u003cem\u003eSporothrix brasiliensis\u003c/em\u003e in Domestic Cats in Magallanes, Chile: A One-Health-Approach Study. \u003cem\u003eJ Fungi (Basel)\u003c/em\u003e. 2023;9(2):226. doi:10.3390/jof9020226\u003c/li\u003e\n\u003cli\u003eCognialli RCR, C\u0026aacute;ceres DH, Bastos F de AGD, et al. Rising Incidence of \u003cem\u003eSporothrix brasiliensis\u003c/em\u003e Infections, Curitiba, Brazil, 2011-2022. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e. 2023;29(7):1330-1339. doi:10.3201/eid2907.230155\u003c/li\u003e\n\u003cli\u003ePoester VR, Xavier MO, Munhoz LS, et al. \u003cem\u003eSporothrix brasiliensis\u003c/em\u003e Causing Atypical Sporotrichosis in Brazil: A Systematic Review. \u003cem\u003eJ Fungi (Basel)\u003c/em\u003e. 2024;10(4):287. doi:10.3390/jof10040287\u003c/li\u003e\n\u003cli\u003eArenas R, S\u0026aacute;nchez-Cardenas CD, Ramirez-Hobak L, Ru\u0026iacute;z Arriaga LF, Vega Memije ME. Sporotrichosis: From KOH to Molecular Biology. \u003cem\u003eJ Fungi (Basel)\u003c/em\u003e. 2018;4(2):62. doi:10.3390/jof4020062\u003c/li\u003e\n\u003cli\u003eOrofino-Costa R, Macedo PM de, Rodrigues AM, Bernardes-Engemann AR. Sporotrichosis: an update on epidemiology, etiopathogenesis, laboratory and clinical therapeutics. \u003cem\u003eAn Bras Dermatol\u003c/em\u003e. 2017;92(5):606-620. doi:10.1590/abd1806-4841.2017279\u003c/li\u003e\n\u003cli\u003eCognialli R, Bloss K, Weiss I, Caceres DH, Davis R, Queiroz-Telles F. A lateral flow assay for the immunodiagnosis of human cat-transmitted sporotrichosis. \u003cem\u003eMycoses\u003c/em\u003e. 2022;65(10):926-934. doi:10.1111/myc.13516\u003c/li\u003e\n\u003cli\u003eSpruijtenburg B, Meis JF, Verweij PE, de Groot T, Meijer EFJ. Short Tandem Repeat Genotyping of Medically Important Fungi: A Comprehensive Review of a Powerful Tool with Extensive Future Potential. \u003cem\u003eMycopathol\u003c/em\u003e. 2024;189(5):72. doi:10.1007/s11046-024-00877-8\u003c/li\u003e\n\u003cli\u003eZhang Y, Hagen F, Stielow B, et al. Phylogeography and evolutionary patterns in \u003cem\u003eSporothrix\u003c/em\u003e spanning more than 14 000 human and animal case reports. \u003cem\u003ePersoonia\u003c/em\u003e. 2015;35:1-20. doi:10.3767/003158515X687416\u003c/li\u003e\n\u003cli\u003eHamer EC, Moore CB, Denning DW. Comparison of two fluorescent whiteners, Calcofluor and Blankophor, for the detection of fungal elements in clinical specimens in the diagnostic laboratory. \u003cem\u003eClin Microbiol Infect\u003c/em\u003e. 2006;12(2):181-184. doi:10.1111/j.1469-0691.2005.01321.x\u003c/li\u003e\n\u003cli\u003eR\u0026uuml;chel R, Schaffrinski M. Versatile fluorescent staining of fungi in clinical specimens by using the optical brightener Blankophor. \u003cem\u003eJ Clin Microbiol\u003c/em\u003e. 1999;37(8):2694-2696. doi:10.1128/JCM.37.8.2694-2696.1999\u003c/li\u003e\n\u003cli\u003eJanke D. Fluorescence microscopy of fungi in the human corneum. \u003cem\u003eKlin Wochenschr\u003c/em\u003e. 1951;29(17-18):326-327. doi:10.1007/BF01651187\u003c/li\u003e\n\u003cli\u003eHoll\u0026auml;nder H, Keilig W, Bauer J, Rothemund E. A reliable fluorescent stain for fungi in tissue sections and clinical specimens. \u003cem\u003eMycopathol\u003c/em\u003e. 1984;88(2-3):131-134. doi:10.1007/BF00436443\u003c/li\u003e\n\u003cli\u003eLi PP, Zhao XH, Yang JX. Development of a Rapid Diagnostic Method for Sporotrichosis. \u003cem\u003eInt Arch Allergy Immunol\u003c/em\u003e. Published online October 25, 2024:1-8. doi:10.1159/000541465\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6991836/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6991836/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSporotrichosis is an implantation mycosis with a high incidence in Brazil. Diagnosing human sporotrichosis poses significant challenges, which can lead to increased morbidity and prolonged treatment duration. Direct examination of fresh biopsies using Blankophor represents a valuable tool for rapid diagnosis, offering high sensitivity.\u003c/p\u003e","manuscriptTitle":"Fluorescent staining using Blankophor for the diagnosis of sporotrichosis on fresh biopsies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 12:52:57","doi":"10.21203/rs.3.rs-6991836/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-14T07:14:40+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-14T05:23:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Mycopathologia","date":"2025-08-02T20:37:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-01T14:13:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycopathologia","date":"2025-07-31T15:39:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f099f10f-bfcf-41ac-a7e2-776fada2e11c","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:01:46+00:00","versionOfRecord":{"articleIdentity":"rs-6991836","link":"https://doi.org/10.1007/s11046-025-01041-6","journal":{"identity":"mycopathologia","isVorOnly":false,"title":"Mycopathologia"},"publishedOn":"2026-01-06 15:57:35","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-08-21 12:52:57","video":"","vorDoi":"10.1007/s11046-025-01041-6","vorDoiUrl":"https://doi.org/10.1007/s11046-025-01041-6","workflowStages":[]},"version":"v1","identity":"rs-6991836","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6991836","identity":"rs-6991836","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}