JAC
Journal of Antimicrobial Chemotherapy (1998) 42, 389–392
In-vitro activities of amphotericin B, itraconazole and voriconazole against 150 clinical and environmental Aspergillus fumigatus isolates Paul E. Verweija*, Marieke Mensinka, Antonius J. M. M. Rijsa, J. Peter Donnellya,b, Jacques F. G. M. Meisa and David W. Denningc a
Department of Medical Microbiology and bDivision of Hematology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands; cDepartment of Medicine, University of Manchester, Hope Hospital, Salford, UK The in-vitro activity of amphotericin B, itraconazole and voriconazole against 130 clinical and 20 environmental Aspergillus fumigatus isolates was tested with an agar dilution method using RPMI 1640 medium. Itraconazole-susceptible (AF71) and -resistant (AF90) isolates were included in each test and all isolates were tested in duplicate. Geometric mean MIC values (ranges) at 48 h and 72 h were, respectively, amphotericin B, 0.91 (0.25–2) and 1.50 (1–4) mg/L; itraconazole, 0.25 (0.06–1) and 0.48 (0.125–8) mg/L; and voriconazole, 0.56 (0.25–8) and 0.78 (0.25–8) mg/L. The reproducibility of the results was high for all drugs. In-vitro resistance of A. fumigatus to the tested antifungal agents was uncommon and the MICs of itraconazole were half those of voriconazole (P < 0.01).
Introduction Invasive aspergillosis is a devastating disease with a high morbidity and mortality rate. The success of treatment is limited and overall only 34% of patients show a favourable response.1 Since the majority of patients who become infected are severely immunocompromised, the choice of antifungal agent is often critical in order successfully to control the infection at least until the recovery of the bone marrow. It is common practice to administer antifungal agents such as amphotericin B desoxycholate empirically to patients suspected of invasive aspergillosis,2 because of the difficulty in establishing the diagnosis. It is assumed that Aspergillus spp. are generally susceptible, but a reliable and meaningful in-vitro susceptibility test is not available for amphotericin B and it was only recently that a method was developed for testing the activity of antifungal azoles against Aspergillus fumigatus. The method was validated by correlating in-vitro test results with in-vivo outcome of infection.3 Using this method, itraconazole resistance among clinical A. fumigatus isolates was documented.4 However, the frequency of resistance among A. fumigatus to antifungal azoles such as itraconazole and the new broad-spectrum triazole voriconazole (UK-109,496) is unknown. We have investigated the frequency of resistance among
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130 clinical and 20 environmental A. fumigatus isolates to amphotericin B, itraconazole and voriconazole by an agar dilution method. The reproducibility of this assay was also assessed.
Materials and methods Antifungal drugs Amphotericin B (Bristol Myers Squibb, Woerden, The Netherlands), voriconazole (UK-109,496; Pfizer Central Research, Sandwich, UK) and itraconazole (Janssen-Cilag, Tilburg, The Netherlands) were obtained as standard powders. Amphotericin B was dissolved at 3.2 g/L in 10 mL of dimethylsulphoxide (Merck, Meppel, The Netherlands). Voriconazole powder was dissolved at 3.2 g/L in 1 mL dimethylformamide (Merck) and 9 mL of sterile distilled water, and itraconazole was dissolved at 3.2 g/L in a 50:50 solution of 20% (v/v) acetone in water and 20% (v/v) hydrochloric acid (specific gravity 1.18; Merck) in water. Each drug was stored in 600 L aliquots at –80°C until use.
Isolates One hundred and fifty A. fumigatus isolates were used in this study. These isolates were recovered at the University
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389 © 1998 The British Society for Antimicrobial Chemotherapy
P. E. Verweij et al. Hospital in Nijmegen, The Netherlands, and included 130 isolates from 65 patients, and 20 isolates from the hospital environment. The patient isolates included 77 recovered from tissue samples, 28 from broncho-alveolar lavage fluid and 25 from sputum. The isolates were identified by colony appearance, growth at 48°C and the morphology of the conidia and conidiophores. The reproducibility of the method of testing susceptibility to amphotericin B, itraconazole and voriconazole was determined by testing each isolate in duplicate. The same subcultures were used, but the inoculum, antifungal drug solutions and agar plates were prepared separately, and susceptibility testing was performed on a different day.
