1Department of Medical Microbiology, University Medical Center St Radboud, PO Box 9101, 6500 HB Nijmegen; 2Canisius-Wilhelmina Hospital, PO Box 9015, 65000 GS Nijmegen, The Netherlands
Received 17 September 2001; returned 14 December 2001; revised 14 January 2002; accepted 27 January 2002.
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Abstract |
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Introduction |
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Intravenous amphotericin B is the treatment of choice for zygomycosis. Nevertheless, the use of conventional amphotericin B is limited by its severe side effects, and clearly new therapeutics are needed. It has been shown recently that mild heating of amphotericin B could greatly reduce its toxicity to mammalian cells in vitro.2 Moreover, it has been demonstrated that heated amphotericin B is less toxic in vivo in mice, allowing the use of higher drug doses than those of conventional amphotericin B.3 Heated amphotericin B maintained in vitro antifungal activity against Candida albicans2,4 and Cryptococcus neoformans,3 and showed in vivo therapeutic activity in different models of candidiasis and cryptococcosis.3,4 Nevertheless, the efficacy of heated ampho-tericin B in vitro and in vivo is unknown in filamentous fungi.
Like amphotericin B, nystatin is a polyene antibiotic that is active against a broad spectrum of fungi. However, toxicity associated with parenteral administration has not allowed its use for intravenous treatment. Recently, a liposomal formulation of nystatin, less toxic than the free drug, has been developed. Liposomal nystatin has previously been reported to be active in vitro against medically important yeasts and filamentous fungi,5,6 as well as in vivo in animal models of fungal infections.79
The aims of this study were (i) to compare the activity of unheated and heated amphotericin B, and (ii) to test the activity of nystatin against strains belonging to different genera of Zygomycota.
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Materials and methods |
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A total of 36 isolates from our private collection, mostly of clinical origin, were tested. These comprised 15 Rhizopus spp. (eight R. oryzae and seven R. microsporus), 10 Absidia corymbifera, six Mucor spp. (three M. hiemalis, one M. circinelloides, one M. racemosus and one M. rouxii), three Rhizomucor spp. (two R. pusillus and one R. miehei), one Cunninghamella bertholletiae and one Apophysomyces elegans.10
All isolates were cultured from frozen stock on Sabouraud dextrose agar (Difco Laboratories, Detroit, MI, USA) sup-plemented with 0.02% chloramphenicol for 7 days at 35°C to ensure purity and viability. Two reference strains, Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were included to ensure quality control.
Antifungal agents
Amphotericin B (Bristol-Myers Squibb, Woerden, The Netherlands) and nystatin (Gist-Brocades, Delft, The Nether-lands) were obtained as standard powder and dissolved in dimethyl sulphoxide to produce stock solutions of 5 and 3.2 mg/mL, respectively.
Stock heated amphotericin B was prepared as described by Gaboriau et al.2 by heating the solution of amphotericin B for 20 min at 70°C.
Susceptibility testing
Isolates were grown on Sabouraud dextrose agar for 7 days at 30°C and stock spore suspensions were prepared by washing over the surface of the slants with 2 mL of sterile saline containing 0.05% Tween 80. For A. elegans, sporulation was obtained by culturing the mycelium in sterile distilled water supplemented with 0.1% yeast extract (Difco) for 10 days at 37°C. Spore suspensions were counted with a haematocytometer and then diluted into RPMI 1640 to a concentration of 2 x 104 spores/mL (two times final concentration). Inoculum sizes were checked by quantitative colony counts on Sabouraud dextrose agar. MICs were determined by a microdilution method based on the National Committee for Clinical Laboratory Standards (NCCLS) M38P document.11 Each well of the microdilution plates containing 100 µL of the drug concentrations (at twice their final concentration) was inoculated with 100 µL of the inoculum suspension (also at twice the final concentration). The final concentrations of the antifungal agents were 0.01516 mg/L and the final inocu-lum concentration was 104 spores/mL. Microplates were incubated at 35°C for 24 h, then read visually with the aid of a reading mirror. The MICs of all drugs were defined as the lowest concentration showing complete growth inhibition. MIC determination was carried out in duplicate and results were within two dilutions in 97% of the cases.
