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Background: Chromoblastomycosis (CBM) is a chronic infection of skin and subcutaneous tissue. CBM cases have been reported in local literature from Pakistan with heterogenous demographic, diagnostic and therapeutic information. The objective of this study is to share the experience of CBM from a large tertiary care hospital laboratory in Pakistan., Method: This was a retrospective observational study. Histopathology and microbiology data of suspected CBM between 2016 and 2022 was retrieved. Patients' demographics, site of involvement, histopathological findings and positive microbiology cultures were assessed. Literature search on Google Scholar, PubMed and PakMediNet was done between 1990 and 2023 with multiple terms., Result: A total of 16 CBM cases were identified; 14 were histopathology positive and two were both histopathology and culture positive. The median age was 21 years, and 11 patients were male. The predominant site was lower extremities followed by the face. Severe acanthosis, hyperkeratosis and granuloma with sclerotic bodies were observed in all histopathology slides. Alternaria spp. and Phialophora spp. were isolated from two culture-positive cases. A total of nine cases of CBM were reported from Pakistan in PubMed non-indexed journal., Conclusion: CBM is not a commonly thought of disease when evaluating skin lesions in Pakistan. A high index of suspicion when assessing patients who may have a history of trauma, exposure to soil and suggestive lesions is reasonable. An integrated approach between clinicians, histopathologist and microbiologist is required to do early identification and therapeutic interventions., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Chromoblastomycosis (CBM), a chronic fungal infection affecting the skin and subcutaneous tissues, is predominantly caused by dematiaceous fungi in tropical and subtropical areas. Characteristically, CBM presents as plaques and nodules, often leading to scarring post-healing. Besides traditional diagnostic methods such as fungal microscopy, culture, and histopathology, dermatoscopy and reflectance confocal microscopy can aid in diagnosis. The treatment of CBM is an extended and protracted process. Imiquimod, acting as an immune response modifier, boosts the host's immune response against CBM, and controls scar hyperplasia, thereby reducing the treatment duration. We present a case of CBM in Guangdong with characteristic reflectance confocal microscopy manifestations, effectively managed through a combination of itraconazole, terbinafine, and imiquimod, shedding light on novel strategies for managing this challenging condition., (© 2024. The Author(s).)

Hyalohyphomycosis and phaeohyphomycosis are groups of mycoses caused by several agents and show different clinical manifestations. We report a case of an immunocompromised patient who presented rare manifestations of opportunistic mycoses: mycetoma-like hyalohyphomycosis on his right foot caused by Colletotrichum gloeosporioides, followed by cutaneous phaeohyphomycosis on his right forearm caused by Exophiala oligosperma. Further to the rarity of this case, the patient's lesion on the foot shows that the clinical aspects of mycetomas could falsely appear in other fungal infections similar to hyalohyphomycosis. We also show that the muriform cells that were seen in the direct and anatomopathological examination of the skin are not pathognomonic of chromoblastomycosis, as observed in the lesion of the patient's forearm.

