Synergistic Activities of Fluconazole and Voriconazole with Terbinafine against Four Candida Species Determined by Checkerboard, Time-Kill, and Etest Methods

Abstract

The in vitro activities of fluconazole or voriconazole plus terbinafine were evaluated against 20 Candida isolates by the checkerboard, time-kill, and Etest methods. Synergism (C. albicans, C. glabrata, and C. tropicalis) and indifference (C. krusei) were observed. Correlation among methods was good. The Etest is a suitable method to determine drug interactions.The checkerboard and time-kill methods to determine in vitro interactions between drugs are time-consuming and cumbersome for use in clinical laboratories. In order to find a method that facilitates synergistic studies, our aim was dual: (i) to assess the in vitro activities of voriconazole (VRC) and fluconazole (FLC) combined with terbinafine (TRB) against four Candida spp. (resistant or susceptible to FLC and/or TRB) by the checkerboard and time-kill methods and (ii) to compare the results of these methods with those obtained by an Etest-agar dilution technique.Twenty blood isolates (Table 1) were tested. C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were included for quality control.Stock solutions of VRC, FLC (Pfizer, Barcelona, Spain), and TRB (Novartis, Barcelona, Spain) were prepared with the appropriate solvent (dimethyl sulfoxide for VRC and TRB and distilled water for FLC). The final concentrations were 0.002 to 2 g/ml for VRC, 0.06 to 64 g/ml for FLC, and 0.25 to 16 g/ml for TRB. MICs of drugs alone or in combination were determined by the NCCLS M27-A2 method (12) and corresponded to the lowest concentration that showed prominent (Ն50%) growth inhibition and by the Etest method as described below.Drug interactions were assessed by the following three methods described below: broth microdilution checkerboard, timekill, and Etest.(i) Broth microdilution checkerboard. The broth microdilution checkerboard method was performed by using the fractional inhibitory concentration (FIC) index, which is defined as the sum of the MIC of each drug when used in combination divided by the MIC of the drug when used alone. For computation of FIC indices, off-scale MICs were raised to the next highest MIC; synergistic and antagonistic FIC indices were defined as Յ0.5 and Ͼ4, respectively.(ii) Time-kill studies. One isolate of each species was selected, and tests were conducted as previously described (RPMI 1640 medium, 10 5 -CFU/ml inoculum, and 5-ml volume) (8). The drug concentrations tested alone were as follows: VRC, 16 and 1 g/ml; FLC, 32 and 2 g/ml; and TRB, 8 and 2 g/ml. For the combinationsVRC/TRB and FLC/TRB, the drug concentrations were as follows: VRC/TRB, 16/2, 1/2, and 1/8 g/ml; and FLC/TRB, 32/2, 32/8, 2/2, and 2/8 g/ml. At 0, 3, 6, 24, and 48 h, aliquots were removed to determine the number of CFU per milliliter. Synergy was defined as a Ն2-log 10 decrease in CFU per milliliter for a combination compared to the killing with the most active drug alone and an increase of Ն2 log 10 as antagonism. Experiments were conducted in duplicate and on 2 separate days.(iii) Etest studies. RPMI 1640 agar with 2% dextrose and 1, 2, and 8 g of TRB per ml, pre...

Evaluation of the best method to assess antibiotic potentiation by phytochemicals against Staphylococcus aureus

“…On the contrary, the agreement of time-kill assay with the other 3 methods was low. In general, the agreements obtained in the literature are diverse (Bonapace et al, 2000;Cantón et al, 2005;Lewis et al, 2002;Martin, 2010;Orhan et al, 2005;White et al, 1996). However, a variety of investigators found disagreement between checkerboard and time-kill results (Cappelletty and Rybak, 1996;Chang et al, 2007;Visalli et al, 1988;White et al, 1996).…”

“…This approach is based on some synergistic definitions applied for combinations involving 2 antimicrobials using checker-board (Mackay et al, 2000;Rand et al, 1996), Etest (Cantón et al, 2005;Lewis et al, 2002;Sopirala et al, 2010) and time-kill assay (Eliopoulos and Eliopoulos, 1988;NCCLS/CLSI, 1999). Table 1 presents the scheme proposed to classify an interaction as negative, indifferent, additive, or potentiation.…”

In vitro activities of antifungal agents alone and in combination against fluconazole-susceptible and -resistant strains of Candida dubliniensis

“…8,12 Thus, combining antifungal agents has been suggested as an alternative strategy, and the use of terbinafine in combination with azoles has been suggested as a potential therapeutic option. [4][5][6]13 Despite promising results obtained through some antifungal associations against Candida spp., only the combination of caspofungin with terbinafine has already been tested against C. dubliniensis. 14 Differential effects were detected when C. albicans and C. dubliniensis were exposed to the same antifungal combination; it is acceptable the evidence of negative effects against C. dubliniensis.…”

“…While the combination of azoles with amphotericin B has provided controversial results, 3 most of studies have found additive or synergistic effect when azoles were combined with terbinafine against C. albicans. [4][5][6] Other Candida spp. have also been evaluated, 5 but little is known about the effects of antifungal combinations against Candida dubliniensis.…”

“…[4][5][6] Other Candida spp. have also been evaluated, 5 but little is known about the effects of antifungal combinations against Candida dubliniensis.…”