Data analysis
MICs for 50% (MIC50) and 90% (MIC90) of the isolates tested were determined for genera for which 5 and
10 isolates were available, respectively. For calculation, the high off-scale MICs of >16 mg/L were converted to the next highest concentration of 32 mg/L. The difference in the distributions of MICs was determined with a one-way ana-lysis of variance after transformation of MICs to log2 values. Statistical significance was defined as P
0.05.
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Results |
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No statistically significant differences were found between the antifungal activities of heated and non-heated amphotericin B against the zygomycete isolates. Amphotericin B MICs obtained without and after heating were within ±1 log2 dilution step in 100% of the strains.
Table 1 summarizes the in vitro activity of the three antifungal agents tested. Amphotericin B had MIC values of <2 mg/L for most of the strains. Rhizopus spp. were significantly less susceptible to amphotericin B than Mucor spp. (P < 0.001) and A. corymbifera (P < 0.001). For two strains (one C. bertholletiae and one A. elegans), amphotericin B showed high MICs of 2 mg/L. Nystatin was active against most of the strains, with an overall geometric mean MIC of 1.59 mg/L. Nevertheless, nystatin was significantly less active than amphotericin B (P < 0.001) and this difference was noted for all the genera. The two strains with elevated amphotericin B MICs were also less susceptible to nystatin.
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Discussion |
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Entrapment of polyenes into liposomes is another way to reduce their toxicity and to increase their therapeutic index. Recently, a liposomal formulation of nystatin has been developed. Liposomal nystatin was shown to be less toxic than free nystatin and to retain activity in vitro against medically important yeasts5 and Aspergillus fumigatus, including itraconazole-resistant isolates.5,6 The in vivo efficacy of liposomal nystatin was also demonstrated against experimental disseminated and pulmonary Aspergillus infections in animals,79 including infection with an isolate of A. fumigatus that had reduced susceptibility to amphotericin B.9
Published reports of the in vitro activity of liposomal nystatin against other opportunistic filamentous fungi are very scarce: it had a poor in vitro activity against Scedosporium apiospermum and Scedosporium prolificans. In this study, we have tested nystatin against a broad range of Zygomycota. The results show that although nystatin exhibits higher MICs than amphotericin B, it is active against most of the strains.
The high amphotericin B MICs for one C. bertholletiae and one A. elegans are in accordance with previous in vitro studies and clinical reports. Cunninghamella infections mostly develop in immunocompromised patients and are often fatal with an overall mortality of 79%. These infections seem to be unresponsive to amphotericin B therapy.1 Although infections with A. elegans occur predominantly in immunocompetent patients, response to amphotericin B therapy is variable10 and many of the patients required surgical interventions. Further studies of these rare but severe infections in animal models are needed to evaluate the beneficial effect of amphotericin B therapy and the potential of new antifungal drugs.
In conclusion, our data show the following. (i) Mild heating of amphotericin B did not alter the in vitro antifungal activity of the drug against Zygomycota. Further animal experiments are needed to assess the potential of heated amphotericin B for the treatment of zygomycosis. (ii) Nystatin was active against most of the strains. The new liposomal formulation of nystatin should be evaluated for use in the treatment of zygomycosis.
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Acknowledgements |
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Footnotes |
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Members are listed in the Acknowledgements.
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References |
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2 . Gaboriau, F., Cheron, M., Petit, C. & Bolard, J. (1997). Heat-induced superaggregation of amphotericin B reduces its in vitro toxicity: a new way to improve its therapeutic index. Antimicrobial Agents and Chemotherapy 41, 234551.[Abstract]
3 . Petit, C., Cheron, M., Joly, V., Rodrigues, J. M., Bolard, J. & Gaboriau, F. (1998). In-vivo therapeutic efficacy in experimental murine mycoses of a new formulation of deoxycholate-amphotericin B obtained by mild heating. Journal of Antimicrobial Chemotherapy 42, 77985.[Abstract]
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Denning, D. W. & Warn, P. (1999). Dose range evaluation of liposomal nystatin and comparisons with amphotericin B and amphotericin B lipid complex in temporarily neutropenic mice infected with an isolate of Aspergillus fumigatus with reduced susceptibility to amphotericin B. Antimicrobial Agents and Chemotherapy 43, 25929.
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11 . National Committee for Clinical Laboratory Standards. (1998). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-forming Filamentous Fungi: Proposed Standard M-38P. NCCLS, Wayne, PA.