Detection of circulating Paracoccidioides brasiliensis antigen in urine of paracoccidioidomycosis patients before and during treatment

For the diagnosis and follow-up of paracoccidioidomycosis patients undergoing therapy, we evaluated two methods (immunoblotting and competition enzyme immunoassay) for the detection of circulating antigen in urine samples. A complex pattern of reactivity was observed in the immunoblot test. Bands of 70 and 43 kDa were detected more often in urine samples from patients before treatment. The immunoblot method detected gp43 and gp70 separately or concurrently in 11 (91.7%) of 12 patients, whereas the competition enzyme immunoassay detected antigenuria in 9 (75%) of 12 patients. Both tests appeared to be highly specific (100%), considering that neither fraction detectable by immunoblotting was present in urine samples from the control group. gp43 remained present in the urine samples collected during the treatment period, with a significant decrease in reactivity in samples collected during clinical recovery and increased reactivity in samples collected during relapses. Reactivity of some bands was also detected in urine specimens from patients with 'apparent cure.' The detection of Paracoccidioides brasiliensis antigens in urine appears to be a promising method for diagnosing infection, for evaluating the efficacy of treatment, and for detecting relapse.

Involvement of the major glycoprotein (gp43) of Paracoccidioides brasiliensis in attachment to macrophages

The yeast form of Paracoccidioides brasiliensis, the causative agent of a deep mycosis in humans, is known to be phagocytized by, and to multiply inside, macrophages. In this work we describe the involvement of gp43, a major antigenic protein of P. brasiliensis, in the initial steps of attachment of the fungus to macrophages. Anti-gp43 F(ab) polyclonal fragments were capable of inhibiting phagocytosis in a concentrationdependent manner. Sheep red blood cells sensitized with purified gp43 were more endocytized than SRBC alone, and this process was also inhibited by anti-gp43 F(ab) fragments. Inhibition tests indicated the involvement of fucose and mannose residues in the phagocytosis of the fungus and of SRBC-gp43 by macrophages. Taken together, these results suggest that gp43 may be involved in the adherence and uptake of the fungus by murine peritoneal macrophages, and that this binding may be dependent on monosaccharide residues that are part of the gp43 glycoprotein. © 1998 ISHAM.

Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Biosynthesis of chondroitinase and hyaluronidase by different strains of Paracoccidioides brasiliensis

The biosynthesis of chondroitinase and hyaluronidase by different isolates of Paracoccidioides brasiliensis was investigated in 20 strains isolated from patients (17 strains), a penguin (Pygocelis adeliae, one strain), an armadillo (Dasypus novemcinctus, one strain) and the environment (dog food, one strain). All the P. brasiliensis isolates studied had the ability to produce chondroitinase and hyaluronidase, although differences in colony morphology and enzyme production were detected among them. These results suggest that further investigations should be carried out in the clinical field in order to clarify the potential role of P. brasiliensis enzyme production in the pathogenesis of paracoccidioidomycosis.

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: Reversal by holotransferrin not by apotransferrin

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.

Interação de Paracoccidioides brasiliensis com células endoteliais

Paracoccidioidomycosis has a variety of clinical manifestations and Paracoccidioides brasiliensis, the causative agent, may infect many tissues, most importantly the lungs. Migration of pathogenic yeasts to the endothelial cell layer is considered a prerequisite for multiple organ invasion and dissemination of the fungus. In this study of the adhesion of P. brasiliensis to endothelial cells in vitro, we investigated whether this adhesion could represent a mechanism of dissemination. To this end, as well as using conventional optical microscopy, an alternative in vivo technique was developed, to detect the presence of fungal cells in umbilical cords embedded in paraffin wax. An experiment on the migration of P. brasiliensis through an endothelial cell monolayer was carried out, and the migration of yeast cells was greater, and took less time, in control wells with no cells. The fungus crossed the monolayer, but, compared to control wells, the migration-rate was about 30% lower. This shows that the monolayer only partially blocked migration of the fungus. In these experiments, we had great difficulty finding P. brasiliensis adhered to the cell monolayer, when it was examined at different times, suggesting that migration of the fungus across the endothelial layer is very fast, and cannot normally be observed in cell culture in vitro. Thus, P. brasiliensis can cross the endothelium rapidly and probably invades deeper tissue.

