Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis.

We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis.     

Exocellular components of Paracoccidioides brasiliensis: identification of a specific antigen.

Yeast forms of Paracoccidioides brasiliensis grown in liquid medium produced exocellular components. Immunodiffusion reactions and immunoprecipitations of 131I-radiolabeled antigenic components with sera from patients having paracoccidioidomycosis (PCM) were used to monitor the isolation of specific constituents. Components having the main antigenic activity (fCon A) were isolated by exclusion from a Bio-Gel P30 column, followed by successive binding of eluted material to a Sepharose-concanavalin A column, and elution. The product contained, from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, a minor 43,000-molecular-weight (MW) component (gp43), a polydisperse high-MW glycoconjugate, and a diffusely migrating 55,000-MW glycoprotein (gp55). Other components, including a 72,000-MW glycoprotein, were irregularly expressed. The high-MW glycoconjugate complex contained, on the basis of methylation and 13C nuclear magnetic resonance data, a branched structure of mainly mannopyranosyl units. These were nonreducing ends, 6-O-, 2-O-, and 2,6-di-O-substituted, and the specific rotation of +16 degrees indicated that the glycosidic configurations of the units were alpha and beta in a ratio of ca. 1:1 (concanavalin A binding indicated that nonreducing ends or 2-O-substituted units or both of alpha-D-mannopyranose were present). A small proportion of nonreducing end units of D-galactopyranose were also present in this polysaccharide. gp55 is a glycoprotein containing a complex carbohydrate moiety with fucose, mannose, galactose, and glucose, either as terminal nonreducing units or substituted in positions indicated by methylation data. Both PCM and normal human sera precipitated the high-MW glycoconjugate from 131I-labeled fCon A preparations, whereas gp55 was unreactive with human sera. gp43 was a specific antigenic component of P. brasiliensis culture filtrates which could be isolated in a pure form by gel filtration column chromatography (Sephadex G150) or by Sepharose-patient immunoglobulin G affinity chromatography. 131I-labeled gp43 reacted equally well with 10 PCM sera and hyperimmune rabbit serum against the band E antigen of Yarzabal at a 10(-3) dilution. At the same dilution, no reaction was detected with sera from normal individuals and from patients with other mycoses. Similarly, only PCM sera and the hyperimmune anti-E serum gave precipitin lines with gp43 in the less sensitive immunodiffusion tests. gp43 consisted of three components, with pI 6.7, 6.4 and 6.2, all of which reacted with PCM serum.     

Polymorphism in the gene coding for the immunodominant antigen gp43 from the pathogenic fungus Paracoccidioides brasiliensis

The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM). It is protective against murine PCM and is a putative virulence factor. The gp43 gene of Paracoccidioides brasiliensis B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp. Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points. In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17 P. brasiliensis isolates after sequencing two PCR products from each fungal sample. The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp. Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein. The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms. The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots. The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine.     

Isolation and purification of two immunodominant antigens from Nocardia brasiliensis.

Two immunogenic proteins from a crude extract of Nocardia brasiliensis were purified to homogeneity. A 61-kDa protein (P61) was isolated from a 50% ammonium sulfate precipitate in two steps. Initially, P61 was obtained by electroelution in a 10% nondenatured preparative polyacrylamide gel electrophoresis (PAGE). In a second step, the eluate from the nondenatured gel was run in a 12% sodium dodecyl sulfate (SDS) preparative polyacrylamide gel. After elution, a single band was demonstrated by SDS-PAGE and Western blot (immunoblot). Also, a 24-kDa immunogenic protein (P24) was isolated by gel filtration in a Sephadex G-100 column and then by electroelution in a 12% nondenatured polyacrylamide gel. In a previous paper, we showed by Western blot assays that these proteins are recognized by the sera of mycetoma patients and not by sera from mycobacterial-infected or healthy individuals. We consider these proteins to be good candidates for the study of the host-parasite relationship in nocardial infections. The possible clinical application of these purified antigens in a serological diagnosis is discussed.     

Identification of a 33-kilodalton immunodominant antigen of Trypanosoma congolense as a cysteine protease.

