Molecular epidemiology of Fonsecaea pedrosoi using mitochondrial DNA analysis.

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) from 49 clinical Fonsecaea pedrosoi isolates (18 isolates from Japan, 17 from Madagascar, 7 from Argentina, 5 from Venezuela, 1 from Costa Rica and 1 unknown) was studied. The 49 isolates were composed of 20 isolates of Type 1, 16 of Type 2, 12 of Type 4 and 1 of a new mtDNA type, Type 7, which was closely related to Type 2. On the bases of the results of 120 isolates of the present (49 isolates) and previous (71 isolates) studies, F. pedrosoi was classified into seven mtDNA types and according to the relationship between mtDNA types and geographic origins: in Japan and probably in China, Type 1 isolates; in Zaire and Madagascar, Type 2; in Central and South America, Type 4 and Type 1. These results indicated that the geographical origin of F. pedrosoi isolate could be roughly inferred from its mtDNA type.     

Isolation and partial characterization of an adhesin from Fonsecaea pedrosoi.

We showed previously that mannose and N-acetylglucosamine (GlcNAc) residues are involved in the process of adhesion of Fonsecaea pedrosoi, the causative agent of chromoblastomycosis, to epithelial cells. It was then suggested that lectin-like molecules would be involved in the interaction. In the present study, we used fluorescein isothiocyanate-labeled neoglycoproteins (bovine serum albumin [BSA]-mannose and BSA-GlcNAc) to analyze the presence of sugar-binding proteins on the surface of conidia of F. pedrosoi grown at 28 and 37 degrees C. Binding of the neoglycoproteins was measured using flow cytometry. Fungal conidia expressed high levels of binding sites for BSA-mannose and BSA-GlcNAc when grown at 37 degrees C rather than 28 degrees C. Binding was inhibited by previous incubation of the conidia in the presence of chloroquine and trypsin. Chloroquine treatment also inhibited the interaction of fungal conidia with Chinese hamster ovary cells. Extracts from the conidia, obtained using a mechanical cell homogenizer, were purified by affinity chromatography using mannose-agarose or GlcNAc-agarose column. Polyacrylamide gel electrophoresis of the purified material from both columns showed a single protein band of 50 kDa, suggesting that the same lectin-like protein recognizes both carbohydrates.     

Exoantigen test for Cladosporium bantianum, Fonsecaea pedrosoi, and Phialophora verrucosa.

Exoantigens from 10-day-old cultures of 100 isolates of pathogenic and saprophytic dematiaceous fungi were analyzed by the exoantigen test. Antisera to Cladosporium bantianum ATCC 10958, Fonsecaea pedrosoi CDC AMO-B06, and Phialophora verrucosa CDC AMO-C12 were prepared in New Zealand rabbits immunized with soluble antigens from 1-month-old cultures. Absorbed and nonabsorbed antisera and exoantigens from the same organisms were used as reference reagents. Serologic reactions were analyzed in terms of the presence or absence of lines of identity or nonidentity. These reactions allowed presumptive differentiation of C. bantianum, F. pedrosoi, and Phialophora verrucosa from other dematiaceous fungi, including Cladosporium spp. (28 isolates), Exophiala spp. (18 isolates), Fonsecaea spp. (17 isolates). Lecythophora hoffmannii (4 isolates), Phaeoannellomyces werneckii (3 isolates), Phialophora spp. (17 isolates), Wangiella dermatitidis (9 isolates), and Rhinocladiella spp. (4 isolates).     

