Paracoccidioides spp. and Histoplasma capsulatum: Current and New Perspectives for Diagnosis and Treatment.

The thermally-dimorphic systemic fungal group includes several important human pathogens: Blastomyces dermatitides, Coccidioides immitis and C. posadasii, Histoplasma capsulatum, Paracoccidioides brasiliensis, P. lutzii, and Talaromyces (Penicillium) marneffei. They usually are geographically restricted and have natural habitats in soil or in plants, and when fungal propagules invade mammalian host by inhalation, they initiate an inflammatory reaction that can result in self-resolution of the infection or cause an acute or chronic disease. In the setting of the AIDS pandemic and the developments in modern medicine, such as immunosuppressive therapy in cancer surgery patients and in transplantation and autoimmune diseases, the incidence of endemic mycoses has progressively increased. Another important factor of the increased incidence of systemic mycoses in certain regions is the progressive devastation of tropical and subtropical forests. In this review, we focus on two of the most important systemic mycoses: paracoccidioidomycosis and histoplasmosis, and their major characteristics in epidemiology, clinical aspects and laboratorial diagnosis.

Prevalence of Candida spp., xerostomia, and hyposalivation in oral lichen planus--a controlled study.

OBJECTIVE: To determine the frequency of Candida spp., xerostomia, and salivary flow rate (SFR) in three different groups: patients with OLP (OLP group), patients with oral mucosal lesions other than OLP (non-OLP group), and subjects without oral mucosal lesions (control group). MATERIAL AND METHODS: Xerostomia as well as SFR was investigated in the three groups. Samples for isolation of Candida spp. were collected from OLP lesions (38 patients), non-OLP lesions (28 patients), and healthy subjects (32 subjects). RESULTS: There was no statistically significant difference regarding the frequency of xerostomia and hyposalivation among the three groups (P > 0.05). A higher prevalence for colonization by Candida spp. was found in the healthy subject as compared to that of patients with OLP (P = 0.03) and non-OLP (P = 0.02) groups. Low SFR was not a factor for colonization by Candida spp. CONCLUSIONS: Xerostomia and hyposalivation occur with similar frequency in subjects with and without oral lesions; also, the presence of oral lesions does not increase the susceptibility to colonization by Candida spp. It seems that any study implicating Candida spp. in the malignant transformation of oral lesions should be carried out mostly on a biochemical basis, that is, by testing the capability of Candida spp. to produce carcinogenic enzyme.

Usefulness of matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry for identifying clinical Trichosporon isolates.

Trichosporon spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for therapeutic management, but conventional identification based on biochemical traits is hindered by the lack of updates to the species databases provided by the different commercial systems. In this study, 93 strains, or isolates, belonging to 16 Trichosporon species were subjected to both molecular identification using IGS1 gene sequencing and matrix-assisted laser desorption ionisation-time-of-flight (MALDI-TOF) analysis. Our results confirmed the limits of biochemical systems for identifying Trichosporon species, because only 27 (36%) of the isolates were correctly identified using them. Different protein extraction procedures were evaluated, revealing that incubation for 30 min with 70% formic acid yields the spectra with the highest scores. Among the six different reference spectra databases that were tested, a specific one composed of 18 reference strains plus seven clinical isolates allowed the correct identification of 67 of the 68 clinical isolates (98.5%). Although until recently it has been less widely applied to the basidiomycetous fungi, MALDI-TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level.

Infliximab partially impairs the anti-Mycobacterium tuberculosis immune responses of severe psoriasis patients with positive tuberculin skin-test.

BACKGROUND: Infliximab and etarnecept are now widely used for treating severe psoriasis. However, these drugs, especially infliximab, increased the risk of tuberculosis reactivation. Surprisingly, epidemiological data suggest that the tuberculosis rate in patients taking infliximab in São Paulo State, Brazil, is similar to that of some developed, non-endemic countries. OBJECTIVE: The aim of this study was to better understand the effect of infliximab on Mycobacterium tuberculosis (Mtb) immune responses of psoriasis patients in an endemic setting (Brazil). METHODS: We evaluated the tuberculosis-specific immune responses of severe psoriasis patients and healthy individuals, both tuberculin skin test (TST) positive, in the presence/absence of infliximab. Patients had untreated severe psoriasis, no co-morbidities affecting the immune responses and a TST >10 mm. Healthy TST(+) (>10 mm) individuals were evaluated in parallel. PBMC cultures from both groups were stimulated with different Mycobacterium tuberculosis (Mtb) antigens (ESAT-6, 85B and Mtb lysate) and phytohemagglutinin, with or without infliximab (5 µg/mL). Parameters evaluated were TNF-α, IFN-γ and IL-10 secretion by ELISA, overnight IFN-γ ELISpot and lymphocyte proliferative response (LPR). RESULTS: Infliximab almost abolished TNF-α detection in PBMC supernatants of both groups. It also significantly reduced the LPR to phytohemagglutinin and the Mtb antigens as well as the IFN-γ levels secreted into day 5 supernatants in both groups. There was no concomitant exaggerated IL-10 secretion that could account for the decreases in these responses. ELISpot showed that, contrasting with the central-memory responses above, infliximab did not affect effector-memory INF-γ-releasing T-cell numbers. CONCLUSIONS: Infliximab affected some, but not all aspects of the in vitro antituberculosis immune responses tested. The preserved effector-memory responses, putatively related to exposure to environmental mycobacteria, may help to explain the lower than expected susceptibility to tuberculosis reactivation in our setting.

