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Purpose: In-house Paracoccidioides spp. antigens are commonly used in the serological diagnosis of paracoccidioidomycosis (PCM). The sensitivity and specificity of a commercial Paracoccidioides spp. antigen was assessed for PCM serological testing., Method: Counterimmunoelectrophoresis and double immunodiffusion were used to evaluate the Paracoccidioides ID Antigen® reagent in sera from PCM cases and patients with other diseases., Results: All active PCM sera (n=24) were reactive using counterimmunoelectrophoresis (sensitivity = 100%), including 11 cases of infection by P. brasiliensis sensu stricto and one by P. americana. Fifteen (88%) out of 17 sera from patients on treatment or cured were reactive, including one case of P. lutzii infection. One to three bands of antigen-antibody precipitate were observed on the agarose gel, with a predominance of two to three bands in the test with untreated PCM sera or at the beginning of antifungal therapy. All sera from patients with histoplasmosis (n=7), aspergillosis (n=5), and other diseases (n=27) tested negative (specificity = 100%). The overall sensitivity and specificity using the commercial antigen and double diffusion test were 75% and 100%, respectively., Conclusion: The commercial antigen performed satisfactorily and may contribute to the dissemination of the use of serological tests for the PCM diagnosis., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2021 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Paracoccidioidomycosis caused by Paracoccidioides lutzii is endemic in the Midwest of Brazil and its clinical spectrum is still little known due to the recent identification of this fungal species. A patient resident in Southeast Brazil, but who had lived for many years in the Midwest region, presented with skin injuries, chronic cough and bilateral adrenal involvement. Paracoccidioides spp. was isolated in culture from a skin lesion biopsy. This isolate was later identified as P. lutzii using gene sequencing. A favorable initial response to treatment with itraconazole was observed, but a few weeks later, the patient developed respiratory failure and worsening of lung lesions. Evaluation by computed tomography and echocardiography were suggestive of pulmonary arterial hypertension, and a bronchoscopic biopsy showed peribronchial remodeling. The patient completed the antifungal treatment but maintained the respiratory dysfunction. The reported case shows that P. lutzii can be isolated from patients in a geographic area far from the place of infection acquisition and that, as P. brasiliensis , it can cause adrenal injury and cardio-respiratory complications as a consequence of excessive necrosis and fibrosis.

The thermodimorphic pathogenic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiologic causes of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Galectin-3 (Gal-3), an animal β-galactoside-binding protein, modulates important roles during microbial infections, such as triggering a Th2-polarized immune response in PCM. Herein, we demonstrate that Gal-3 also plays other important roles in P. brasiliensis infection. We verified that Gal-3 levels are upregulated in human and mice infections and established that Gal-3 inhibited P. brasiliensis growth by inhibiting budding. Furthermore, Gal-3 affected disruption and internalization of extracellular vesicles (EVs) from P. brasiliensis by macrophages. Our results suggest important protective roles for Gal-3 in P. brasiliensis infection, indicating that increased Gal-3 production during P. brasiliensis infection may affect fungal growth and EV stability, thus promoting beneficial effects that could influence the course of PCM. The finding that Gal-3 has effects against P. brasiliensis together with previously reported effects against Cryptococcus neoformans suggests that molecule has a general antifungal role in innate defenses against fungal pathogens. IMPORTANCE Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Although the immune mechanisms to control PCM are still not fully understood, several events of the host innate and adaptive immunity are crucial to determine the progress of the infection. Mammalian β-galactoside-binding protein galectin-3 (Gal-3) plays significant roles during microbial infections and has been studied for its immunomodulatory roles, but it can also have direct antimicrobial effects. We asked whether this protein plays a role in Paracoccidioides brasiliensis We report herein that Gal-3 indeed has direct effects on the fungal pathogen, inhibiting fungal growth and reducing extracellular vesicle stability. Our results suggest a direct role for Gal-3 in P. brasiliensis infection, with beneficial effects for the mammalian host., (Copyright © 2019 Hatanaka et al.)

Paracoccidioidomycosis is a systemic fungal disease associated with agricultural activities. Its incidence and prevalence are underestimated because of the lack of reporting in several Brazilian states. If paracoccidiodomycosis is not diagnosed and treated early and adequately, endemic fungal infection may result in serious sequelae. In addition to the Paracoccidioides brasiliensis (P. brasiliensis) complex, the appearance of a new species, Paracoccidioides lutzii (P. lutzii), in Rondônia state, where the disease has reached epidemic levels, and in the country's Midwest region and Pará state, are challenges to diagnosis and to the urgent availability of antigens that are reactive with patients' sera. These guidelines aim to update the first Brazilian consensus on paracoccidioidomycosis by providing evidence-based recommendations for bedside patient management. The guidelines provide data on etiology, epidemiology, immunopathogenesis, diagnosis, treatment and sequelae, with emphasis on diagnosis and treatment, as well as current recommendations and challenges in this field of knowledge.

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.

