Comparison of the antifungal activity of the pyrimidine analogs flucytosine and carmofur against human-pathogenic dematiaceous fungi.

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.

Successful management of chromoblastomycosis utilizing conventional antifungal agents and imiquimod therapy.

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.

Draft Genome Sequence of the Rhinocladiella similis Clinical Isolate CBS 149759.

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.

Treatment responses in patients with chromoblastomycosis to itraconazole in Madagascar.

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.

Chromoblastomycosis (CBM) is a chronic fungal infection of the skin and subcutaneous tissue commonly seen in tropical and subtropical areas. This study mainly discusses the therapeutic while also describing the epidemiological, clinical, and pathological features of CBM in Madagascar.

Molecular epidemiology and clinical-laboratory aspects of chromoblastomycosis in Mato Grosso, Brazil.

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 ß-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.

New possibilities for chromoblastomycosis and phaeohyphomycosis treatment: identification of two compounds from the MMV Pathogen Box® that present synergism with itraconazole.

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.

Comparative genomics of opportunistic Phialophora species involved in divergent disease types.

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.

Chromoblastomycosis in the Amazon region, Brazil, caused by Fonsecaea pedrosoi, Fonsecaea nubica, and Rhinocladiella similis: Clinicopathology, susceptibility, and molecular identification.

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 ß-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.

Melanin: Quantification and protection against oxidative stress in chromoblastomycosis agents.

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.

Case report: Fever- pneumonia- lymphadenectasis- osteolytic- subcutaneous nodule: Disseminated chromoblastomycosis caused by phialophora.

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.

New Molecular Markers Distinguishing Fonsecaea Agents of Chromoblastomycosis.

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.

Case Report: A Case of Chromoblastomycosis Caused by Fonsecaea pedrosoi in Vietnam.

BACKGROUND: Chromoblastomycosis is a chronic fungal infection of the skin and subcutaneous tissues caused by different melanized fungi. The disease occurs worldwide, particularly in tropical and subtropical regions but not reported in Vietnam. A 47-year-old women was admitted to hospital 103, Hanoi, Vietnam, with a 10-year lasting lesion on backside of her right shank. Diagnosis of chromoblastomycosis was made after discovery of a muriform cell in histopathological examination. A black, slow-growth fungus was isolated and identified as Fonsecaea pedrosoi after molecular analysis. After 1-month treatment with itraconazole, the lesion has significant improvement. CONCLUSION: This is the first case of chromoblastomycosis caused by Fonsecaea pedrosoi reported in Vietnam.

Chromoblastomycosis Caused by Fonsecaea nubica: First Report in Northern China and Literature Review.

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.

Combination of Amphotericin B and Terbinafine against Melanized Fungi Associated with Chromoblastomycosis.

Our in vitro studies showed that a combination of amphotericin B and terbinafine had synergistic effects against the majority of melanized fungi associated with chromoblastomycosis (CBM) and similar infections, including those with Cladophialophora carrionii, Cladophialophora arxii, Exophialadermatitidis, Exophialaspinifera, Fonsecaea monophora, Fonsecaea nubica, Fonsecaea pedrosoi, and Phialophora verrucosa. This drug combination could provide an option for the treatment of severe or unresponsive cases of CBM, particularly in cases due to species of Fonsecaea and Cladophialophora.

Identification of chromoblastomycosis agents by PCR based reverse line blot (PCR-RLB) hybridization assay.

Chromoblastomycosis is one of the most prevalent implantation fungal infections caused by melanized fungi, affecting individuals with certain risk factors with high morbidity due to its recalcitrant nature. It is difficult to identify the etiological agents and thus a suitable reproductive molecular identification method applicable in developing countries has been investigated. We report the identification of four different fungal causative agents of chromoblastomycosis by reverse line blotting hybridization (RLB) based on biotin-labeled PCR products and amine labeled probes to hybridize. Sixty five reference strains, including type strains, i.e. Fonsecaea pedrosoi, F. monophora, F. nubica, and Phialophora verrucosa, obtained from the CBS-KNAW were included in this study. Internal transcribed spacer 1 (ITS1) regions of relevant species were aligned and adjusted using BIONUMERICS v. 4.61 in order to design four specific probes to identify informative nucleotide polymorphisms. The final identification of these species by RLB assay was concordant with ITS sequencing and showed 100% specificity with no cross hybridization, able to identify all tested strains. The time and cost were less compare to other routine identification methods such as sequencing. This assay allows sensitive and specific simultaneous detection and identification of a different fungal species.

Black yeasts in the omics era: Achievements and challenges.