Chromoblastomycosis is a fungal chronic disease, which affects humans, especially in cutaneous and subcutaneous tissues. There is no standard treatment for Chromoblastomycosis, and it is a therapeutic challenge, due natural resistance of their causative agents, inadequate response of patients and common cases of relapse. Protocols for determination of antifungal drugs susceptibility are not standardized for chromoblastomycosis agents and endpoint definition is usually based on visual inspection, which depends on the analyst, making it sometimes inaccurate. We presented a colorimetric and quantitative methodology based on resazurin reduction to resofurin to determine the metabolic status of viable cells of Fonsecaea sp. Performing antifungal susceptibility assay by a modified EUCAST protocol allied to resazurin, we validated the method to identify the minimum inhibitory concentrations of itraconazole, fluconazole, amphotericin B, and terbinafine for eight Fonsecaea clinical isolates. According to our data, resazurin is a good indicator of metabolic status of viable cells, including those exposed to antifungal drugs. This work aimed to test resazurin as an indicator of the metabolic activity of Fonsecaea species in susceptibility assays to antifungal drugs. Species of this genus are the main causative agents of Chromoblastomycosis, which affects humans., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Chromoblastomycosis (CBM) and pheohyphomycosis (PHM) are the most common implantation mycoses caused by dematiaceous fungi. In the past, flucytosine (5-FC) has been used to treat CBM, but development of resistance is common. Carmofur belongs to the same class as 5-FC and has in vitro inhibitory activity against the main agents of CBM and PHM. The aim of this study was to compare the action of these two pyrimidine analog drugs against CBM and PHM agents. The minimum inhibitory concentration (MIC) and the selectivity index based on cytotoxicity tests of these two drugs against some agents of these mycoses were determined, with carmofur presenting a higher selectivity index than 5-FC. Carmofur demonstrated here synergistic interactions with itraconazole and amphotericin B against Exophiala heteromorpha, Fonsecaea pedrosoi, Fonsecaea monophora, and Fonsecaea nubica strains. Additionally, carmofur plus itraconazole demonstrated here synergism against a Phialophora verrucosa strain. To evaluate the development of carmofur resistance, passages in culture medium containing subinhibitory concentrations of this pyrimidine analog were carried out, followed by in vitro susceptibility tests. Exophiala dermatitidis quickly developed resistance, whereas F. pedrosoi took seven passages in carmofur-supplemented medium to develop resistance. Moreover, resistance was permanent in E. dermatitidis but transient in F. pedrosoi. Hence, carmofur has exhibited certain advantages, albeit accompanied by limitations such as the development of resistance, which was expected as with 5-FC. This underscores its therapeutic potential in combination with other drugs, emphasizing the need for a meticulous evaluation of its application in the fight against dematiaceous fungi., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Chromoblastomycosis is an implantation mycosis of the skin caused by certain species of melanised fungi. A man in his 50s, born in Kerala but living in England for 14 years, presented with a nodular lesion on his left buttock, which had been present for 20 years. Biopsy revealed muriform cells and fungal culture isolated Fonsecaea spp , consistent with a diagnosis of chromoblastomycosis. Treatment with oral terbinafine was initiated and changed to itraconazole based on results of antifungal susceptibility. Drug intolerance and low drug levels of itraconazole necessitated change to voriconazole and topical terbinafine. Despite long-term combined therapy, the lesions worsened, and the patient opted for surgical excision abroad. Recurrence was evident at surgical sites and combined therapy continues. Chromoblastomycosis is an insidious and burdensome neglected tropical disease. Within non-endemic countries, diagnosis remains challenging. A travel history and appropriate fungal investigations are vital., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Chromoblastomycosis is a fungal disease presented with local warty papule, plaque, and verrucous nodules. In addition, the incidence and drug resistance of chromoblastomycosis are increasing each year worldwide. Photodynamic therapy is a promising method to treat mycoses. The purpose of this study was to evaluate the effect of new methylene blue (NMB)-induced PDT on multidrug-resistant chromoblastomycosis in vitro. We isolated one wild-type strain pathogen from one clinical patient diagnosed with chromoblastomycosis for over 27 years. The pathogen was identified by histopathology, the morphology of fungal culture, and genetic testing. Drug susceptibility testing was performed on the isolate. It was cultured with logarithmic growth phase spore in vitro and incubated with different concentrations of NMB for 30 min, and received illumination by red light-emitted diode with different light doses. After photodynamic treatment, the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were conducted. The pathogen was Fonsecaea nubica, and it was resistant to itraconazole, terbinafine, amphotericin B, voriconazole andcaspofungin. At the same NMB concentration, the sterilization efficiency of NMB-photodynamic therapy (PDT) on F. nubica increased with increasing light intensity; F. nubica was completely killed at 25 µmol/L NMB with a light dose of 40 J/cm 2 or 50 µmol/L NMB and light doses of ≥ 30 J/cm 2 . SEM and TEM observed ultrastructural changes after PDT. NMB-PDT inactivates the survival of multidrug-resistant F. nubica in vitro; it therefore has the potential to become an alternative or adjuvant treatment for refractory chromoblastomycosis., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Rhinocladiella similis is a melanized fungi involved in chromoblastomycosis. R. similis genome has never been sequenced, therefore we propose the first draft genome of R. similis., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The experimental rodent models for the fungal disease are a handy tool for understanding host-fungus interactions. To Fonsecaea sp., one of the causative agents of chromoblastomycosis, there is an extra challenge because the animals preferably used show a spontaneous cure; so until now, there is no model to reproduce the long-term disease similar to human chronic disease. In this chapter, we described an experimental model using rats and mice with a subcutaneous route, with the checkpoints of acute-like and chronic-like lesion analysis comparable with human lesions, the fungal burden, and the lymphocytes investigation., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Chromoblastomycosis is a disease caused by melanized fungi, primarily belonging to the genera Fonsecaea and Cladophialophora, mainly affecting individuals who are occupationally exposed to soil and plant products. This research aimed to determine the clinical, epidemiological and laboratory characteristics of chromoblastomycosis in the state of Mato Grosso, Brazil., Materials and Methods: Patients diagnosed with chromoblastomycosis treated at the Júlio Müller University Hospital, Cuiabá, Brazil, from January 2015 to December 2020, whose isolates were preserved in the Research Laboratory of the Faculty of Medicine of the Federal University of Mato Grosso. Isolates were identified by partly sequencing the Internal Transcribed Spacer (ITS) and β-tubulin (BT2) loci. AFLP fingerprinting was used to explore the genetic diversity. Susceptibility to itraconazole, voriconazole, 5-fluorocytosine, terbinafine and amphotericin B was determined by the broth microdilution technique., Results: Ten patients were included, nine were male (mean age = 64.1 years). Mean disease duration was 8.6 years. Lesions were mainly observed in the lower limbs. Predominant clinical forms were verrucous and scarring. Systemic arterial hypertension and type II diabetes mellitus were the predominant comorbidities. Leprosy was the main concomitant infectious disease. Fonsecaea pedrosoi was the unique aetiological agent identified with moderate genetic diversity (H = 0.3934-0.4527; PIC = 0.3160-0.3502). Antifungal agents with the highest activity were terbinafine, voriconazole and itraconazole., Conclusion: Chromoblastomycosis is affecting the poor population in rural and urban areas, mainly related to agricultural activities, with F. pedrosoi being the dominant aetiologic agent. All isolates had low MICs for itraconazole, voriconazole and terbinafine, confirming their importance as therapeutic alternatives for chromoblastomycosis., (© 2022 The Authors. Mycoses published by Wiley-VCH GmbH.)

