JAC Advance Access originally published online on September 29, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1295-1298; doi:10.1093/jac/dkl395
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Combined antifungal therapy in a murine infection by Candida glabrata
Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili Carrer Sant Llorenç 21, 43201, Reus, Spain
*Corresponding author. Tel: +34-977-759359; Fax: +34-977-759322; E-mail: franciscojavier.pastor{at}urv.cat
Received 28 June 2006; returned 12 August 2006; revised 24 August 2006; accepted 9 September 2006
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Abstract |
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Objectives: To develop proper treatments for patients who do not respond to current antifungal treatments, we tested new combinations of antifungal drugs for treating disseminated infections by Candida glabrata in a murine model.
Methods: Mice were rendered neutropenic by intraperitoneal cyclophosphamide and intravenous 5-fluorouracil administration. The animals were infected intravenously with 2 x 108 cfu of C. glabrata. The efficacies of micafungin combined with amphotericin B, fluconazole or flucytosine, and of amphotericin B combined with fluconazole were evaluated by survival and tissue burden reduction.
Results and Conclusions: Micafungin plus amphotericin B was the most effective combination at reducing tissue burden. Micafungin at 10 mg/kg combined with amphotericin B at 0.75, 1.5 or 3 mg/kg prolonged survival with respect to the monotherapies, but only the second combination showed a synergistic effect in reducing fungal load in spleen and kidney. Amphotericin B at 1.5 mg/kg combined with micafungin at 5, 10 or 20 mg/kg reduced tissue burden with respect to the monotherapies, but the effects of the three combinations were very similar. These results suggest that amphotericin B in combination with micafungin is promising for the treatment of disseminated C. glabrata infections.
Keywords: candidiasis , animal models , micafungin , fluconazole , flucytosine
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Introduction |
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Candida glabrata is an opportunist pathogen that mainly affects severely immunocompromised patients, causing disseminated and frequently fatal infections.1 Amphotericin B is recommended for the treatment of non-Candida albicans systemic infections2 but its toxicity limits its use at high doses in patients in a critical condition.3 Many isolates of C. glabrata have shown an innate resistance to fluconazole, and treatment often fails.4 In a recent study using a murine model of disseminated infection by C. glabrata we demonstrated that although amphotericin B showed the best results, few differences exist in the activity of amphotericin B, fluconazole, flucytosine and micafungin and that none of them completely resolved the infection.5 Combined therapy could be a therapeutic alternative, but it has been poorly explored. There has been some experience in combining amphotericin B with other drugs that can allow the reduction of the doses of this antifungal and/or shorten the duration of therapy.6 In a recent murine study, the combination of this drug with caspofungin showed synergistic in vivo activity, although the doses of amphotericin B tested were considerably high.7
Here, we have tested combinations of the four drugs mentioned above in a murine model of disseminated infection by C. glabrata in order to evaluate whether combined therapy can improve the results obtained with the monotherapies.
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Materials and methods |
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Fungus
C. glabrata strain FMR 8489 was used in the study. The inoculum size was adjusted to the desired concentration by haemocytometer counts, and viability was confirmed by serial plating on Sabouraud dextrose agar.
In vitro susceptibility testing
The in vitro antifungal susceptibility of the fungus to the four drugs was tested using a reference microdilution method.8
Animals
Male OF1 mice (30 g) were used in accordance with the procedure standards approved by the Animal Welfare Committee of the Rovira i Virgili University.
Drugs
The drugs tested were flucytosine (Sigma-Aldrich Corp., St Louis, MO, USA), amphotericin B (Fungizona), fluconazole (Pfizer Inc. Madrid, Spain) and micafungin (Fujisawa Pharmaceutical Co. Ltd, Osaka, Japan). Amphotericin B was administered at doses of 0.75, 1.5 or 3 mg/kg of body weight/dose once daily, intraperitoneally; flucytosine was administered at 80 mg/kg three times a day (240 mg/kg/day), orally; fluconazole was administered at 40 mg/kg twice a day (80 mg/kg/day), orally; and micafungin was administered at 2.5, 5 or 10 mg/kg twice a day (5, 10 or 20 mg/kg/day), subcutaneously.
Immunosuppression
Mice were immunosuppressed by a single intraperitoneal injection of 200 mg/kg of cyclophosphamide plus a single intravenous injection of 150 mg/kg of 5-fluorouracil, on the day of infection.
