a School of Medicine, Section of Infectious Diseases, University of Manchester, Hope Hospital, Salford, Manchester M6 8HD, UK; b Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain; c Department of Infectious Diseases and Tropical Medicine, North Manchester General Hospital, Delaunays Road, Manchester M8 6RB, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The use of azole antifungal agents for the treatment of zygomycosis is not supported by clinical data and has barely been evaluated in in vivo models. In vitro, this group of antifungals is inefficacious with most, but not all, zygomycetes,2,46 and in vivo against Rhizopus spp.2,7,8
Compared with some zygomycetes, Absidia spp. have been shown to be much more susceptible to azoles in vitro;46 however, no data in vivo are available. Absidia spp. cause some cases of human zygomycosis, generally in immunocompromised hosts, and they are the most frequent agents of zygomycosis among lower animals. In this study we tested and compared in vitro and in vivo susceptibility data for amphotericin B and itraconazole for the first time against Absidia corymbifera murine infection.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Male CD-1 mice, 45 weeks old and weighing between 18 and 20 g were purchased from Charles River UK Ltd (Margate, UK) and randomized into groups of 10. Animals were immunosuppressed with cyclophosphamide (Sigma Aldrich, Poole, UK) administered intravenously via the lateral tail vein to all animals at a dose of 200 mg/kg. A state of profound neutropenia was achieved 3 days after administration and lasted for 4 days.
The isolates were grown on Sabouraud dextrose agar (Oxoid, Basingstoke, UK) plus chloramphenicol (Sigma) (SAB + C) for 10 days. The spores were harvested in 25 mL of sterile phosphate-buffered saline0.05% Tween 80 (Sigma) (PBSTween), with the flask shaken gently, stored at 4°C and used within 3 days. On the day of infection, the stock solution was adjusted to an inoculum that would give an LD90 (4.5 x 104 and 1.1 x 105 cfu/mL for CM-537 and CM-1074, respectively), according to preliminary studies based on viability counts. Three days after immunosuppression all animals were infected with 0.15 mL of the LD90 suspension, via the tail vein (day 0). The inoculum was rechecked from the remaining conidial suspension after the animals were infected.
Deoxycholate amphotericin B and itraconazole were dissolved in 5% dextrose and an aqueous solution of 2-hydroxypropyl-ß-cyclodextrin, respectively, as described previously.9 Doses employed were 5 and 0.5 mg/kg for amphotericin B and 75 and 25 mg/kg for itraconazole. Amphotericin B was administered via ip injection (0.15 mL) od at 24, 48 and 96 h and 7 days post-infection. Itraconazole was given by gavage (0.15 mL) tds on days 1 and 2 and bd on days 37.
A separate group of four uninfected, cyclophosphamide pre-treated mice, was treated with the 75 and 25 mg/kg itraconazole doses and then serum was collected and analysed on day 4, 3 h after the morning dosage, by bioassay, as described previously.9
On day 11 of the experiment, all surviving mice were humanely killed. The lungs, brain, liver and kidneys were removed and transferred into 2 mL of PBSTween and homogenized in a tissue grinder (Polytron, Kinematica AG, Luzern, Switzerland) for c. 1530 s and then diluted 10-fold. One hundred microlitres of the neat and diluted suspensions were then transferred to SAB + C plates and the liquid spread over the surface of the plates. Histology was not done. Plates were incubated at 37°C and colony counts were recorded from all plates that showed growth in 3 days.
Mortality and culture data were analysed by the Mann Whitney U-test or the KruskallWallis test if all values in one group were identical. Two-sided P values are given. Mice that died before day 10 were assumed to have organ counts at least as high as the highest counts in surviving mice in the calculation of culture result statistics. All data analysis was carried out with the computer package Arcus Quik Stat (Addison Wesley Longman Ltd)
In vitro susceptibility testing was carried out with a methodology derived from published work from our laboratory that validated in vivo a method of detecting itraconazole resistance in Aspergillus fumigatus but failed to do so for amphotericin B.9 Two final inocula were compared. The main differences from the NCCLS recommendations9 were that: itraconazole was dissolved at a stock concentration of 3200 mg/L in acetone and hydrochloric acid and then diluted down in medium; doubling dilutions instead of batch dilutions of the drug were used; spores were harvested by shaking the inoculated flask gently; the inoculum suspension densities were counted with an improved Neubauer haemocytometer; the final inoculum in the wells was 5 x 105 or 5 x 103 cfu/mL; microdilution plates were incubated at 37°C; the MICs were read visually and were defined as the lowest drug concentration with no growth.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
In our murine model, both A. corymbifera strains were responsive to amphotericin B. Mortality was 0% and 10% at 5 and 0.5 mg/kg, respectively, with both strains (P 0.0004 versus controls). CM-537 was inhibited by MICs of 0.12 and 0.25 mg/L at 5 x 103 and 5 x 105 cfu/mL, respectively, and CM-1074 by 0.25 mg/L with both inocula.
