Synergistic Effect of Antituberculosis Drugs and Azoles In Vitro against Histoplasma capsulatum var. capsulatum

Abstract

This study evaluated in vitro interactions of antituberculosis drugs and triazoles against Histoplasma capsulatum. Nine drug combinations, each including an antituberculosis drug (isoniazid, pyrazinamide, or ethambutol) plus a triazole (itraconazole, fluconazole, or voriconazole), were tested against both growth forms of H. capsulatum. Stronger synergistic interactions were seen in isoniazid or pyrazinamide plus triazoles for the mold form and ethambutol plus voriconazole for the yeast-like form. Further studies should evaluate these combinations in vivo.Previously we demonstrated the inhibitory effect of some antituberculosis drugs alone or combined with antifungals against the pathogen Coccidioides posadasii (5, 6). Stronger synergistic interactions were seen in the combinations including ethambutol (ETB) plus triazoles as well as pyrazinamide (PZA) plus itraconazole (ITR). Based on these results, the purpose of this study was to investigate the effect of these combinations against the dimorphic pathogen Histoplasma capsulatum var. capsulatum-the etiological agent of American histoplasmosis, regarded as the most frequent systemic fungal infection worldwide (1, 7) and an important opportunistic infection among AIDS patients (7,8,13,14). The emergence of resistance to fluconazole (FLC) as a cause of failure during treatment of histoplasmosis in patients with AIDS indicates a need for studies seeking new therapeutic options for this mycosis (13).A total of 18 strains of Histoplasma capsulatum var. capsulatum (henceforth called H. capsulatum) isolated in Brazil were included in the study. Most of the strains were recovered from AIDS patients with histoplasmosis and were isolated from bone marrow puncture (n ϭ 10) and peripheral blood (n ϭ 6). Two strains isolated from cutaneous ulcers of domestic felines with disseminated histoplasmosis were also tested. The strains belong to the fungal collection of the Specialized Medical Mycology Center (CEMM) of the Federal University of Ceará, Brazil. All procedures were performed inside a biosafety level 3 laboratory.For macrobroth testing, inoculum preparation was carried out as described by Li et al. (9) with minor modifications. First, the H. capsulatum strains were grown on brain heart infusion agar (BHI; BD Diagnostics) at 28°C for 7 days. Sterile 0.9% saline was added to the agar slant, and the cultures were gently scraped with cotton swabs. The suspensions were read at 530 nm and adjusted to 90 to 95% transmittance. The suspension was then diluted 1:10 with RPMI 1640 medium (Sigma Chemical Co.) containing L-glutamine and without sodium bicarbonate and buffered to pH 7.0 with 0.165 M MOPS (morpholinepropanesulfonic acid; Sigma Chemical Co.) to obtain an inoculum of approximately 0.5 ϫ 10 3 to 2.5 ϫ 10 4 CFU ⅐ ml Ϫ1 . Inoculum preparation for microbroth tests was performed as described by Brilhante et al (3). The suspensions were diluted with RPMI medium buffered to pH 7.0 with MOPS to obtain an inoculum of approximately 0.5 ϫ 10 3 to 2.5 ϫ 10 4 CFU ⅐ ml Ϫ1 . The densit...

Synthesis and Antifungal Activity In Vitro of Isoniazid Derivatives against Histoplasma capsulatum var. capsulatum

“…Some of these compounds have been described as more efficient than standard antibiotics (27). Considering the antifungal potential of INH previously described against the dimorphic pathogens C. posadasii (14) and H. capsulatum (10), in the present study, INH-derived hydrazones were evaluated against H. capsulatum in both yeast-like and filamentous forms. Synthetized hydrazone compounds showed antifungal activity, although all but one were effective at high concentrations only.…”

“…Further experimentation was performed only on the drugs that presented the lowest MIC values for both forms. INH derivatives were tested from 3.9 to 2,000 g/ml, based on H. capsulatum susceptibility to INH; AMB and ITC were tested from 0.002 to 1.00 g/ml and from 0.0001 to 0.0625 g/ml, respectively (10).…”

“…The MICs of the hydrazones were based on a previous study with INH and were defined as the lowest drug concentration that caused 80% inhibition of visible fungal growth (10) For AMB, the MIC was defined as the lowest drug concentration that prevented any discernible growth (100% inhibition). The minimum fungicidal concentration (MFC) was defined as the lowest concentration that inhibited fungal growth completely after seeding 0.1 ml of the fungal suspension at concentrations above the MIC onto potato agar for 15 days at 35°C (10).…”

“…Previous studies have shown the antifungal potential of antituberculosis drugs against the highly virulent pathogens H. capsulatum var. capsulatum and Coccidioides posadasii (10,14). We also evaluated the in vitro synergism of one of these isoniazid analogs, N=-(1-phenylethylidene)isonicotinohydrazide [N=-(1-phenyl)], with amphotericin B in both the planktonic and biofilm forms of H. capsulatum var.…”

“…In agents of severe infections, such as Histoplasma capsulatum (5), Cryptococcus gattii (6), and Coccidioides species (7), reduced susceptibility to antifungals has been detected worldwide. Therefore, many studies have been conducted to search for new antifungal drugs (8)(9)(10).…”