Infection
Mice were challenged with 2 x 108 cfu in 0.2 mL into the lateral tail vein.5
Experimental design
Three sequential experiments were performed. In the first we evaluated the efficacy of micafungin combined with amphotericin B, fluconazole or flucytosine, and amphotericin B combined with fluconazole, in reducing tissue burden in kidney and spleen. Results were compared with those obtained with these drugs administered alone. In the second and third experiments we evaluated the efficacy of micafungin plus amphotericin B, which was the combination that yielded the best results in the first experiment, first varying the doses of amphotericin B and then those of micafungin. Results were compared with those from the monotherapies in prolonging the survival and reducing tissue burden in kidney and spleen.
Experiment 1
Twenty-four hours after challenge, groups of 10 mice were randomly assigned to one of the following treatment groups: micafungin 10 mg/kg/day, amphotericin B 1.5 mg/kg/day, fluconazole 80 mg/kg/day, flucytosine at 240 mg/kg/day, micafungin 10 mg/kg/day plus amphotericin B 1.5 mg/kg/day, micafungin 10 mg/kg/day plus fluconazole 80 mg/kg/day, micafungin 10 mg/kg/day plus flucytosine 240 mg/kg/day and amphotericin B 1.5 mg/kg/day plus fluconazole 80 mg/kg/day. Control animals received no treatment. The therapy lasted for 5 days. Monotherapies were performed not only for comparison with combined treatments, but also to confirm the results of a previous study.5 Five of the surviving mice randomly chosen were killed 1 day after the completion of treatment. Spleen and kidneys were aseptically removed, and the entire organs were homogenized in 1 mL of sterile saline. Serial 10-fold dilutions of the homogenates were plated on Sabouraud dextrose agar, incubated at 35°C and examined daily for 3 days.
Experiment 2
Twenty-four hours after challenge, groups of 20 mice were randomly assigned to one of the following treatment groups: amphotericin B 0.75 and 3 mg/kg/day, and micafungin 10 mg/kg/day plus amphotericin B 0.75, 1.5 or 3 mg/kg/day. Therapy lasted for 5 days. A control group received no treatment. The efficacy of these treatments was evaluated by survival and tissue burden studies and was compared with those obtained in the first experiment. For survival studies, mice were checked daily for 15 days after challenge. Tissue burden was performed as described above.
Experiment 3
Twenty-four hours after challenge, groups of 20 mice were randomly assigned to one of the following treatment groups: micafungin 20 mg/kg/day, and amphotericin B 1.5 mg/kg/day plus micafungin 5 or 20 mg/kg/day. Therapy lasted for 5 days. A control group received no treatment. The efficacy of these treatments was evaluated by survival and tissue burden studies and was compared with those obtained in the first experiment. Survival and tissue burden studies were performed as described above.
Statistics
Mean survival time was estimated by the Kaplan–Meier method and compared among groups using the log-rank test. cfu counts were analysed using the Mann–Whitney U-test. Graph Pad 4.00 and SPSS 11.5 for Windows were used.
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Results |
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The MICs of the drugs tested were as follows: amphotericin B, 2 mg/L; micafungin, 0.25 mg/L; fluconazole, 8 mg/L; and flucytosine, <0.06 mg/L.
Experiment 1
Results are shown in Figure 1. Amphotericin B significantly reduced the fungal load in both organs versus the control group, while flucytosine only did so in kidney. Micafungin and fluconazole were ineffective in this model. All the combinations tested were able to reduce the fungal load with respect to the control in at least one organ. The combination of micafungin plus amphotericin B showed the best results and was the only one that showed a synergistic effect, significantly reducing the cfu counts with respect to the monotherapies in both organs.
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Experiment 2
Results are shown in Figure 2(a–c). All the combinations of micafungin with amphotericin B significantly prolonged the survival, with a rate over 75% in all the cases. The results of all the combinations were significantly better than those of the corresponding monotherapies (P < 0.05), but with no differences among them. Although the two new combinations reduced fungal load with respect to the control in both organs, they did not show a synergistic effect, their results being worse than those obtained with micafungin at 10 mg/kg/day plus amphotericin B at 1.5 mg/kg/day in the first experiment.
