No 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...

The in vitro activities of caspofungin plus amphotericin B against 50 Candida glabrata isolates were evaluated by the time-kill, disk diffusion, and Etest methods. In vitro experiments showed a positive interaction. Even though each of these methods uses different conditions and endpoints, the results of the different methods frequently agreed.Candida glabrata is an opportunistic pathogen that mainly affects severely immunocompromised patients, causing disseminated and frequently fatal infections (9). Many isolates of C. glabrata have shown innate resistance to fluconazole, and treatment often fails. Combined therapy could be a therapeutic alternative, but it has been poorly explored (7).Caspofungin (CAS), an echinocandin, inhibits fungal cell wall synthesis. Amphotericin B (AMB) targets fungal ergosterol, the main component of the fungal cell membrane (5). With their different mechanisms of action, these two drugs could be effective in combination. In this study, we hypothesized that the combination of CAS with AMB could have an advantage against C. glabrata over monotherapy with either drug.Fifty strains of C. glabrata were isolated from clinical samples at our laboratory. Candida parapsilosis ATCC 22019 was included for quality control (4). Antifungal susceptibility testing was performed, following both the broth microdilution (4) and Etest (Etest technical guide 4; AB Biodisk, Solna, Sweden) methods. The final concentrations were 0.03 to 2.0 g/ml of AMB and 0.0625 to 64 g/ml of CAS. MICs were read after 48 h of incubation. The Etest was performed on RPMI 1640 agar plates as recommended (Etest technical guide 4) (1). For CAS, an 80% inhibition in growth was used as the MIC endpoint (microcolonies were ignored), and for AMB, the MIC endpoint was defined as the lowest concentration with complete (100%) growth inhibition (1).For the time-kill studies, the drugs alone and in combination were used at 1ϫ MIC (1.0 g/ml for both drugs). The numbers of CFU were determined at 0, 2, 6, and 24 h. The limit of detection was 50 CFU/ml. Fungicidal activity was considered to have been achieved when the number of CFU per milliliter was Ͻ99.9% compared with the initial inoculum size. Synergy and antagonism were defined, respectively, as a Ն100-fold increase or decrease in killing compared with that achieved with the most active single agent. If there was less than a 100-fold change, the interaction was considered indifferent (3). For the antifungal combination studies, two types of Etest methods were used. For the first method (Etest-1; described in reference 5), synergy was defined as a decrease of Ն3 dilutions, indifference as a decrease of Ͻ2 dilutions, and antagonism as an increase of Ն3 dilutions, respectively, in the resultant MIC. The second method (Etest-2) was carried out as described in a previous study (10). The fractional inhibitory concentration (FIC) index was calculated as follows: ⌺FIC ϭ FIC A ϩ FIC B, where FIC A is the MIC of the combination/the MIC of drug A alone, and FIC B is the MIC of the combination/the...

We evaluated the postantifungal effects (PAFEs) of caspofungin (CAS), voriconazole (VOR), amphotericin B (AmB), and the combinations of CAS + VOR and CAS + AmB against 30 clinical Candida krusei isolates at 0.25, 1 and 4 times the MIC of each individually and in the indicated combinations. Antifungals were removed after 1 hour and colony counts were performed at 0, 2, 6, 24, and 48 h. VOR did not display any measurable PAFE regardless of antifungal concentrations, while AmB and CAS exhibited dose-dependent PAFE. The most effective agent producing a prolonged PAFE in this study was CAS. Although the combination of CAS with VOR generated longer PAFEs at 0.25 and 1 times their respective MICs in comparison with CAS alone, this combination was indifferent rather than synergistic. However, the combination of CAS with AmB at 4 times their MICs exhibited the best performance, reducing the colony counts during the 48 h after removal of drugs and resulted in synergic interaction in respect to 20 (67%) isolates. Consequently, CAS has a prolonged PAFE in vitro against C. krusei isolates, and the combination of AmB + CAS may increase significantly the efficacy of CAS. Our data may be useful in optimizing dosing regimens for these agents and their combinations, although further studies are needed to explore the clinical usefulness of our results.

Objective. Biofilms have been shown to play a major role in the pathogenesis of otolaryngologic infections. However, very limited studies have been undertaken to demonstrate the presence of biofilms in tissues from patients with chronic otitis media (COM) with or without cholesteatoma. Our objective is to study the presence of biofilms in humans with chronic suppurative and nonsuppurative otitis media and cholesteatoma. Study Design. In all, 102 tissue specimens (middle ear, mastoid tissue, and ossicle samples) were collected during surgery from 34 patients. Methods. The samples were processed for the investigation of biofilms by scanning electron microscopy (SEM). Results. Our research supports the hypothesis in which biofilms are involved in chronic suppurative otitis media, cholesteatoma, and, to a lesser degree, chronic nonsuppurative otitis media. There were higher rates in hypertrophic and granulated tissue samples than in normal mucosa. In addition, the presence of biofilms was significantly higher in the middle ear mucosa compared with the mastoid and ossicle samples. Conclusion. In the clinic, the careful use of topical or systemic antimicrobials is essential, and, during surgery, hypertrophic tissue must be carefully removed from normal tissue.