{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T15:24:52Z","timestamp":1780413892390,"version":"3.54.1"},"reference-count":42,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2024,2,18]],"date-time":"2024-02-18T00:00:00Z","timestamp":1708214400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Institute Evandro Chagas (IEC), Department of Health Surveillance, Ministry of Health","award":["CAPES\u201488887.840006\/2023-00"],"award-info":[{"award-number":["CAPES\u201488887.840006\/2023-00"]}]},{"name":"Coordena\u00e7\u00e3o de Aperfei\u00e7oamento de Pessoal de N\u00edvel Superior\u2014Brasil","award":["CAPES\u201488887.840006\/2023-00"],"award-info":[{"award-number":["CAPES\u201488887.840006\/2023-00"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>Chromoblastomycosis (CBM) and phaeohyphomycosis (FEO) are infections caused by melanized filamentous fungal agents, primarily found in tropical and subtropical regions. Both infections pose significant challenges for the correct identification of the causative agent due to their morphological similarity, making conventional methods of morphological analysis highly subjective. Therefore, molecular techniques are necessary for the precise determination of these species. In this regard, this study aimed to contribute to a new methodology based on PCR-RFLP for the identification of agents causing CBM and FEO. Sequences from the Internal Transcribed Spacer (ITS) region were used to identify potential restriction enzyme sites in silico, followed by in vitro validation using the selected restriction enzymes. The obtained results were compared with species identification through morphological analyses and sequencing. The results demonstrated that the PCR-RFLP applied in this study accurately identified two major agents of chromoblastomycosis, Fonsecaea pedrosoi and Fonsecaea monophora, as well as Cladophialophora bantiana and Exophiala dermatitidis, both causative agents of phaeohyphomycosis. In this context, the proposed assay can complement current methods for identifying these species, aiding in diagnosis, and contributing to the proper management of these infections.<\/jats:p>","DOI":"10.3390\/jof10020159","type":"journal-article","created":{"date-parts":[[2024,2,19]],"date-time":"2024-02-19T04:39:36Z","timestamp":1708317576000},"page":"159","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Identification of Chromoblastomycosis and Phaeohyphomycosis Agents through ITS-RFLP"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2605-3755","authenticated-orcid":false,"given":"Gabriel S. M.","family":"Sousa","sequence":"first","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Biologia de Agentes Infecciosos e Parasit\u00e1rios, Instituto de Ci\u00eancias Biol\u00f3gicas, Universidade Federal do Par\u00e1, Bel\u00e9m 66075-750, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5353-719X","authenticated-orcid":false,"given":"Rodrigo S.","family":"De Oliveira","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Micoses Superficiais e Sist\u00eamicas, Se\u00e7\u00e3o de Bacteriologia e Micologia, Instituto Evandro Chagas, Ananindeua 67030-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1244-4026","authenticated-orcid":false,"given":"Alex B.","family":"De Souza","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Micoses Superficiais e Sist\u00eamicas, Se\u00e7\u00e3o de Bacteriologia e Micologia, Instituto Evandro Chagas, Ananindeua 67030-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruan C.","family":"Monteiro","sequence":"additional","affiliation":[{"name":"Laboratory of Emerging Fungal Pathogens, Universidade Federal de S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elaine P. T. E.","family":"Santo","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Micoses Superficiais e Sist\u00eamicas, Se\u00e7\u00e3o de Bacteriologia e Micologia, Instituto Evandro Chagas, Ananindeua 67030-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5648-4118","authenticated-orcid":false,"given":"Luciano C.","family":"Franco