A 33-kDa protein of Trypanosoma congolense is a major antigen in infected cattle and the production of antibody to this antigen appeared to correlate with enhanced resistance to trypanosomiasis [4]. Immunoelectron microscopy using a monoclonal antibody (mAb 4C5) raised against the 33-kDa antigen showed a lysosomal localisation, similar to that of a previously described 32-kDa cysteine protease of T. congolense. Both mAb 4C5 and anti-33 kDa antibody from infected cattle bound on Western blots to the cysteine protease that had been purified by affinity chromatography on cystatin-Sepharose. Sepharose-coupled mAb 4C5 was used to affinity purify the antigen from bloodstream forms of T. congolense. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the affinity-purified antigen had a molecular mass of 33 kDa under non-reducing conditions, and 40 kDa under reducing conditions. Anti-33-kDa antibody from infected cattle bound to both non-reduced and reduced affinity-purified antigen on Western blots. Serum from a rabbit immunised with the biochemically purified enzyme also bound the affinity-purified antigen. The affinity-purified antigen displayed proteolytic activity in fibrinogen-containing SDS-PAGE and against Azocoll. It hydrolysed benzyloxycarbonyl-Phe-Arg-7-amino-methyl coumarin (Z-Phe-Arg-NHMec) with a Km similar to that of the biochemically purified enzyme. Proteolytic and peptidolytic activities of the antigen were inhibited by the inhibitors of cysteine proteases, cystatin and trans-epoxysuccinyl-L-leucyl-amido (4-guanidino)butane (E-64). On two-dimensional gel electrophoresis, the antigen displayed similar characteristics to those of the biochemically purified enzyme. We conclude that the 33-kDa antigen of T. congolense and the cysteine protease are the same molecule.     

An immunodominant 90-kilodalton Aspergillus fumigatus antigen is the subunit of a catalase.

We have identified, purified, and characterized structurally and functionally a 90-kDa immunodominant antigen associated with the water-soluble fraction of Aspergillus fumigatus. This antigen is recognized by 90.3% of serum samples from patients with aspergilloma and should be considered either by itself or better in combination with other purified antigens as a candidate for developing a standardized immunoassay for the detection of aspergilloma. p90 is a glycoprotein containing at least two two N-linked sugar chains of 2 and 5 kDa, respectively, which are not necessary for its reactivity with aspergilloma serum samples. Using specific anti-p90 rabbit serum, we have demonstrated that under native conditions, p90 exists in oligomeric form and has associated catalase activity. This activity is resistant to extreme temperatures (> 60 degrees C), reducing agents (40 mM dithiothreitol), high concentrations of denaturing agents such as 8 M urea and 8% sodium dodecyl sulfate, and treatments with ethanol-chloroform-water (5:3:10 [vol/vol]) mixtures.     

Induction of antigen-specific T suppressor cells by soluble Paracoccidioides brasiliensis antigen.

In naturally acquired paracoccidioidomycosis, patients have depressed in vivo and in vitro cell-mediated immune (CMI) responses to Paracoccidioides brasiliensis antigen. In addition, it has been reported that these patients have significant levels of circulating paracoccidioidal antigen in their sera. The primary purpose of this investigation was to assess the effects of P. brasiliensis antigen on the CMI responses in a mouse model. On the basis of findings with other fungal agents, we predicted that circulating paracoccidioidal antigen may be inducing suppressor cells which modulate the CMI response. In this study, we show (i) that a soluble P. brasiliensis culture filtrate antigen (Pb.Ag) emulsified in complete Freund adjuvant and injected subcutaneously into mice induces reasonably high levels of delayed-type hypersensitivity (DTH) in CBA/J mice; (ii) that Pb.Ag elicits DTH reactions specific for P. brasiliensis when injected into footpads of immunized mice; and (iii) that an intravenous injection of Pb.Ag induces a population of lymph node and spleen cells which, upon adoptive transfer, suppress the afferent limb of the DTH response to paracoccidioidal antigen. The afferent suppressor cells can be detected in spleens as early as 5 days after Pb.Ag treatment, are present in significant numbers by 7 days in both spleens and lymph nodes, and are virtually absent by 14 days. In contrast, at 14 days after antigen injection, efferent suppressor cells were detected in spleens and lymph nodes. The Pb.Ag-induced afferent suppressor cells specifically inhibit the antiparacoccidioidal DTH response. They are nylon wool-nonadherent cells, and their activity is abrogated by anti-Thy-1 and complement treatment, indicating that they are T lymphocytes. The phenotype of these afferent suppressor T cells is L3T4+ Lyt-1+2- I-J+. The Pb.Ag-specific suppressor cells described in this paper are similar to the Ts1 cells in the azobenzenearsonate, 4-hydroxy-3-nitrophenyl acetyl, and cryptococcal models of suppression of the DTH response and to the afferent suppressor cells in the dinitrofluorobenzene contact sensitivity system.     