Susceptibility testing Antifungal susceptibility was determined by an agar dilution method with RPMI 1640 agar supplemented with 2% glucose (Gibco, Breda, The Netherlands) and 0.03% (w/v) L-glutamine and buffered with 0.165 M 3-[Nmorpholino]propanesulphonic acid (Sigma, Amsterdam, The Netherlands).3 Doubling dilutions of amphotericin B, itraconazole and voriconazole to give final concentrations in agar from 64 to 0.03 mg/L were prepared in 2 mL volumes of sterile water. Molten agar (18 mL) was added, mixed, poured into Petri dishes and allowed to set. A. fumigatus isolates were subcultured on to Sabouraud glucose agar slopes and incubated for 3 days at 28°C. Spores were harvested in 3 mL of phosphate-buffered saline (PBS) containing 0.05% Tween 80, and heavy particles were allowed to settle for 5 min. Spores were collected from the upper part of the suspension and vortexed for 10 s. The density of the inoculum was estimated by the turbidities of the spore suspensions at 530 nm using a spectrophotometer (Spectronic 20D, Spectronic, Rochester, NY, USA), and confirmed by plating on Sabouraud glucose agar to determine the cfu/mL. Spore suspensions were adjusted to a density of 107 conidia/mL with PBS. Plates were inoculated with 22 spore suspensions from 20 isolates together with one itraconazolesusceptible isolate (AF71) and one itraconazole-resistant isolate (AF90) using a multi-point inoculator (Denley multipoint inoculator A400; UK) and incubated at 35°C. MICs were read at 48 and 72 h and were defined as the lowest concentration of drug inhibiting visible growth.
Results All A. fumigatus isolates produced visible growth at 48 and 72 h of incubation. MICs of amphotericin B, itraconazole and voriconazole are shown in the Table and were dependent on the time at which MICs were read (P 0.001). The MICs of itraconazole for the control isolates were 0.125–0.25 mg/L and 0.25–0.5 mg/L for AF71 at 48 and 72 h respectively, and 64 mg/L for AF90 at both 48 and 72 h of incubation. The MICs of voriconazole and amphotericin B for both control isolates were 0.5–1 and 1–4 mg/L respectively at 48 h. Overall, the MICs of itraconazole were approximately half those of voriconazole, and these differences were statistically significant at both 48 and 72 h (P 0.01). There was no difference between the MICs for clinical and environmental isolates. No isolates were identified with an MIC of 64 mg/L against itraconazole. One isolate (AZN 3075) had an MIC of 8 mg/L at 72 h, but the duplicate MIC was 1 mg/L. Twelve A. fumigatus isolates had been cultured from consecutive sputum specimens from a patient with cystic fibrosis over a 25 month period. The MIC of itraconazole among these isolates was 0.25–0.5 mg/L. Despite treatment with itraconazole (200 mg three times a day) for more than 16 months there was no evidence for the development of resistance among the A. fumigatus isolates. The MIC of voriconazole for one isolate (AZN 606; CBS 612.97) was 8 mg/L after 48 and 72 h of incubation, which was approximately four dilution steps higher than the geometric mean MIC of voriconazole. The MIC of itraconazole for this isolate was 0.5–1 mg/L. These MICs were reproducible on retesting. The isolate was cultured from the sputum of a kidney transplant recipient who died of disseminated aspergillosis; it produced white colonies when grown on Sabauroud glucose agar and incubated at 30°C. The strain had not caused invasive disease since an A. fumigatus isolate which produced greyish-green colonies was cultured from the lung at autopsy. This isolate had an MIC of 0.5 mg/L for itraconazole and 1 mg/L for voriconazole. The levels of agreement between duplicate MICs at 48 and 72 h of incubation, respectively, were 90.6 and 94.6% for amphotericin B; 100 and 98.7% for itraconazole, and 99.3 and 98.7% for voriconazole.
Data analysis The geometric mean of duplicate log2 MICs was calculated for each antifungal agent. Discrepancies among MICs of no more than one dilution step were used to calculate the percent agreement. The significance of the differences in the distributions of voriconazole and itraconazole MICs and those between MICs read at 48 and 72 h were determined by the Wilcoxon rank-sum test. A P value of 0.01 was considered a statistically significant difference.
Discussion To be of any value, an in-vitro susceptibility test must be able to detect resistance; ideally, the results of the test should also correspond to the outcome of treatment. Although many attempts have been made to develop meaningful and reproducible in-vitro susceptibility tests
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In-vitro susceptibility of Aspergillus fumigatus Table. In-vitro activity of amphotericin B, itraconazole and voriconazole against 150 A. fumigatus isolates as determined by an agar dilution method after 48 and 72 h of incubation MIC (mg/L) 48 h Antifungal agenta
range
MIC50
MIC90
Amphotericin B
0.25–2 0.25–1 0.06–1 0.06–1 0.25–4 0.25–8
1 0.5 0.125 0.25 0.25 0.25
1 0.5 0.25 0.25 0.5 0.25
Itraconazole Voriconazole
72 h geometric mean 0.91 0.81 0.25 0.19 0.56 0.43
range 1–4 0.5–2 0.125–8 0.125–1 0.25–8 0.25–8
MIC50
MIC90
geometric mean
1 1 0.5 0.125 0.5 0.5
1 1 0.5 0.25 0.5 0.5
1.50 1.39 0.48 0.32 0.78 0.68
a
For each antifungal agent, the MIC values determined in one series of experiments are given on the first line and those from a duplicate series on the second line.