Effect of prostaglandins on the production of H2O2 and cytokines that modulate the fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Human monocytes lack fungicidal activity against high virulent strains of Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, even after IFN-γ activation. However, monocytes treated with indomethacin (INDO) or INDO plus IFN-γ effectively killed this fungus, suggesting an inhibitory role of prostaglandins in this process. Thus, the purpose of this work was to test if this regulatory effect of prostaglandin was associated with alterations on H2O2 production and/or on modulatory cytokines levels, such as TNF-α, IL-10, and IL-6. Peripheral blood monocytes obtained from 10 healthy donors were incubated for 18 hours in the presence or absence of IFN-γ, INDO, or IFN-γ plus INDO, and further challenged with a high virulent strain of P. brasiliensis (Pb18) for 4 hours. Then, the monocytes cultures were evaluated for H2O2 release and fungicidal activity calculated by counting the colony forming units after plating. Moreover, on supernatants of the same cultures, TNF-α, IL-10, IL-6, and PGE2 concentrations were evaluated by ELISA. Monocytes treated with INDO or INDO plus IFN-γ presented higher fungicidal activity associated with the release of higher levels of H2O2 and TNF-α, but lesser levels of PGE2, when compared to nontreated cells. However, the levels of IL-10 and IL-6 were similar between treated and nontreated cells. The results suggest that human monocytes when challenged with high virulent strains of P. brasiliensis produce prostaglandins that inhibit the fungicidal activity of these cells by reducing H2O2 and TNF-α levels.

Chloroquine inhibits Paracoccidioides brasiliensis survival within human monocytes by limiting the availability of intracellular iron

The mechanisms used by Paracoccidioides brasiliensis (Pb 18) to survive into monocytes are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens, including P. brasiliensis, whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Chloroquine, by virtue of its basic properties, has been shown to prevent release of iron from holotransferrin by raising endocytic and lysosomal pH, and thereby interfering with normal iron metabolism. Then, in view of this, we have studied the effects of CHLOR on P. brasiliensis multiplication in human monocytes and its effect on the murine paracoccidioidomycosis. CHLOR induced human monocytes to kill P. brasiliensis. The effect of CHLOR was reversed by FeNTA, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. CHLOR treatment of Pb 18-infected BALB/c mice significantly reduced the viable fungi recovery from lungs, during three different periods of evaluation, in a dose-dependent manner. This study demonstrates that iron is of critical importance to the survival of P. brasiliensis yeasts within human monocytes and the CHLOR treatment in vitro induces Pb 18 yeast-killing by monocytes by restricting the availability of intracellular iron. Besides, the CHLOR treatment in vivo significantly reduces the number of organisms in the lungs of Pb-infected mice protecting them from several infections. Thus, CHLOR was effective in the treatment of murine paracoccidioidomycosis, suggesting the potential use of this drug in patients' treatment.

Pro- and anti-inflammatory cytokines produced by human monocytes challenged in vitro with Paracoccidioides brasiliensis

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1β), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1β, IL-6, IL-10 and TNF-α. Pb18 induced higher levels of IL-1β, IL-6, and IL-10 than Pb265. IL-8 and TGF-β1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.

Evidence for the role of calcineurin in morphogenesis and calcium homeostasis during mycelium-to-yeast dimorphism of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25°C to 37°C and is critical for pathogenicity. Intracellular Ca 2+ levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca 2+ with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca 2+ accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of P. brasiliensis. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca 2+ homeostasis and decreased mRNA transcription of the CCH1 gene for the plasma membrane Ca 2+ channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca 2+/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca 2+ homeostasis in P. brasiliensis and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on P. brasiliensis reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Induction and secretion of elastinolytic and proteolytic activity in cultures of Paracoccidioides brasiliensis

P. brasiliensis parasitizes various human tissues and proteinases exported by this fungus may allow it to metabolize and invade host tissues. The influence of the culture medium on the production of proteinases by P. brasiliensis isolates was studied and the export of these enzymes was followed as a function of culture time. The fungus was grown in neopeptone, BSA, elastin or collagen medium. The culture medium was assayed for azocollytic, elastinolytic and caseinolytic activity. Proteolytic activity was also analysed by electrophoresis of the culture medium on gelatin and casein substrate gels. P. brasiliensis expressed relatively high levels of azocoll, elastin and casein degrading activity in all types of medium, except in neopeptone medium. Generally, expression of azocollytic activity peaked during the third week of culture and caseinolytic activity during the fourth week of culture. Azocollytic activity was highest at pH 4.0 and caseinolytic activity at pH 8.0. Elastinolytic activity was also highest at pH 8.0. This activity, as well as the others, may provide the fungus with a source of carbon and nitrogen and may also be responsible for the invasion of host tissues, such as pulmonary elastic fiber, by P. brasiliensis.

