Post-antifungal effect of amphotericin B and voriconazole against Aspergillus fumigatus analysed by an automated method based on fungal CO2 production: dependence on exposure time and drug concentration

Erja Chryssanthou1,* and Jan Sjölin2

1 Department of Clinical Microbiology L202, Karolinska University Hospital, S-171 76 Stockholm; 2 Section of Infectious Diseases, Department of Medical Sciences, University Hospital, Uppsala, Sweden

Received 3 June 2004; returned 30 July 2004; revised 30 August 2004; accepted 12 September 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The post-antifungal effect (PAFE) of amphotericin B and voriconazole, either alone or in combination, on Aspergillus fumigatus was studied using an automated system based on fungal CO2 production.

Methods: Conidia of A. fumigatus were exposed to concentrations of 1–10 x MIC of amphotericin B and 1–40 x MIC of voriconazole for 1, 2 and 4 h. After a washing step, exposed and control conidia were inoculated into Pedi-BacT culture bottles. CO2 production was automatically monitored until the bottles signalled positive. The difference in time span for positive signals in drug-exposed and control bottles was used to calculate PAFE.

Results: There was a linear relationship between inoculum size and time to positive signal (r2=0.99). The precision of duplicate analyses was 1.5%. Longer exposure times increased the amphotericin B-induced PAFE (P<0.001), whereas concentrations above the MIC did not. Voriconazole after 4 h of exposure induced a short dose-independent PAFE. The combination with amphotericin B did not prolong the PAFE over that caused by amphotericin B alone.

Conclusions: This automated method can be used for determination of PAFE. In contrast to Candida spp., in which amphotericin B-induced PAFE is mainly related to the area under the curve, the effect on A. fumigatus was more dependent on the exposure time. This implies that pharmacodynamic data obtained from Candida experiments cannot be directly extrapolated to Aspergillus.

Keywords: azoles , antifungals , PAFE , polyenes , susceptibility testing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Post-antifungal effect (PAFE) is a pharmacodynamic parameter that refers to the suppression of fungal regrowth persisting after short exposure to, and subsequent removal of, an antifungal drug. Knowledge of PAFE could be helpful in optimizing dosage regimens. The post-antifungal effects of polyenes, azoles, echinocandins and 5-fluorocytosine against Candida spp. have previously been relatively extensively reported.13 Because of a lack of appropriate methods, only a few investigations have hitherto studied the PAFE against filamentous fungi such as Aspergillus spp. Recently, Vitale et al. and Manavathu et al. demonstrated that amphotericin B induced PAFE in different Aspergillus spp.4,5 Vitale et al. analysed fungal growth by a turbidimetric method, whereas Manavathu et al. used a radiometric assay in which incorporation of labelled amino acids was measured. We have recently developed and evaluated an automated method for measuring PAFE of amphotericin B against Candida spp., based on monitoring of CO2 production by the BacT/Alert culture system.6 The aim of this study was to apply this automated method for determination of the PAFEs of amphotericin B and voriconazole alone and in combination against Aspergillus fumigatus. The effects of exposure time, drug concentration and area under the curve (AUC) were analysed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Aspergillus strain, antifungal agents and susceptibility testing

A. fumigatus CBS 13361 strain was used. Amphotericin B (Fungizone, Bristol-Myers Squibb, Bromma, Sweden) and voriconazole (Pfizer Pharmaceuticals, New York, NY, USA) were dissolved in sterile water and dimethylsulphoxide, respectively, as stock solutions of 5 g/L and stored at –70°C until required. The MICs of amphotericin B and voriconazole were determined by broth macrodilution modification according to the National Committee for Clinical Laboratory Standards guidelines, M38-A.7 The MIC was 0.5 mg/L for both amphotericin B and voriconazole.

Determination of PAFE

A. fumigatus was cultured on potato dextrose agar (PDA) at 37°C. After incubation for 2–3 days, the conidia were counted in a haemocytometer and quantified to a range of 1–3 x 106 cfu/mL by colony counts on PDA plates.

PAFE tests were carried out using RPMI-1640 medium (Sigma) supplemented with 1% dextrose and buffered with MOPS. Aliquots (10 mL) of conidial suspension were added to either 10 mL of RPMI medium alone (control) or amphotericin B and voriconazole diluted in RPMI to final concentrations of 1.0, 2.5, 5.0 and 10, and 2.5, 10 and 40 times the MIC, respectively. Test cultures were vortexed and viable counts were made by plating 0.1 mL of 10–3 dilutions on PDA plates in duplicate. Cultures were then placed on the shaker and agitated for 1, 2 or 4 h (voriconazole only 4 h) at 37°C. The PAFEs induced by combinations of amphotericin B (2.5 x MIC) plus voriconazole (2.5 x MIC) and amphotericin B (10 x MIC) plus voriconazole (40 x MIC) were determined after 4 h exposure. Drugs were removed by filtering the cultures through 0.45 µm membrane filters (Sartorius, Goettingen, Germany). The filters were washed with 100 mL PBS, transferred into tubes with 20 mL fresh PBS, and vortexed gently to dislodge conidia from the filters. The control cultures were treated in the same way. The control was divided into an undiluted and diluted (1:2 in PBS) portion in order to obtain a control with an inoculum as close as possible to the viable count of the cultures exposed to antifungal drugs. Viable counts of exposed and control cultures were carried out as above. Aliquots (2 mL) of each culture were injected into Pedi-BacT Aerobic Pediatric culture bottles (bioMérieux, Gothenburg, Sweden). Triplicate Pedi-BacT bottles for each exposed culture and controls were used on each occasion. Incubation of the bottles in the Bact/Alert Microbial Detection System and calculation of PAFE were done as described previously. Experiments were repeated on three separate occasions.

