R. Negroni

Preparacion y estudio de un antigeno celular de Paracoccidioides brasiliensis, util para pruebas cutaneas

SummaryA cellular antigen from Paracoccidioides brasiliensis, obtained from the supernate of the disrupted yeast phase, was studied. It was prepared by a method similar to that for obtaining the spherulin from Coccidioides immitis. This antigen is composed of 55 μg/mg of polysaccharides, determined by the phenol method, and 7·8 μg/mg of protein, determined by Folin-Ciocalteus technique.In vitro tests of antigenicity were done by immunodiffusion and complement fixation. The optimum dilution of the antigen was 0·5 mg/ml determined by microtiter complement fixation.The in vivo standardization of the cellular paracoccidioidin was performed on three groups of patients. The first was composed of 56 patients with mycologically proved paracoccidioidomycosis. The disease was active in 32 and clinically healed in 24 of this group. The second was composed of 15 cases of histoplasmosis with strong histoplasmin skin tests. The third group were 70 patients with no mycotic pulmonary diseases who had come from different...

Itraconazole and flucytosine+itraconazole combination in the treatment of experimental cryptococcosis in hamsters.

Summary. The efficacy of two different daily doses of itraconazole (ITRA) and the combination of flucytosine (5‐FC) with ITRA in the treatment of an experimental model of cryptococcosis in hamsters was studied. Five groups of 20 animals each were inoculated by the intracardiac route with 105 cells of Cryptococcus neoformans. Treatment started 3 days after the infection, and was administered by gavage for 30 days. ITRA was applied at a daily dose of 25 mg kg‐1 or 50 mg kg‐1 and the combination of 5‐FC and ITRA was given at 75 mg kg‐1 day‐1 or 50 mg kg‐1 day‐1 respectively. One group of 20 hamsters received the vehicle and was used as a control group. Treatment evaluation was based on the following parameters: number of surviving animals 60 days after the infection; presence of encapsulated yeasts on microscopic examination of wet preparations of brain, lungs, liver, spleen and kidneys at necropsy; and brain qualitative (massive seeding) and quantitative cultures (determination of colony forming units, CFU). ITRA 50 (50 mg kg day‐1) was the most effective treatment according to the studied parameters; 70% of brain cultures became negative and 95% of the treated hamsters survived to the end of the study period. ITRA efficacy was dose dependent. The combination of ITRA with 5‐FC was less effective than administering the drugs separately; the reason for this finding is not known. The results obtained in this study should encourage the use of high doses of ITRA in cases of disseminated cryptococcosis in humans.

Flucytosine+fluconazole association in the treatment of a murine experimental model of cryptococcosis.

The efficacy of flucytosine (5-FC) and fluconazole (FLU) association in the treatment of a murine experimental model of cryptococcosis, was evaluated. Seven groups of 10 Balb C mice each, were intraperitoneally inoculated with 10(7) cells of Cryptococcus neoformans. Six groups were allocated to receive 5-FC (300 mg/kg) and FLU (16 mg/kg), either combined and individually, by daily gavage beginning 5 days after the infection, for 2 and 4 weeks. One group received distilled water and was used as control. The evaluation of treatments was based on: survival time; macroscopic examination of brain, lungs, liver and spleen at autopsy; presence of capsulated yeasts in microscopic examination of wet preparations of these organs and cultures of brain homogenate. 5-FC and FLU, individually or combined, significantly prolonged the survival time of the treated animals with respect to the control group (p < 0.01). Animals treated for 4 weeks survived significantly longer than those treated for 2 weeks (p < 0.01). No significant differences between the animals treated with 5-FC and FLU combined or separately were observed in the survival time and morphological parameters. The association of 5-FC and FLU does not seem to be more effective than 5-FC or FLU alone, in the treatment of this experimental model of cryptococcosis.