Paracoccidioidomycosis should be differentiated from other opportunistic diseases in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients who live in Latin America. Laboratory investigation can begin with serological tests, which are rapid and efficient. In the present study, double immunodiffusion (DID), counterimmunoelectrophoresis (CIEP) and an enzyme linked immunosorbent assay (ELISA) tests were assessed for the detection of anti-Paracoccidioides brasiliensis antibodies in 40 patients coinfected with HIV. The results were compared to those obtained for 75 non-HIV-infected patients with endemic paracoccidioidomycosis. Anti-P. brasiliensis antibodies were detected in 65% (DID), 79% (CIEP) and 95% (ELISA) of the patients with HIV/AIDS, significantly lower rates than those detected in cases of endemic paracoccidioidomycosis, which were 89%, 99% and 100%, respectively. The reactive sera of HIV-infected patients also showed lower anti-P. brasiliensis antibody titres than those of non-HIV-infected patients. Despite the lower intensity of the specific humoral response, serological tests are useful for the diagnosis of opportunistic paracoccidioidomycosis in the HIV/AIDS population. We suggest optimization of the laboratory diagnosis by combining the ELISA test with CIEP or DID.

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS(-/-)) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS(-/-) mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS(-/-) mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS(-/-) mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS(-/-) mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-gamma and TNF-alpha) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis. To investigate the role of interleukin (IL)-12 in this disease, IL-12p40-/- deficient mice (IL-12p40-/-) and wild type mice (WT) were infected intravenously with viable yeast cells of P. brasiliensis 18 isolate. We found that, unlike WT mice, IL-12p40-/- mice did not control fungal proliferation and dissemination and succumbed to infection by day 21 after inoculation. Additionally, IL-12p40-/- mice presented a higher number of granulomas/mm2 in lung tissue than WT mice, and showed unorganized granulomas containing high numbers of yeast cells. Moreover, IL-12p40-/- mice did not release detectable levels of IFN-gamma, but they produced high levels of IL-10, as well as IgG1 antibody. Additionally, splenocytes from both infected IL-12p40-/- and WT mice exhibited a suppressed Con-A-induced T cell proliferative response. Our findings suggest that the IL-12p40 subunit mediates resistance in paracoccidioidomycosis by inducting IFN-gamma production and a Th1 immune response.

KM(+) is a mannose-binding lectin from Artocarpus integrifolia that induces interleukin (IL)-12 production by macrophages and protective T helper 1 immune response against Leishmania major infection. In this study, we performed experiments to evaluate the therapeutic activity of jackfruit KM(+) (jfKM(+)) and its recombinant counterpart (rKM(+)) in experimental paracoccidioidomycosis. To this end, jfKM(+) or rKM(+) was administered to BALB/c mice 10 days after infection with Paracoccidiodes brasiliensis. Thirty days postinfection, lungs from the KM(+)-treated mice contained significantly fewer colony-forming units and little to no organized granulomas compared to the controls. In addition, lung homogenates from the KM(+)-treated mice presented higher levels of nitric oxide, IL-12, interferon-gamma, and tumor necrosis factor-alpha, whereas higher levels of IL-4 and IL-10 were detected in the control group. With mice deficient in IL-12, Toll-like receptor (TLR) 2, TLR4, or TLR adaptor molecule MyD88, we demonstrated that KM(+) led to protection against P. brasiliensis infection through IL-12 production, which was dependent on TLR2. These results demonstrated a beneficial effect of KM(+) on the severity of P. brasiliensis infection and may expand its potential use as a novel immunotherapeutic molecule.

Paracoccidioidomycosis, a debilitating pulmonary mycosis, is caused by the dimorphic fungus Paracoccidioides brasiliensis. The infection results in the formation of granulomas containing viable yeast cells that are the fungal sources for disease reactivation. Because CD4+CD25+ regulatory T cells (Tregs) are in the lesions of patients with paracoccidioidomycosis, the migration of Treg cells is dependent on the axis chemokine-chemokine receptors, and CCR5 ligands are produced in P. brasiliensis-induced lesions, we investigated the role of CCR5 in the control of the infection. The results showed that CCR5-/- mice are more efficient in controlling fungal growth and dissemination and exhibited smaller granulomas than wild-type (WT) mice. In the absence of CCR5, the percentage of CD4+CD25+ T cells expressing Foxp3, glucocorticoid-induced TNFR (GITR), CD103, CD45low, and CTLA-4 in the granulomas was significantly decreased. Interestingly, P. brasiliensis infection resulted in an absence of T cell proliferation in response to Con A in WT but not CCR5-/- mice that was abrogated by anti-CTLA-4 mAb and anti-GITR mAb. Moreover, the adoptive transfer of CD4+CD25+ but not CD4+CD25- T cells from infected WT to infected CCR5-/- mice resulted in a significant increase in fungal load. Overall, CCR5 is a key receptor for the migration of Treg cells to the site of P. brasiliensis infection, leading to down-modulation of effector immune response and the long-term presence of the fungus in the granulomas. Thus, a tight control of Treg cell migration to the granulomatous lesions could be an important mechanism for avoiding exacerbation and reactivation of the disease.