The highest control counts were from the liver, and were quite low from kidneys. For both strains counts were significantly lower than in the controls with both amphotericin B treatments for liver (P < 0.0001) and kidneys (CM-537 P < 0.0133; CM-1074 P < 0.0008).
Itraconazole
Itraconazole was less effective than the lower amphotericin B dose in the in vivo experiments with both strains. A dose effect of itraconazole was observed. Mortality was 40% at 75 mg/kg and 60% at 25 mg/kg with isolate CM-1074 (itraconazole MIC 1 mg/L) (P 0.0353 versus controls), and 60% and 100% with isolate CM-537 (MIC 2 mg/L) (not significant versus controls), respectively. Itraconazole MICs varied somewhat depending on the inoculum employed. The higher the inoculum used, the higher the MICs. MICs for CM-537 were 0.5 and 2 mg/L and for CM-1074 were 0.25 and 1 mg/L, at 5 x 103 and 5 x 105 cfu/mL of final inocula, respectively.
For both strains, counts were not significantly lower than in the controls in any itraconazole treatment groups for any organ except liver with itraconazole at 25 mg/kg in the CM-1074 model (P = 0.0436). For both liver and kidneys in the CM-1074 model counts were higher with the higher itraconazole dose.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amphotericin B is an effective drug against A. corymbifera infection in this model. It has in vitro activity against Absidia spp.5 We obtained low amphotericin B MICs for both strains (0.120.25 mg/L). This is consistent with our in vivo results. In other mice models of Rhizopus spp. infection, amphotericin B was also effective, although to a lesser extent with lower doses.7,8,10
Itraconazole was not efficacious against Rhizopus infection in guinea pig and murine models, and neither were other azoles, ketoconazole, fluconazole or saperconazole in a guinea pig model.2,7 These data are consistent with the high MICs determined in vitro for that species with itraconazole, ketoconazole, miconazole and saperconazole.46 Nevertheless, azole MICs were low for Absidia spp. in the same studies. Goldani & Sugar11 reported the triazole SCH 42427 to be active in a murine model of pulmonary Rhizopus oryzae infection. In our animal model of A. corymbifera infection, itraconazole treatment was inferior to amphotericin B, although the response was better than with the control group. This correlates with the intermediate response observed in vitro. The highest MICs were associated with the less responsive strain, although differences in MICs were small and overall efficacy mediocre.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Van Cutsem, J., Van Gerven, F., Fransen, J. & Janssen, P. A. J. (1989). Treatment of experimental zygomycosis in guinea pigs with azoles and with amphotericin B. Chemotherapy 35, 26772.[ISI][Medline]
3 . Chick, E. W., Evans, J. & Baker, R. D. (1958). Treatment of experimental mucormycosis (Rhizopus oryzae infection) in rabbits with amphotericin B. Antibiotics and Chemotherapy 8, 3949.
4
.
Otená
ek, M. (1992). Susceptibility of clinical isolates of fungi to saperconazole. Mycopathologia 118, 17983.[ISI][Medline]
5
.
Otená
ek, M. & Buchta, V. (1994). In vitro susceptibility to 9 antifungal agents of 14 strains of Zygomycetes isolated from clinical specimens. Mycopathologia 128, 1357.[ISI][Medline]
6 . Van Cutsem, J., Van Gerven, F. & Janssen, P. A. J. (1987). Activity of orally, topically, and parenterally administered itraconazole in the treatment of superficial and deep mycoses: animal models. Reviews of Infectious Diseases 9, Suppl. 1, 1532.
7
.
Odds, F. C., Van Gerven, F., Espinel-Ingroff, A., Bartlett, M. S., Ghannoum, M. A., Lancaster, M. V. et al. (1998). Evaluation of possible correlations between antifungal susceptibilities of filamentous fungi in vitro and antifungal treatment outcomes in animal infection models. Antimicrobial Agents and Chemotherapy 42, 2828.
8
.
Sugar, A. M. & Liu, X.-P. (2000). Combination antifungal therapy in treatment of murine pulmonary mucormycosis: roles of quinolones and azoles. Antimicrobial Agents and Chemotherapy 44, 20046.
9
.
Johnson, E. M., Oakley, K. L., Radford, S. A., Moore, C., Warn, P., Warnock, D. W. et al. (2000). Lack of correlation of in vitro amphotericin B susceptibility testing with outcome in a murine model of Aspergillus infection. Journal of Antimicrobial Chemotherapy 45, 8593.
10 . Law, D., Moore, C. B. & Denning, D. W. (1994). A new bioassay for serum itraconazole concentrations using hydroxyitraconazole standards. Antimicrobial Agents and Chemotherapy 38, 15616.[Abstract]
11 . Goldani, L. Z. & Sugar, A. M. (1994). Treatment of murine pulmonary mucormycosis with SCH 42427, a broad-spectrum triazole antifungal drug. Journal of Antimicrobial Chemotherapy 33, 36972.[ISI][Medline]
Received 2 January 2001; returned 6 April 2001; revised 29 May 2001; accepted 5 July 2001