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Experiment 3
Since the best results of the two previous experiments were those obtained with the combination of micafungin at 10 mg/kg/day plus amphotericin B at 1.5 mg/kg/day, we tested amphotericin B at 1.5 mg/kg/day with micafungin at 5 or 20 mg/kg/day in order to evaluate if varying the doses of the last drug would improve the previous results. Due to the inefficacy of micafungin at 10 mg/kg/day given alone, monotherapy with micafungin at 5 mg/kg/day was not tested. Figure 2(d–f) shows that no differences were found among the combinations assayed, results in all cases being significantly better than those obtained with the monotherapies (P < 0.05).
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Discussion |
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Combined therapy in experimental C. glabrata infections, especially using the two standard drugs amphotericin B and fluconazole, has already been studied by other authors.9 In the present study, this combination was not able to improve the results obtained with amphotericin B alone. By contrast the combination amphotericin B plus micafungin showed excellent results. All the doses tested showed a synergistic effect in prolonging survival versus the monotherapies. Other investigators, using a similar murine model, had previously reported the efficacy of the combinations of amphotericin B with echinocandins, although evaluating only kidney tissue burden.7,10 Barchiesi et al.7 recently reported a dose-dependent effect of amphotericin B when combined with caspofungin, the highest dose of both drugs being 3 mg/kg/day. However, in our case, in the combination amphotericin B plus micafungin, amphotericin B at 1.5 mg/kg worked better than amphotericin B at 3 mg/kg in reducing fungal load in spleen. Our results are similar to those of Olson et al.,10 who also obtained a significant reduction of tissue burden using a high dose of liposomal amphotericin B combined with micafungin at 2.5 mg/kg/day, although they used a considerably lower fungal inoculum than we did. Lipid formulations of amphotericin B are less toxic than amphotericin B deoxycholate, but its availability is limited due to the cost. We obtained similar results using amphotericin B at small toxic doses. Here, we chose a strain of C. glabrata that practically did not respond to micafungin in our previous study5 to evaluate the potential efficacy of this drug in combination. The present study confirmed those results.
In conclusion, our data demonstrate a favourable in vivo interaction between amphotericin B and echinocandins against C. glabrata murine infections confirming the results of other authors.7,10 This association constitutes a promising therapeutic approach; but further work is needed to ascertain its clinical relevance.
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None to declare.
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Acknowledgements |
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This work was supported by a grant from Fondo de Investigaciones Sanitarias from the Ministerio de Sanidad y Consumo of Spain (PI 050031).
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References |
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1 Patterson TF. (2005) Advances and challenges in management of invasive mycoses. Lancet 366:1013–25.[CrossRef][Web of Science][Medline]
2 Spellberg BJ, Filler SG, Edwards JE. (2006) Current treatment strategies for disseminated candidiasis. Clin Infect Dis 42:244–51.[CrossRef][Web of Science][Medline]
3 Kleinberg M. (2006) What is the current and future status of conventional amphotericin B? Int J Antimicrob Agents 27:Suppl 1, 12–6.
4
Fidel PL Jr, Vazquez JA, Sobel JD. (1999) Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev 12:80–96.
5
Mariné M, Serena C, Fernández-Torres B, et al. (2005) Activities of flucytosine, fluconazole, amphotericin B, and micafungin in a murine model of disseminated infection by Candida glabrata. Antimicrob Agents Chemother 49:4757–9.
6 Lewis RE and Kontoyiannis DP. (2001) Rationale for combination antifungal therapy. Pharmacotherapy 21:149–64S.[CrossRef][Web of Science][Medline]
7
Barchiesi F, Spreghini E, Fothergill AW, et al. (2005) Caspofungin in combination with amphotericin B against Candida glabrata. Antimicrob Agents Chemother 49:2546–9.
8 National Committee for Clinical Laboratory Standards. (1990) Reference Method for Broth Dilution Antifungal Susceptibility of Yeasts—Second Edition: Approved Standard M27-A2(Wayne, PA, USA, NCCLS).
9
Atkinson BA, Bouthet C, Bocanegra R, et al. (1995) Comparison of fluconazole, amphotericin B and flucytosine in treatment of a murine model of disseminated infection with Candida glabrata in immunocompromised mice. J Antimicrob Chemother 35:631–40.
10
Olson JA, Adler-Moore JP, Smith PJ, et al. (2005) Treatment of Candida glabrata infection in immunosuppressed mice by using a combination of liposomal amphotericin B with caspofungin or micafungin. Antimicrob Agents Chemother 49:4895–902.
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