Black yeasts (BY) comprise a group of polyextremotolerant fungi, mainly belonging to the order Chaetothyriales, which are capable of colonizing a wide range of extreme environments. The tolerance to hostile habitats can be explained by their intrinsic ability to survive under acidic, alkaline, and toxic conditions, high temperature, low nutrient availability, and osmotic and mechanical stress. Occasionally, some species can cause human chromoblastomycosis, a chronic subcutaneous infection, as well as disseminated or cerebral phaeohyphomycosis. Three years after the release of the first black yeast genome, the number of projects for sequencing these organisms has significantly increased. Over 37 genomes of important opportunistic and saprobic black yeasts and relatives are now available in different databases. The whole-genome sequencing, as well as the analysis of differentially expressed mRNAs and the determination of protein expression profiles generated an unprecedented amount of data, requiring the development of a curated repository to provide easy accesses to this information. In the present article, we review various aspects of the impact of genomics, transcriptomics, and proteomics on black yeast studies. We discuss recent key findings achieved by the use of these technologies and further directions for medical mycology in this area. An important vehicle is the Working Groups on Black Yeasts and Chromoblastomycosis, under the umbrella of ISHAM, which unite the clinicians and a highly diverse population of fundamental scientists to exchange data for joint publications.

Chromoblastomycosis in Taiwan: A report of 30 cases and a review of the literature.

Chromoblastomycosis (CBM) is an implantation mycosis characterized by the presence of pigmented muriform cells in tissue. CBM is endemic in Taiwan, but only three formal cases have been reported to date because of underreporting. To describe and update its epidemiologic features, we report a series of 30 cases between 2003 and 2016 at a single medical center. Patients were predominately male (2.75:1). The mean age of onset was 65.9 years, and disease duration ranged from 2 months to 20 years. Diabetes was the most common comorbidity, and extremities were the most frequent sites of involvement. The lesions presented as papuloplaque, verrucous, cicatricial, targetoid, or mixed types. The dermoscopic features were variable, including red dots, white vague areas, black globules, and sand-like patterns. Among 10 Fonsecaea isolates further identified by sequencing the ITS regions of ribosomal DNA, nine were F. monophora and one was F. nubica. All but one patient received either systemic antifungal agents, surgical excision, or both. Surgical excision achieved a higher complete remission rate than the other forms of treatment did.

Successful treatment of chromoblastomycosis of 10-year duration due to Fonsecaea nubica.

We report a case of chromoblastomycosis due to the presence of large plaque and verrucous hyperplasia lesions on the left upper limb, with elbow abnormal activities, in a 56-year-old male. The diagnosis of chromoblastomycosis was based on gross and microscopic morphologies, histopathological examination and clinical manifestation. Molecular tools were applied to identifying the causative agent Fonsecaea nubica, which is rarely reported to be associated with chromoblastomycosis. The patient was initially treated orally with terbinafine (250 mg/day) and itraconazole (200 mg/day), subsequently patient received thermotherapy (45-50°C, 3 h/day) for 1 month. The patient was successfully cured. A literature review was performed to assess general features, treatment and outcome of chromoblastomycosis due to F.  nubica. All the 5 reviewed patients were male, over 30 years old and their lesions occurred after traumatic inoculation.

High-resolution melting analysis assay for identification of Fonsecaea species.

BACKGROUND: Chromoblastomycosis (CBM) is a chronic fungal disease. In China, the principle etiologic agent was a group of dematiaceous fungi, including Fonsecaea monophora, Fonsecaea nubica, and Cladophialophora carrionii. Although the Fonsecaea species have similar morphology, their pathogenicity is quite different. This study aims to establish a new solution for early identification of Fonsecaea species because of their distinctive potential infection risk. METHODS: Five reference strains and 35 clinical isolates from patients with CBM, preserved in our laboratory, were used in this study. The universal primer ITS1 and ITS2 were chosen to amplify the highly conserved regions of rDNA. High-resolution melting (HRM) analysis was performed using the LIGHTCYCLER® 96 System. All the amplicons were verified by direct sequencing and the sequence were aligned with those in GenBank by BLAST analysis. RESULTS: We successfully differentiated the five strains according to their different Tm values and curve shapes. The 35 clinical isolates from patients were identified as 24 strains for F. monophora and 11 strains for F. nubica, which is consistent with the DNA sequencing results. CONCLUSION: It is the first time to use HRM analysis for identification of Fonsecaea species. Since the CBM etiologic agent in South China is mainly F. monophora and F. nubica, this strategy is sufficient to be applied in the clinical examination with high accuracy, speed, and throughput.