Rapid and reliable method for production of a specific Paracoccidioides brasiliensis immunodiffusion test antigen

Previously published methods to produce Paracoccidioides brasiliensis antigens for serological tests have yielded antigens of inconsistent quality and have involved the use of special semisynthetic media and growth periods of 1 to 3 months to yield suitable reagents. A simple procedure that uses commercially available potato glucose agar and either SABHI broth (Difco Laboratories) or Trypticase soy broth (BBL Microbiology Systems) inoculated with the mycelial form of P. brasiliensis consistently yielded high-titer antigens in 2 weeks or less. This new method permits the almost exclusive production of an antigen identical to the specific E antigen described by Yarzabal (Yarzabal et al., Sabouradia 14:275-280, 1976) and the apparently equivalent specific antigen 1 described by Restrepo and Moncada (A. Restrepo and L. H. Moncada, Appl. Microbiol. 28:138-144, 1974). In the immunodiffusion test, the rapidly produced antigen demonstrated a sensitivity of 90% by detecting antibody in sera from 103 of 114 proven cases of paracoccidioidomycosis. The specificity of this antigen was 100% because none of 139 sera from patients with heterologous mycotic diseases demonstrated diagnostic precipitins against the P. brasiliensis antigen. In the complement fixation tests, the rapidly produced antigen was not as suitable as the one prepared by the method of Restrepo-Moreno and Schneidau (A. Restrepo-Moreno and J. D. Schneidau, Jr., J. Bacteriol. 93:1741-1748, 1967).     

Antibody isotypes to a Paracoccidioides brasiliensis somatic antigen in sub-acute and chronic form paracoccidioidomycosis

This report describes the differences in isotype antibody reactivity against a crude Paracoccidioides brasiliensis antigenic preparation in the sub-acute (SAF) and chronic (CF) forms of paracoccidioidomycosis before treatment. IgG antibodies were detected in all patients, with a slightly but not significantly higher reactivity in the SAF. IgG1 antibodies were present, frequently at high levels, in both forms, whereas IgG3 was always low or absent. IgG2 antibodies were detectable in most patients, but at high levels in only a few CF patients. IgG4 was found mainly in SAF patients, whereas IgA was detected almost only in CF patients, probably due to a Th2 pattern of immune response in the more severe SAF, and the characteristic mucosal involvement of the CF, respectively. Immunoblot analysis showed that, in addition to the 43-kDa immunodominant fraction, other less well-characterised fractions were also recognised differentially by the isotypes and deserve further investigation.     

Paracoccidioides brasiliensis 87-kilodalton antigen, a heat shock protein useful in diagnosis: characterization, purification, and detection in biopsy material via immunohistochemistry

The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with P. brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.     

The 93-kilodalton protein of Borrelia burgdorferi: an immunodominant protoplasmic cylinder antigen.

Using immunoblots, we identified proteins of Borrelia burgdorferi recognized by sera from 62 patients with either acute or chronic Lyme disease. In all groups studied, the 41-kDa flagellar protein and a relatively minor 93-kDa protein (p93) were the most commonly recognized antigens in patients with acute and chronic disease due to B. burgdorferi. A murine monoclonal antibody (MAb 181.1) was developed against p93, and the antigen was detected by immunoblot analysis in four European and American strains of B. burgdorferi. On two-dimensional gel electrophoresis, p93 had an apparent pI of 6.8. Immunoelectronmicroscopy with MAb 181.1 demonstrated that p93 is located within the protoplasmic cylinder compartment of the organism. The gene encoding p93 was retrieved from a phage expression library. The derived amino acid sequence of p93 confirmed chemical characterization of the antigen, including its amino-terminal peptide sequence. The derived amino acid sequence predicted it to be predominantly alpha helical. A prominent antigenic domain located at the carboxy portion of the protein was recognized by human and rabbit polyclonal antisera and human (MAb D4) and mouse (MAb 181.1) MAbs.     

Molecular detection and identification of Paracoccidioides brasiliensis.

Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi.     

Identification of a Mycobacterium bovis BCG 45/47-kilodalton antigen complex, an immunodominant target for antibody response after immunization with living bacteria.

