1 Center for Medical Mycology, Department of Dermatology, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106-5028; 2 University Hospitals of Cleveland, Cleveland, OH 44106, USA
Received 25 September 2002; returned 1 December 2002; revised 26 February 2003; accepted 26 February 2003
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Abstract |
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Keywords: disseminated candidiasis, antifungal, therapy
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Introduction |
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This study evaluates the activity of CFG+AMB against an azole-resistant C. albicans isolate using in vitro and in vivo methodologies. No antagonistic interactions were observed between the two agents, which showed a trend towards additivity.
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Materials and methods |
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Candida albicans strain 12-99, a clinical isolate obtained from a patient for whom fluconazole therapy failed, was used. Antifungal susceptibility testing was performed according to M27-A methodology.4 Before each experiment, C. albicans was grown overnight at 37°C in Sabouraud dextrose broth (SDB; Difco Laboratories, Detroit, MI, USA). Blastospores were washed twice and suspended in sterile normal saline (NS; 0.85%); their number was determined using a haemocytometer, and confirmed by quantitative culturing.
A chequerboard technique was used to evaluate drugdrug interactions. Different concentrations of CFG and AMB in RPMI 1640 medium were combined into wells of microtitre plates so that the concentration of each agent increased simultaneously. Two rows, consisting of serial dilutions of the individual drugs alone, were also included. The highest concentrations of CFG and AMB used were 128 and 64 mg/L, respectively. Wells were inoculated with C. albicans (between 0.5 and 2.5 x 103 cfu/mL) and plates incubated at 35°C for 48 h. Growth in each well was observed visually. The fractional inhibitory concentration index (FICI) was calculated. The activity was expressed as synergic when the FICI was < 0.5 and antagonistic when the FICI was > 4.0; FICI < 1 was regarded as a positive and FICI > 1 as a negative interaction.
The Institutional Animal Care and Use Committee approved the protocol for our murine model. Six- to eight-week-old male, BALB/c mice (Charles River, Wilmington, MA, USA) were housed in cages (three mice or fewer/cage) and acclimatized for 5 days before fungal challenge. Each mouse was inoculated with 0.1 mL of C. albicans 12-99 (prepared in sterile NS from an overnight, 37°C, SDB culture) via the lateral tail vein. To determine the optimal challenge inoculum, three groups of mice (five mice/group) were infected intravenously with different concentrations of C. albicans (5 x 105, 1 x 106 or 3 x 106 blastospores).
In subsequent experiments, the animals were infected with 5 x 105 cells in 100 µL of NS. They were divided into seven mice/group and received their therapies intraperitoneally 2 h post-challenge. In preliminary experiments, drug dosages that were below an effective dose when used singly were determined. The following dosages were evaluated: CFG 0.0005, 0.001 and 0.002 mg/kg; and AMB 0.008, 0.016 and 0.032 mg/kg daily for 14 days. Based on these experiments, the following dosages were selected for subsequent drugdrug interaction studies: CFG 0.002 mg/kg and AMB 0.016 mg/kg. Efficacy was evaluated by monitoring survival and tissue burden. Animals were monitored daily for evidence of infection and its severity, and deaths were noted. Each experiment was performed twice (totalling 14 animals/group) to determine the survival pattern. For determination of tissue fungal burden, seven mice per group were killed; their kidneys and brain were removed aseptically, weighed and homogenized. Diluted samples of homogenates were cultured on agar plates (at 37°C for 48 h), and the number of cfu counted and expressed as cfu/g of tissue.
Differences in survival were assessed by the KaplanMeier method, while the mean cfu were compared using the MannWhitney U-test (mean ± S.E.). A P value of <0.05 was considered statistically significant.
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Results |
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Experiments directed at determining the optimal challenge inoculum, showed that mice infected with 1 x 106, or 3 x 106 cells of C. albicans exhibited clinical signs of infection (e.g. reduced activity, ruffled hair) on day 2, and all died within 23 days. Mice inoculated with 5 x 105 cells showed signs of infection on day 4, and survived until day 7. Thus, 5 x 105 cells/animal was selected as the optimal challenge inoculum.
Treating animals with a CFG+AMB combination significantly prolonged survival compared with infected, untreated controls (P = 0.006) (Figure 1). Treatment of mice with AMB+CFG, even at low dosage (0.016 and 0.002 mg/kg), also tended to prolong survival. Survival rates for untreated controls, CFG, AMB and CFG+AMB groups at day 21 were 0%, 22%, 50% and 72%, respectively. Thus, combining CFG with AMB increased the survival rate compared with the control group. Although animals treated with a CFG+AMB combination survived longer than those treated with AMB alone (72% versus 50%, respectively) this difference was not significant (P = 0.36).
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Discussion |
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The underlying mechanisms of synergic or additive effects for CFG+AMB are unknown. It is likely that inhibition of (1,3)-ß-D-glucan formation by CFG leads to cell wall damage. This would allow AMB easier access to the fungal cell membrane, where it binds to membrane ergosterol, resulting in pore formation and cell lysis.5 In this study, we focused on in vivo evaluation of the combined effects of CFG and AMB against an azole-resistant C. albicans strain. We did not include an azole-sensitive strain; such evaluation was beyond the scope of this study. Extending this work to cover more strains should be undertaken in the future.
In conclusion, our data demonstrate no evidence of antagonism between CFG and AMB when combined, and interactions in vivo and in vitro have tended to be favourable. Further work is needed to ascertain the clinical relevance of our findings.
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Acknowledgements |
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Footnotes |
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References |
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2
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Abruzzo, G. K., Gill, C. J., Flattery, A. M., Kong, L., Leighton, C., Smith, J. G. et al. (2000). Efficacy of the echinocandin caspofungin against disseminated aspergillosis and candidiasis in cyclophosphamide-induced immunosuppressed mice. Antimicrobial Agents and Chemotherapy 44, 23108.
3 . Hossain, M. A. & Ghannoum, M. A. (2000). New investigational antifungal agents for treating invasive fungal infections. Expert Opinion on Investigational Drugs 9, 1797813.[ISI][Medline]
4 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard M27-A. NCCLS, Wayne, PA, USA.
5 . Franzot, S. P. & Casadevall A. (1997). Pneumocandin L-743,872 enhances the activities of amphotericin B and fluconazole against Cryptococcus neoformans in vitro. Antimicrobial Agents and Chemotherapy 41, 3316.[Abstract]
6 . Manavathu, E., Ganesan L. T., Cutright J. L. & Chandrasekar P. H. (2001). In vitro antifungal activity of voriconazole in two-drug combination with micafungin, caspofungin and amphotericin B. In Program and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2001. Abstract J-125, p. 364. American Society for Microbiology, Washington, DC, USA.
7 . Flattery, A. M., Bartizal, K., Gill, C. J., Kong, L., Leighton, C., Pikounis, V. B. et al. (1998). Preclinical efficacy of MK-991 in combination with amphotericin B or fluconazole in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis. In Program and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract J-61, p. 468. American Society for Microbiology, Washington, DC, USA.