The migration of leukocytes to inflammatory sites elicited by Paracoccidioides brasiliensis is supposed to be coordinated by cytokines and chemokines. Here, we investigated the role of intercellular adhesion molecule-1 (ICAM-1) in recruiting inflammatory cells to lungs of mice infected with P. brasiliensis and in determining the outcome of the disease. Expression of ICAM-1 was up-regulated on T lymphocytes after infection with the fungus, and its expression was dependent on interferon-gamma, tumor necrosis factor-alpha, and interleukin-12. Moreover, the absence of ICAM-1 resulted in high susceptibility to the infection and delayed formation of granulomatous lesions. In addition, the absence of ICAM-1 resulted in increased growth and dissemination of fungus, decreased number of CD3+CD4+ and CD3+CD8+ T cells, and increased production of interleukin-4 in the inflammatory site. The organization of a granulomatous reaction in mice deficient of ICAM-1 was delayed, starting only on day 60 after infection, whereas in wild-type mice it was complete on day 30 of infection. These data show that ICAM-1 is effectively involved in cellular migration and in the organization of the granulomatous lesion caused by the fungus P. brasiliensis.

Paracoccidioides brasiliensis components interact with host cells and can influence the pathogenesis of paracoccidioidomycosis (PCM). Among the components released by P. brasiliensis, gp 43 and a heavily glycosylated antigen with MM>160 kDa are the most recognized by serum antibodies from patients with PCM. In order to isolate the high MM glycoconjugate, we carried out affinity chromatography of a crude exoantigen preparation on immobilized jacalin. The bound fraction (JBE, jacalin binding exoantigen) consisted of a major antigen of high MM and frequently of an additional 70-kDa minor protein. This protein, designated paracoccin, exhibited selective binding to immobilized GlcNAc, a property that was used for its purification. The structural data of paracoccin obtained by mass spectrometry of tryptic peptides did not match any known protein. Anti-paracoccin serum localized the lectin on the surface of P. brasiliensis yeasts, especially in the budding regions. Paracoccin was able to interact with laminin in a dose-dependent manner. This interaction was inhibited by GlcNAc, followed by D-glucose and D-mannose, but not by D-galactose, N-acetyl-galactosamine or L-fucose. Interestingly, paracoccin induced both resident and elicited mouse peritoneal cavity macrophages to release high and persistent levels of TNF-alpha in vitro, a fact that was associated with high nitric oxide production in elicited cells. Because binding to laminin can favor yeast adhesion and invasion of host tissues, and overproduction of NO has been associated with suppression of cell immunity, paracoccin is suggested to play an important role in PCM pathogenesis.

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis (PCM). Here, we describe the microsatellite patterns observed in a collection of P. brasiliensis random sequence tags. We identified 1,117 microsatellite patterns in about 3.8 Mb of unique sequences (0.47% of the total DNA used in the analysis). The majority of these microsatellites (87.5%) are found in noncoding sequences. We used two polymorphic microsatellites located on noncoding and coding sequences, as well as two microsatellites located on introns, as molecular markers to discriminate P. brasiliensis isolates, to look for relationships between the genetic background of the strains and the types of human disease they cause. We did not observe any correlation between the clinical form of human PCM and four simple sequence repeat patterns analyzed.

Serum antibodies against antigens of Paracoccidioides brasiliensis have been one of the major diagnostic indicators of paracoccidioidomycosis (PCM). In the present study, released antigen preparations (exoAg) obtained from P. brasiliensis isolates were characterized in terms of their protein components electrophoretically detectable and recognizable by sera (IgG) of patients. Among five different isolates (DGO, C-9, BAT, Pb-18 and B-339) the electrophoretic profiles of exoAg varied greatly. A total of 28 different components were detected, 11 of them shared by all isolates. The most representative preparation was BAT-exoAg, which presented the largest number of protein bands (23) and the highest frequency of reacting bands (19) with sera from patients with active PCM (n = 40). Six bands reacted with more than 20% of sera. Independently of clinical forms, the sera recognized the 43-kDa (97% of tested sera), 160-kDa (78% of tested sera) and 70-kDa (60% of tested sera) antigens more frequently. Sera from patients with severe forms of acute (n = 14) or chronic (n = 10) PCM recognized a greater number of antigens, with a higher frequency, than those from moderate forms. The most pronounced reduction in reactivity was provided by sera of patients that became asymptomatic at the beginning of treatment. Remnant reactivity with BAT-exoAg was detected after clinical recovery, especially with those of 43, 70 and 160 kDa. The latter presented a stable recognition frequency (60%) during the entire follow-up, allowing us to suppose that the IgG reactivity against the 160-kDa antigen constitutes a possible persistent marker of P. brasiliensis infection.