Leprosy in transplant recipients: report of a case after liver transplantation and review of the literature.

Leprosy still is an important public health problem in several parts of the world including Brazil. Unlike the diseases caused by other mycobacteria, the incidence and clinical presentation of leprosy seems little affected in immunosuppressed patients. We report the first case, to our knowledge, of a liver transplant patient who developed multi-bacillary leprosy. The patient presented with papules and infiltrated plaques with loss of sensation suggestive of leprosy 3.5 years after living-related liver transplantation for autoimmune hepatitis. A skin biopsy showing non-caseating macrophagic granulomas, neuritis, and intact acid-fast bacilli on Fite-Faraco stain, confirmed the diagnosis of borderline lepromatous leprosy. The donor of the liver did not show any evidence of leprosy. During follow-up, the patient presented 2 episodes of upgrading leprosy type I reactions, 1 mild before leprosy treatment, and 1 moderate 3 months after receiving standard multi-drug treatment (rifampicin, clofazimine, and dapsone). These reactions were accompanied by increase in liver function tests, especially of canalicular enzymes. This reaction occurred despite the patient's triple immunosuppression regimen. The moderate reaction was successfully treated with further immunosuppression (prednisone, 0.5 mg/kg). Currently, the patient is asymptomatic, off leprosy medication, with routine liver transplant follow-up. The dilemmas in diagnosis and management of such a case are discussed and the literature on leprosy in transplant recipients is reviewed.

Tissue and serum immune response in chronic hepatitis C with mild histological lesions.

The immunopathogenesis of chronic hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro and anti-inflammatory activity. We investigated the expression of inflammatory cells and cytokines in the liver and serum of 51 chronically HCV infected patients and compared them to data from two sets of normal controls: 51 healthy blood donors and 33 liver biopsies of healthy liver donors. We also assessed the relationship between selected cytokines and cell populations in hepatic compartments and the disease stage. Compared with controls, hepatitis C patients had a greater expression of portal TNF-alpha, TGF-beta and CD4(+) and acinar IFN-gamma, TNF-alpha, IL-1beta and IL-4, as well as a higher serum concentration of IL-2, IL-10 and TGF-beta. Significant positive correlations were found between portal CD4+ and TNF-alpha, portal CD8(+) and TGF-beta, portal CD45(+)RO and TNF-alpha, acinar CD45(+)RO and IFN-gamma and acinar CD57(+) and TGF-beta. In conclusion, we have shown that (i) in this sample of predominantly mild disease, the immune response was associated with a pro-inflammatory response pattern, (ii) CD4(+) T-lymphocytes played a major role in orchestrating the immune response and (iii) these events primarily took place in the portal space.

Tissue and serum immune response in chronic hepatitis C with mild histological lesions

The immunopathogenesis of chronic hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro and anti-inflammatory activity. We investigated the expression of inflammatory cells and cytokines in the liver and serum of 51 chronically HCV infected patients and compared them to data from two sets of normal controls: 51 healthy blood donors and 33 liver biopsies of healthy liver donors. We also assessed the relationship between selected cytokines and cell populations in hepatic compartments and the disease stage. Compared with controls, hepatitis C patients had a greater expression of portal TNF-á, TGF-â and CD4+ and acinar IFN-ã, TNF-á, IL-1â and IL-4, as well as a higher serum concentration of IL-2, IL-10 and TGF-â. Significant positive correlations were found between portal CD4+ and TNF-á, portal CD8+ and TGF-â, portal CD45+RO and TNF-á, acinar CD45+RO and IFN-ã and acinar CD57+ and TGF-â. In conclusion, we have shown that (i) in this sample of predominantly mild disease, the immune response was associated with a pro-inflammatory response pattern, (ii) CD4+ T-lymphocytes played a major role in orchestrating the immune response and (iii) these events primarily took place in the portal space.

