We evaluated the in vitro activities of voriconazole, itraconazole, and terbinafine against 30 clinical isolates of Pythium insidiosum using a checkerboard macrodilution method. The combined activity of terbinafine plus itraconazole or plus voriconazole was synergic against 17% of the strains. Antagonism was not observed.Pythium insidiosum is classified in the kingdom Stramenopila, class Oomycetes (3). It causes pythiosis, a disease mainly diagnosed in horses, dogs, and humans (14). Human pythiosis was first documented in 1985 (3). Since then, several cases have been reported, with high rates of morbidity and mortality (12). It is found mostly in Thailand, and two factors contribute to importance of pythiosis in that country: the prevalence of -thalassemia and the presence of large flooded areas used for agriculture (18). Combinations of antifungal agents have been poorly studied in medical mycology, and their activities against P. insidiosum are almost unknown (17).The aim of the present study was to investigate the in vitro activity of terbinafine (TRB) combined with itraconazole (ITC) and of TRB combined with voriconazole (VRC) against 30 isolates of Pythium insidiosum from animal pythiosis by using a macrodilution methodology based on the M38-A technique (10).This study included 28 Brazilian P. insidiosum strains obtained from animal pythiosis (horses, dogs, and sheep) and two standard strains (ATCC 58637 and CBS 101555). The identities of the isolates were confirmed by a PCR-based assay (13). The inocula consisted of P. insidiosum zoospores obtained as previously described (11). These were counted in a hemacytometer and diluted in RPMI 1640 broth containing L-glutamine and buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid, yielding a final concentration of 2 ϫ 10 3 to 3 ϫ 10 3 zoospores/ml. Candida parapsilosis (ATCC 22019) and Aspergillus flavus (ATCC 204304) were used as quality control organisms (10).The antifungal agents tested were TRB (Novartis) at 1 to 64 mg/liter, ITC (Sigma Pharma) at 0.125 to 32 mg/liter, and VRC (Pfizer) at 0.125 to 32 mg/liter. The interactions of the combinations (TRB-ITC and TRB-VRC) were evaluated by using the checkerboard technique according to the broth macrodilution design (2). The range of drug concentrations for use in the checkerboard assay was the same used in individual tests. Aliquots (50 l) containing different concentrations of each antifungal agent (seven of TRB and nine of triazole agents) were placed in tubes to provide 63 drug combinations; 0.9 ml of inoculum was added to each tube. The interactions were interpreted as synergistic (fractional inhibitory concentration index [FICI] Յ 0.5), indifferent (0.5 Ͻ FICI Յ 4), or antagonistic (FICI Ͼ 4) based on the respective FICI (5), using the following formula: FICI ϭ (MIC A in combination/MIC A) ϩ (MIC B in combination/MIC B). Off-scale MICs were converted to the next higher dilution for calculation purposes.MIC-1 and MIC-0 were used as the reading criteria for TRB and were determined as the lowest dru...