Filho","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Micoses Superficiais e Sist\u00eamicas, Se\u00e7\u00e3o de Bacteriologia e Micologia, Instituto Evandro Chagas, Ananindeua 67030-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0956-4553","authenticated-orcid":false,"given":"Silvia H. M.","family":"Da Silva","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Micoses Superficiais e Sist\u00eamicas, Se\u00e7\u00e3o de Bacteriologia e Micologia, Instituto Evandro Chagas, Ananindeua 67030-000, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,2,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1055\/s-0039-3400957","article-title":"Phaeohyphomycosis","volume":"41","author":"Arcobello","year":"2020","journal-title":"Semin. Respir. Crit. Care Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1128\/CMR.00032-16","article-title":"Chromoblastomycosis","volume":"30","author":"Santos","year":"2017","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Santos, D.W.C.L., Azevedo, C.d.M.P.e.S.d., Vicente, V.A., Queiroz-Telles, F., Rodrigues, A.M., de Hoog, G.S., Denning, D.W., and Colombo, A.L. (2021). The Global Burden of Chromoblastomycosis. PLoS Neglected Trop. Dis., 15.","DOI":"10.1371\/journal.pntd.0009611"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Enbiale, W., Bekele, A., Manaye, N., Seife, F., Kebede, Z., Gebremeskel, F., and van Griensven, J. (2023). Subcutaneous Mycoses: Endemic but Neglected among the Neglected Tropical Diseases in Ethiopia. PLoS Neglected Trop. Dis., 17.","DOI":"10.1371\/journal.pntd.0011363"},{"key":"ref_5","unstructured":"WHO (2022). Ending the Neglect to Attain the Sustainable Development Goals: A Strategic Framework for Integrated Control and Management of Skin-Related Neglected Tropical Diseases."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Agarwal, R., Singh, G., Ghosh, A., Verma, K.K., Pandey, M., and Xess, I. (2017). Chromoblastomycosis in India: Review of 169 Cases. PLoS Neglected Trop. Dis., 11.","DOI":"10.1371\/journal.pntd.0005534"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"myab062","DOI":"10.1093\/mmy\/myab062","article-title":"Chromoblastomycosis in Latin America and the Caribbean: Epidemiology over the Past 50 Years","volume":"60","author":"Guevara","year":"2021","journal-title":"Med. Mycol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"He, Y., Zheng, H.L., Mei, H., Lv, G.X., Liu, W.D., and Li, X.F. (2022). Phaeohyphomycosis in China. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.895329"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1016\/j.medmal.2017.09.011","article-title":"From Phaeohyphomycosis to Disseminated Chromoblastomycosis: A Retrospective Study of Infections Caused by Dematiaceous Fungi","volume":"48","author":"Thomas","year":"2018","journal-title":"Med. Mal Infect."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Liu, S., Zhi, H., Shen, H., Lv, W., Sang, B., Li, Q., Zhong, Y., Liu, Z., and Xia, X. (2022). Chromoblastomycosis: A Case Series from Eastern China. PLoS Neglected Trop. Dis., 16.","DOI":"10.1371\/journal.pntd.0010800"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1155\/2021\/9742832","article-title":"Reviewing the Etiologic Agents, Microbe-Host Relationship, Immune Response, Diagnosis, and Treatment in Chromoblastomycosis","volume":"2021","author":"Passero","year":"2021","journal-title":"J. Immunol. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1590\/abd1806-4841.20187321","article-title":"Chromoblastomycosis: An Etiological, Epidemiological, Clinical, Diagnostic, and Treatment Update","volume":"93","author":"Bittencourt","year":"2018","journal-title":"An. Bras. Dermatol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"884","DOI":"10.1128\/CMR.00019-10","article-title":"Melanized Fungi in Human Disease","volume":"23","author":"Revankar","year":"2010","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1111\/myc.12308","article-title":"Phenotypic and Molecular Identification of Fonsecaea Pedrosoi Strains Isolated from Chromoblastomycosis Patients in Mexico and Venezuela","volume":"58","year":"2015","journal-title":"Mycoses"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Bienvenu, A.L., and Picot, S. (2020). Mycetoma and Chromoblastomycosis: Perspective for Diagnosis Improvement Using Biomarkers. Molecules, 25.","DOI":"10.3390\/molecules25112594"},{"key":"ref_16","unstructured":"De Hoog, G.S., Guarro, J., Gen\u00e9, J., and Figueras, M.J. (2000). Atlas of Clinical Fungi, Universitat Rovira i Virgili Reus. [2nd ed.]