Epithelial cells treated with genistein inhibit adhesion and endocytosis of Paracoccidioides brasiliensis.

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus' transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.

Two patients coinfected with Mycobacterium leprae and human immunodeficiency virus type 1 and naive for antiretroviral therapy who exhibited type 1 leprosy reactions mimicking the immune reconstitution inflammatory syndrome.

Two case reports of patients with human immunodeficiency virus type 1 (HIV-1) infection who developed leprosy are presented. Both developed type 1 leprosy reactions in the absence of antiretroviral therapy. Reactions have been described for a number of HIV-1- and Mycobacterium leprae-coinfected patients and have been considered to be part of an immune reconstitution inflammatory syndrome (IRIS) since the reactions were usually linked to the administration of highly active antiretroviral therapy. The reports of our two patients suggest that the type 1 reactions in patients with leprosy and HIV may not always be an IRIS manifestation but may be akin to the classical reactional state described for the natural course of leprosy infection, which occurs in leprosy patients due to the fluctuations of the antimycobacterial immune response, whether they are coinfected with HIV or not.

The role of gallium-67 scan in defining the extent of disease in an endemic deep mycosis, paracoccidioidomycosis: a predominantly multifocal disease.

The tools currently used to evaluate the extent of paracoccidioidomycosis (PCM) may be of limited value in detecting subclinical lesions. The aim of this study was to verify the role of gallium-67 whole-body scan in evaluating the extent of disease of 65 patients with active PCM. The (67)Ga scan findings were compared with the results of clinical evaluation, chest radiography and/or high-resolution computed tomography (CT), abdominal ultrasound (US) or CT, laryngoscopy, CT or magnetic resonance imaging (MRI) of the head, and technetium-99m methylene diphosphonate bone scan, obtained before treatment. Clinically unsuspected lesions were detected by imaging procedures in 21 patients (32%), mainly in the lungs (n=11), adrenals (n=6), and superficial (n=3) and deep lymph nodes (n=14). (67)Ga scan detected 100% of the cases with subclinical involvement in the lungs. Scintigraphy was superior to chest radiography in demonstrating lung disease (94% vs 81%). The lymphatic lesions were demonstrated by (67)Ga scan in all the clinically suspected cases and in nearly all unsuspected cases, and also revealed more extensive involvement than was clinically suspected in many of them. There was good agreement between (67)Ga scan and the other imaging procedures for the initial detection of thoracic and abdominal lymph nodes and bone involvement. (67)Ga imaging detected most cases of laryngopharyngeal disease with active inflammatory lesions found at indirect laryngoscopy. On the other hand, (67)Ga scan failed to demonstrate most of the adrenal and CNS lesions detected by abdominal US/CT and head CT/MRI. In conclusion, (67)Ga imaging is a useful tool for evaluating the location and extent of suspected and unsuspected lesions in PCM. It could serve as a screening method before the use of other diagnostic procedures, particularly in the detection of lung, superficial and deep lymph node and bone involvement.

Paracoccidioides brasiliensis-reactive antibodies in Brazilian blood donors.

In a survey for primary paracoccidioidomycosis (PCM) infection (and not the clinical disease), two groups of blood donors were analyzed. One study group was drawn from donors living in a rural area where PCM is endemic, and the other group from urban residents of a large city, São Paulo. Anti-Paracoccidioides brasiliensis (Pb) specific antibodies (IgG) in sera were analyzed by ELISA, using crude Pb exoantigens (exoAg) and purified specific Pb 43 kDa glycoprotein (gp43). The results showed that 21% of 700 rural samples and 0.9% of 350 urban samples were positive for exoAg and gp43. To avoid cross-reactions, the sera were adsorbed first with Histoplasma capsulatum antigens and secondly with Leishmania amazonensis antigens. In the first adsorption with H. capsulatum, reactivity to gp43 fell to 12.8% in the rural group and to 0% in the urban group. In the succeeding adsorption with L. amazonensis, this reactivity fell to 12.3% in the rural group. There was a statistically greater proportion of persons with gp43-reactive antibodies in rural group than in the urban group, indicating that rural residents had frequently become exposed to Pb and contracted primary, subclinical PCM. The present report is the first epidemiological study using ELISA to detect antibodies against gp43 in blood donors.

Randomized trial with itraconazole, ketoconazole and sulfadiazine in paracoccidioidomycosis.

