A total of 60 clinical isolates of Paracoccidioides brasiliensis were tested for susceptibility to sulfadiazine and sulfadimethoxyne by the agar dilution technique. A modification of the Mueller-Hinton medium was devised which gave good growth of the yeast form. The minimum inhibitory concentrations for only 51.6% of the isolates were in the range of the recommended blood serum concentration (50 ,ug/ml (7,14).In spite of these observations, there has been no survey of the in vitro susceptibility of the causative agent, Paracoccidioides brasiliensis, to sulfonamides. Earlier studies either employed complex culture media, which could have antagonized the effect of the drug, or made use of the fungus mycelial phase, which is different from the phase found in tissues, that is, the yeast phase of this dimorphic fungus (5,8,15 This report describes the procedure developed for sulfonamide susceptibility testing and presents data on the susceptibility pattern of 60 different isolates of P. brasiliensis to sulfadiazine and sulfadimethoxyne. MATERIALS AND METHODSFungi. Sixty-five isolates of P. brasiliensis were used in this study. All were from clinical sources (Colombian patients) and represented the original isolate at time of diagnosis, with the exception of three which were obtained during relapses. The stock cultures were kept in Sabouraud glucose agar at approximately 25°C. When needed, they were converted to the yeast phase by subculturing in the special media described below and incubated at 36°C. Identity confirmation was obtained by demonstration of dimorphism and by the characteristic microscopical appearance, multiple budding yeast cells, in cultures incubated at 360C. Culture media. It was necessary to find a medium which would not antagonize sulfonamides and which, at the same time, could promote abundant growth of P. brasiliensis. Earlier attempts to culture the fungus in the conventional Mueller-Hinton medium (BBL Microbiology Systems, Cockeysville, Md.) used for sulfonamide susceptibility testing of bacteria, proved unsuccessful (4, 22). A series of experiments was performed by enriching such a medium with various chemicals known to promote growth in the fungus (1,17
Surgical specimens were obtained from a patient who had had life-threatening paracoccidioidomycosis 5 years previously. Residual lesions were found in the mesenteric lymph nodes; there were areas of caseous necrosis separated from the surrounding tissue by a fibrous capsule made of connective tissue. Abundant P. brasiliensis cells, many of which appeared degenerated, were observed in the necrotic material. Primary isolation was possible only.under the microaerophilic conditions offered by fluid media kept stationary at 36 °C. Cultures in solid media were obtained after serial passages and gradual accommodation of the strain to aerobic incubation. It is apparent that P. brasiliensis yeast cells can become accustomed to reduced oxygen tension in the tissues. In this way, they are able to withstand the passing of time without completely losing their viability.Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is isolated regularly from pathological specimens on a diversity of solid media incubated under aerobic conditions at either 37 °C or room temperature I-3, 4, 6]. For primary isolation, liquid media are seldom used 1-3, 4, 6-1. However, Pedroso found that the semianaerobic environment offered by liquid cultures in tall columns was advantageous for the primary isolation of the yeast phase [5]. The latter report, however, is not very detailed, thus hindering complete assessment of the finding.Recent experimental studies revealed that the yeast-like cells of this fungus can accommodate to the microaerophilic conditions present in stationary liquid cultures [9]. Long periods of latency are known to occur in human paracoccidioidomycosis [2] and we therefore wondered whether dormancy of P. brasiliensis, similar to that seen in vitro, might occur in vivo.We recently had the opportunity to study material from walled-off lesions obtained at surgery from a patient who had experienced active paracoccidioidomycosis 5 years previously. This report presents the results of the pertinent mycological and histopathological studies. CASE REPORTIn December 1975, a 17-year-old man presented because of cramping, progressive abdominal pain of approximately 3 months' duration. Two weeks before admission he developed vomiting and for the last 5 clays he had been unable to have bowel movements and his urine output had diminished. Examination showed
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