Introduction: Chromoblastomycosis (CBM) is a chronic fungal infection of the skin and subcutaneous tissue caused by several pigmented fungi. It is frequently found in tropical and subtropical areas like Madagascar. This study primarily discusses the effects of antifungal therapy while also describing the epidemiological, clinical, and pathological features of CBM in our patients., Methods: From March 2013 to January 2019, a descriptive prospective study on CBM patients was undertaken. The study included patients with CBM who had received antifungal treatment for at least 3 months. Itraconazole 200 mg was given to patients every day for ˃3 months. Results were assessed at the 6th and 12th months and classified as major responses, minor responses to treatment, or failure., Results: A total of 29 cases of CBM were included. The mean age of patients was 42.02 years. They primarily worked in rural areas. Infected men were more prevalent. At the end of the 12th month of itraconazole therapy, 3 patients presented major responses, 14 patients had minor responses to treatment, and 12 had been lost to follow-up. The clinical response of CBM to treatment was correlated to the severity and the long course of CBM. When compared with CBM caused by Cladophialophora, CBM caused by Fonsecaea showed a greater clinical response., Conclusion: These findings demonstrated that CBM lesions are recalcitrant and difficult to treat., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Chromoblastomycosis (CBM) is a chronic fungal infection of the cutaneous and subcutaneous tissues caused by brown pigmented fungi. Fonsecaea monophora is one of the most common pathogens of CBM in China. Most formal cases have been reported from Southern China, however, the infection is not uncommon in Eastern China where very few case series are available. To describe the clinical aspects of CBM, we report a series of 11 cases between 2018 and 2021 at a single medical center in Eastern China. The patients were predominately male (n = 9) and the disease duration ranged from 3 months to 20 years. Plaque type lesions were the most common clinical manifestations. There were 7 cases of mild forms and 3 cases of severe forms. Among the 3 severe cases, one case gave up treatment due to economic poverty; one case did not respond to a 1-year systemic treatmen; one case was cured by combination therapy of 10 months. Other cases were cured by treatment with antifungal agents. All cases of direct mycological examination were positive. All isolates were identified by morphology and sequencing of the the ITS regions of ribosomal DNA, Ten were F. monophora and 1 was Cladophialophora carrionii. All cases had been evaluated at other clinics, where 8 cases were misdiagnosed as other diseases. As a neglected tropical disease (NTD), CBM is still a major challenge in the field of dermatology, especially in its severe clinical forms. As an effective and simple diagnostic method of CBM, direct microscopic examination should be further promoted in rural hospitals., Competing Interests: The authors have declared that no competing interests exist.

Background: Black fungi of the Herpotrichiellaceae family are agents of chromoblastomycosis and phaeohyphomycosis. There are few therapeutic options for these infections and it is common to associate antifungal drugs in their treatment., Objectives: To investigate the Medicines for Malaria Venture (MMV) Pathogen Box® for possible compounds presenting synergism with antifungal drugs used to treat black fungal infections., Methods: An initial screening of the Pathogen Box® compounds was performed in combination with itraconazole or terbinafine at sub-inhibitory concentrations against Fonsecaea pedrosoi. Hits were further tested against eight Herpotrichiellaceae using the checkerboard method., Findings: No synergism was observed with terbinafine. MMV687273 (SQ109) and MMV688415 showed synergism with itraconazole against F. pedrosoi. Synergism of these compounds was confirmed with some black fungi by the checkerboard method. SQ109 and itraconazole presented synergism for Exophiala dermatitidis, F. pedrosoi, F. monophora and F. nubica, with fungicidal activity for F. pedrosoi and F. monophora. MMV688415 presented synergism with itraconazole only for F. pedrosoi, with fungicidal activity. The synergic compounds had high selectivity index values when combined with itraconazole., Main Conclusions: These compounds in combination, particularly SQ109, are promising candidates to treat Fonsecaea spp. and E. dermatitidis infections, which account for most cases of chromoblastomycosis and phaeohyphomycosis.