Highly specific and sensitive, immunoblot-detected 54 kDa antigen from Fonsecaea pedrosoi.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by a group of different dematiaceous fungi, first described by Rudolph in 1914. In Brazil there is a clear predominance of Fonsecaea pedrosoi. Sixty sera samples obtained from patients with F. pedrosoi-caused CBM were analysed. Sera obtained from 36 sporothricosis (SPT) patients, 34 cutaneous leishmaniasis (CL) patients and from 48 blood donors (HBD) were used as control. F. pedrosoi metabolic antigen was obtained from F. pedrosoi sample no. 884 (Instituto de Medicina Tropical de S?o Paulo Collection). IE reaction disclosed an anodic migrating arch, which was eluted and used as antigen. Both metabolic and eluate F. pedrosoi antigens were submitted to SDS-PAGE and two fractions, weighing approximately 54 and 66 kDa were identified. The 66-kDa fraction reacted against 43 of 60 CBM (71.7%) sera samples and was recognized by 10 SPT and eight CL sera (15.3%). No reactivity was observed against HBD sera. The 54-kDa fraction reacted against 58 of 60 CBM sera (96.7% sensitivity) and was not recognized by HBD, SPT nor CL sera (100% specificity). Such high sensitivity and specificity levels suggest this antigenic fraction is immunodominant and might prove a useful tool for further studies on F. pedrosoi-caused CBM.     

Secretory aspartyl peptidase activity from mycelia of the human fungal pathogen Fonsecaea pedrosoi: effect of HIV aspartyl proteolytic inhibitors

Fonsecaea pedrosoi is the principal causative agent of chromoblastomycosis, which is a chronic, often debilitating, suppurative and granulomatous mycosis. Very little is known about the hydrolytic enzymes produced by this human fungal pathogen. In the present study, we have identified extracellular proteolytic activity from F. pedrosoi mycelial forms when grown in chemically defined conditions. Secretory aspartyl peptidase activity was measured during 15 days of fungal growth in vitro using bovine serum albumin (BSA) as the soluble substrate and extreme acidic pH (2.0). This activity was totally inhibited by pepstatin A, a classic aspartyl peptidase inhibitor. Conversely, metallo (o-phenanthroline), cysteine (E-64) and serine (PMSF) proteolytic inhibitors failed to restrain proteolytic activity. We also evaluated the effect of four distinct HIV aspartyl peptidase inhibitors on the secretory proteolytic activity of F. pedrosoi mycelia. Indinavir, ritonavir and nelfinavir powerfully inhibited extracellular aspartyl proteolytic activity by approximately 97, 96 and 87%, respectively, whereas saquinavir did not significantly interfere with BSA hydrolysis. Mycelial-derived secretory aspartyl peptidase activity cleaved other proteinaceous substrates, including human albumin, fibrinogen, fibronectin, laminin and type I collagen. As previously reported by our group, conidia also produce secretory aspartyl peptidase. In this sense, we investigated the effect of pepstatin A on F. pedrosoi development. Pepstatin A was able to inhibit the growth of conidium and its transformation into mycelium. Taken together, our results suggest a possible participation of aspartyl peptidases in the essential fungal processes, such as growth, differentiation, nutrition and cleavage of relevant host proteinaceous components.     

Immunochemical characterisation of antigens and growth inhibition of Fonsecaea pedrosoi by species-specific IgG

Antigens of Fonsecaea pedrosoi, the most common agent of chromomycosis, were characterised by immunoprecipitation with a rabbit antiserum raised against the cell-protein extract and serum from an infected patient. Thirteen antigens were commonly detected and, as some of these antigens could be iodinated, they may be present in the fungal cell wall. Purified IgG from the rabbit antiserum was shown to produce a 50-60% inhibition of fungal growth. Some of the antigens characterised may be important in relation to the stimulation of protective immunity against chromomycosis.     

Alternate week and combination itraconazole and terbinafine therapy for chromoblastomycosis caused by Fonsecaea pedrosoi in Brazil.

Patients with long-standing chromoblastomycosis may respond poorly to standard treatments such as amphotericin B, oral antifungals, surgical measures or thermotherapy. The objective of this study was to determine the potential of alternate week and combination therapy with itraconazole and terbinafine in the treatment of poorly responsive, or non-responsive, chromoblastomycosis. Four patients with longstanding chromoblastomycosis (8-23 years) caused by Fonsecaea pedrosoi had responded poorly to standard therapies including monotherapy with the oral antifungal agents. In order to try and improve the response to oral itraconazole and terbinafine, alternate week or combination therapy with itraconazole and terbinafine was initiated. Bloodwork including complete blood count and liver function tests were performed every 3-8 weeks to ensure patient safety. Reduction or resolution of lesions of chromoblastomycosis was noted with alternate week or combination treatment using oral itraconazole and terbinafine. Three of four patients experienced no clinical side-effects; the third reported mild, transient gastric discomfort which responded to antacids. Bloodwork generally remained within normal limits throughout the entire course of treatment with no clinically significant changes. The combination therapy was considered effective in treating the poorly responsive chromoblastomycosis of all four patients. Some success with alternative week therapy was also noted in one patient. The favorable response and lack of significant adverse effects suggests that these regimens may be an option for some patients with chromoblastomycosis.     