Inhibition of human neutrophil apoptosis by Paracoccidioides brasiliensis: Role of interleukin-8

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production. © 2008 The Authors.

Importance of xenarthrans in the eco-epidemiology of Paracoccidioides brasiliensis

Background. Several pathogens that cause important zoonotic diseases have been frequently associated with armadillos and other xenarthrans. This mammal group typically has evolved on the South American continent and many of its extant species are seriously threatened with extinction. Natural infection of armadillos with Paracoccidioides brasiliensis in hyperendemic areas has provided a valuable opportunity for understanding the role of this mammal in the eco-epidemiology of Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Latin America. Findings. This study aimed to detect P. brasiliensis in different xenarthran species (Dasypus novemcinctus, Cabassous spp., Euphractus sexcinctus, Tamandua tetradactyla and Myrmecophaga tridactyla), by molecular and mycological approaches, in samples obtained by one of the following strategies: i) from road-killed animals (n = 6); ii) from naturally dead animals (n = 8); iii) from animals that died in captivity (n = 9); and iv) from living animals captured from the wild (n = 2). Specific P. brasiliensis DNA was detected in several organs among 7/20 nine-banded armadillos (D. novemcinctus) and in 2/2 anteaters (M. tridactyla). The fungus was also cultured in tissue samples from one of two armadillos captured from the wild. Conclusion. Members of the Xenarthra Order, especially armadillos, have some characteristics, including a weak cellular immune response and low body temperature, which make them suitable models for studying host-pathogen interaction. P. brasiliensis infection in wild animals, from PCM endemic areas, may be more common than initially postulated and reinforces the use of these animals as sentinels for the pathogen in the environment. © 2009 Bagagli et al; licensee BioMed Central Ltd.

Detection of Paracoccidioides spp. in environmental aerosol samples

Taking into account that paracoccidioidomycosis infection occurs by inhalation of the asexual conidia produced by Paracoccidioides spp. in its saprobic phase, this work presents the collection of aerosol samples as an option for environmental detection of this pathogen, by positioning a cyclonic air sampler at the entrance of armadillo burrows. Methods included direct culture, extinction technique culture and Nested PCR of the rRNA coding sequence, comprising the ITS1-5.8S-ITS2 region. In addition, we evaluated one armadillo (Dasypus novemcinctus) as a positive control for the studied area. Although the pathogen could not be isolated by the culturing strategies, the aerosol sampling associated with molecular detection through Nested PCR proved the best method for discovering Paracoccidioides spp. in the environment. Most of the ITS sequences obtained in this investigation proved to be highly similar with the homologous sequences of Paracoccidioides lutzii from the GenBank database, suggesting that this Paracoccidioides species may not be exclusive to mid-western Brazil as proposed so far. © 2013 ISHAM.

Molecular and morphological data support the existence of a sexual cycle in species of the genus Paracoccidioides

The genus Paracoccidioides includes the thermodimorphic species Paracoccidioides brasiliensis and P. lutzii, both of which are etiologic agents of paracoccidioidomycosis, a systemic mycosis that affects humans in Latin America. Despite the common occurrence of a sexual stage among closely related fungi, this has not been observed with Paracoccidioides species, which have thus been considered asexual. Molecular evolutionary studies revealed recombination events within isolated populations of the genus Paracoccidioides, suggesting the possible existence of a sexual cycle. Comparative genomic analysis of all dimorphic fungi and Saccharomyces cerevisiae demonstrated the presence of conserved genes involved in sexual reproduction, including those encoding mating regulators such as MAT, pheromone receptors, pheromone-processing enzymes, and mating signaling regulators. The expression of sex-related genes in the yeast and mycelial phases of both Paracoccidioides species was also detected by realtime PCR, with nearly all of these genes being expressed preferentially in the filamentous form of the pathogens. In addition, the expression of sex-related genes was responsive to the putative presence of pheromone in the supernatants obtained from previous cocultures of strains of two different mating types. In vitro crossing of isolates of different mating types, discriminated by phylogenetic analysis of the α-box (MAT1-1) and the high-mobility-group (HMG) domain (MAT1-2), led to the identification of the formation of young ascocarps with constricted coiled hyphae related to the initial stage of mating. These genomic and morphological analyses strongly support the existence of a sexual cycle in species of the genus Paracoccidioides. © 2013, American Society for Microbiology.