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous mycosis caused by black, dimorphic, and filamentous fungi of the Herpothrichiellaceae family, such as species of the genus Fonsecaea . These fungi can switch between the saprophytic forms (conidia and hyphae) and the pathogenic form, the muriform cells (MCs), which is considered an essential mechanism for fungal virulence. Nearly all types of cells can produce membranous structures formed by a lipid bilayer that communicate extracellularly with other cells, known as "extracellular vesicles" (EVs), which may act as virulence factors, as observed for several species of pathogenic fungi. Our findings demonstrated for the first time that F. pedrosoi, F. nubica , and F. erecta produce EVs in response to nutritional conditions. The EVs varied in sterol and protein contents, size, and morphology. Moreover, the EVs induced different cytokine and nitric oxide release patterns by bone marrow-derived macrophages (BMDMs). The EVs activated IL-1β production, possibly acting as the first signal in inflammasome activation. Unlike the pathogenic species, the EVs isolated from F. erecta did not significantly stimulate TNF and IL-10 production in general. Overall, these results demonstrated that different species of Fonsecaea produce EVs capable of modulating pro- and anti-inflammatory cytokine and nitric oxide production by BMDMs and that growth conditions affected the immunomodulatory capacities of the EVs as well as their size, content, and morphology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Las-Casas, Marina, de Castro, Coelho, Báo, de Hoog, Vicente, Fernandes and Bocca.)

Chromoblastomycosis is a fungal infection of the skin and subcutaneous tissue, of chronic evolution, caused by dematiaceous fungi. The disease occurs worldwide, mainly in tropical and subtropical regions, but in regions like Chile there is only one report of a human case more than 30 years ago. We present the case of a 46-year-old Haitian man, resident in Chile, with verrucous plaques in the right anterior tibial area of one year of evolution. The diagnosis of chromoblastomycosis was confirmed when muriform cells and dematiaceous colonies were observed in the histopathological analysis and the direct microscopy, respectively. After six months of treatment with systemic antimycotics and cryotherapy, complete remission of the lesions was achieved.

Introduction: The three fungal skin neglected tropical diseases (NTD) - mycetoma, chromoblastomycosis and sporotrichosis - currently lack prioritization and support to establish drug discovery programs in search for novel treatment options. This has made the efforts to identify novel drugs for these fragmented skinNTDs., Areas Covered: To help escalate the discovery of novel drugs to treat these fungal skinNTDs, the authors have prepared an overview of the compounds with activity against fungal skinNTDs by analyzing data from individual drug discovery studies, including those performed on the Medicines for Malaria Venture (MMV) open access boxes., Expert Opinion: The authors were unable to identify studies in which causative agents of all three skinNTDs were included, indicating that an integrated approach is currently lacking. From current available data, the azoles and iodoquinol were the only compounds with activity against causative agents from the three different fungal skinNTDs. Fungal melanin inhibition enhanced the activity of antifungal agents. For mycetoma, the fenarimols, aminothiazoles and benzimidazole carbamates are currently being investigated in the MycetOS initiative. To come to a more integrated approach to identify drugs active against all three fungal skinNTDs, compounds made in the MycetOS initiative could also be explored for chromoblastomycosis and sporotrichosis.

Chromoblastomycosis (CBM) is a chronic disease caused by several species of dematiaceous fungi. In this study, a regional collection of 45 CBM cases was conducted in Guangdong, China, a hyper-endemic area of CBM. Epidemiology findings indicated that the mean age of cases was 61.38 ± 11.20 years, long duration ranged from 3 months to 30 years, and the gender ratio of male to female was 4.6:1. Thirteen cases (29%) declared underlying diseases. Verrucous form was the most common clinical manifestation ( n = 19, 42%). Forty-five corresponding clinical strains were isolated, and 28 of them (62%) were identified as F. monophora ; the remaining 17 (38%) were identified as F. nubica through ITS rDNA sequence analysis. Antifungal susceptibility tests in vitro showed low MICs in azoles (PCZ 0.015-0.25 μg/ml, VCZ 0.015-0.5 μg/ml, and ITZ 0.03-0.5 μg/ml) and TRB (0.015-1 μg/ml). Itraconazole combined with terbinafine was the main therapeutic strategy used for 31 of 45 cases, and 68% ( n = 21) of them improved or were cured. Cytokine profile assays indicated upregulation of IL-4, IL-7, IL-15, IL-11, and IL-17, while downregulation of IL-1RA, MIP-1β, IL-8, and IL-16 compared to healthy donors ( p < 0.05). The abnormal cytokine profiles indicated impaired immune response to eliminate fungus in CBM cases, which probably contributed to the chronic duration of this disease. In conclusion, we investigated the molecular epidemiological, clinical, and laboratory characteristics of CBM in Guangdong, China, which may assist further clinical therapy, as well as fundamental pathogenesis studies of CBM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Sun, Li, Cai, Chen, Liu, Huang, Xie, Zeng and Xi.)