Forty-two patients with active paracoccidioidomycosis were randomized to receive itraconazole (50-100 mg d(-1)), ketoconazole (200-400 mg d(-1)) or sulfadiazine (100-150 mg kg d(-1) up to 6 g d(-1)) for 4-6 months, followed by slow release sulfa until negativity of serological tests. All 14 patients in itraconazole and sulfadiazine groups and 13 in the ketoconazole group showed an adequate clinical response to the chemotherapy. One patient in the latter group showed treatment failure according to clinical and mycological criteria. The test of the hypothesis that the drugs reduced antibody levels up to ten months of treatment showed a p value equal to 0.0001 for itraconazole, 0.017 for ketoconazole and 0.0012 for sulfadiazine; this reduction was similar for the three groups. In this first randomized study for the treatment of paracoccidioidomycosis we could not show superiority of any one regimen over the others in the clinical and serological responses of patients with the moderately severe form of the disease.

Different kinetics in anti-cytomegalovirus immunity reconstitution evaluated by lymphocyte proliferation and IFN-gamma production in allogeneic and autologous bone marrow transplantation.

Allogeneic bone marrow transplantation (ALLOBMT) is associated with an increased risk of cytomegalovirus (CMV) morbidity compared to autologous BMT (AUTOBMT). To investigate this, we evaluated AUTOBMT and ALLOBMT patients regarding anti-CMV immune reconstitution at 1 and 4 months after BMT and on the day after first CMV antigenemia detection. Intermittent ganciclovir preemptive therapy was prompted by antigenemia of >or=2 cells. One month after transplant, AUTOBMT recipients already displayed larger CD8+ T cell numbers than ALLOBMT recipients, but comparably small CD4+ T cell numbers. Most AUTOBMT patients had positive CMV antigen (CMV-Ag)-induced lymphoproliferation (86%) and IFN-gamma secretion (86%), whereas this was infrequently seen in ALLOBMT patients (20 and 10%, respectively). This early AUTOBMT immune reconstitution was associated with a lower frequency of CMV reactivation up to +4 months in AUTOBMT (21%) than ALLOBMT patients (91%). At +4 months, most ALLOBMT recipients had also recovered CMV-Ag immune responses. At first antigenemia detection, all 3 AUTOBMT recipients already displayed anti-CMV immune functions and 2 cleared the infection without therapy, whereas of the 10 ALLOBMT recipients only 1 had positive lymphoproliferation. In the latter group, none had IFN-gamma secretion or cleared the infection without therapy. Thus, differences in anti-CMV immune reconstitution may help to explain the contrasting rates of CMV morbidity between ALLOBMT and AUTOBMT patients.

Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis.

Patients with paracoccidioidomycosis (PCM) display a certain degree of immunecompromise characterized by lymphocyte hyporesponsiveness to the main Paracoccidioides brasiliensis antigen (gp43). To determine whether cytokines are involved in this state, we evaluated the secretion of IL-2, IL-10 and IFN-gamma by peripheral blood mononuclear cells (PBMC) from patients with the acute (AF) and chronic (CF) forms of PCM and from healthy, P. brasiliensis-sensitized subjects. gp43-stimulated PBMC from healthy subjects produced substantial amounts of IL-2, IFN-gamma and IL-10, whereas PBMC from AF and CF patients produced low levels of IL-2 and IFN-gamma but substantial amounts of IL-10. Phytohaemagglutinin-induced cytokine secretion was comparable among AF and CF patients and healthy subjects, suggesting integrity of non-specific cellular immune mechanisms in PCM. gp43-pulsed adherent cells, but not non-adherent cells, were the main source of IL-10. Moreover, IL-2 and IFN-gamma secretion correlated inversely with the amount of specific antibodies produced by patients and healthy subjects. Our results suggest that the imbalance in cytokine production of patients with PCM plays a role in the gp43-hyporesponsiveness and the marked (non-protective) antibody production of these patients.

Paracoccidioidomycosis: a model for evaluation of the effects of human immunodeficiency virus infection on the natural history of endemic tropical diseases.

The interaction of human immunodeficiency virus (HIV) infection with endemic tropical diseases has become a major concern, but its mechanisms are still poorly understood. Paracoccidioidomycosis (PCM), a South America endemic deep mycosis, may provide an interesting model to investigate this interaction, as clinical-epidemiological features of most HIV-PCM-coinfected patients are difficult to classify into the standard acute and chronic forms of PCM. Such patients have presented clinical features indicative of an uncontrolled infection with lymphohematogenous dissemination, similar to the more severe, acute form. However, this infection probably resulted from reactivated latent foci that, in nonimmunocompromised hosts, leads to the less severe chronic form, characterized by mucosal lesions. We propose that a new outcome of the Paracoccidioides brasiliensis-host interaction is induced by concomitant HIV infection. This outcome probably reflects an impaired anti-P. brasiliensis immune response during coinfection that is similar to that seen in the acute form, although the patients have a chronic P. brasiliensis infection.

