Long-term outcome of neuroparacoccidioidomycosis treatment

INTRODUCTION: Neuroparacoccidioidomycosis (NPCM) is a term used to describe the invasion of the central nervous system by the pathogenic fungus Paracoccidioides brasiliensis. NPCM has been described sporadically in some case reports and small case series, with little or no focus on treatment outcome and long-term follow-up. METHODS: All patients with NPCM from January 1991 to December 2006 were analyzed and were followed until December 2009. RESULTS: Fourteen (3.8%) cases of NPCM were identified out of 367 patients with paracoccidioidomycosis (PCM). A combination of oral fluconazole and sulfamethoxazole/trimethoprim (SMZ/TMP) was the regimen of choice, with no documented death due to Paracoccidioides brasiliensis infection. Residual neurological deficits were observed in 8 patients. Residual calcification was a common finding in neuroimaging follow-up. CONCLUSIONS: All the patients in this study responded positively to the association of oral fluconazole and sulfamethoxazole/trimethoprim, a regimen that should be considered a treatment option in cases of NPCM. Neurological sequela was a relatively common finding. For proper management of these patients, anticonvulsant treatment and physical therapy support were also needed.

Bilateral paracoccidioidomycotic iliopsoas abscess associated with ileo-colonic lesion

This case report shows the clinical development of a patient with systemic paracoccidioidomycosis presenting with lymphatic-intestinalmanifestation. The patient initially had a substantial clinical improvement but had a recrudescence after six months of sulfamethoxazoletrimethoprim oral treatment, with the emergence of feverish syndrome, lumbar pain, and intermittent claudication, characterizing a bilateral iliopsoas muscle abscess, necessitating clinicosurgical therapeutics.

The influence of carbohydrates in the interaction of Paracoccidioides brasiliensis with CCL-6 cells in vitro

INTRODUCTION: Little is known about the early events in the interaction between Paracoccidioides brasiliensis and its host. To understand the effect of carbohydrates in the interaction between the fungus and epithelial cell in culture, we analyzed the influence of different carbohydrate solutions on the adhesion of P. brasiliensis yeast cells to CCL-6 cells in culture. METHODS: Fungal cells were cultivated with the epithelial cell line, and different concentrations of D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine, D-galactosamine, sorbitol and fructose were added at the beginning of the experiment. Six hours after the treatment, the cells were fixed and observed by light microscopy. The number of P. brasiliensis cells that were adhered to the CCL-6 monolayer was estimated. RESULTS: The number of adhesion events was diminished following treatments with D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine and D-galactosamine as compared to the untreated controls. Sorbitol and fructose-treated cells had the same adhesion behavior as the observed in the control. P. brasiliensis propagules were treated with fluorescent lectins. The FITC-labeled lectins WGA and Con-A bound to P. brasiliensis yeast cells, while SBA and PNA did not. CONCLUSIONS: The perceptual of adhesion between P. brasiliensis and CCL-6 cells decreased with the use of D-mannose, N-acetyl-glucosamine and D-glucosamine. The assay using FITC-labeled lectins suggests the presence of N-acetyl-glucosamine, α-mannose and α-glucose on the P. brasiliensis cell surface. An enhanced knowledge of the mediators of adhesion on P. brasiliensis could be useful in the future for the development of more efficient and less harmful methods for disease treatment and control.

Optimization of a mouse immunization protocol with Paracoccidioides brasiliensis antigens

The objectives of the present study were to optimize the protocol of mouse immunization with Paracoccidioides brasiliensis antigens (Rifkind's protocol) and to test the modulation effect of cyclophosphamide (Cy) on the delayed hypersensitivity response (DHR) of immunized animals. Experiments were carried out using one to four immunizing doses of either crude particulate P. brasiliensis antigen or yeast-cell antigen, followed by DHR test four or seven days after the last immunizing dose. The data demonstrated that an immunizing dose already elicited response; higher DHR indices were obtained with two or three immunizing doses; there were no differences between DHR indices of animals challenged four or seven days after the last dose. Overall the inoculation of two or three doses of the yeast-cell antigen, which is easier to prepare, and DHR test at day 4 simplify the original Rifkind's immunization protocol and shorten the duration of the experiments. The modulation effect of Cy on DHR was assayed with administration of 2.5, 20 and 100 mg/kg weight at seven day intervals starting from day 4 prior to the first immunizing dose. Only the treatment with 2.5 mg Cy increased the DHR indices. Treatment with 100 mg Cy inhibited the DHR, whereas 20 mg Cy did not affect the DHR indices. Results suggest an immunostimulating effect of low dose of Cy on the DHR of mice immunized with P. brasiliensis antigens.