Background: Black opportunists Phialophora verrucosa complex species can cause different disease types in competent and in immunocompromised individuals, but are remarkably overrepresented in CARD9-related infections., Objectives: To better understand the ecology and potential pathogenicity of opportunistic Phialophora species and reveal eventual genetic parameters associated with the behaviour in vivo and genetic profiles in patients with CARD9 immunodeficiency., Methods: Genomes of 26 strains belonging to six species of the Phialophora verrucosa complex were sequenced. Using multilocus analysis, all environmental and clinical strains were identified correctly. We compared the genomes of agents from different disease types among each other including CARD9 immunodeficiency., Results: We obtained genome sizes of the 26 Phialophora strains ranged between 32 and 37 MB. Some species showed considerable intraspecific genomic variation. P americana showed the highest degree of variability. P verrucosa was variable in CAZy enzymes, whereas P americana varied in PKS-related genes. Phialophora species, particularly P verrucosa, are relatively frequent in patients with CARD9-related immunodeficiency. Different mutations in the CARD9 gene seem to increase susceptibility for infection by different groups of species, that is either Candida, dermatophytes or black fungi. A number of patients with chromoblastomycosis revealed an as yet unknown CARD9 mutation. TNFα impairment was prevalent in patients with CARD9 infections, while CBM patients were invariably IFNγ., Conclusions: From genomic investigations, the known virulence factors between clinical and environmental strains did not reveal any significant difference. Phialophora complex has an equal chance to cause infection in humans, either healthy or CARD9-impaired., (© 2021 Wiley-VCH GmbH.)

Fonsecaea monophora, which is very similar to Fonsecaea pedrosoi in morphological features, has been commonly misdiagnosed as F. pedrosoi. Like F. pedrosoi, F. monophora has been also identified as a predominant pathogen of Chromoblastomycosis (CBM). Melanin has been recognized as a virulence factor in several fungi, however, it is still largely unknown about the biological role of melanin and how melanin is synthesized in F. monophora. In this study, we identified two putative polyketide synthase genes (pks), AYO21&#95;03016 (pksA) and AYO21&#95;10638, by searching against the genome of F. monophora. AYO21&#95;03016 and AYO21&#95;10638 were further targeted disrupted by Agrobacterium tumefaciens-mediated transformation (ATMT). We discovered that pksA gene was the major polyketide synthase required for melanin synthesis in F. monophora, rather than AYO21&#95;10638. Phenotypic analysis showed that, knocking out of the pksA gene attenuated melanogenesis, growth rate, sporulation ability and virulence of F. monophora, as compared with wild-type and complementation strain (pksA-C). Furthermore, the ΔpksA mutant was confirmed to be more sensitive to the oxidative stress, extreme pH environment, and antifungal drugs including itraconazole (ITC), terbinafine (TER), and amphotericin B (AMB). Taken together, these findings enabled us to comprehend the role of pksA in regulating DHN-melanin pathway and its effect on the biological function of F. monophora., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Fonsecaea pedrosoi is one of the main agents of chromoblastomycosis, a chronic subcutaneous mycosis. Itraconazole (ITC) is the most used antifungal in its treatment, however, in vitro antifungal susceptibility tests are important to define the best therapy. These tests are standardized by the Clinical and Laboratory Standards Institute (CLSI), but these protocols have limitations such as the high complexity, cost and time to conduct. An alternative to in vitro susceptibility test, which overcomes these limitations, is FTIR. This study determined the minimum inhibitory concentration (MIC) of itraconazole for F. pedrosoi, using FTIR and chemometrics. The susceptibility to ITC of 36 strains of F. pedrosoi was determined according to CLSI and with the addition of tricyclazole (TCZ), to inhibit 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Strains were grown in Sabouraud agar and prepared for Attenuated Total Reflection (ATR)/FTIR. Partial least squares (PLS) regression was performed using leave-one-out cross-validation (by steps of quintuplicates), then tested on an external validation set. A coefficient of determination (R²) higher than 0.99 was obtained for both the MIC-ITC and MIC-ITC+TCZ ATR/PLS models, confirming a high correlation of the reference values with the ones predicted using the FTIR spectra. This is the first study to propose the use of FTIR and chemometric analyses according to the M38-A2 CLSI protocol to predict ITC MICs of F. pedrosoi. Considering the limitations of the conventional methods to test in vitro susceptibility, this is a promising methodology to be used for other microorganisms and drugs., Competing Interests: The authors have declared that no competing interests exist.

