Combined use of Paracoccidioides brasiliensis recombinant rPb27 and rPb40 antigens in an enzyme-linked immunosorbent assay for immunodiagnosis of paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it's usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by 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 good results. In the present study combinations of the previously described 27-kDa recombinant antigen (rPb27) and a recombinant 40-kDa-molecular-mass antigen (rPb40) from this fungus, that was identified by Goes et al. (2005) through the AST strategy as a homolog of Neurospora crassa calcineurin B, were used in an indirect enzyme linked immunosorbent assay (ELISA) for diagnosis and follow-up of patients with PCM. The complete coding cDNA of rPb40 and rPb27 were cloned into a pET-21a and a pET-DEST 42 plasmid, respectively, expressed in E. coli with a his-tag and purified by affinity chromatography. Among 109 PCM serum samples analyzed, a homogeneous IgG response to these proteins was observed. 62 serum samples from patients with other diseases, 18 from patients with other mycosis and 23 from healthy individuals were also studied. Detection of anti-rPb27 and anti-rPb40 antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 96% with a specificity of 100% in relation to control normal human sera and to sera from patients with other systemic mycosis and 93.5% to sera from patients with diverse infections. The use of this two proteins combination provided an excellent immunodiagnosis assay with great values of sensitivity and specificity, even in relation to sera from patients with other mycosis, making possible the standardization of a new methodology to diagnose this important mycosis, with a good confiability and reprodutibility.     

Immunological response to cell-free antigens of Paracoccidioides brasiliensis: relationship with clinical forms of paracoccidioidomycosis.

Sera from patients with the acute (AF) and chronic (CF) forms of paracoccidioidomycosis (PCM) were tested against Paracoccidioides brasiliensis cell-free antigens by Western blot (immunoblot). The CFA preparation contained components ranging in molecular mass from 18 to 102 kDa. The immunoglobulin G (IgG) reactivity profiles were similar for patients with both forms of the disease, and the 43-kDa component was recognized by 100% of the sera. IgM antibodies from the AF- and the CF-PCM sera recognized 21 and 20 components, respectively, the AF-PCM sera reacting preferentially with components with molecular masses above 50 kDa. None of the AF-PCM sera (IgM) reacted with the 43-kDa component, and only 10% of the CF-PCM sera recognized this molecule. The IgA response was more significant in the CF-PCM group than in the AF-PCM group, and the 43- and 74-kDa components were the most reactive ones (about 40% each). Our results showed that the cell-free antigen preparation is very appropriate for the immunoblotting analysis of PCM sera, and they also showed that the detection of IgG anti-gp43 is the best marker for the diagnosis and the following up of patients with the acute or the chronic form of the disease.     

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.     

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.     

Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to extracellular matrix proteins

Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, beta-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.     

Estrogens inhibit mycelium-to-yeast transformation in the fungus Paracoccidioides brasiliensis: implications for resistance of females to paracoccidioidomycosis

Evidence that disease due to the thermally dimorphic fungus Paracoccidioides brasiliensis occurs post-puberty predominantly in males led us to hypothesize that hormonal factors critically affect its pathogenesis. We show here that estrogens inhibit mycelial- to yeast-form transformation of P. brasiliensis in vitro. Transformation of three isolates was inhibited to 71, 33, and 19% of the control values in the presence of 10(-10), 10(-8), and 10(-6) M 17 beta-estradiol, respectively. The synthetic estrogen diethylstilbestrol was active but less potent than estradiol, whereas testosterone, 17 alpha-estradiol, tamoxifen, and corticosterone were inactive. This function was specifically inhibited, since yeast-to-mycelium transformation, yeast growth, and yeast reproduction by budding were unaffected by 17 beta-estradiol. Of note is the fact that mycelium-to-yeast transformation occurs as the first step in vivo in the establishment of infection. The cytosol of the three isolates studied possesses a steroid-binding protein which has high affinity for 17 beta-estradiol. We believe that this binding protein represents a P. brasiliensis hormone receptor which can also recognize mammalian estrogens. We hypothesize that the ability of estrogen to decrease or delay mycelium-to-yeast transformation at the initial site of infection contributes to or is responsible for the marked resistance of females, and that the binder described is the molecular site of action.     