Virulence attenuation and phenotypic variation of Paracoccidioides brasiliensis isolates obtained from armadillos and patients

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features.

Mortality due to systemic mycoses as a primary cause of death or in association with AIDS in Brazil: a review from 1996 to 2006

Deaths caused by systemic mycoses such as paracoccidioidomycosis, cryptococcosis, histoplasmosis, candidiasis, aspergillosis, coccidioidomycosis and zygomycosis amounted to 3,583 between 1996-2006 in Brazil. When analysed as the underlying cause of death, paracoccidioidomycosis represented the most important cause of deaths among systemic mycoses (~ 51.2%). When considering AIDS as the underlying cause of death and the systemic mycoses as associated conditions, cryptococcosis (50.9%) appeared at the top of the list, followed by candidiasis (30.2%), histoplasmosis (10.1%) and others. This mortality analysis is useful in understanding the real situation of systemic mycoses in Brazil, since there is no mandatory notification of patients diagnosed with systemic mycoses in the official health system.

Induction of apoptosis in A549 pulmonary cells by two Paracoccidioides brasiliensis samples

Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.

Simultaneous infection of human host with genetically distinct isolates of Paracoccidioides brasiliensis

This study is the first report on genetic differences between isolates of Paracoccidioides brasiliensis from a single patient. We describe a simultaneous infection with genetically distinct isolates of P. brasiliensis in a patient with chronic paracoccidioidomycosis. The clinical isolates were obtained from lesions in different anatomical sites and were characterised by random amplified polymorphic DNA (RAPD) analysis. The RAPD technique can be helpful for distinguishing between clinical isolates. Different random primers were used to characterise these clinical isolates. The RAPD patterns allowed for differentiation between isolates and the construction of a phenetic tree, which showed more than 28% genetic variability in this fungal species, opening new possibilities for clinical studies of P. brasiliensis. Based on these results and preliminary clinical findings, we suggest that different genotypes of P. brasiliensis might infect the same patient, inducing the active form of the disease.

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Interleukin (IL)-15 is a pleiotropic cytokine that regulates the proliferation and survival of many cell types. IL-15 is produced by monocytes and macrophages against infectious agents and plays a pivotal role in innate and adaptive immune responses. This study analyzed the effect of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with Paracoccidioides brasiliensis (Pb18), the agent of paracoccidioidomycosis. Peripheral blood monocytes were pre-incubated with IL-15 and then challenged with Pb18. Fungicidal activity was assessed by viable fungi recovery from cultures after plating on brain-heart infusion-agar. Superoxide anion (O2-), hydrogen peroxide (H2O2), tumour necrosis factor-alpha (TNF-α), IL-6, IL-15 and IL-10 production by monocytes were also determined. IL-15 enhanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by addition of anti-IL-15 monoclonal antibody. A significant stimulatory effect of IL-15 on O2- and H2O2 release suggests that fungicidal activity was dependent on the activation of oxidative metabolism. Pre-treatment of monocytes with IL-15 induced significantly higher levels of TNF-α, IL-10 and IL-15 production by cells challenged with the fungus. These results suggest a modulatory effect of IL-15 on pro and anti-inflammatory cytokine production, oxidative metabolism and fungicidal activity of monocytes during Pb18 infection.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Computed tomography findings of paracoccidiodomycosis in musculoskeletal system

Objective: To evaluate musculoskeletal involvement in paracoccidioidomycosis at computed tomography. Materials and Methods: Development of a retrospective study based on a review of radiologic and pathologic reports in the institution database. Patients with histopathologically confirmed musculoskeletal paracoccidioidomycosis and submitted to computed tomography were included in the present study. The imaging findings were consensually described by two radiologists. In order to avoid bias in the analysis, one patient with uncountable bone lesions was excluded from the study. Results: A total of seven patients were included in the present study. A total of 18 bone lesions were counted. The study group consisted of 7 patients. A total number of 18 bone lesions were counted. Osteoarticular lesions were the first manifestation of the disease in four patients (57.14%). Bone lesions were multiple in 42.85% of patients. Appendicular and axial skeleton were affected in 85.71% and 42.85% of cases, respectively. Bone involvement was characterized by well-demarcated osteolytic lesions. Marginal osteosclerosis was identified in 72.22% of the lesions, while lamellar periosteal reaction and soft tissue component were present in 5.55% of them. One patient showed multiple small lesions with bone sequestra. Conclusion: Paracoccidioidomycosis can be included in the differential diagnosis of either single or multiple osteolytic lesions in young patients even in the absence of a previous diagnosis of pulmonary or visceral paracoccidioidomycosis