Objective: To describe the clinical aspects of chromoblastomycosis (CBM) presented by patients who had received incomplete antifungal treatment before consultation., Methods: A prospective study of patients with clinically suspected CBM was performed between 2013 and 2018 in the Department of Dermatology at the University Hospital Antananarivo, and during consultation campaigns., Results: Patients develop CBM over a period of more than 10 years, and many will have already received antifungals prescribed by general practitioners before consulting with a dermatologist. Such treatment obviously modifies the clinical presentation. From the 63 CBM patients in this large study, we describe 12 patients who received oral antifungals (terbinafine, griseofulvine, itraconazole, fluconazole) before consultation. The most frequent clinical aspect presented by these patients was cicatricial lesions, which are characteristically smooth and non-elevated, and enlarge by peripheral extension, with atrophic scarring at the center., Conclusion: Our study is the first to show that cicatricial lesions are a clinical aspect presented by CBM patients who received antifungals before presentation., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Melanized fungi and black yeasts in the family Herpotrichiellaceae (order Chaetothyriales) are important agents of human and animal infectious diseases such as chromoblastomycosis and phaeohyphomycosis. The oligotrophic nature of these fungi enables them to survive in adverse environments where common saprobes are absent. Due to their slow growth, they lose competition with common saprobes, and therefore isolation studies yielded low frequencies of clinically relevant species in environmental habitats from which humans are thought to be infected. This problem can be solved with metagenomic techniques which allow recognition of microorganisms independent from culture. The present study aimed to identify species of the family Herpotrichiellaceae that are known to occur in Brazil by the use of molecular markers to screen public environmental metagenomic datasets from Brazil available in the Sequence Read Archive (SRA). Species characterization was performed with the BLAST comparison of previously described barcodes and padlock probe sequences. A total of 18,329 sequences was collected comprising the genera Cladophialophora, Exophiala, Fonsecaea, Rhinocladiella and Veronaea, with a focus on species related to the chromoblastomycosis. The data obtained in this study demonstrated presence of these opportunists in the investigated datasets. The used techniques contribute to our understanding of environmental occurrence and epidemiology of black fungi.

Chromoblastomycosis (CBM) is a chronic worldwide subcutaneous mycosis, caused by several dimorphic, pigmented dematiaceous fungi. It is difficult to treat patients with the disease, mainly because of its recalcitrant nature. The correct activation of host immune response is critical to avoid fungal persistence in the tissue and disease chronification. CD4+ T cells are crucial for the development of protective immunity to F. pedrosoi infection. Here, we investigated T helper cell response dynamics during experimental CBM. Following footpad injection with F. pedrosoi hyphae and conidia, T cells were skewed towards a Th17 and Th1 phenotype. The Th17 population was the main Th cell subset found in the infected area during the early stages of experimental murine CBM, followed by Th1 predominance in the later stages, coinciding with the remission phase of the disease in this experimental model. Depletion of CD25+ cells, which leads to a reduction of Treg cells in the draining lymph node, resulted in decline in fungal burden after 14 days of infection. However, fungal cells were not cleared in the later stages of the disease, prolonging CBM clinical features in those animals. IL-17A and IFN-γ neutralization hindered fungal cell elimination in the course of the disease. Similarly, in dectin-2 KO animals, Th17 contraction in the course of experimental CBM was accompanied by fungal burden decrease in the first 14 days of infection, although it did not affect disease resolution. In this study, we gained insight into T helper subsets' dynamics following footpad injections of F. pedrosoi propagules and uncovered their contribution to disease resolution. The Th17 population proved to be important in eliminating fungal cells in the early stages of infection. The Th1 population, in turn, closely assisted by Treg cells, proved to be relevant not only in the elimination of fungal cells at the beginning of infection but also essential for their complete elimination in later stages of the disease in a mouse experimental model of CBM., Competing Interests: The authors have declared that no competing interests exist.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 μM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 μM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 μM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 μM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection., Competing Interests: The authors have declared that no competing interests exist.

Chromoblastomycosis (CBM) is a fungal infection caused by fungi belonging to the order Chaetothyriales, and caused mainly by Fonsecaea pedrosoi. The classic treatment, based on itraconazole and/or terbinafine as well as physical approaches, is considered complex and ineffective due to the high rate of relapses. Thus, new strategies are needed to manage CBM; in this regard, the present work reports the evolution of lesions in patients successfully treated with imiquimod. Of note, classic treatment was not effective in healing the lesions of two of them, but single topical treatment with imiquimod healed the lesions., (© 2020 Japanese Dermatological Association.)