Species identification and strain typing of Fonsecaea pedrosoi using ribosomal RNA gene internal transcribed spacer regions.

The restriction fragment length polymorphism (RFLP) in the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene (rDNA) was analyzed on Fonsecaea pedrosoi isolates kept in the Department of Dermatology, Kanazawa Medical University, Japan. On the bases of the RFLP patterns with Dde I and Msp I, 131 isolates were classified into 5 types (D1-D5) and 4 types (M1-M4), respectively. Combining the RFLP patterns with Dde I and Msp I, the isolates were further classified into 6 rDNA-types which corresponded to the 6 mtDNA-types reported by Kawasaki et al. based on the mtDNA-RFLP patterns, except for a single strain of mtDNA-type 7, which was indistinguishable from mtDNA-type 2. The strains of each rDNA-type formed a clade on the phylogenetic tree constructed from sequences of the ITS regions. ITS-RFLP analysis discriminated F. pedrosoi from 11 other species of pathogenic phaeoid fungi except F. compacta. These results strongly suggest that the typing based on ITS-RFLP is reliable and that F. pedrosoi and F. compacta are conspecific. Compared with mtDNA-RFLP analysis, ITS-RFLP analysis is less tedious, permits simultaneous analysis of many samples and gives equivalent results rapidly. This analysis is therefore useful for typing or epidemiologically investigating F. pedrosoi and for differentiating it from other dematiaceous fungi.     

Extensive chromoblastomycosis caused by Fonsecaea pedrosoi successfully treated with a combination of amphotericin B and itraconazole.

Chromoblastomycosis is a chronic infection caused by dematiaceous (dark-colored) fungi which affect the skin and subcutaneous tissues, and is characterized by a wide variety of clinical and dermatological features including papillomatous, verrucous and vegetating lesions. Although it has been described world-wide, most cases originate in tropical and sub-tropical areas. In general, present treatments of the disease are unsatisfactory as one of the most common etiologic agents, Fonsecaea pedrosoi is difficult to manage from a therapeutic point of view. We report a case of extensive chromoblastomycosis of 22 years duration caused by F. pedrosoi and review the clinical course, diagnosis and management of this disease.     

Differentiation of Fonsecaea pedrosoi mycelial forms into sclerotic cells is induced by platelet-activating factor

Platelet-activating factor (PAF) has been shown to induce the differentiation of several cell types. In this work, we evaluated the effects of PAF on the formation of sclerotic cells of Fonsecaea pedrosoi, the major causative agent of chromoblastomycosis. Cell differentiation was evaluated by light and electron microscopy, which showed that treatment of mycelial forms with PAF results in the generation of sclerotic bodies with typical morphological characteristics. Biochemical features of PAF-induced sclerotic cells were also analyzed and compared with those from sclerotic forms induced by propranolol, a previously described differentiating agent of F. pedrosoi. Chemical analyses of lipid and carbohydrate components from PAF- or propranolol-induced sclerotic bodies revealed that palmitic, stearic, oleic and linoleic acids were the major fatty acid components, while glucose, mannose, galactose and rhamnose were detected as the principal sugar constituents in these cells. Surface carbohydrate components of PAF- and propranolol-induced sclerotic cells were also evaluated, by flow cytometry analysis with twelve different lectins. The profile of reactivity of PAF- or propranolol-induced fungal cells with lectins was also very similar. Hydrolysis of the synthetic substrate p-nitrophenylphosphate by fungal cells demonstrated that the addition of PAF or propranolol to the mycelial cultures similarly promotes a significant increase in ecto-phosphatase activity. These results indicate that the differentiation of F. pedrosoi mycelial cells induced by PAF generates authentic sclerotic forms, as confirmed by the analysis of morphological and biochemical attributes. Since PAF is synthesized in normal conditions by the human host, these observations may have a correlation with the differentiation of F. pedrosoi in vivo.     