for filamentous fungi,5 only recently has an assay been described which allows detection of resistance to antifungal azoles in A. fumigatus and which has been validated by correlating the in-vitro result to in-vivo outcome.3 By using this method and by including itraconazolesusceptible and -resistant control isolates, we found no isolates resistant to itraconazole. One isolate exhibited an MIC of 8 mg/L for itraconazole, but this could not be confirmed at retesting. Most of the isolates tested were cultured from specimens obtained at diagnosis or from specimens obtained at autopsy from patients who failed to respond to amphotericin B treatment and died. None of the isolates were resistant to itraconazole. Even among 12 A. fumigatus isolates cultured from the sputum of a cystic fibrosis patient who had been treated with itraconazole for more than a year, no development of resistance or replacement of itraconazole-susceptible isolates by itraconazoleresistant isolates was apparent. Voriconazole is a novel broad-spectrum triazole which is fungicidal against a wide range of fungal organisms6 including Aspergillus spp.7 and other opportunistic moulds.8 The drug has been shown to have good efficacy in animal models9 and in the treatment of patients with acute invasive aspergillosis. In the present study, the MICs of voriconazole were approximately one dilution step higher than those of itraconazole, confirming previous results obtained with the same method.7 However, others have found comparable in-vitro activities of voriconazole and itraconazole against A. fumigatus or superior in-vitro activity of voriconazole by using a modified microdilution method proposed by the National Committee for Clinical Laboratory Standards.9 In the latter study it was also shown that voriconazole was more effective in vivo than itraconazole in increasing survival in a rat model of invasive infection with A. fumigatus.9 These reported
differences in efficacy may result from the variable absorption of itraconazole as opposed to good bioavailability of voriconazole. Therefore, comparative clinical trials are warranted in order to compare the efficacy and toxicity of these azoles in the treatment of invasive aspergillosis. The present study also showed that voriconazole was active in vitro against the itraconazoleresistant A. fumigatus control isolate, but this observation remains to be confirmed in an animal model. Resistance of A. fumigatus to voriconazole has not been described and among the isolates we tested only one had an MIC of 8 mg/L. Further studies are currently being performed to characterize this isolate and to establish its pathogenicity in vivo, since little is known about the taxonomy and pathogenicity of white mutant strains of A. fumigatus. The results of the present study demonstrate an excellent level of agreement between duplicate MICs of itraconazole and voriconazole at both 48 and 72 h. Our results also indicate that MICs obtained by the agar dilution method might be read and forwarded to the clinician at 48 h since the resistant control A. fumigatus isolate showed an MIC of 64 mg/L at 48 h. In-vitro susceptibility testing for amphotericin B remains a problem and the agar dilution method cannot be used to test this compound despite the good reproducibility. We recently observed resistance of an A. fumigatus isolate to amphotericin B in an animal model of invasive aspergillosis, and the resistance was not detected by this agar dilution method or by other in-vitro susceptibility tests.10 Since amphotericin B remains the standard treatment for invasive aspergillosis, it is clear that the development of a meaningful in-vitro test to detect resistance to amphotericin B among A. fumigatus needs to be addressed in the near future.
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7. Oakley, K. L., Moore, C. B. & Denning, D. W. (1996). In vitro activity of voriconazole against Aspergillus spp. and comparison with amphotericin B and itraconazole. In Program and Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, 1996. Abstract F81, p. 114. American Society for Microbiology, Washington, DC. 8. Radford, S. A., Johnson, E. M. & Warnock, D. W. (1997). In-vitro studies of activity of voriconazole (UK-109,496), a new triazole antifungal agent, against emerging and less-common mold pathogens. Antimicrobial Agents and Chemotherapy 41, 841–3. 9. Murphy, M., Bernard, E. M., Ishimaru, T. & Armstrong, D. (1997). Activity of voriconazole (UK-109,496) against clinical isolates of Aspergillus species and its effectiveness in an experimental model of invasive pulmonary aspergillosis. Antimicrobial Agents and Chemotherapy 41, 696–8. 10. Verweij, P. E., Oakley, K. L., Morrissey, J., Morrissey, G. & Denning, D. W. (1998). In vitro activity of LY303366 against amphotericin B susceptible and resistant Aspergillus fumigatus in a murine model of invasive aspergillosis. Antimicrobial Agents and Chemotherapy 42, 873–8.
Received 23 October 1997; accepted 17 March 1998
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