JAC Advance Access originally published online on January 28, 2008
Journal of Antimicrobial Chemotherapy 2008 61(3):755-757; doi:10.1093/jac/dkn005
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Research letters |
In vitro activities of combinations of amphotericin B, posaconazole and four other agents against Rhizopus
1 Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain 2 Hospital Universitari de Sant Joan, Reus, Spain
* Corresponding author. Tel: +34-977-759359; Fax: +34-977-759322; E-mail: josep.guarro{at}urv.cat
Keywords: zygomycosis , itraconazole , Rhizopus oryzae , Rhizopus microsporus var. rhizopodiformis
Zygomycosis is a severe, life-threatening fungal infection that occurs mainly in patients with diabetes mellitus, or with haematological malignancies, after organ transplantation, in conditions of iron overload or those treated with immunosuppressive drugs. Rhizopus oryzae, followed by Rhizopus microsporus var. rhizopodiformis, are the most frequently involved organisms, accounting for 
75% of all cases. Amphotericin B is the drug of choice for the treatment of zygomycosis, but overall mortality rate is high. Recently, posaconazole has shown to be active as salvage therapy for disseminated zygomycosis,1 but few studies still exist and the search of novel therapeutic strategies is still necessary. Combination of antifungal agents with different active mechanisms could be an interesting therapeutic option for improving clinical outcomes. Few data exist on the in vitro effect of combinations of antifungal drugs against zygomycetes. Only associations of amphotericin B with flucytosine and of terbinafine with either amphotericin B or triazoles have been evaluated.2,3 The interaction between amphotericin B and azoles against zygomycetes has not been investigated. There is recent evidence for synergistic interaction between the echinocandin drug caspofungin and amphotericin B lipid complex in a murine model of disseminated R. oryzae infection.4 However, in vitro studies on interactions of echinocandins with other antifungals against Rhizopus spp. have not been performed. The aim of this study was to investigate the in vitro interaction of amphotericin B and micafungin, a recently developed echinocandin, and of each with posaconazole, itraconazole, voriconazole or ravuconazole against Rhizopus.
Nineteen clinical isolates were tested, comprising 9 isolates of R. oryzae and 10 of R. microsporus var. rhizopodiformis. The isolates were grown on potato dextrose agar plates and incubated at 30°C for 4–7 days. Inocula were prepared following the CLSI (formerly NCCLS) M38-A document. The spore suspensions were adjusted to a final concentration of 4 x 103–5 x 104 cfu/mL with a haemocytometer. Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were included as quality controls.
The following drugs were tested: amphotericin B (USP, Rockville, MD, USA), posaconazole (Schering-Plough Europe, Brussels, Belgium), itraconazole (Janssen Pharmaceutica, Beerse, Belgium), voriconazole (Pfizer Inc., Madrid, Spain), ravuconazole (Bristol-Myers Squibb Company, New Brunswick, NJ, USA) and micafungin (Astellas Pharma Inc., Tokyo, Japan). All were obtained as pure powders from their respective manufacturers and dissolved in DMSO, apart from micafungin, which was dissolved in water.
The MIC of all drugs was defined as the lowest concentration that produced a 100% growth inhibition after 24 h of incubation at 35°C. Drug interactions were assessed by a chequerboard microdilution method that also included the determination of the MIC of each drug alone on the same plate, by using the parameters outlined in CLSI. Antifungal agents were placed in rows or in columns of the trays to test the different combinations. The fractional inhibitory concentration index (FICI) was used to classify drug interaction. Nearly 80% of the tests were repeated, and interactions demonstrated mainly the same tendencies.
Of the 171 tests performed, synergistic interaction was observed in 25 (14.6%) cases, and for the rest, we obtained an indifferent effect. No antagonistic interactions were observed in any case. Amphotericin B plus posaconazole or itraconazole were the combinations that presented the highest percentage of synergistic interaction, 47.4% and 42.1%, respectively, being more frequent against R. microsporus var. rhizopodiformis (6/10 and 5/10, respectively) than against R. oryzae (3/9 for both combinations) (Table 1). Amphotericin B combined with ravuconazole showed synergism for 21.1% of the isolates. Interactions of amphotericin B with voriconazole or micafungin and of micafungin with itraconazole were mainly indifferent; they were synergistic in only 5.3% to 10.6% of the tests. Combinations of micafungin with posaconazole, voriconazole or ravuconazole showed indifferent interactions.
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In murine models of disseminated zygomycosis, posaconazole has demonstrated only limited efficacy.5 However, Spellberg et al.4 have recently benefited by using the combination of amphotericin B lipid complex and caspofungin with diabetic mice infected with R. oryzae. Over the last few years, posaconazole has shown efficacy as salvage therapy for invasive zygomycosis.1 Eventually, the activity of posaconazole alone, added to its synergistic effect when combined with amphotericin B, could render this antifungal association particularly attractive. The poor results that we have obtained with associations of micafungin with other antifungals against Rhizopus spp. are in contrast to previous in vitro studies, in which the combination of micafungin with amphotericin B produced synergistic effects against Scedosporium spp.6
In summary, our results demonstrated that the combination of amphotericin B with posaconazole or itraconazole showed a remarkable degree of synergism against Rhizopus isolates. Further in vivo studies are warranted to explore the potential role of these combinations for the treatment of zygomycosis.
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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 05003).
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None to declare.
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1 Greenberg RN, Mullane K, van Burik, et al. Posaconazole as salvage therapy for zygomycosis. Antimicrob Agents Chemother (2006) 50:126–33.
2 Dannaoui E, Afeltra J, Meis JFGM, et al. In vitro susceptibilities of zygomycetes to combinations of antimicrobial agents. Antimicrob Agents Chemother (2002) 8:2708–11.
3 Gómez-López A, Cuenca-Estrella M, Mellado E, et al. In vitro evaluation of combination of terbinafine with itraconazole or amphotericin B against Zygomycota. Diagn Microbiol Infect Dis (2003) 45:199–202.[CrossRef][Web of Science][Medline]
4
Spellberg B, Fu Y, Edwards JE Jr, et al. Combination therapy with amphotericin B lipid complex and caspofungin acetate of disseminated zygomycosis in diabetic ketoacidotic mice. Antimicrob Agents Chemother (2005) 49:830–2.
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Barchiesi F, Spreghini E, Santinelli A, et al. Posaconazole prophylaxis in experimental systemic zygomycosis. Antimicrob Agents Chemother (2007) 51:73–7.
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Yustes C, Guarro J. In vitro synergistic interaction between amphotericin B and micafungin against Scedosporium spp. Antimicrob Agents Chemotherapy (2005) 49:3498–500.
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