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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.

The fungal pathogen Penicillium marneffei produces melanin-like pigment in vitro. The synthetic pathway of melanin and its possible influence in the protective yeast cells surviving within macrophage cells are not known. In this work, P. marneffei produced brown black pigment in the presence of L-DOPA and black particles were extracted from yeast cells treated with proteolytic enzymes, denaturant and concentrated hot acid. Kojic acid inhibited the brown-black pigment production of P. marneffei yeast grown on brain heart infusion agar. Transmitting electron microscopy showed spherical granular electron-dense particles with an average diameter of 100 nm in a beaded arrangement in the innermost cell wall. Electron-paramagnetic resonance revealed that the black particles contain a stable free radical compound. The UV-visible and Fourier transform infrared spectra of particles extracted from P. marneffei and synthetic DOPA-melanin showed a high degree of similarity. Melanized yeast cells decreased phagocytosis by macrophage cells and increased resistance to intracellular digestion in vitro. These results indicate that P. marneffei can synthesize DOPA-melanin or melanin-like compounds in vitro and suggest that the DOPA-melanin pathway is associated with cell wall structure and enhances the resistance to phagocytosis by macrophages.

Global distribution patterns of Cladophialophora carrionii, agent of human chromoblastomycosis in arid climates of Africa, Asia, Australia, Central-and South-America, were compared with similar data of the vicarious Fonsecaea spp., agents of the disease in tropical rain forests. Population diversities among 73 C. carrionii strains and 60 strains of three Fonsecaea species were analyzed for rDNA ITS, partial β-tubulin, and amplified fragment-length polymorphism (AFLP) fingerprints. Populations differed significantly between continents. Lowest haplotype diversity was found in South American populations, while African strains were the most diverse. Gene flow was noted between the African population and all other continents. The general pattern of Fonsecaea agents of chromoblastomycosis differed significantly from that of C. carrionii and revealed deeper divergence among three differentiated species with smaller numbers of haplotypes, indicating a longer evolutionary history.

Chromoblastomycosis is a chronic cutaneous and subcutaneous mycosis. The management of this infection continues to be challenging because there is no consensus on the therapeutic regimen. We report here a case of a 69-year-old male patient with cauliflower-like lesions on his left leg and foot. He had already been treated with itraconazole at a dose of 200 mg/day for 5 months, with mycological cure for all the affected areas. However, the lesions relapsed at both sites, and treatment with itraconazole was resumed at the dose previously used. Initially, direct mycological examination, cultural, and microculture slide observation were performed. Afterward, sequencing of the ITS1-5.8S rDNA-ITS2 region of the fungal DNA and evaluation of its susceptibility to antifungal agents alone and in combination were performed. In direct mycological examination, the presence of sclerotic cells was verified, and the fungus was identified as Fonsecaea based on cultural and microscopic examinations. Identification as Fonsecaea monophora was confirmed after sequencing of the ITS region and phylogenetic analysis. The isolate was susceptible to itraconazole and terbinafine. The combinations of amphotericin B and terbinafine and terbinafine and voriconazole were synergistic. The use of drugs for which the causative agent is susceptible to singly or in combination may be an alternative for the treatment of mycosis. Furthermore, the identification of the agent by molecular techniques is important for epidemiological purposes. To the best of our knowledge, this is the first case of relapsed chromoblastomycosis caused by F. monophora in Brazil.

Fonsecaea pedrosoi is the principal etiologic agent of chromoblastomycosis, a fungal disease whose pathogenic events are poorly understood. Treatment of the disease presents poor effectiveness and serious side effects. The disease is epidemiologically important in several regions, which has stimulated studies focused on the biology and pathogenic potential of its major causative agent. In this review, we summarize the current knowledge on the biological aspects of F. pedrosoi, including cell differentiation and pathogenic mechanisms during the interaction of fungi with different hosts' elements.

We report two cases of chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in otherwise healthy Cuban males. Direct microscopic examination of biopsies revealed muriform cells, the hallmark of chromoblastomycosis. The suspected agents were recovered in culture, identified on the basis of morphological criteria and confirmed by sequencing of the internal transcribed spacer regions of rDNA. Final treatment consisted of surgical excision. The patients were successfully cured since there was no relapse after a follow-up of more than a year. In vitro antifungal susceptibility testing of both isolates showed that itraconazole and posaconazole had potent activity. High MICs of amphotericin B (2 μg/ml), fluconazole (>64 μg/ml), anidulafungin (8 μg/ml) and caspofungin (8 μg/ml) were found.