Chromoblastomycosis (CBM) is a chronic cutaneous or subcutaneous mycosis that is prevalent worldwide. Though CBM tends not to be fatal, it is difficult to treat and complications can include chronic, marked lesions, lymphatic damage, and neoplastic transformation. Fonsecaea monophora, as a new species segregated from Fonsecaea pedrosoi, is the predominant causative pathogen of CBM in southern China. However, research about F. monophora has been limited, which may be due to a lack of an effective genetic manipulation system for F. monophora. In this study, we successfully established a random insertional mutagenesis system by Agrobacterium tumefaciens-mediated transformation (ATMT) in F. monophora for the first time. In order to improve the efficiency of ATMT, various co-culture conditions were optimized, including: acetosyringone (AS) concentrations, co-culture duration, ratio of bacteria to conidia, and the A. tumefaciens strains. In addition, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to identify the transferred DNA (T-DNA) flanking sequences of the F. monophora transformants. The valuable transformants obtained in this study will be used for research in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chromoblastomycosis is a neglected disease characterized by cutaneous, subcutaneous or disseminated lesions. It is considered an occupational infectious disease that affects mostly rural workers exposed to contaminated soil and vegetal matter. Lesions mostly arise after a traumatic inoculation of herpotrichiellaceous fungi from the Chaetothyriales order. However, the environmental niche of the agents of the disease remains obscure. Its association with insects has been predicted in a few studies. Therefore, the present work aimed to analyze if social insects, specifically ants, bees, and termites, provide a suitable habitat for the fungi concerned. The mineral oil flotation method was used to isolate the microorganisms. Nine isolates were recovered and phylogenetic analysis identified two strains as potential agents of chromoblastomycosis, i.e., Fonsecaea pedrosoi CMRP 3076, obtained from a termite nest (n = 1) and Rhinocladiella similis CMRP 3079 from an ant exoskeleton (n = 1). In addition, we also identified Fonsecaea brasiliensis CMRP 3445 from termites (n = 1), Exophiala xenobiotica CMRP 3077 from ant exoskeleton (n = 1), Cyphellophoraceae CMRP 3103 from bees (n = 1), Cladosporium sp. CMRP 3119 from bees (n = 1), Hawksworthiomyces sp. CMRP 3102 from termites (n = 1), and Cryptendoxyla sp. from termites (n = 2). The environmental isolate of F. pedrosoi CMRP 3076 was tested in two animal models, Tenebrio molitor and Wistar rat, for its pathogenic potential with fungal retention in T. molitor tissue. In the Wistar rat, the cells resembling muriform cells were observed 30 d after inoculation., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Chromoblastomycosis is a chronic subcutaneous disease caused by human contact with melanized fungi occurring mainly in tropical and subtropical zones worldwide. This study assessed 12 patients with chromoblastomycosis from Rondônia, Brazil, Amazon region. In sum, 83.3% were men, 41.6% were from Monte Negro city, median age was 52.9 years, and median time to disease progression was 12.2 years. Lesions were located on the lower limbs (75%), and verruciform was prevalent form (66.6%). After 3 years of treatment with itraconazole, two patients were considered cured. The etiological agents were identified by the molecular sequence of the ribosomal internal transcribed spacer ITS1, 5.8S, and ITS2 region and β-tubulin genes. Eight strains were identified as Fonsecaea pedrosoi, two were F. nubica, and two were Rhinocladiella similis. The antifungal activity of five drugs was evaluated, and the most active drug was terbinafine (range minimal inhibitory concentration [MIC] 0.015-0.12 μg/ml), itraconazole (range MIC 0.03-0.5 μg/ml) and voriconazole (range MIC 0.06-0.5 μg/ml). The highest MIC was 5-fluorocytosine (range MIC 2-32 μg/ml), and amphotericin B (range MIC 0.25-2 μg/ml). In conclusion, the present study expanded the epidemiological disease database and described for the first time F. nubica and R. similis as chromoblastomycosis agents in the Brazilian Amazon region. Our results confirmed the importance of using molecular methods to identify the melanized fungi and stimulate the recognition of the disease in other places where no cases have been reported., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Competing Interests: None

