In Vitro Activity of Terbinafine Combined with Caspofungin and Azoles against Pythium insidiosum

Abstract

In this text we evaluated the in vitro antifungal activities of terbinafine combined with caspofungin, miconazole, ketoconazole, and fluconazole against 17 Pythium insidiosum strains by using the microdilution checkerboard method. Synergistic interactions were observed with terbinafine combined with caspofungin (41.2% of the strains), fluconazole (41.2%), ketoconazole (29.4%), and miconazole (11.8%). No antagonistic effects were observed. The combination of terbinafine plus caspofungin or terbinafine plus fluconazole may have significant therapeutic potential for treatment of pythiosis.Pythiosis is a life-threatening infectious disease in humans and animals that is caused by the aquatic oomycete Pythium insidiosum (9). Horses are the most frequently infected animals, and equine pythiosis typically involves ulcerative granulomas (8). In humans, the infection occurs as ophthalmic, subcutaneous, and systemic forms, which are frequently associated with ␣-and ␤-thalassemia (5, 7). Pythiosis therapy, which is based on amphotericin B or azoles, has been ineffective or controversial, and the associated prognosis for human and equine pythiosis is poor (5,7,8,9,12). Therefore, surgical procedures, including amputation, are often effective, but disease reoccurrence rates are unfortunately high (7).Combinations of antifungal agents against pythiosis have not been thoroughly studied, and therefore, such in vitro combinatory activities against P. insidiosum require attention (1, 6).The purpose of this study was to investigate the in vitro activity of terbinafine (TRB) combined with caspofungin (CAS), miconazole (MNZ), ketoconazole, and fluconazole (FLC) against 17 strains of Pythium insidiosum isolated from animals.A total of 15 Brazilian P. insidiosum strains isolated from equines with pythiosis and two standard strains (ATCC 58637 and CBS 101555) were tested. All strains were maintained in cornmeal agar, and strain identification was confirmed by a PCR-based assay (4).The susceptibility of the P. insidiosum strains to the antifungal agents was tested by microdilution, based on protocol M38-A2 (2). The inoculum consisted of P. insidiosum zoospores obtained following zoosporogenesis. Cell numbers of zoospores were counted on a hemocytometer; zoospores were diluted in RPMI 1640 containing L-glutamine and buffered to pH 7.0 with 0.165 M MOPS (morpholinepropanesulfonic acid) to obtain a final concentration range of 2 ϫ 10 3 to 3 ϫ 10 3 zoospores/ml (10). The combinations of TRB (Novartis) plus CAS (Merck), TRB plus MNZ (Labware), TRB plus ketoconazole (Janssen), and TRB plus FLC (Pfizer) were evaluated using the checkerboard technique, according to the broth microdilution design (2,14). In the individual tests, 100 l of each drug concentration was plated in microplate wells and an equal volume of the inoculum was added to each well. In the combination tests, the antifungals were plated at a 4ϫ concentrate of 50 l of drug A plus 50 l of drug B and 100 l of the inoculum, resulting in a final 1ϫ drug concentration of each compound....

Pythiosis cutánea en equinos tratados con acetonida de triamcinolona. Parte 1. Caracterización clínica

“…Much has been studied on the treatment of equine cutaneous pythiosis, from traditional antifungals, state-of-the-art antimycotics, antibiotic therapy, surgery and immunotherapy; however, none of the treatments studied have given satisfactory results in terms of producing a complete solution for the disease (5,17,21,22). Se ha estudiado mucho sobre el tratamiento de la pythiosis cutánea equina, desde antimicóticos tradicionales, antimicóticos de última generación, antibioticoterapia, cirugía e inmunoterapia, sin embargo, ninguno de los tratamientos estudiados ha dado resultados satisfactorios en el sentido de obtener una solución completa para la enfermedad (5,17,21,22).…”

“…Se ha estudiado mucho sobre el tratamiento de la pythiosis cutánea equina, desde antimicóticos tradicionales, antimicóticos de última generación, antibioticoterapia, cirugía e inmunoterapia, sin embargo, ninguno de los tratamientos estudiados ha dado resultados satisfactorios en el sentido de obtener una solución completa para la enfermedad (5,17,21,22).…”

“…Asimismo Pereira et al (21), testaron la carposfungina in vitro con una acción del 63%, sin embargo al ser testada in vivo mostró reducción de las lesiones, pero cuando el tratamiento fue suspendido el crecimiento de las lesiones reinició. Por otro lado, Cavalheiro et al (22) y Argenta (24), estudiaron in vitro antimicóticos de última generación en combinación de voriconazol con terbinafina e itraconazol con terbinafina demostrando sinergismo contra 17% de las muestras de ese estudio. Biava et al (25) informaron resultados pocos satisfactorios e inferiores al 25% de recuperación en estudios donde fueron testadas la anfotericina B, yoduro de sodio y de potasio al 10%, flurocitosina, ketoconazol asociados o no a la cirugía, así como la anfotericina B sistémica combinada con compresas de gasas embebidas en soluciones de anfotericina B y dimetilsulfóxido (DMSO).…”

“…Los antimicóticos de última generación como la carposfungina (un inhibidor de la síntesis de β-glucano) mostraron reducción de las lesiones, pero cuando el tratamiento fue suspendido el crecimiento de las lesiones reinició (21); Combinaciones de itraconazol y terbinafina o voriconazol y terbinafina, presentaron algún grado de desempeño contra aislados de P. insidiosum (22,24). Asimismo,…”

“…Similarly, Pereira et al (21) tested in vitro carposfungin with an action of 63%; however, when tested in vivo, it showed a reduction in lesions, but when the treatment ceased the growth of the lesions restarted. On the other hand, Cavalheiro et al (22) and Argenta (24) studied state-of-theart in vitro antifungals in combination with voriconazole with terbinafine and itraconazole with terbinafine, showing synergism against 17% of the samples from that study. Biava et al (25) reported unsatisfactory results and a recovery of less than 25% in studies where amphotericin B, 10% sodium and potassium iodide, flurocytosine, ketoconazole, and nonsurgery were tested, as well as systemic amphotericin B combined with gauze pads embedded in solutions of amphotericin B and dimethyl sulfoxide (DMSO).…”