Correlation between antigenemia of Paracoccidioides brasiliensis and inhibiting effects of plasma in patients with paracoccidioidomycosis.

Metabolites produced by pathogenic fungi may be involved in the pathogenesis of fungal infections consequently altering the defence mechanisms of the host. In this study the levels of Paracoccidioides brasiliensis antigens detected in the plasma of patients with paracoccidioidomycosis correlated with the suppression index detected by the low mitogenic response of peripheral blood mononuclear cells (PBMC) to phytohaemaglutinin (PHA). This inhibitory effect on lymphoproliferation was observed in the plasma of 58% of the patients, suggesting the presence of inhibitory factors. Plasma samples from paracoccidioidomycosis patients having or not having inhibitory factors showed no significant effect on chromosomes of lymphocytes from healthy individuals. However, these plasmas had a suppressive activity on the blastogenic response of these lymphocytes stimulated with PHA, that was independent of a cytotoxic effect. P. brasiliensis antigens added to the proliferative response of PBMC from healthy individuals stimulated or not stimulated with PHA showed a dose-dependent suppressor effect, reproducing the inhibitory effect of patients' plasma. We suggest that the antigens of P. brasiliensis present in the plasma of patients, even at low concentrations, can play an important role in the reduction of the cellular immune response and in the genesis of the immunoregulatory disturbances observed in paracoccidioidomycosis.     

Detection of Paracoccidioides brasiliensis by PCR in biopsies from patients with paracoccidioidomycosis: correlation with the histopathological pattern

Paracoccidioidomycosis, a systemic mycosis, is rarely diagnosed in its initial phase and can remain latent for up to 40 years. Although PCR is sensitive for the identification of Paracoccidioides brasiliensis (Pb) in different samples, no study using paraffin-embedded human tissue has been published. The size of the amplicon, the fixation method and the time of the storage may affect the reaction. Recently the more sensitive Primer-Extension-Preamplification (PEP)-Nested-PCR has been used for amplification of small samples. Our aims were to detect Pb in paraffin embedded biopsies using (PEP)-Nested-PCR and to correlate the data with histopathological parameters. Analyses were carried out in 107 biopsies from tegument, lymph node, lung and tongue. The fungal DNA was detected in 29.9% of the biopsies by (PEP)-nested-PCR against 5% of Nested-PCR. The positivity correlated with numbers of fungi and fungal viable cells, and there was no correlation with the granuloma pattern.     

The naked-tailed armadillo Cabassous centralis (Miller 1899): a new host to Paracoccidioides brasiliensis. Molecular identification of the isolate.

The natural habitat of Paracoccidioides brasiliensis remains undefined but the repeated demonstration of infection by this fungus in the nine-banded armadillo Dasypus novemcinctus has opened interesting research avenues. We report here the isolation of this fungus from the spleen of a naked-tailed armadillo Cabassous centralis (Miller 1899) captured in a coffee farm localized in the Colombian endemic area for paracoccidioidomycosis. This particular isolate was identified by its dimorphism and also by comparison of the PbGP43 gene and ribosomal internal transcribed spacer regions (ITS) with recognized P brasiliensis strains. This finding extends the range of naturally acquired infections in mammals of the family Dasypodidae and confirms the existence of this human pathogen in areas where human paracoccidioidomycosis is known to occur.     

Adjuvant effect of synthetic oligodeoxyribonucleotides (CpG-ODN) from the Paracoccidioides brasiliensis gp43 gene on the Th2-Th1 immunomodulation of experimental paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis. Immunostimulatory effects of P. brasiliensis DNA and CpG-oligodeoxyribonucleotides (CpG-ODN) have shown a Th2-Th1 immunomodulation of the isogenic murine model of susceptibility, which develops a progressive and disseminating disease. In this study, we investigated the optimum time interval and doses of CpG-ODN which are able to induce Th2-Th1 immunomodulation. The optimum concentrations for the induction of a decrease in antibody production were 0.5 and 1 microg. Mice immunized twice with CpG-ODN and gp43 (5 and 7 days before the challenge) showed a 60% higher chance of survival compared with the control group (nonimmunized), and an increase in Th1 isotype (IgG2a) was also observed. In vitro assays of naive and preimmunized mice showed discrete cellular proliferation when stimulated by suitable concentrations of CpG-ODN. Type 1 cytokines interleukin-12 (IL-12) and interferon-gamma were increased in cell culture supernatants, but no significant difference was found in Th2 IL-4 cytokines in stimulated or nonstimulated cell cultures. Concerning the Th2-Th1 kinetics in experimental PCM models by adjuvant effect of CpG-ODN, there are still many questions to be answered and clarified. However, the gathering of data obtained in this investigation has led us to suggest that the modulation of Th2-Th1 in experimental PCM depends on time and CpG-ODN concentration.     