Knowledge of natural ecology is essential for a better understanding of pathogenicity and opportunism in black yeast-like fungi. Although etiological agents of diseases caused by these fungi are supposed to originate from the environment, their isolation from nature is difficult. This is probably due to their oligotrophic nature, low competitive ability, and, overall, insufficient data on their natural habitat. We obtained environmental samples from mangrove areas where mortalities by lethargic crab disease (LCD) are reported and areas without disease recorded. Isolation of chaetothyrialean black yeasts and relatives was performed using a highly selective protocol. Species-specific primers were used to determine if these isolates represented Exophiala cancerae or Fonsecaea brasiliensis , two proven agents of LCD, in order to test hypotheses about the origin of the disease. Isolates, identified by morphology as Fonsecaea - or Exophiala -like, were tested specific diagnostic markers for the fungi associated with LCD. Although several black fungi were isolated, the main causative agent of the LCD, E. cancerae , was not found. Molecular markers for F. brasiliensis revealed 10 positive bands for isolates from biofilms on mangrove leaves, branches, and aerial roots, of which four were confirmed by ITS sequencing. The absence of E. cancerae in environmental samples suggests that the species is dependent on the crab, as a genuine pathogen, different from F. brasiliensis , which is probably not dependent on the host species, U. cordatus . However, we did not attempt isolation from the marine water, which may represent the pathway of dispersion of the black yeast species between neighbor mangroves.

Chromoblastomycosis is a chronic infectious disease of the skin and subcutaneous tissue. However, the host-defence response to this fungal infection has not been investigated thoroughly. This study was carried out to analyse the sequential events and the change of local cytokine release in a murine model infected with Fonsecaea pedrosoi in footpad. The anti-inflammatory Th2 cytokine IL-10 demonstrated an upward trend up to 7 days post infection followed by a steady decline. The titers of TNF-α (a pro-inflammatory Th1 cytokine) increased up to 7 days post infection followed by a relatively steady-state until full recovery. The anti-inflammatory cytokine IL-4 showed a similar pattern as TNF-α. The pro-inflammatory cytokine IFN-γ did not increased until 7 days post infection, while demonstrated an upward trend up to 30 days when the mice reached a full recovery from infection.

Chromoblastomycosis is a chronic and progressive deep mycosis that is usually found in tropical and subtropical areas. Fonsecaea pedrosoi is considered its most frequent etiologic agent and causes a typical granulomatous inflammatory response, whose degree reflects the immune status of the host. Since macrophages play a fundamental role in the control of the infection, this study aimed at investigating the production of oxygen reactive specimens, the phagocytic capacity and the production of nitric oxide (NO) by macrophages employing in vitro assays and an in vivo model of chromoblastomycosis. Our results demonstrated that, during the infection, peritoneal macrophages show an increased phagocytic capacity and H2O2 production, but also a reduced ability to produce NO. Moreover, F. pedrosoi stimulated H2O2 production in vitro but not the synthesis of NO. The incubation of IFNgamma and LPS-stimulated macrophages with melanin, obtained from the fungus, inhibited NO production. Examination of the liver and spleen of infected animals, at day 30 or 60 following inoculation, showed a progressive increase in the number and size of granulomas, indicating that macrophages are properly mobilized and activated. Our data suggest that the inability of the host to clear F. pedrosoi, leading to a chronic disease, is due, at least in part, to the inhibition of NO synthesis by macrophages by fungus-produced melanin.

The cell divisions cycle 42 ( Cdc42 ) gene has been characterized in the fungi, such as Candida albicans , Penicillium marneffei , and Wangiella ( Exophiala ) dermatitidis , which plays important roles during growth and development. The partial cDNA sequence of Cdc42 of Fonsecaea monophora ( F. monophora ), designated FmCdc42 , was obtained using degenerate primers based on the conserved domain of the other fungi Cdc42 . Then the complete cDNA sequence of FmCdc42 was obtained by 5′ and 3′ RACE. The full-length cDNA is 1,510 bp in size which had an open reading frame (ORF) of 582 bp, encoding 193 amino acid residues. The predicted molecular mass of FmCdc42 is 21.5 kDa with an estimated theoretical isoelectric point of 5.67. The deduced amino acid sequence of FmCdc42 shows 99% identity to that of Wangiella ( Exophiala ) dermatitidis. 5 exons and 4 introns are identified within the 1,617 bp FmCdc42 genomic DNA sequence of F. monophora . The ORF could be subcloned into the pCDNA6/myc-His B expression vector. The recombinant protein about 27.5 kD infusion protein had high expression level in Vero cells with SDS-PAGE and Western blot analysis. Quantitative real time RT-PCR revealed that FmCdc42 was the highest expression in the sclerotic bodies’ stage compared with that in the mycelia and conidia stages, which indicated that the FmCdc42 may be involved in formation of F. monophora sclerotic bodies.