Paracoccidioides brasiliensis infection in dogs from Western Brazilian Amazon

The objective of the study was to evaluate Paracoccidioides brasiliensis infection in urban dogs from the municipality of Monte Negro, Rondonia, Western Brazilian Amazon. The serum samples (n=126) were analyzed by indirect ELISA and the immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively. A positivity of 54.8% was observed only in the ELISA test and no statistical difference was observed in the seroprevalence in relation to age or sex. This is the first paracoccidioidomycosis survey carried out with dogs from the Western Brazilian Amazon. The higher positivity rates of P. brasiliensis infection observed in this study suggest that veterinarians must be alert to detect new cases of natural disease in dogs living in paracoccidioidomycosis endemic areas.

Cytokines produced by susceptible and resistant mice in the course of Paracoccidioides brasiliensis infection

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-a and TGF-ß) and type-1 (IL-2 and IFN-g) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected ip with P. brasiliensis produced early and sustained levels of IFN-g and IL-2; type-2 cytokines (IL-4 and IL-5) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-g concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-ß and low levels of TNF-a. In contrast, macrophages from A/Sn animals released high levels of TNF-a associated with a small production of TGF-ß. The in vivo depletion of IFN-g not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.A animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-a and IFN-g followed by a sustained secretion of IL-2 and IFN-g plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-a and IFN-g associated with production of IL-5, IL-10 and TGF-ß characterizes the progressive disease of susceptible animals.

The low efficiency of dendritic cells and macrophages from mice susceptible to Paracoccidioides brasiliensis in inducing a Th1 response

In the present study we evaluated T cell proliferation and Th lymphokine patterns in response to gp43 from Paracoccidioides brasiliensis presented by isolated dendritic cells from susceptible and resistant mice. T cell proliferation assays showed that dendritic cells from susceptible mice were less efficient than those from resistant mice. The pattern of T cell lymphokines stimulated by dendritic cells was always Th1, although the levels of IL-2 and IFN-gamma were lower in T cell cultures from susceptible mice. To determie whether different antigen-presenting cells such as macrophages and dendritic cells stimulated different concentrations of Th1 lymphokines, the production of IFN-gamma and IL-2 was measured. It was observed that dendritic cells were more efficient than macrophages in stimulating lymphoproliferation in resistant mice. However, no significant difference was observed for IFN-gamma or IL-2 production. When cells from susceptible mice were used, macrophages were more efficient in stimulating lymphoproliferation than dendritic cells, but no difference was observed in the production of Th1 cytokine. Taken together, these results suggest the lower efficiency of dendritic cells and macrophages from B10.A mice in stimulating T cells that secrete Th1 lymphokines in vitro, an effect that may be involved in the progression of the disease in vivo.

Micoses sistémicas

Este trabalho teve como objetivo a análise das micoses sistémicas, no que respeita à sua identificação, morfologia dos agentes patogénicos que lhes dão origem, onde são endémicas, os sintomas que apresentam, os métodos de diagnóstico e as terapêuticas disponíveis para cada patologia. As micoses sistémicas são infeções causadas por fungos patogénicos primários que têm o trato respiratório como porta de entrada, e a partir daí podem disseminar-se por todo o organismo. Os agentes antifúngicos são usados no tratamento destas infeções, em que, no caso das infeções sistémicas, predominam o uso de cetoconazol (polieno), fluconazol e itraconazol (azóis). Existem ainda estudos que demonstram que as vacinas atenuadas podem ser usadas na profilaxia destas infeções. Com este trabalho conclui-se que as infeções fúngicas estão longe de ser extintas, uma vez que se verifica cada vez mais a existência de resistências por parte dos fungos aos fármacos, sendo o diagnóstico e a terapêutica usada dois parâmetros fundamentais para um tratamento eficaz.