."},{"key":"ref_17","unstructured":"Monteiro, R.C. (2017). Epidemiologia Molecular e Caracteriza\u00e7\u00e3o Fenot\u00edpica de Fungos Dem\u00e1ceos Agentes de Cromoblastomicose no Estado do Par\u00e1, com Relato de Fungos Neurotr\u00f3picos na Regi\u00e3o Amaz\u00f4nica, Disserta\u00e7\u00e3o de Mestrado, Universidade Federal do Par\u00e1."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Sun, J., Najafzadeh, M.J., Gerrits van den Ende, A.H.G., Vicente, V.A., Feng, P., Xi, L., and de Hoog, G.S. (2012). Molecular Characterization of Pathogenic Members of the Genus Fonsecaea Using Multilocus Analysis. PLoS ONE, 7.","DOI":"10.1371\/annotation\/8fef18d2-c01c-4f66-9b75-ecbfac5c82a5"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1160","DOI":"10.1093\/bib\/bbx108","article-title":"MAFFT Online Service: Multiple Sequence Alignment, Interactive Sequence Choice and Visualization","volume":"20","author":"Katoh","year":"2018","journal-title":"Brief. Bioinform."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1093\/mmy\/myv083","article-title":"Identifying Paracoccidioides Phylogenetic Species by PCR-RFLP of the Alpha-Tubulin Gene","volume":"54","author":"Roberto","year":"2016","journal-title":"Med. Mycol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3688","DOI":"10.1093\/nar\/gkg526","article-title":"NEBcutter: A Program to Cleave DNA with Restriction Enzymes","volume":"31","author":"Vincze","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Borman, A.M., Fraser, M., Patterson, Z., Linton, C.J., Palmer, M., and Johnson, E.M. (2022). Fungal Infections of Implantation: More Than Five Years of Cases of Subcutaneous Fungal Infections Seen at the UK Mycology Reference Laboratory. J. Fungi, 8.","DOI":"10.3390\/jof8040343"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1016\/j.ijid.2020.09.1479","article-title":"Clinical Aspects of Previously Treated Chromoblastomycosis: A Case Series from Madagascar","volume":"101","author":"Sendrasoa","year":"2020","journal-title":"Int. J. Infect. Dis."},{"key":"ref_24","unstructured":"Ariani, T., Rizal, Y., and Ledika Veroci, R. (2023). Clinical and Mycological Spectrum of Chromoblastomycosis, Available online: https:\/\/www.jpad.com.pk\/index.php\/jpad\/article\/view\/2399."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Revankar, S.G. (2015). Phaeohyphomycosis in Transplant Patients. Fungi, 2.","DOI":"10.3390\/jof2010002"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"869","DOI":"10.1093\/qjmed\/hcz190","article-title":"Exophiala Dermatitis and Exacerbation of Chronic Obstructive Pulmonary Disease","volume":"112","author":"Gu","year":"2019","journal-title":"QJM"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"An, L., Jia, G., Tan, J., Yang, L., Wang, Y., and Li, L. (2023). Analysis of the Synergistic Antifungal Activity of Everolimus and Antifungal Drugs against Dematiaceous Fungi. Front. Cell. Infect. Microbiol., 13.","DOI":"10.3389\/fcimb.2023.1131416"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Coelho, R.A., Brito-Santos, F., Figueiredo-Carvalho, M.H.G., Silva, J.V.d.S., Gutierrez-Galhardo, M.C., do Valle, A.C.F., Zancop\u00e9-Oliveira, R.M., Trilles, L., Meyer, W., and Freitas, D.F.S. (2018). Molecular Identification and Antifungal Susceptibility Profiles of Clinical Strains of Fonsecaea Spp. Isolated from Patients with Chromoblastomycosis in Rio de Janeiro, Brazil. PLoS Neglected Trop. Dis., 12.","DOI":"10.1371\/journal.pntd.0006675"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"E0169","DOI":"10.1590\/0037-8682-0169-2021","article-title":"Cryptococcosis by Cryptococcus Neoformans\/Cryptococcus Gattii Species Complexes in Non-Hiv-Infected Patients in Southeastern Brazil","volume":"54","author":"Nascimento","year":"2021","journal-title":"Rev. Soc. Bras. Med. Trop."