Development of natural culture media for rapid induction of Fonsecaea pedrosoi sclerotic cells in vitro

Fonsecaea pedrosoi is the main agent of chromoblastomycosis, a skin disease presenting verrucous lesions, in which round, thick-walled sclerotic cells are found. In vitro induction of sclerotic cells is time-consuming (20 to 45 days) and temperature dependent. We present two new natural media that reduce the sclerotic-cell induction time to only 2 days.     

Structure, cellular distribution, antigenicity, and biological functions of Fonsecaea pedrosoi ceramide monohexosides

Monohexosylceramides (CMHs, or cerebrosides) have been reported as membrane and cell wall constituents of both pathogenic and nonpathogenic fungi, presenting remarkable differences in their ceramide moiety compared to mammalian CMHs. Current evidence suggests that CMHs are involved in fungal differentiation and growth and contribute to host immune response. Here we describe a structural diversity between cerebrosides obtained from different forms of the human pathogen Fonsecaea pedrosoi. The major CMH species produced by conidial forms displayed the same structure previously demonstrated by our group for mycelia, an N-2'-hydroxyhexadecanoyl-1-beta-d-glucopyranosyl-9-methyl-4,8-sphingadienine. However, the major cerebroside species purified from sclerotic cells carries an additional hydroxyl group, bound to its long-chain base. The structural difference between cerebrosides from mycelial and sclerotic cells was apparently not relevant for their antigenicity, since they were both recognized at similar levels by sera from individuals with chromoblastomycosis and a monoclonal antibody to a conserved cerebroside structure. Preincubation of fungal cells with anti-CMH monoclonal antibodies had no effect on the interaction of F. pedrosoi sclerotic cells with murine macrophages. In contrast to what has been described for other fungal species, sclerotic bodies are resistant to the antifungal action of anti-CMH antibodies. Immunofluorescence analysis showed that recognition of sclerotic cells by these antibodies only occurs at cell wall regions in which melanization is not evident. Accordingly, melanin removal with alkali results in an increased reaction of fungal cells with anti-CMH antibodies. Our results indicate that cerebroside expression in F. pedrosoi cells is associated with dimorphism and melanin assembly on the fungal cell wall.     

Melanin from Fonsecaea pedrosoi induces production of human antifungal antibodies and enhances the antimicrobial efficacy of phagocytes

Fonsecaea pedrosoi is a fungal pathogen that produces melanin. The functions of melanin and its possible influence in the protective immunological response during infection by F. pedrosoi are not known. In this work, treatment of F. pedrosoi mycelia with proteases and glycosidases followed by a denaturing agent and hot concentrated acid left a black residue. Scanning electron microscopy demonstrated that this processed melanized residue resembled very closely the intact mycelium in shape and size. Melanin particles were also isolated from culture fluids of conidia or sclerotic forms of F. pedrosoi. Secreted melanins were reactive with sera from infected human patients, suggesting that F. pedrosoi synthesizes melanin in vivo. The antibodies against melanin were purified from patients' sera and analyzed by indirect immunofluorescence. They reacted with sclerotic cells from patients' lesions as well as with sclerotic bodies cultivated in vitro, conidia, mycelia, and digested residues. Treatment of F. pedrosoi with purified antibodies against melanin inhibited fungal growth in vitro. The interaction of F. pedrosoi with phagocytes in the presence of melanin resulted in higher levels of fungal internalization and destruction by host cells, which was accompanied by greater degrees of oxidative burst. Taken together, these results indicate that melanin from F. pedrosoi is an immunologically active fungal structure that activates humoral and cellular responses that could help the control of chromoblastomycosis by host defenses.     