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.     

Gel-filtration chromatography of Salmonella protein antigens and their implication in immunodiagnosis

Crude Barber protein (Bp) antigens were prepared from Salmonella typhi, S. krefeld and S. derby by an original method that has been described previously. These antigens were subjected to gel-filtration chromatography using Sephadex G-200. A sharp peak that eluted together with the void volume was thus separated from a broad second peak that eluted from the column at positions equivalent to 118,000 to 12,000 daltons. The proteins eluted in the latter peak were arbitrarily divided into 5 fractions and, together with the first peak, subjected to polyacrylamide gel electrophoresis and immunoprecipitation with both homologous and heterologous rabbit antisera. The extent of immunological cross reactivities was determined by enzyme-linked immunosorbent assay. The preliminary results obtained by this technique showed species-specific protein antigens to have molecular weights ranging between 36,000 and 68,000 daltons.     

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. Of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.     

Identification of a 26-kDa protein fraction as an important antigen for application in the immunodiagnosis of strongyloidiasis

Strongyloidiasis caused by the intestinal nematode Strongyloides stercoralis typically occurs in the asymptomatic form. The definitive diagnosis is usually done by detection of larvae on fecal samples. However, as the parasite load is often low in most cases, microscopy is not usually sensitive and specific, and diagnosis becomes extremely difficult. Thus, development of reliable serological methods is imperative. In the present study, a diversity of epitopes from S. stercoralis larva were characterized by analysis of reactivity with serum samples obtained from individuals with and without the infection by using Western blot technique. A total of 91 serum samples belonging to 5 groups were analyzed. Different reactivity profiles were observed, representing recognition of proteins with molecular mass varied from 6 to 129 kDa. A protein band of approximately 26 kDa presented a high frequency of reactivity with serum samples from the strongyloidiasis patients group (18/23). Reactivity with this protein band was also observed in only 7 of 64 non-infected individuals or individuals infected with other helminthes. Reactivity with 2 other bands, 1 of approximately 33 kDa and a duplet of approximately 21 kDa, were also found in high frequency (17/23 and 9/23, respectively). However, reactivity with these bands was also observed in all the other serum groups studied. The results indicate that the 26-kDa band maybe be an important tool for the development of diagnostic techniques for strongyloidiasis.     

Isolation of species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii for immunodiagnosis and immunoprophylaxis

A simple procedure for the selective isolation of the protective species-specific protein antigens (SPAs) of Rickettsia typhi and Rickettsia prowazekii was developed to permit use of the SPAs in the immunodiagnosis and immunoprophylaxis of typhus infections. Although the SPAs were readily extracted from lysozyme- or detergent-treated rickettsiae, as measured by rocket immunoelectrophoresis, other polypeptides were also present, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, both water and seven buffers, each at a 10 mM concentration and pH 7.6, were nearly equally effective in the selective release of the SPAs from whole cells by extraction for 30 min at 45 degrees C. High-ionic-strength buffers and MgCl2 abolished this SPA release, thus suggesting that divalent cations were important in the binding of the SPAs to the cell envelope. The efficacy of the dilute buffer extraction procedure for isolation of large amounts of SPAs was tested by further characterization of the supernatants obtained by centrifugation (200,000 x g) of two successive tris-(hydroxymethyl)aminomethane-hydrochloride buffer (Tris) extracts. With this procedure, between 10 and 15 mg of SPA was obtained from 100 mg of purified rickettsiae. Although low-molecular-weight ribonucleic acid fragments were released into the Tris extracts in significant amounts, only the SPAs were detected, in significant quantities, as measured by polyacrylamide gel electrophoresis and rocket immunoelectrophoresis. The Tris extracts contained the same major and minor SPA polypeptides as those observed previously in SPA preparations obtained by extensive diethylaminoethyl-cellulose column chromatography, but the Tris SPAs were more satisfactory antigens in an enzyme-linked immunosorbent assay.     