Increased protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by a previous immunization with living avirulent mycobacteria, usually Mycobacterium bovis BCG. Only a transient and marginal protection is obtained after immunization with bacterial extracts or dead bacteria. Both living and heat-killed bacteria share a number of common antigens. In order to identify mycobacterial molecules which are dominant antigens during immunization with living bacteria, a two-step selection method was used. Two groups of guinea pigs were immunized either with living or with heat-killed BCG. Sera were then collected and used to select and counterselect antigens present in BCG culture filtrates. Each major fraction eluted from a series of high-pressure liquid chromatography columns (gel filtration, DEAE, and reverse-phase chromatography) was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on polyvinylidene difluoride sheets. The molecules present on twin immunoblots were stained with antibodies raised in guinea pigs immunized either with living or with heat-killed BCG. Cross-reactive antigens stained in twin immunoblots were eliminated. Major antigens interacting with antibodies raised after immunization only with living bacteria were further purified. A complex of 45- and 47-kDa major molecules (45/47-kDa complex) was thus identified and further purified. The complex was found to interact only with antibodies present in sera of guinea pigs immunized with living bacteria and not at all with antibodies raised after immunization with dead bacteria. The 45/47-kDa antigen complex molecules were resolved on two-dimensional electrophoresis in three major and seven minor proteins detected with silver staining. All the molecules interacted with the antibodies present in sera of guinea pigs immunized with living BCG. The three major proteins (two at 47 kDa and one at 45 kDa) were amino-terminal sequenced. The sequence A-P-E-P-A-P-P-V-P-P-A-A-A-A-P-P-A, which was not previously reported, was the same for these three molecules. By using a competitive enzyme-linked immunosorbent assay, the concentrations of the 45/47-kDa antigen complex were measured in BCG culture filtrates, freeze-dried BCG, and dried heat-killed BCG; they were, respectively, 2, 0.01, and 0.001% of the total mass. The low or very low values compared with the high antibody concentration emphasized the ability of the 45/47-kDa complex delivered through live BCG to trigger an antibody response.     

Random sequencing of Paracoccidioides brasiliensis genes.

Paracoccidioides brasiliensis genome has been reported as having a size of about 30 Mb. By digestion of genomic DNA from strain IVICPb 73 (ATCC 32071), we have constructed a DNA library with an insert size average of 8 kb in Escherichia coli XL1 Blue. We have fully sequenced 7 clones comprising 51,022 bp which represent 20 putative protein-coding sequences (seven of them, partial) and one tRNA. The 20 coding sequences cover 46% of the total 51,022 bp with introns present in 10 out of the 20 sequences. Database similarity analysis reveals the presence of genes conserved in other fungal species and higher organisms, including humans.     

Agrobacterium tumefaciens-mediated transformation of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, an important mycosis endemic to Latin America. As the tools to study gene function in P. brasiliensis are only in the early stage of development, there is presently no system that allows for both the delivery and integration of exogenous nucleic acids into its genome. We report in this paper the transformation of the yeast phase of P. brasiliensis (ATCC-60855) with Agrobacterium tumefaciens (GV3101) carrying the vector pAD1625. The microorganisms were co-cultivated for 2 days and then incubated for 10 days at 35 degrees C on selective media. PCR and dot-blot targeted at a fragment of 222 bp from the hph (hygromycin phosphotransferase) gene which confers Hygr confirmed the transformation of P. brasiliensis.     

Combined use of Paracoccidioides brasiliensis recombinant 27-kilodalton and purified 87-kilodalton antigens in an enzyme-linked immunosorbent assay for serodiagnosis of paracoccidioidomycosis

The diagnosis of paracoccidioidomycosis (PCM) has relied on the identification of the host's humoral response by using a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable and may yield optimum results. Accordingly an indirect enzyme-linked immunosorbent assay (ELISA) using combinations of the previously described 27-kDa recombinant antigen and the 87-kDa heat shock protein were used for diagnosis and follow-up of patients with PCM. A total of 37 patients classified according to their clinical presentations (7 with the acute or subacute form of the disease, 22 with the chronic form of the disease, and 8 with the chronic unifocal form) were studied. Eighteen of these patients were also evaluated at every follow-up appointment. Forty serum samples from patients with other diseases and 50 serum samples from healthy individuals were also studied. Detection of anti-27-kDa and anti-87-kDa antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 92% with a specificity of 88% in comparison with normal human sera and 90% in comparison with the heterologous sera. These results demonstrated a significant increase in sensitivity and specificity compared to results when the antigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer clear advantages over the use of single antigens when diagnosing PCM.     

