Indirect fluorescent test for detection of anti-Paracoccidioides brasiliensis antibodies using coated bentonite particles as antigen

An indirect fluorescent test was developed for detecting antibodies to Paracoccidioides brasiliensis using bentonite particles as antigen (Bent-IF). The bentonite particles were coated with P. brasiliensis polysaccharide antigen and tested with sera from paracoccidioidomycosis patients (36 sera), normal blood donors (32 sera) and patients with non-mycotic diseases (29 sera). The titres given by the positive sera were compared with those of complement fixation (CF), immunodiffusion (ID) and immunofluorescent test using yeast forms of the fungus as antigen (conventional-IF). All normal blood donors' sera gave a negative Bent-IF, conventional-IF, ID and CF tests. All paracoccidioidomycosis sera were reactive in conventional-IF and gave concordant results in Bent-IF. There was no correlation between CF and Bent-IF titres. 27·6% of sera from patients with non-mycotic diseases gave weak titres in both IF-tests. The present data indicate that the Bent-IF is a sensitive and simple serodiagnostic technique comparable with the conventional P. brasiliensis antibody test. © 1983.

The effect of Ketoconazole on experimental paracoccidioidomycosis in the Syrian hamster: immunological and histopathological study

The effect of Ketoconazole (KTZ) on the hamster experimental intratesticular paracoccidioidomycosis was studied employing different treatment schedules. KTZ long course treatment beginning at an early stage of the infection was effective in preventing fungal proliferation, dissemination to lymph nodes, spleen and kidneys, and in maintaining low levels of humoral and cellular specific immune responses. KTZ short course treatment starting at an advanced stage of disease resulted in a more severe histopathological picture without significant changes in the immunological profile. The drug prolonged the life span of hamsters infected with Paracoccidioides brasiliensis, but did not prevent mortality. Toxic necrosis of the bone marrow occurred in normal animals receiving 120 mg/kg/day of KTZ but with lower doses no morphologic alterations were observed in heart, lungs, kidneys, adrenals, spleen, liver, intestine or bone marrow. © 1984 Dr W. Junk Publishers.

Serology of paracoccidioidomycosis. II. Correlation between class-specific antibodies and clinical forms of the disease

Untreated and previously treated patients with paracoccidioidomycosis were studied for: (i) serum levels of total IgG, IgM and IgA immunoglobulins, by radial immunodiffusion and Paracoccidioides brasiliensis (Pb) antibodies, by indirect immunofluorescence; (ii) correlation between their levels with the clinical forms of the disease; (iii) correlation between the serum titres obtained by tube precipitin with those of anti-Pb IgG, IgM and IgA. In the untreated group, serum IgG levels were significantly increased in patients with the more systemic forms of the disease, especially the acute progressive form. Serum IgA levels were significantly increased in all patients with no statistical difference between clinical forms. Serum IgM levels were normal in all patients. Anti-Pb IgG, IgA and IgM were detected in 97·5%, 32·5% and 45·0% of all cases, respectively. There was a sharp tendency towards higher levels of anti-Pb IgG among those with the acute progressive form (83·4%) in relation to the chronic, more localized forms, mixed form (68·0%) and isolated organic form (55·5%). In the untreated and previously treated group sera, there was positive correlation between the level of anti-Pb IgG and positivity for the tube precipitin test, suggesting that the precipitin-type antibodies are of the IgG class. Broadly, the present data demonstrate a polyclonal activation of the humoral immune system in paracoccidioidomycosis, with a positive relationship between serological results and severity of the disease. © 1984.

Skin and pulmonary models using coated bentonite particles for the study of the inflammation evoked by Paracoccidioides brasiliensis antigens in previously immunized mice

Bentonite particles coated with polysaccharide antigen or crude soluble antigen of Paracoccidioides brasiliensis were injected intradermally or intravenously in mice. In control animals that were not pre-immunized with P. brasiliensis antigens, coated and uncoated bentonite caused minimal and nonspecific inflammation around the cutaneous injection site or around the bentonite thrombi in small lung vessels after intravenous injection. However, in mice previously immunized with P. brasiliensis antigens, the coated bentonite particles boosted the humoral and cellular immune responses to P. brasiliensis and evoked intense inflammatory reactions. Twelve days after intradermal injection, the inflammatory reaction around the bentonite was rich in neutrophils, macrophages, lymphocytes and plasma cells associated with young granulation tissue. In intravenously injected mice, the pulmonary inflammation was maximal at day 2, and was characterized by a florid neutrophilic and macrophagic cellular infiltration around bentonite thrombi; in some foci, there was incipient organization to mature granuloma. However, in both models, there was no formation of epithelioid granulomata, demonstrating that in paracoccidioidomycosis cellular immunity alone, without the presence of intact micro-organisms, may not be enough for the development of this type of granuloma.