High frequency of Paracoccidioides brasiliensis infection in armadillos (Dasypus novemcinctus): an ecological study.

The fungus Paracoccidioides brasiliensis has been isolated from nine-banded armadillos (Dasypus novemcinctus) in different regions where paracoccidiodomycosis (PCM) is endemic. The link between PCM and these animals has provided the first valuable clue in the effort to elucidate the ecological niche of P. brasiliensis. The present study was aimed at correlating P. brasiliensis infection in armadillos with local ecological features and, if possible, the presence of the fungus in the soil in the Botucatu hyperendemic area of PCM. In this region the mean temperature ranges from 14.8 to 25.8 degrees C and the annual average precipitation is 1520 mm. The sites where 10 infected animals (positive group) were collected were studied and compared with the sites where five uninfected animals were found. The occurrence of the fungus in soil samples collected from the positive armadillos' burrows and foraging sites was investigated by the indirect method of animal inoculation. Environmental data from the sites of animal capture, such as temperature, rainfall, altitude, vegetation, soil composition, presence of water and proximity of urban areas, were recorded. All 37 soil samples collected from the sites had negative fungal cultures. Positive animals were found much more frequently in sites with disturbed vegetation, such as riparian forests and artificial Eucalyptus or Pinus forests, in altitudes below 800 m, near water sources. The soil type of the sites of positive animals was mainly sandy, with medium to low concentrations of organic matter. The pH was mainly acidic at all the sites, although the concentrations of aluminum cations (H+Al) were lower at the sites where positive animals were found. Positive armadillos were also captured in sites very close to urban areas. Our data and previous studies indicate that P. brasiliensis occurs preferentially in humid and shady disturbed forests in a strong association with armadillos.     

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).     

Toxoplasma gondii: Uptake of fetuin and identification of a 15-kDa fetuin-binding protein

Lectin-binding studies demonstrated the presence of a 68-kDa glycoprotein in tachyzoites ofToxoplasma gondii harvested from P388D1 macrophage cell cultures but not in tachyzoites maintained in peritoneal cavities of NMRI mice. This protein was identified as the embryonic protein fetuin that regularly is contained in fetal calf serum, a component of cell-culture media. Uptake of fetuin byT. gondii was demonstrated by intracellular localization of this protein. As shown by latex agglutination and immunofluorescence, no specific binding of fetuin to the parasite's surface was detected. Using affinity chromatography of fetuin-agarose, it was demonstrated that fetuin bound specifically to a 15-kDa antigen of tachyzoites. As revealed by inhibition studies with sialic acid and the lectinSambucus nigra agglutinin, the 15-kDa protein probably recognized glycan structures of fetuin.     

Randomly amplified polymorphic DNA as a valuable tool for epidemiological studies of Paracoccidioides brasiliensis

Randomly amplified polymorphic DNA (RAPD) has been successfully used to detect genetic variations among isolates of Paracoccidioides brasiliensis. However, the usefulness of this technique for assessing important parasitic properties is still unconfirmed. In the present work we further investigated the applicability of RAPD in revealing important intrinsic and extrinsic features of this fungus associated with geographical origin, time of isolation, source of clinical specimen, clinical forms of human disease and also in vitro and in vivo susceptibility to antimicrobial and antifungal drugs. The RAPD patterns allowed us to distinguish all of the analyzed strains, which included 26 clinical isolates, 2 animal isolates, and 1 environmental isolate of P. brasiliensis obtained from different geographic regions, confirming the strong discriminating power of this technique. A phenetic tree, build from the RAPD data, showed that although the two nonclinical Brazilian strains were set together the majority of the clinical Brazilian strains were randomly distributed through different sub-branches of a major cluster without any correlation to any of the parameters analyzed. A second major cluster, however, has grouped isolates from Mato Grosso and Roraima (Brazil) that not only were susceptible in vitro to trimethoprim-sulfamethoxazole but also produced a good in vivo response. These results open new vistas for epidemiological and clinical studies of P. brasiliensis.