Differential antibody isotype expression to the major Paracoccidioides brasiliensis antigen in juvenile and adult form paracoccidioidomycosis.

We investigated the relationship between antibody response to the major Paracoccidioides brasiliensis antigen, a 43-kDa glycoprotein, and the two paracoccidioidomycosis (PCM) clinical presentations, the juvenile and the adult forms. Total immunoglobulin G (IgG), IgG isotypes, and IgA anti-gp43 antibodies were determined by enzyme-linked immunosorbent assay in patients'sera. Juvenile PCM patients had higher (P =.003) IgG anti-gp43 levels than adult form patients. IgG1 subclass levels, however, were comparable between the two clinical forms. Patients with the juvenile form had higher (P <. 001) IgG4, but lower (P =.03) IgG2 levels than patients with the adult form. The IgG4 isotype, regulated by interleukin 4, was found in all juvenile form patients but in only 12% of the adult form patients. In contrast, high levels of the IgG2 isotype, regulated by interferon-gamma, were found in 41% of the adult PCM patients, mainly those with a more benign disease, but in only 12% of the juvenile patients. IgG3 was either absent or detected at low levels. These results demonstrate, for the first time, specific IgG4 antibodies in the humoral immune response of patients with an endemic deep mycosis and suggest that the switch to the IgG subclasses in PCM is regulated by the patients' T-helper subset (Th-1 or Th-2) dominant cytokine profile. A possible role for IgG4 in the immunopathogenesis of the juvenile, more severe form of the disease is discussed. Finally, IgA was found mainly in adult form patients, probably as a result of the chronic mucosal antigenic stimulation characteristic of this form.

MRI of head and neck paracoccidioidomycosis.

Paracoccidioidomycosis, caused by the dimorphic fungus Paracoccidioides brasiliensis, is the most important systemic mycosis in Latin America. Imported cases have been reported in North America, Asia and Europe, in individuals who lived in endemic areas, sometimes many years before the development of clinical manifestations. The disease causes cutaneous and/or respiratory tract mucosal lesions as well as lymph node enlargement. Involvement of the oropharynx and/or the larynx, either alone or in association with pulmonary involvement, is one of the commonest clinical presentations. On MRI, the major features are mucosal lesions, usually hypointense on T1 weighted images and hyperintense on T2 weighted or fat suppressed images, affecting the oral cavity, oropharynx and larynx, with head and neck lymph node enlargement. Differential diagnosis includes other granulomatous infectious diseases, especially tuberculosis, and cancers such as squamous cell carcinoma and lymphomas.

In vitro inhibitory activity of tumor necrosis factor alpha and interleukin-2 of human immunoglobulin preparations.

Human immunoglobulin preparations have been used in a number of clinical settings with good results, although in many of them the mechanism of action is not yet known. One possible mechanism is the modulation of cytokine activity. This study investigated the presence of inhibitory activity in intravenous immunoglobulin (IVIg) and F(ab')2 fragment preparations to two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2). Cytotoxic activity of human recombinant TNF-alpha or TNF-alpha secreted by peripheral blood mononuclear cells (PBMC) on L929 cells and the proliferative activity of the IL-2 on CTLL-2 cells were examined. Human serum albumin (HSA) was used as control. F(ab')2 inhibited, in a dose-dependent fashion, the TNF-alpha activity secreted by PBMC serial dilutions or, at the higher concentrations (25 and 10 mg/ml), recombinant TNF-alpha activity. In contrast, IVIg was able to inhibit only at 25 and 10 mg/ml the TNF-alpha activity secreted by any PBMC dilution tested, and did not inhibit the recombinant TNF-alpha activity. With IL-2, however, even HSA was able to inhibit its proliferative activity, possibly through a carrier effect. The IVIg inhibition of IL-2 activity was not different from that of HSA, but F(ab')2, at 12.5 mg/ml, was capable of inhibiting significantly more the IL-2 activity than HSA. Our results suggest an anticytokine effect of the immunoglobulin preparations that this activity may be mainly mediated by variable regions of the immunoglobulins, and that the more pronounced effect of F(ab')2 may be due to its greater molar concentration compared to intact IgG molecules.