Synergistic Activities of Three Triazoles with Caspofungin against Candida glabrata Isolates Determined by Time-Kill, Etest, and Disk Diffusion Methods

Abstract

Combinations of voriconazole, fluconazole, and itraconazole with caspofungin were evaluated against 50 Candida glabrata isolates by the time-kill, disk diffusion, and Etest methods. The majority of antifungal combinations were indifferent. By the time-kill method, synergistic activity was detected with eight (16%) of the caspofungin-voriconazole and seven (14%) of the caspofungin-fluconazole combinations, but synergy was not seen with the caspofungin-itraconazole combination. Further comparisons of the Etest and disk diffusion synergy techniques with the time-kill method are warranted.Candida glabrata infections are common in immunocompromised hosts and difficult to treat since they are often resistant to azole antifungal agents (2, 10, 12). The echinocandin caspofungin (CAS) inhibits the synthesis of 1,3-␤-D-glucan, an essential cell wall compound (19). Voriconazole (VORI), fluconazole (FLU), and itraconazole (ITRA) are triazole antifungal agents and inhibit the synthesis of ergosterol by inhibiting the enzyme lanosterol 14␣-demethylase (3). As these drugs act on different targets, it is important to look for combinations of drugs that might be synergistic. The main objective of our study was to test for synergistic activities of VORI, FLU, and ITRA combined with CAS against C. glabrata isolates. The other objectives were to compare results from different synergy testing methods, namely, the time-kill method, Etest, and disk diffusion method, and to evaluate the CLSI and Etest methods in terms of categorical agreement.Fifty clinical isolates were tested. Candida parapsilosis ATCC 22019 was included for quality control (6). Stock solutions of VORI (Pfizer, Barcelona, Spain), CAS (Merck & Co., Inc., West Point, PA), FLU (Pfizer, Barcelona, Spain), and ITRA (Jansen-Cilag) were prepared with the appropriate solvent (dimethyl sulfoxide for VORI and ITRA and distilled water for CAS and FLU). The final concentrations were 0.03 to16 g/ml for VORI, 0.0625 to 64 g/ml for CAS and FLU, and 0.5 to 8 g/ml for ITRA. MICs of drugs were determined by the CLSI broth microdilution method (BMD) (6) and corresponded to the lowest concentration that showed prominent (Ն50%) growth inhibition. MICs were read after 24 and 48 h of incubation and were evaluated (5, 6, 9). The Etest was performed on RPMI 1640 agar plates as recommended by the manufacturer, and MICs were compared to the reference BMD MICs (AB Biodisk, Solna, Sweden) (1).Synergy testing was performed using the time-kill, Etest, and disk diffusion methods. All testing was carried out in duplicate.For the time-kill studies, each isolate was tested against drugs alone and in combination at concentrations equal to each drug's MIC to correlate with the Etest. The numbers of CFU were determined at 0, 2, 6, and 24 h. The limit of detection was 50 CFU/ml. Synergy and antagonism were defined, respectively, as a Ն100-fold increase or decrease in killing compared with the killing of the most active single agent. If the change was less than 100-fold, the interaction was considered indi...

Synergy of β-Lactams with Vancomycin against Methicillin-Resistant Staphylococcus aureus: Correlation of Disk Diffusion and Checkerboard Methods

“…The disk diffusion test was previously shown to be a valid method by which to detect synergy for combinations of vancomycin and ␤-lactam antibiotics against staphylococci with reduced susceptibility to vancomycin (8) and to detect synergistic activities of triazoles with caspofungin against Candida glabrata (12). It is more rapid, less expensive, and less labor intensive than the more commonly used time-kill or checkerboard method.…”

“…The current study was designed to determine the utility of a modified disk diffusion (MDD) test to assess synergy and antagonism for combinations of vancomycin and ␤-lactam antibiotics among clinical isolates of MRSA (8,12). The CLSI checkerboard method was used as the reference for the determination of synergy.…”

Fluconazole Assists Berberine To Kill Fluconazole-Resistant Candida albicans

“…The combinations of antimicrobial agents against drug-resistant Candida have been studied (12)(13)(14)(15). Members of our group have shown that concomitant use of berberine (BBR; an alkaloid with a long history of medicinal use in traditional Chinese medicine [16]) and FLC is highly efficacious in killing FLC-resistant C. albicans in vitro, and the fractional inhibitory concentration (FIC) index ranges from 0.017 to 0.127 (17,18).…”

Evaluation of paper gradient concentration strips for antifungal combination testing of Candida spp.

“…There are no studies evaluating MTS performance for in vitro combination testing of Candida spp., whereas few studies used Etest method. [11][12][13][14] None of these studies present a quantitative correlation between the gradient concentration strip and microdilution method. When C. glabrata isolates were tested against amphotericin B+caspofungin combination with Etest strips placed simultaneously at 90°a ngle, the diffusion of the drugs through the agar was different resulting in dissimilar ellipse patterns and the agreement with the time-kill method was only 52%.…”

“…By contrast, other already introduced to the market products are made of plastic (non-porous), impregnated with a predefined concentration gradient of antifungal drug across 15 twofold dilutions of a conventional dilution method. Although gradient concentration strip diffusion assays have been used for in vitro antifungal combination testing against Candida spp., [11][12][13][14] a head-to-head comparison with chequerboard method exploring their reproducibility and agreement using different in vitro parameters is lacking. Furthermore, there are no studies evaluating the performance of the new paper strips for in vitro combination testing of Candida spp.…”