Correlation between cell-mediated immunity and clinical forms of paracoccidioidomycosis

Cellular immune response to specific and non-specific stimulants was investigated, both in vivo and in vitro, in 29 healthy controls and in 53 previously untreated patients with the chronic isolated organic form (CIOF), the chronic mixed form (CMF) and the acute progressive form (APF) of paracoccidioidomycosis. The study included skin tests to Paracoccidioides brasiliensis antigen (PbAg) and phytohaemagglutinin (PHA), DNCB sensitization, determination of T lymphocytes and complement rosette-forming cells, lymphocyte transformation and leucocyte migration inhibition tests using PbAg and PHA. Patients displayed staggered cutaneous response to PHA and to PbAg, with marked decrease in intensity in the APF group. DNCB sensitization test and proliferative response of lymphocytes to PHA and PbAg were severely depressed in most of the patients. Leucocyte migration inhibition indices to PbAg were highly positive, while response to PHA was slightly decreased regardless of the clinical form. The number of T lymphocytes was reduced in most of patients and in them the number of complement-rosette forming cells was normal. The distribution of patients according to a suppression index, based in the results of the tests employed, revealed a tendency towards an increased degree of cellular immunosuppression from the least severe (CIOF) to the most severe (APF) clinical form of the disease. On the whole, the present study demonstrated a gamut of immunological reactivity in paracoccidioidomycosis. © 1985.

Ketoconazole in the treatment of experimental murine paracoccidioidomycosis

In a murine model of chronic disseminated paracoccidioidomycosis (strain 18; intravenous route), Ketoconazole (200 mg/kg in 0.2% agar) was given daily by gavage in three different schedules. Continuous treatment from an early stage of infection (day 3) up to week 20 was the most effective protocol, leading to remission of histopathological lesions and of both humoral and cellular anti-P. brasiliensis immune response, and clearance of the fungus in lungs; only 1 treated animal at week 20 showed pulmonary granulomas, although less extensive than control mice. Continuous treatment from early stage up to week 8, followed by a 16 week-period of drug discontinuity, caused remission of lesions in all but 3 treated mice which showed active pulmonary paracoccidioidomycosis similar to controls (14.2% of unresponsiveness to treatment). The continuous Ketoconazole protocol since a late stage of infection (week 4) up to week 20 produced a slower remission of lesions and immune response when compared with the first drug schedule. In this model of paracoccidioidomycosis, Ketoconazole showed no detectable side-effects and was a very effective drug especially in a prolonged administration protocol from an early stage of infection.

Host-parasite relationships in paracoccidioidomycosis

A viewpoint of host-parasite relationships in paracoccidioidomycosis is presented. The characteristics of the fungus which are important to the host-parasite interaction are discussed. Aspects of inhibition of mycelium-to-yeast transformation by estrogens acting at receptors on the fungal wall and in the cytoplasm, and the role of polysaccharide components of the cell wall in virulence are reviewed. The natural mechanisms of host defense are also examined, including phagocytosis, complement system, natural-killer cells and genetic control of resistance and susceptibility. Finally, a discussion of granuloma morphogenesis and its relationship to the humoral and cellular anti-P. brasiliensis immune response is presented.

Experimental pulmonary paracoccidioidomycosis in the Syrian hamster: morphology and correlation of lesions with the immune response.

A model for pulmonary paracoccidioidomycosis in the hamster is described. The disease was induced by intratracheal inoculation of 1.7 x 10(5) viable yeast forms of P. brasiliensis. Lung histopathology, dissemination lesions and humoral and cellular immune responses were investigated at intervals up to 24 weeks after infection. Humoral immunity was studied by immunodiffusion and complement fixation tests. Cell-mediated immunity was evaluated in vitro by the macrophage migration inhibition test in the presence of phytohaemagglutinin and P. brasiliensis soluble antigen, and in vivo by the paracoccidioidin test. Thirty out of 35 infected animals (85.7%) developed pulmonary paracoccidioidomycosis. Dissemination lesions were observed in regional lymph nodes (82.8%), liver (8.5%) and spleen (5.7%). Lung involvement was mainly around bronchi and vessels. Regional lymph nodes were severely involved from the fourth week on, acquiring a pseudotumoral aspect at later stages. Specific antibodies were detected from the fourth week on, with titres increasing progressively. The cellular immune response to phytohaemagglutinin was intact throughout the experiment and the response to P. brasiliensis antigen was already detectable by the second week and remained positive to the end of the experiment. The skin test became positive from the fourth week on. Inoculation by the intratracheal route represents a highly effective way of infecting hamsters with P. brasiliensis, with the induction of localized disease, good antibody production and intact cell immunity.

