|Home | About | Journals | Submit | Contact Us | Français|
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Over the last two decades, Candida albicans has been replaced by the non-albicans Candida species, especially C. glabrata, as the cause of candidemia and invasive candidiasis (36).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).
The 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).
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.
Against both fluconazole-resistant and fluconazole-susceptible strains, caspofungin plus fluconazole displayed synergistic activity, with an FICI of 0.12. In combination with voriconazole, the FICI was 0.56 against the fluconazole-resistant and voriconazole-resistant strains. The FICI for caspofungin plus fluconazole for the fluconazole-susceptible strain was 0.185 (Table (Table22).
The FICI for caspofungin with amphotericin B was 1.06 when evaluated against a caspofungin- and amphotericin B-susceptible strain. Caspofungin in combination with amphotericin B against C. glabrata demonstrated indifference (Table (Table22).
Caspofungin has demonstrated excellent in vitro fungicidal activity against C. glabrata. Caspofungin has shown activity against a wide variety of yeasts and molds (8, 9, 16, 17, 33). The results show that caspofungin demonstrated MIC and MFC ranges similar to those seen with amphotericin B. The MIC ranges for caspofungin against the C. glabrata isolates ranged from 0.125 to 1 μg/ml, similar to what has been described in prior in vitro studies (23, 25, 34).
In this study, we compared the MICs of caspofungin against C. glabrata to those of voriconazole, fluconazole, and amphotericin B. The fungicidal activity of caspofungin was demonstrated against both fluconazole-resistant and voriconazole-resistant strains of C. glabrata. The MIC90s for caspofungin and amphotericin B against C. glabrata were 2 and 1 μg/ml, respectively. On the other hand, the MIC90s for fluconazole and voriconazole against C. glabrata were 128 and 32 μg/ml, respectively. In addition, we were able to show that caspofungin had a fungicidal activity similar to that of amphotericin B against all 50 isolates of C. glabrata that were evaluated.
The TKA evaluating caspofungin against fluconazole-susceptible and -resistant C. glabrata strains showed a 99.9% kill in 4 to 6 h. Caspofungin at a concentration of 1 μg/ml was found to be fungicidal in 4 h, whereas caspofungin at a concentration of 4 μg/ml was fungicidal in 2 h. Excellent fungicidal activity was observed with caspofungin against C. glabrata within 2 to 4 h for both the fluconazole-resistant and fluconazole-susceptible strains.
Combination assays demonstrated excellent synergistic activity with caspofungin and either fluconazole or voriconazole and indifference with amphotericin B. Most studies evaluating combination antifungal therapy have been able to demonstrate synergy with caspofungin and azoles against Aspergillus species and C. albicans (3, 24, 29, 30). It is important to note that the current experiments used the checkerboard methodology based on the M27-A2 methods that recommended the echinocandin MICs to be read at 48 h. In contrast, the new CLSI M27-A3 methodology recommends that echinocandin MICs be read at 24 h.
In conclusion, caspofungin demonstrates excellent in vitro fungicidal activity against all strains of C. glabrata, with excellent fungicidal activity within 2 h, a result not seen with azole antifungals. In addition, caspofungin also demonstrated promising synergistic activity with the combination of caspofungin and either fluconazole or voriconazole against strains of C. glabrata. These results reiterate the need for future research to focus on fungicidal and synergistic activity of caspofungin and possibly the other echinocandins. Caspofungin, with its excellent safety profile and fungicidal activity against C. glabrata isolates, should be considered a pivotal antifungal agent for infections due to C. glabrata.
Published ahead of print on 19 October 2009.