Absence of CD4+ T cells impairs host defence of mice infected with Fonsecaea pedrosoi

Chromoblastomycosis is a human chronic, often debilitating, suppurative, granulomatus mycosis of the skin and subcutaneous tissues beginning after inoculation trauma. It occurs worldwide, but is more frequently observed in tropical countries such as Brazil. Some studies have focused on fungus-host interaction, showing a predominantly cell-mediated immune response, with the activation of macrophages involved in fungus phagocytosis. Immunization with live conidia produced a high influx of CD4 T cells into the draining lymph node. The sensitized T cells proliferate in vitro when restimulated with specific antigen and preferentially produce IFN- gamma. To better characterize the role played by T cells on the chromoblastomycosis infection we used mice deficient for CD4 and CD8. Data determined by CFU counts associated with decreased DTH and IFN-gamma production of infected mice clearly demonstrated that, during experimental F. pedrosoi infection, absence of CD4(+) cells induces a more severe disease.     

Association of IgG immunoglobulin and subclasses level with the severity of chromoblastomycosis due to Fonsecaea pedrosoi and therapeutic response to itraconazole

Chromoblastomycosis (CBM) is a chronic, suppurative, granulomatous mycosis of the skin and subcutaneous tissues. The aim of this study was to evaluate the association between IgG antibody levels and the severity of CBM and therapeutic response of patients to itraconazole. A longitudinal study was conducted in patients with CBM due to Fonsecaea pedrosoi and in healthy subjects with chromomycin skin test (CST)+. The dosage of anti-F. pedrosoi IgG antibody performed in 47 healthy individuals with CST+ showed positivity in 97.5 %, with an average titer of 2,109 [standard deviation (SD)?+?3,676)] and a mean optical density (OD) of 1.174 (SD?+?0.456), showing positive correlation with the induration area of the CST (mm²). The level of antibodies in 55 patients with CBM expressed in OD and titration showed that, before treatment, patients with severe disease had higher levels of IgG, IgG1, IgG2, and IgG3 when compared with moderate or mild disease (p?IgG, IgG1, and IgG2 were reduced after treatment (p?IgG titers in patients with rapid response (p?p?F. pedrosoi in the daily lives of these subjects, with prospects of preventive measures for the target population. The immunological analysis shows that the antibody anti-F. pedrosoi did not exhibit a protective role against infection caused by this agent.     

Listeria cell wall fraction. Characterization of in vitro adjuvant activity.

We have previously described a crude cell wall fraction of Listeria monocytogenes (LCWF) which induces resistance to listeria infection in mice, is a murine B-cell mitogen and is an immunological adjuvant. Data reported here show that LCWF, which is effective over a wide dose range, exerts its adjuvant action on early events in the induction of an immune response. Moreover, LCWF stimulates nonadherent cells to respond to sheep red cells when adherent cells are severely depleted or absent, suggesting that LCWF can therefore act directly on lymphocytes present within the non-adherent population.     

Fonsecaea pedrosoi infection induces differential modulation of costimulatory molecules and cytokines in monocytes from patients with severe and mild forms of chromoblastomycosis

The host defense mechanism in chromoblastomycosis has not been thoroughly investigated. It has been suggested that cell-mediated immunity in patients with long-standing chromoblastomycosis is somehow impaired. As a result, these individuals became unable to develop an efficient immune reaction. Many studies have shown that monocyte-derived macrophages exhibit critical activities in immunity to microorganisms. Moreover, the ability of cells from the monocytic lineage to process and present antigens, to produce cytokines, and to provide costimulatory signals confirms their pivotal role in the initiation of specific immune responses. In the present study, it was observed that monocytes from patients with a severe form of disease had a higher production of IL-10 and a lower expression of HLA-DR and costimulatory molecules when stimulated with specific antigen or LPS. Immune modulation with recombinant IL-12 or anti-IL-10 can restore the antigen-specific Th1-type immune response in chromoblastomycosis patients by up-regulating HLA-DR and costimulatory molecules in monocytes. Therefore, our data show that monocytes from patients with different clinical forms of chromoblastomycosis present distinct phenotypic and functional profiles. This observation suggests possible mechanisms that control the T cell response and influence their role in the development of pathology.