Assessment of the In Vitro Kinetic Activity of Caspofungin against Candida glabrata

Echinocandins have become the drug of choice in infections caused by Candida glabrata. The objective of this study was to evaluate the in vitro activity of caspofungin alone and in combination against C. glabrata. In vitro assays demonstrated that caspofungin alone showed excellent fungicidal activity against C. glabrata, including fluconazole-resistant strains. The combination of caspofungin and azole antifungals showed potential synergy against C. glabrata. Overall, caspofungin demonstrated excellent in vitro activity, alone and in combination, against strains of C. glabrata.Over the last two decades, Candida albicans has been replaced by the non-albicans Candida species, especially C. glabrata, as the cause of candidemia and invasive candidiasis (36). Fluconazole resistance and reduced susceptibility to azole antifungals, especially fluconazole, may explain the increasing prevalence of infections due to C. glabrata. The increasing prevalence of fungal infections due to C. glabrata has created the need for more-effective antifungals (12). For the past few decades, fluconazole and amphotericin B have been the drugs of choice in the management of candidiasis (23, 31). However, amphotericin B has well-known side effects, such as nephrotoxicity, and the azoles, especially fluconazole, are fungistatic, and there is documented primary and secondary resistance to them (23). Additionally, recent epidemiologic studies have shown that up to 25% of C. glabrata isolates have been found to be intrinsically resistant to fluconazole (27).Caspofungin belongs to the echinocandin class of antifungals (10, 18, 22). Several in vitro studies have demonstrated the excellent in vitro activity of caspofungin against many non-albicans Candida species, especially C. glabrata, including those that are fluconazole resistant (6, 25-28). Several clinical trials have also demonstrated excellent response rates to caspofungin in cases of C. glabrata infection (2, 19, 35).Despite the numerous in vitro and in vivo studies evaluating the antifungal activity of caspofungin against C. glabrata, there is a lack of data evaluating the fungicidal activity of caspofungin (1, 4-6, 11). It would be beneficial to evaluate and compare the fungicidal activity of caspofungin and that of conventional antifungal agents against C. glabrata.Recently, combination antifungal therapy has received increased attention. Most of the literature has evaluated combination therapy in cases of invasive aspergillosis. Due to the different mechanisms of action of caspofungin and other antifungals, combination antifungal therapy has excellent potential and may play a key role in the treatment of fungal infections. Several studies have demonstrated synergy, whereas others have reported indifference and occasionally antagonism (20, 33). Few studies have evaluated the effect of caspofungin in combination with other antifungals against Candida species (13, 14). Most in vitro combination studies evaluating caspofungin have used C. albicans or Aspergillus spp. as the prototype (7, 8, 32).The goal of this study was to evaluate the in vitro activity of caspofungin against clinical isolates of C. glabrata and compare the results with those for standard antifungal agents. Kinetic in vitro studies using time-kill assays (TKA) were used to evaluate both strains of C. glabrata. In addition, we also evaluated the antifungal activity of caspofungin with either fluconazole, voriconazole, or amphotericin B.Fifty isolates of C. glabrata were obtained from clinical samples from patients seen in a chronic vaginitis clinic. Quality control isolates were used in each testing batch and were obtained from American Type Culture Collection (ATCC; Rockville, MD). Quality control isolates included ATCC 90028 (C. albicans) and 6258 (C. krusei). All in vitro assays were performed in duplicate.Caspofungin was obtained as powder from Merck Research Laboratories, Rahway, NJ, and amphotericin B (AmB), fluconazole (Flz), and voriconazole (Vcz) were obtained from their respective manufacturers.In vitro susceptibility testing was done using broth microdilution assays in RPMI 1640 medium using CLSI M27-A2 guidelines (21). The minimal fungicidal concentration (MFC) was defined as the lowest concentration of an antifungal needed to produce a 99.9% kill.TKA were performed as previously described (11, 15). Two different C. glabrata isolates were evaluated, one fluconazole-susceptible strain (MIC, 1 μg/ml) and one fluconazole-resistant strain (MIC, >64 μg/ml). The MIC of caspofungin for these two isolates was 1 μg/ml. Time-kill curves for caspofungin and fluconazole using two different concentrations were compared. The concentrations of caspofungin tested were 1 and 4 μg/ml, while the concentrations of fluconazole were 8 and 128 μg/ml. The lowest limit of accurate and reproducible detectable colony counts was 100.Synergy studies were done using the checkerboard broth microdilution method. Drug interactions were assessed with a checkerboard titration, based on CLSI recommendations (21). The fractional inhibitory concentration index (FICI) was calculated for each combination. The FICI was calculated as FICI = MIC A combination/MIC A alone + MIC B combination/MIC B. A FICI of <0.5 indicates a synergistic effect, >0.5 to <1 indicates an additive effect, 1 to 2 indicates indifference, and >2 indicates an antagonistic effect. Synergy allows a >4-fold reduction in the MICs of individual drugs, compared to the MIC of the combination (20).The MIC of caspofungin against C. glabrata ranged from 0.125 to 1 μg/ml, whereas the MIC of fluconazole against C. glabrata ranged from 2 to 64 μg/ml; the MIC of voriconazole ranged from 0.03 to 16 μg/ml, and the MIC for amphotericin B ranged from 0.5 to 1 μg/ml. The MIC₉₀ of caspofungin, amphotericin B, and voriconazole was 1 μg/ml, whereas the MIC₉₀ of fluconazole was 32 μg/ml (Table ​(Table11).

TABLE 1.

MIC range, MIC₅₀, MIC₉₀, and MFC of caspofungin, amphotericin B, fluconazole, and voriconazole against 50 C. glabrata isolates

Antifungal	MIC range (μg/ml)	MIC50 (μg/ml)	MIC90 (μg/ml)	MFC (μg/ml)
Caspofungin	0.125-1	0.5	2	4
Amphotericin B	0.5-1	0.5	1	1
Voriconazole	0.03-16	0.125	1	>32
Fluconazole	2-64	4	32	>128

Open in a separate windowThe mean fungicidal activity of caspofungin was established at 4 μg/ml, compared to an MFC of 0.125 to 1 μg/ml for amphotericin B, >32 μg/ml for voriconazole, and >128 μg/ml for fluconazole. The MIC₉₀ of caspofungin was 2 μg/ml, compared with 1 μg/ml for amphotericin B. The MIC₉₀s of voriconazole and fluconazole were 32 μg/ml and 128 μg/ml, respectively (Table ​(Table11).The time-kill assays evaluating the fluconazole-susceptible strain revealed continuous growth of C. glabrata when fluconazole was used, even at a concentration of 128 μg/ml. In contrast, with caspofungin at 4 μg/ml, the TKA revealed a 99.9% fungicidal activity at 4 to 6 h, and there was a 99.9% fungicidal activity at 4 h with caspofungin at a concentration of 1 μg/ml (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.Time-kill assays evaluating caspofungin against fluconazole-susceptible (A) and fluconazole-resistant (B) C. glabrata. Flz, fluconazole; Cfgn, caspofungin; QC, quality control isolates.As expected, the TKA performed with the fluconazole-resistant strain revealed a 99.9% fungicidal activity at 4 to 6 h for both concentrations of caspofungin (Fig. ​(Fig.1B).1B). There was no difference in fungicidal activity levels between the different concentrations of caspofungin.