In Vitro Effect of Sulfamethoxazole-Trimethoprim against Histoplasma capsulatum var. capsulatum

This study evaluated the in vitro effect of sulfamethoxazole-trimethoprim against Histoplasma capsulatum var. capsulatum isolated from HIV-positive patients. The drugs were tested by microdilution testing in accordance with the CLSI guidelines. All of the strains were inhibited by sulfamethoxazole-trimethoprim, with MIC ranges of 0.039 (sulfamethoxazole)/0.0078 (trimethoprim) mg/ml to 0.625/0.125 mg/ml for mycelial forms and 0.0025/0.0005 to 0.02/0.004 mg/ml for yeast-like forms. However, in vivo studies are necessary to evaluate the significance of these results.Histoplasmosis is an infection caused by the dimorphic fungus Histoplasma capsulatum. It is endemic in the Americas (3) and is currently one of the most important systemic infections in Brazil (6). In a retrospective study carried out in Ceará state (northeastern Brazil) from 1995 to 2004, 164 histoplasmosis cases were found in HIV-positive patients (4).The treatment of histoplasmosis depends on the infection''s severity and clinical manifestation, along with individual risk factors (9, 13). Because of the increase in histoplasmosis, particularly in HIV-positive patients, as well as the development of antifungal resistance associated with refractory and repeated infections (13), there is a need for seeking new therapeutic options for this mycosis. Therefore, this study aimed at testing the in vitro activity of sulfamethoxazole-trimethoprim (SMX-TMP) against H. capsulatum var. capsulatum strains isolated from AIDS patients.A total of 84 clinical strains of H. capsulatum isolated from two different biogeographic regions in Brazil were included in this study. Of them, 68 came from Ceará (northeastern semiarid region) and 16 from southeastern states (a subtropical region). The strains were obtained from the collection of the Specialized Medical Mycology Center of Ceará Federal University and were handled in our level 3 biosecurity laboratory.For each strain, a combined solution of SMX-TMP (Roche, Brazil) was used at a concentration range of 0.0025 mg/ml SMX/0.0005 mg/ml TMP and 20/4 mg/ml. The inocula were prepared as described by Li et al. (10), with some modifications. Briefly, H. capsulatum strains were cultured in the mycelial phase onto brain heart infusion (BHI) agar for 7 days at 28°C, and then 2 ml of sterile saline was added to each culture. The surfaces of the mycelia were harvested with a swab. To obtain yeast cells, isolates were cultured on agar BHI with sheep blood (10%) at 35°C and were maintained through weekly passages (7). The supernatant was read in a spectrophotometer at 530 nm, and the transmittance was adjusted to 90 to 95%. The suspensions then were diluted to obtain an inoculum of 0.5 × 10³ to 2.5 × 10⁴ CFU/ml, as demonstrated by quantitative colony counts on Sabouraud dextrose agar (10). Susceptibility tests were carried out as described by Nakai et al. (11), except that the readings were performed after 7 or 4 days for mycelial and yeast growth, respectively. For SMX-TMP, the MIC was defined as the lowest concentration at which no visible growth was observed (14). The differences in the MICs between northeastern and southeastern strains were evaluated by Student''s t test (P < 0.05). Susceptibility control tests were performed with amphotericin B (AMB) against 84 and 7 H. capsulatum strains in mycelial and yeast-like forms, respectively. SMX-TMP quality control was carried out with Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.All H. capsulatum strains were inhibited by SMX-TMP, with MICs ranging from 0.039/0.0078 to 0.625/0.125 mg/ml for the mycelial forms and 0.0025/0.0005 to 0.02/0.004 mg/ml for the yeast-like forms. The SMX-TMP MICs for strains from northeastern Brazil presented geometric means of 0.1544 mg/ml for sulfamethoxazole and 0.0309 mg/ml for trimethoprim for the mycelial forms and 0.0141 mg/ml for sulfamethoxazole and 0.0028 mg/ml for trimethoprim for yeast-like forms. The geometric means for the strains from southeast Brazil were 0.1152 mg/ml for sulfamethoxazole and 0.0230 mg/ml for trimethoprim for the mycelial forms and 0.0079 mg/ml for sulfamethoxazole and 0.0016 mg/ml for trimethoprim for yeast-like forms (Table