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1773","DOI":"10.3855\/jidc.17077","article-title":"ITS-RFLP Optimization for Dermatophyte Identification from Clinical Sources in Alagoas (Brazil) versus Phenotypic Methods","volume":"16","author":"Silva","year":"2022","journal-title":"J. Infect. Dev. Ctries"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Fontecha, G., Montes, K., Ortiz, B., Galindo, C., and Braham, S. (2019). Identification of Cryptic Species of Four Candida Complexes in a Culture Collection. J. Fungi, 5.","DOI":"10.20944\/preprints201911.0293.v1"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Maubon, D., Garnaud, C., Ramarozatovo, L.S., Fahafahantsoa, R.R., Cornet, M., and Rasamoelina, T. (2022). Molecular Diagnosis of Two Major Implantation Mycoses: Chromoblastomycosis and Sporotrichosis. J. Fungi, 8.","DOI":"10.3390\/jof8040382"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"37","DOI":"10.15578\/squalen.v11i2.213","article-title":"Internal Transcribed Spacer (ITS) as Dna Barcoding to Identify Fungal Species: A Review","volume":"11","author":"Fajarningsih","year":"2016","journal-title":"Squalen Bull. Mar. Fish. Postharvest Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Hoang, M.T.V., Irinyi, L., Chen, S.C.A., Sorrell, T.C., and Meyer, W. (2019). Dual DNA Barcoding for the Molecular Identification of the Agents of Invasive Fungal Infections. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.01647"},{"key":"ref_35","unstructured":"Xu, J. (2016). Fungal DNA Barcoding1. Genome, Canadian Science Publishing."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"R\u00e9blov\u00e1, M., Untereiner, W.A., and R\u00e9blov\u00e1, K. (2013). Novel Evolutionary Lineages Revealed in the Chaetothyriales (Fungi) Based on Multigene Phylogenetic Analyses and Comparison of ITS Secondary Structure. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0063547"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.simyco.2017.01.001","article-title":"Exploring the Genomic Diversity of Black Yeasts and Relatives (Chaetothyriales, Ascomycota)","volume":"86","author":"Teixeira","year":"2017","journal-title":"Stud. Mycol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1080\/13693780802527178","article-title":"Genetic Diversity and Species Delimitation in the Opportunistic Genus Fonsecaea","volume":"47","author":"Najafzadeh","year":"2009","journal-title":"Med. Mycol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"7963","DOI":"10.12998\/wjcc.v9.i27.7963","article-title":"Exophiala Dermatitidis","volume":"9","author":"Usuda","year":"2021","journal-title":"World J. Clin. Cases"},{"key":"ref_40","first-page":"10","article-title":"Pediatric Phaeohyphomycosis: A 44-Year Systematic Review of Reported Cases","volume":"12","year":"2022","journal-title":"J. Pediatr. Infect. Dis. Soc."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Le Govic, Y., Demey, B., Cassereau, J., Bahn, Y.S., and Papon, N. (2022). Pathogens Infecting the Central Nervous System. PLoS Pathog., 18.","DOI":"10.1371\/journal.ppat.1010234"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Ganesan, V., Hallur, V., Velvizhi, S., and Rajendran, T. (2023). Cerebral Phaeohyphomycosis Due to Cladophialophora Bantiana: Case Report and Systematic Review of Cases. Infection.","DOI":"10.1007\/s15010-023-02126-4"}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/10\/2\/159\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:01:31Z","timestamp":1760104891000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/10\/2\/159"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,18]]},"references-count":42,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2024,2]]}},"alternative-id":["jof10020159"],"URL":"https:\/\/doi.org\/10.3390\/jof10020159","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,18]]}}}