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous fungal infection caused by certain dematiaceous fungi (usually Fonsecaea, Phialophora, or Cladophialophora). Histologically, CBM is characterized by the presence of medlar bodies. However, the diagnosis is difficult because of the rarity of these pathognomonic presentations and the wide variety of presentations. Treatment of these infections is challenging as it lacks standardization. Herein, we report a case of chromoblastomycosis caused by Phialophora, in a 42-year-old immunocompetent male agriculturist from the humid and subtropical region of southern China. He had a 3-month history of pneumonia with intermittent fever, coughing, and expectoration. The infection subsequently spread to the bone and lymph nodes forming deep lesions and eventually resulting in osteolysis and lymphadenectasis. These subcutaneous nodules were observed after 9 months. Antifungal treatment was administered for 20 months leading to clinical improvement before the patient was lost to follow-up. This case is unique because such deep lesions are rare in immunocompetent individuals and because the initial onset was associated with pneumonia., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by melanotic fungi, affecting mainly rural workers in tropical and subtropical regions. Secondary bacterial infections (SBIs) in CBM lesions bring complications to the disease, but little is known about the agents involved. Fungal and bacterial identification and epidemiological profile of 50 patients with CBM were analyzed in this study. Bacteria were tested for susceptibility to antibacterial drugs. Fonseacea pedrosoi and Rhinocladiella aquaspersa were the fungal agents isolated. 88% of the patients presented SBI. Gram-positive bacteria coinfected mainly upper limbs, and Gram-negative bacteria were more isolated from lower limbs. Streptococcus pyogenes and mixed bacterial microbiota were associated with severe lesions. Staphylococcus aureus was associated with mixed infections and consequently with the severity of the infection. Resistance to β-lactams and methicillin was detected. Our results emphasize the necessity of bacterial culture and susceptibility testing as part of routine monitoring CBM cases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The authors report a case of 40-year-old male patient with a five-year history of chromoblastomycosis on his right leg. Diagnosis was performed by direct 40% KOH exam of skin scales, culture with micro- and macromorphologic analysis, and genotypic characterization (sequencing of a fragment of the ITS region and phylogenetic analysis) of the isolated fungus. Rhinocladiella aquaspersa was identified as the etiological agent. Initially, the treatment was with oral itraconazole 200mg/day for one year. However, the presence of "sclerotic cells" with filaments ("Borelli spiders") resulted in a change of medical treatment: a higher dose of itraconazole (400mg/day) and surgery, achieving clinical and mycological cure in one year. This is the first report of chromoblastomycosis caused by R. aquaspersa in Guatemala., (Copyright © 2019 Sociedade Brasileira de Dermatologia. Published by Elsevier España, S.L.U. All rights reserved.)

The species belonging to the genus Fonsecaea are the main causative agents of chromoblastomycosis. The invasive potential of Fonsecaea differs significantly among its various sibling species. Moreover, the lack of clarity on the virulence and availability of precise markers to distinguish and detect Fonsecaea species is attributed to the different ways of dissemination and pathogenicity. Therefore, the present study aimed to propose new molecular tools to differentiate between sibling species causing chromoblastomycosis. We used an infection model of chromoblastomycosis in BALB/c to study species-specific molecular markers for the in vivo detection of Fonsecaea species in biological samples. Specific primers based on the CBF5 gene were developed for Fonsecaea pedrosoi, Fonsecaea monophora, Fonsecaea nubica, and Fonsecaea pugnacius. In addition, a padlock probe was designed for F. pugnacius based on ITS sequences. We also assessed the specificity of Fonsecaea species using in silico, in vitro, and in vivo assays. The results showed that markers and probes could effectively discriminate the species in both clinical and environmental samples, enabling bioprospecting of agents of chromoblastomycosis, thereby elucidating the infection route of the disease.

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous infection caused by melanized fungal species. We quantified the extractable melanin of 77 strains of CBM agents distributed within five genera. Moreover, resistance to oxidative stress was evaluated in strains exposed or not to the melanin inhibitor tricyclazole. The median percentage of melanin mass extracted from dry fungal mass varied from 0.69 (Rhinocladiella similis) to 3.81 (Phialophora americana). Inhibition of melanin synthesis decreased survival rates to hydrogen peroxide. Together, these data highlight the importance of melanin in CBM agents.

Chromoblastomycosis is found worldwide with higher incidence in tropical and subtropical regions. Fonsecaea spp. is one of the major causative agents of this disease. First case of chromoblastomycosis due to Fonsecaea nubica in Northern China is reported in a 75-year-old Chinese male. We firstly summarized molecular identification methods of Fonsecaea spp. and all the strains of F. nubica reported in the literature. Sequencing of internal transcribed spacer alone and/or combined with actin (ACT1), partial cell division cycle (CDC42) and partial beta-tubulin (BT2) were most commonly used to identify species, while lactase (Lac), homogentisate (HmgA) and polyketide synthase (PKS1) were also used in some cases. Most strains were isolated from South America and Eastern China. Five clinical cases of chromoblastomycosis due to F. nubica from Asia and Europe were also reviewed. All the five patients were male, over 30 years old, and their lesions occurred after trauma.