JAC Advance Access originally published online on February 13, 2008
Journal of Antimicrobial Chemotherapy 2008 61(4):877-879; doi:10.1093/jac/dkn012
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Original research |
Micafungin combined with fluconazole, an effective therapy for murine blastoschizomycosis
Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain
* Corresponding author. Tel: +34-977-759359; Fax: +34-977-759322; E-mail: josep.guarro{at}urv.cat
Received 5 September 2007; returned 29 October 2007; revised 5 December 2007; accepted 3 January 2008
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Abstract |
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Objectives: To study the efficacy of micafungin combined with fluconazole in a murine model of disseminated blastoschizomycosis.
Methods: Mice were treated with fluconazole at 40 or 80 mg/kg/day given orally, with micafungin at 10 mg/kg/day given subcutaneously or with a combination of micafungin with fluconazole at the doses described above. Treatment began 1 day after infection and continued for 6 days post-infection. Tissue burden studies were performed 1 day after the treatment finished. The experiments were performed with three different strains.
Results: The two combinations tested significantly improved the survival of mice with respect to the control group and reduced the tissue burden significantly with respect to the control and their respective monotherapies in most organs tested. All animals that received the combination of micafungin with fluconazole at 80 mg/kg/day survived up to the end of the experiment.
Conclusions: The combination of micafungin with fluconazole is promising for the treatment of disseminated blastoschizomycosis.
Keywords: Blastoschizomyces capitatus , echinocandins , combined therapy
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Introduction |
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Blastoschizomyces capitatus is an uncommon but frequently fatal cause of invasive infections in immunocompromised patients, particularly in those with haematological malignancies.1 In a previous study, we observed that fluconazole at high doses (80 mg/kg/day) was the most effective treatment in a systemic murine infection by B. capitatus.2 However, the use of a single drug to treat systemic blastoschizomycosis has not been completely satisfactory.3,4 Despite the administration of amphotericin B or fluconazole, this infection is almost always fatal.3 In another study, we also tested the activity of amphotericin B combined with micafungin, voriconazole or flucytosine, but any of them could improve the efficacy of fluconazole at high doses.5 As micafungin combined with fluconazole was effective in the treatment of murine trichosporonosis, caused by another basidiomycetous yeast,6 and of a murine Candida glabrata infection,7 we have evaluated here the efficacy of this combination in a murine model.
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Materials and methods |
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Three clinical strains of B. capitatus were used (Table 1). The isolates were stored at –80°C, and prior to testing, they were subcultured on Sabouraud dextrose agar (SDA) at 35°C. The resulting suspensions were adjusted to the desired inoculum based on the haemocytometer counts and by serial plating on SDA to confirm viability.
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The in vitro antifungal susceptibility of the strains was tested by a reference microdilution method.8 The interactions of the drugs were assessed by a chequerboard method.9 We have used an MIC-2 endpoint criterion, defined as the lowest concentration at which there was 50% inhibition of growth when compared with a drug-free control, for all the drugs and their combinations.
Male OF1 mice weighing 30 g (Charles River, Criffa S.A., Barcelona, Spain) were used in this study. Animals were housed under standard conditions. All animal care procedures were supervised and approved by the Universitat Rovira i Virgili Animal Welfare Committee. Animals were immunosuppressed by intraperitoneal administration of a single dose of 200 mg of cyclophosphamide per kilogram plus intravenous administration of 150 mg of 5-fluorouracil per kilogram the day of infection.10 Mice were challenged with 2 x 106 cfu in 0.2 mL into lateral tail vein. Groups of 15 mice were randomly established for each strain and each treatment. Ten mice were randomly chosen for survival and five for tissue burden studies, the latter being identified before the study started. The treatments tested were fluconazole (Pfizer Inc., Madrid, Spain) at doses of 20 or 40 mg/kg twice a day orally (40 or 80 mg/kg/day); micafungin (Astellas Pharma Inc., Tokyo, Japan) at 5 mg/kg twice a day subcutaneously (10 mg/kg/day); and the combinations of both drugs at the doses described earlier. All treatments began 24 h after challenge and the therapy lasted for 6 days. Mice survival was evaluated daily for 30 days after challenge. One day after the treatment finished, liver, spleen and kidneys were aseptically removed and were homogenized in 1 mL of sterile saline. Serial 10-fold dilutions of the homogenates were plated on SDA and incubated for 48 h at 35°C. The number of cfu per gram of tissue was calculated.
Mean survival time was estimated by the Kaplan–Meier method. The conservative Bonferroni inequality approach was used to keep the error rate to 5% for the set of 15 tests (significance P < 0.003). Cfu counts were analysed using one-way ANOVA followed by Tukey's pairwise comparison test to compare all pairs of results within a group with an overall error rate of 5%.
