Division of Infectious Diseases, Department of Internal Medicine, Wayne State University School of Medicine, Detroit, MI 48201, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To address the question of prolonged antifungal therapy contributing to resistance emergence in Aspergillus, we recovered six isolates from patients receiving amphotericin B for invasive aspergillosis. These isolates were characterized and tested for resistance to amphotericin B in vitro. The results were reviewed in the context of the clinical profile of these patients and the final outcome. For comparison, we tested the antifungal susceptibility of Aspergillus isolates obtained from patients with no prior antifungal exposure. We also examined the possibility of selecting amphotericin B-resistant mutants by passaging Aspergillus isolates on plates containing various concentrations of the drug.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We identified six patients with fatal invasive aspergillosis treated at Harper Hospital, Wayne State University School of Medicine between 1996 and 1999. Three had culture-proven aspergillosis before treatment, whereas the remaining three were treated based on characteristic clinical and radiological findings. All six isolates available for testing were obtained from the six patients after they had received amphotericin B treatment for a variable duration.
Aspergillus isolates
The clinical isolates of A. fumigatus and Aspergillus niger used in this investigation were isolated from bronchial wash fluid and sputum at the Microbiology Laboratory of the Detroit Medical Center, Detroit, MI, USA. Working cultures of the isolates were maintained on Sabouraud dextrose agar slants at 4°C. Thirty-five Aspergillus isolates from patients with no prior exposure to any antifungal treatment were also examined (controls).
Antifungal drugs tested
The antifungal agents used in this study were obtained as pure powders from the manufacturers. Itraconazole (R51 211, batch no. STAN-9304-005-1) was obtained from Janssen Pharmaceutica, Beerse, Belgium; voriconazole (batch no. 25381-57-8) from Pfizer Inc., New York, NY, USA; amphotericin B (batch no. 20-914-29670) from Squibb Institute for Medical Research, Princeton, NJ, USA; and posaconazole (batch no. 97-56592-X-208) from Schering-Plough Research Institute, Kenilworth, NJ, USA. All antifungals were dissolved in dimethyl sulfoxide at a concentration of 1 g/L and stored at 20°C. The frozen stock was thawed at room temperature and gently vortexed before use. The concentrations of various drugs used for MIC studies ranged from 0.0625 to 16 mg/L. Where applicable, comparable concentrations of dimethyl sulfoxide were used as solvent controls.
MIC determination
The in vitro susceptibilities of various isolates of Aspergillus species to antifungal agents were determined by a broth microdilution method similar to the NCCLS M38-P protocol4 using peptone yeast extract glucose (PYG) medium [Bacto-peptone (Difco Chemicals, Detroit, MI, USA), 1 g yeast extract, 1 g glucose, 3 g/L distilled water], since RPMI 1640 is unsuitable for detecting amphotericin B resistance. Briefly, fresh conidia were collected from various Aspergillus isolates and suspended in PYG medium at a density of 2 x 104 conidia/mL. Twice the required concentration of the drugs was prepared in the same medium (0.1 mL) by serial dilution in 96-well round bottom microtitre plates (Rainin Instruments Company, Woburn, MA, USA) and inoculated with an equal volume (0.1 mL) of the conidial suspension. The microtitre plates were incubated at 35°C for 48 h and scored for visible growth with the aid of a viewing mirror. The MIC, defined as the lowest concentration of drug that produces no visible growth, was determined in duplicate with the experiment repeated once.
In vitro resistance selection
Conidial suspensions prepared from a susceptible clinical isolate of A. fumigatus (ATCC 208966) were plated (1 x 106 conidia/plate) on PYG agar containing amphotericin B (816 mg/mL) and incubated at 35°C for 6 days. Colonies that grew on plates containing amphotericin B were further tested for resistance by in vitro susceptibility testing.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
All six Aspergillus isolates were susceptible to amphotericin B; MICs ranged from 0.125 to 0.5 mg/L. The range of MICs of amphotericin B for Aspergillus isolates from 35 patients with no prior exposure to the antifungal agent was similar (Table 2). All isolates, from patients and controls, were equally susceptible to itraconazole and the newer triazoles voriconazole and posaconazole (Table 2
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amphotericin B has been used to treat fungal infections for over 40 years. Despite this long period of use, clinical resistance among Aspergillus species remains rare.8 It has been hypothesized that disruption of the complex interaction between amphotericin B and the plasma membrane requires multiple changes in the cell wall, making secondary resistance a challenge for the fungus and hence a rare event.3,8,9
All six patients in this study were severely immunocompromised, with high dose steroids and/or neutropenia playing a prominent role in five of the patients. Besides host factors, pharmacokinetics of amphotericin B may have played a role in the clinical outcome. Christiansen et al.10 proposed that the poor therapeutic activity of amphotericin B in vivo may be because the effective concentration of drug at the site of infection is considerably less than the apparent concentration. They hypothesized that amphotericin B binds tissue components such as cholesterol and lipoproteins in addition to the intended target, ergosterol. The poor outcome seen in four of the patients in this study with cerebral aspergillosis may be explained by subtherapeutic concentrations of amphotericin B in the CNS as a result of both tissue binding and poor penetration.
Our study indicates that use of amphotericin B does not select for resistant mutants during therapy for invasive aspergillosis. In clinical practice, recovery of Aspergillus isolates is infrequent from patients receiving treatment, hence the limited data. These preliminary findings need validation with a larger number of Aspergillus isolates from patients with prolonged exposure to amphotericin B.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Manavathu, E. K., Alangaden, G. J. & Chandrasekar, P. H. (1998). In-vitro isolation and antifungal susceptibility of amphotericin B-resistant mutants of Aspergillus fumigatus. Journal of Antimicrobial Chemotherapy 41, 6159. [Abstract]
3 . Seo, K., Akiyoshi, H. & Ohnishi, Y. (1999). Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus. Microbiology and Immunology 43, 101725. [ISI][Medline]
4 . National Committee for Clinical and Laboratory Standards. (1998). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi: Proposed Standard M38-P. NCCLS, Wayne, PA.
5
.
Johnson, E. M., Oakley, K. L., Radford, S. A., Moore, C. B., 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.
6
.
Mosquera, J., Warn, P. A., Morrissey, J., Moore, C. B., Gil-Lamaignere, C. & Denning, D. W. (2001). Susceptibility testing of Aspergillus flavus: inoculum dependence with itraconazole and lack of correlation between susceptibility to amphotericin B in vitro and outcome in vivo. Antimicrobial Agents and Chemotherapy 45, 145662.
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 . Vanden Bossche, H., Warnock, D. W., Dupont, B., Kerridge, D., Sen Gupta, S., Improvisi, L. et al. (1994). Mechanisms and clinical impact of antifungal drug resistance. Journal of Medical and Veterinary Mycology 32, 189202.
9 . Vanden Bossche, H., Marichal, P. & Odds, F. C. (1994). Molecular mechanisms of drug resistance in fungi. Trends in Microbiology 2, 393400. [Medline]
10 . Christiansen, K. J., Bernard, E. M., Gold, J. W. & Armstrong, D. (1985). Distribution and activity of amphotericin B in humans. Journal of Infectious Diseases 152, 103743. [ISI][Medline]
Received 27 March 2001; returned 5 July 2001; revised 16 August 2001; accepted 30 August 2001