Histopathological evolution of experimental paracoccidioidomycosis in Wistar rats

Twenty Wistar rats were inoculated, by the intracardiac route, with 0.5 ml each of a yeast phase suspension of Paracoccidioides brasiliensis Utero strain. The rats were sacrificed at regular intervals post-infection, at which time their lungs, heart, liver, spleen and kidneys were removed, fixed and stained for study. The parameters of interest for the lung specimens were: (a) extent of the lesions; (b) number of fungi; (c) presence of a lymphomononuclear halo. Extrapulmonary lesions were also sought. Until the fourth month post-infection, the lesions were progressive in nature, contained great numbers of viable fungi, and were surrounded by an important lymphomononuclear halo which tended to be confluent. At four and a half months p.i., the extent of the pulmonary lesions was reduced, the granulomas were less compact with fewer viable fungi, macrophages showed microvacuolation, and the lymphomononuclear halo was less pronounced. Extrapulmonary lesions, which were frequently identified in the first months post-infection, diminished from the seventh month onwards. The histological characteristics of extrapulmonary lesions were always the same as those found in the lungs. Infection tended to be controlled by the animals from the fourth month, but without complete resolution of the lesions.

Experimental coccidioidomycosis in hamsters. Disease kinetics and death curve in relation to infective dose

A study of experimental coccidioidomycosis in hamsters (Mesocricetus auratus) is presented. Two experiments were conducted on 75 animals inoculated intracardially with the mycelial form of Coccidioides immitis. The first research (experiment I) studied the kinetics of experimental disease in 15 hamsters inoculated with 300 C. immitis arthroconidia. The parameters studied were: (a) presence of macroscopic lesions in the brain, lungs, liver, spleen and kidneys; (b) microscopic identification of spherules in wet mount preparations of these specimens; (c) samples from all organs cultured at 37 °C on Sabouraud glucose agar; (d) blood cultures drawn every 24 h during the first week and subsequently every 48 h and (e) histopathological studies of all organs. The second experiment (experiment II) determined the relationship between the inoculum size and death curve in six groups of 10 animals each, which had received doses of 10, 50, 100, 150, 200 and 300 arthroconidia, respectively. On day 14 post‐inoculation, all the animals underwent skin tests and 1 ml of blood was obtained by cardiac puncture to detect antibodies. Disseminated disease with persistent fungaemia developed in all the studied animals. Coccidioides immitis was recovered from all organs, with the lungs being the first to present disease. Death occurred in all groups, regardless of the dose of arthroconidia and 83.3% died between day 22 and day 28 post‐infection. The use of this model is proposed for the biological standardization of antigens, the study of prophylactic measures and the ‘‘in vivo’’ evaluation of new antifungal treatments.

Efecto de la ciclofosfamida en ratas con paracoccidioidomicosis

Paracoccidioidomycosis is an endemic fungal disease widely distributed throughout Latin America. The potent immunosuppressor cyclophosphamide (CY) has been used to modulate host immune response to Paracoccidioides brasiliensis in an experimental model. Inbred male Buffalo/Sim rats weighing 250-300 g were inoculated with 5 x 10(6) P. brasiliensis cells of the yeast phase form by intracardiac route. One group of animals was treated with 20 mg/kg body weight at days +4, +5, +6, +7, +11 and +12 post-infection (pi.), while a control group was infected alone. No mortality was recorded in either group. Treated rats presented: a) a decrease in granuloma size, which contained less fungal cells; b) a lack of specific antibodies up to 35 days pi., and c) a significant increase in the footpad swelling test (DTH) against paracoccidioidin. Splenic cell transfer from CY-treated P. brasiliensis-infected donors to recipients infected alone led to a significant increase in DTH response in the latter versus untreated infected controls. Likewise, in treated infected recipients transferred with untreated infected donor spleen cells, footpad swelling proved greater than in controls. Thus, it would seem that each successive suppressor T lymphocyte subset belonging to the respective cascade may be sensitive to repeated CY doses administered up to 12 days pi.. Alternatively, such CY schedule may induce the appearance of a T cell population capable of amplifying DTH response.