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Results and discussion |
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The in vitro results are shown in Table 1. The combination of micafungin with fluconazole showed a synergistic interaction against the three strains tested. Figure 1 shows the effects of the different treatments on prolonging mice survival. The monotherapies, in general, showed results similar to those obtained in previous studies,2,5 with no significant differences with respect to the previous works, which confirmed the reproducibility of the model. The combination of micafungin with fluconazole at 40 or 80 mg/kg/day prolonged significantly the survival versus the control group and tended to improve the survival versus monotherapies for the three strains tested, but it was not statistically significant. The combination of micafungin plus fluconazole at 80 mg/kg/day prolonged the survival of all the animals up to the end of the experiment. The efficacy of the monotherapies in reducing the fungal load in the different organs was similar to that shown in previous studies.2,5 Both doses of fluconazole reduced tissue burden significantly compared with control group in the three strains and in the three organs tested (Table 2). Micafungin administered alone was generally ineffective. The efficacy of the combined therapies was similar against the three strains. The combination of micafungin plus fluconazole at 40 mg/kg/day reduced fungal load in all the organs compared with controls and the monotherapies in kidneys and spleen. The combination of micafungin plus fluconazole at 80 mg/kg/day showed the best results as it reduced fungal load compared with controls and monotherapies in all the organs and strains, with the exception of liver in the strain IHEM 5666.
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Combined therapy of amphotericin B with voriconazole or with high-dose fluconazole has been recommended for the treatment of blastoschizomycosis.4 However, in a previous murine study, we proved that voriconazole combined with amphotericin B did not improve the results obtained with fluconazole alone. In contrast, we have demonstrated here that the combination micafungin plus fluconazole is significantly more effective in reducing the fungal loads than the antifungal combinations of amphotericin B plus micafungin, voriconazole or flucytosine, and fluconazole alone. These data suggest that the combination of fluconazole with micafungin, both drugs being less toxic than amphotericin B, merits further investigation for the treatment of disseminated blastoschizomycosis. Our study demonstrated a good correlation between in vitro and in vivo results because micafungin with fluconazole showed synergism in vitro against the three strains tested and, in general, combined therapies showed better results that the monotherapies. Further studies are required to ascertain the real clinical relevance of this combination.
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Funding |
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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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Transparency declarations |
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None to declare.
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References |
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1 Girmenia C, Pagano L, Martino B, et al. Invasive infections caused by Trichosporon species and Geotrichum capitatum in patients with hematological malignancies: a retrospective multicenter study from Italy and review of the literature. J Clin Microbiol (2005) 43:1818–28.
2 Serena C, Mariné M, Marimon R, et al. Effect of antifungal treatment in a murine model of blastoschizomycosis. Int J Antimicrob Agents (2007) 29:79–83.[CrossRef][Web of Science][Medline]
3 Christakis G, Perlorentzou S, Aslanidou M, et al. Fatal Blastoschizomyces capitatus sepsis in a neutropenic patient with acute myeloid leukemia: first documented case from Greece. Mycoses (2005) 48:216–20.[CrossRef][Web of Science][Medline]
4 Martino R, Salavert M, Parody R, et al. Blastoschizomyces capitatus infection in patients with leukemia: report of 26 cases. Clin Infect Dis (2004) 38:335–41.[CrossRef][Web of Science][Medline]
5
Serena C, Rodríguez MM, Mariné M, et al. Combined therapies in a murine model of blastoschizomycosis. Antimicrob Agents Chemother (2007) 51:2608–10.
6 Serena C, Pastor FJ, Gilgado F, et al. Efficacy of micafungin in combination with other drugs in a murine model of disseminated trichosporonosis. Antimicrob Agents Chemother (2005) 51:2608–10.[CrossRef]
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Mariné M, Serena C, Pastor FJ, et al. Combined antifungal therapies in a murine infection by Candida glabrata. J Antimicrob Chemother (2006) 58:1295–8.
8 National Committee for Clinical Laboratory Standards. In: Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Second Edition: Approved Standard M27-A2 (2002) Wayne, PA, USA: NCCLS.
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Johnson MD, MacDougall C, Ostrosky-Zeichner L, et al. Combination antifungal therapy. Antimicrob Agents Chemother (2004) 48:693–715.
10
Graybill JR, Bocanegra R, Najvar LK, et al. Granulocyte colony-stimulating factor and azole antifungal therapy in murine aspergillosis: role of immune suppression. Antimicrob Agents Chemother (1998) 42:2467–73.
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