Paracoccidioidomycosis: study of six cases with ocular involvement

We present 6 patients with ocular involvement due to paracoccidioidomycosis. All cases were confirmed by the finding of Paracoccidioides brasiliensis in histopathological or direct mycologic examination of material from the lesion in the eyelid or conjunctiva. In two cases the bulbar conjunctiva was also involved, in another the cornea, and still another patient developed endophthalmitis. The presence of this mutilating disease which may lead to blindness should be suspected when chronic blepharitis or palpebral ulcerated papular lesions are detected in patients from endemic areas of paracoccidioidomycosis. This etiology should also be suspected in patients with anterior and posterior uveitis after discarding the most frequent causes of this condition. © 1988 Kluwer Academic Publishers.

Detection of the 43,000-molecular-weight glycoprotein in sera of patients with paracoccidioidomycosis

The 43,000-molecular-weight (43K) soluble glycoprotein was detected in sera of patients with paracoccidioidomycosis by the immunoblot technique by using as the probe rabbit monospecific antisera to this fraction. The 43K antigen was present before treatment in sera of patients with the acute (juvenile) form; it started to disappear from circulation after 10 months of chemotherapy, and it was undetectable afer 2 years of treatment. In the chronic cases, the 43K antigen was detected in patients without treatment, and it was absent in the healed cases. The detection of the 43K protein specific to Paracoccidioides brasiliensis may be important for its diagnostic value as well as for modulation of the host immune response.

Antibody response to the 43 kDa glycoprotein of Paracoccidioides brasiliensis as a marker for the evaluation of patients under treatment

Sera of patients with paracoccidioidomycosis contained IgG-, IgA-, and IgM-specific antibodies to a 43 kDa antigen contained in the filtrate of a culture of Paracoccidioides brasiliensis. IgG- and IgA-specific antibodies were present in all observed patients. The IgM response was more frequent in acute cases, and the mean titers of IgG- and IgM-specific antibodies were higher in the acute forms. By the fourth month of chemotherapy, there was a decay of IgG, IgA, and IgM antibody titers to this antigen in acute cases, correlating with clinical improvement. The detection of IgG and IgA antibodies and the sequential determination of antibodies to the 43 kDa glycoprotein may be useful tools for serodiagnosis and evaluation of therapeutic efficacy.

Sputum cytology in the diagnosis of pulmonary paracoccidioidomycosis

The presence of Paracoccidioides brasiliensis was determined in sputum samples from 50 patients with paracoccidioidomycosis using four different techniques: (a) cell-block preparations stained with silver methenamine, (b) direct microbiologie examination, (c) smears stained with Shorr, and (d) smears stained with silver methenamine. Overall, cell-block preparations and smears stained with silver methenamine proved to be the most sensitive techniques, followed by smears stained with Shorr and direct microbiologic examination in decreasing order of sensitivity. Sputum cytology tended to be less positive in patients with interstitial pulmonary lesions as determined by chest X-ray than in patients with alveolar lesions. In addition to its high sensitivity, cell-block preparation technique allows storage of blocks and slides for further studies. © 1991 Kluwer Academic Publishers.

Pulmonary paracoccidioidomycosis in immunized mice

Paracoccidioidomycosis was induced in immunized (IM) and non-immunized (NI) mice. The histopathology, the number of fungi in the lungs, the cellular (footpad test - FPT and macrophage inhibition factor assay - MIF) and humoral (immunodiffusion test) immune response were investigated serially postinfection. In the IM mice, at days 1 and 3, there was intense and predominant macrophagic-lymphocytic alveolitis with loose granulomatous reaction; at day 30, inflammation was mild. In the NI group, up to day 3, the lesions were focal; later there was formation of extensive epithelioid granuloma. The number of fungi in IM mice were always smaller than those of NI group. Immunization alone induced positive FPT and MIF indices with low titer of antibody. After infection, there was a significant decrease of the FPT indices in the IM group, which we interpreted as desensitization due to trapping of sensitized lymphocytes in the lungs. In conclusion, (1) The lesional pattern of pulmonary paracoccidioidomycosis in IM mice was similar to that of a hypersensitivity pneumonitis. This reaction was probably effective in reducing the extension of the infection and decrease the number of fungi. (2) In this model, pulmonary resistance against P. brasiliensis seems to be related to local and systemic delayed-type hypersensitivity reaction. © 1992 Kluwer Academic Publishers.