The role of chlamydospores of Paracoccidioides brasiliensis.

The role of chlamydospores in the conversion process from a mycelial-to-yeast form using the slide culture method was studied. Three clinical isolates and two other isolates from armadillo, belonging to the fungal species Paracoccidioides brasiliensis, were cultured on Sabouraud dextrose agar (SDA), potato dextrose agar (PDA) and brain heart infusion dextrose agar (BHIDA). Initially, the mycelial forms of each isolate were grown at 25 degrees C for 7, 14, 30 or 60 days on slide cultures and then the temperature was shifted to 35 degrees C. Interestingly, the slide cultures of all the isolates at 25 degrees C formed chlamydospores on either SDA or BHIDA, whereas, on PDA medium, aleurioconidia were formed. If the slide cultures on BHIDA were incubated at 35 degrees C for 7 to 14 days, multiple budding forms could be observed. This phenomenon was not evident in the slide cultures of SDA or PDA. The results of this morphological study indicate that in P. brasiliensis, chlamydospores may play an important role in the conversion process from a mycelial-to-yeast form.     

Diagnosis of systemic candidiasis by an enzyme-linked dot immunobinding assay for a circulating immunodominant 47-kilodalton antigen.

A dot immunobinding assay based on the detection of the immunodominant 47-kilodalton (kDa) antigen of Candida albicans is described for the serological diagnosis of systemic candidiasis. It was compared with a reverse passive latex agglutination test and a dot immunobinding assay with total unfractionated hyperimmune serum to C. albicans. Use of the 47-kDa antigen-specific probe increased both the sensitivity and specificity of the assay system. Patients with systemic candidiasis were detected earlier in the course of the infection. The rate of detection of systemic C. albicans infections in neutropenic patients was 77% compared with 55% with total antibody in the dot immunobinding assay and 29% with the latex test. All three assay systems were positive in over 73% of infected patients who were not neutropenic. The 47-kDa antigen-specific probe was relatively specific to C. albicans. Antibody probes to the immunodominant antigens of other yeasts might be incorporated in the same dot immunobinding assay to detect systemic candidiasis caused by other species of yeasts.     

Immunity and hypersensitivity to gp43 antigen in susceptible and resistant mice infected with Paracoccidioides brasiliensis.

Pathogenic mechanisms underlying paracoccidioidomycosis are still poorly understood. A well-established murine model of resistance (mouse lineage A/Sn) and susceptibility (lineage B10.A) to P. brasiliensis pulmonary infection was here employed to compare immune response to gp43, the major antigenic component of the fungus. Mice were infected and their cellular and humoral immunity to gp43 were investigated for up to 16 weeks. In both mouse strains, challenge with gp43 indistinguishably evoked a typical immediate-hypersensitivity response, followed by a 24-h late-phase reaction consistent with the same type of immunological activation. IL-4 was detected in cultures of gp43-stimulated lymph node cells only in susceptible animals 2 weeks post-infection, while IL-5 was found throughout the study in both mouse strains. IL-10 appeared in the supernatants of stimulated cells from resistant and susceptible animals in increasing amounts as infection advanced. Conversely, interferon (IFN)-gamma was produced under gp43 stimulation only by cells from A/Sn animals. The humoral response was characterized by low levels of anti-gp43. Titration of IgG isotypes, however, revealed a predominance of IgG1. IgG2a levels were highest in resistant animals, whereas IgG2b levels were highest in susceptible mice. In conclusion, immunity induced by gp43 exhibits common features in A/Sn and B10.A phenotypes, such as immediate hypersensitivity, late phase reaction and high levels of IL-10, but some differences between the strains are also seen.     

Electrophoretic karyotype of environmental isolates of Paracoccidioides brasiliensis.

Five of the 12 environmental isolates of the dimorphic fungus Paracoccidioides brasiliensis known to date, were analysed by contour-clamped homogeneous electric field gel electrophoresis (CHEF). The electrophoretic pattern was shown to consist of five bands, with molecular size ranging from 3.2 to 10 Mb, a model quite similar to the one found in the clinical isolates previously tested and used here as controls. However, one of the bands in the environmental isolates had a lesser weight (7.2 Mb), than the one corresponding to the clinical counterparts (8.8 Mb). This resulted in a smaller genome, approximately 29.7 Mb. The small differences that were found indicate the presence of chromosome polymorphism in this fungus.