Linearity between the time required for a positive signal and the number of conidia and the precision of duplicate pairs were tested by injecting 2 mL aliquots of serially diluted control cultures from 101 to 106 cfu/mL into Pedi-BacT bottles.

Time–kill curve procedure

Aliquots (10 mL) of the conidial suspension (4 x 105–8 x 105 conidia/mL) were added to 10 mL of RPMI medium alone (control) and to amphotericin B or voriconazole diluted in 10 mL RPMI to the same concentrations as used in the PAFE experiments. Cultures were placed on the shaker and agitated at 37°C. At predetermined time points (0, 1, 2, 4, 8 h) two samples of 0.1 mL were removed from each test suspension, serially diluted, and 0.1 mL aliquots were spread in duplicate on PDA plates and incubated at 37°C overnight. The experiments were conducted on two separate occasions. Time–kill curves of averaged colony counts (log10 cfu/mL) versus time (h) were constructed.

Statistics

Regression analysis was employed to determine whether there was a linear relationship between the time required for a positive signal and the inoculum size. The method error (s) used in the precision analysis was estimated on the basis of duplicates using the formula s={surd}({Sigma}d2/2n), where d is the difference between the duplicates and n the number of duplicate pairs. The precision was calculated as the method error as a percentage of the mean of the determinations. A repeated measures ANOVA was used to analyse the influence of exposure time, drug concentration and AUC on PAFE, and regression analysis was applied to test linearity. A difference between two groups was considered significant when the P value was <0.05. STATISTICA software (StatSoft, Inc., Tulsa, OK, USA) was used in the statistical calculations.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The time–kill curves demonstrated that within 4 h of amphotericin B exposure, there was only a limited concentration-dependent killing, the difference to the control being maximally 0.4 log10 cfu/mL, whereas exposure to voriconazole did not result in any significant killing rate (Figure 1).



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Figure 1. Time–kill curves of amphotericin B (a) and voriconazole (b) against Aspergillus fumigatus.

 
There was a linear relationship between inoculum size and time to positive signal in the Pedi-BacT system (r2=0.99), which with inocula ranging from 101 to 106 cfu/mL occurred after 13.1–31.3 h. The method error of duplicate analyses was 0.32 h, corresponding to a precision of 1.5%.

Amphotericin B induced PAFEs in A. fumigatus at concentrations of 1–10 x MIC ranging from 0.1 to 7.2 h. The PAFE was significantly influenced by exposure time, with the longest exposure time resulting in the longest observed PAFE (P < 0.001) (Figure 2). It was also dependent on the amphotericin B concentration (P < 0.001), but this was solely caused by the relatively low PAFE at the amphotericin B concentration of 1 x MIC. In the range of 2.5–10 x MIC, no concentration dependency could be demonstrated (P=0.36). In the regression analysis, exposure time dependency of the PAFE resulted in an r2 value of 0.72. If the 1 x MIC values were excluded from the calculation, the r2 increased to 0.96. In contrast, when the PAFE was related to amphotericin B concentration or log concentration, r2 values of 0.04 and 0.07, respectively, were obtained. Corresponding r2 values for the AUC were 0.45 and 0.46, respectively.



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Figure 2. Effect of exposure time and concentration of amphotericin B on the post-antifungal effect (mean ± S.E.M.). The solid line depicts the regression line based on all values, whereas the broken line represents the values obtained from concentrations in the interval of 2.5–10 x MIC.

 
Voriconazole induced a significant but short and dose-independent PAFE, ranging from 0.1 to 1.4 h at 2.5–40 x MIC after 4 h of exposure (n=21; P < 0.001). Addition of voriconazole at 2.5 x MIC and 40 x MIC to amphotericin B at 2 x MIC and 10 x MIC, respectively, did not increase the PAFE over that induced by amphotericin B alone.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present method utilizes CO2 production from actively growing hyphae for assessment of fungal growth. This offers an advantage in comparison with turbidimetric methods in which alteration in the frequency of fungal branching may result in changes in the optical density and therefore necessitates microscopic examination. The BacT/Alert system is readily available and does not need a process of radioactive labelling. The accuracy of the present method was very good due to the high linearity between time for signalling and inoculum size. In agreement with previous results,8 fungal killing was limited during the first 4 h, indicating that the use of only two dilutions was sufficient to adjust the control inoculum to that after drug exposure. As has also been found by others,4 microscopic examination revealed that germination did not occur during this period. The precision was higher than that reported for the turbidimetric method.4 For the radiometric assay, no precision data were given.5

In agreement with the other methods used,4,5 a post-antifungal effect of amphotericin B of several hours against A. fumigatus was demonstrated. Although voriconazole alone demonstrated a short PAFE, the addition of voriconazole did not affect the amphotericin B-induced PAFE.

The PAFE of amphotericin B against Candida spp. has been shown to be dependent on the AUC,3 which was recently confirmed using the present method.6 The relationship between the PAFE and the logarithm of the concentration was close to linear for a given exposure time.6 In contrast, our data on A. fumigatus indicated that there is no concentration dependency at concentrations above the MIC value. The concentration dependency found in the studies by Vitale et al. and Manavathu et al. may be due to strain differences or the fact that PAFEs caused by concentrations close to the MIC4,5 were included in the calculations, which then may imply uncertainty of the pharmacodynamic effect of amphotericin B.

In the treatment of experimental invasive candidiasis, high infrequent doses of amphotericin B have been shown to be more effective than if the same total dose was administered more frequently.9 Our result suggests that this cannot be directly extrapolated to Aspergillus infections because the concentration–effect on the PAFE seems to be much less. The frequency of concentration independency among Aspergillus strains, the mechanism behind this and its clinical relevance need further investigation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by grants from Pfizer.


    Footnotes
 
* Corresponding author. Email: erja.chryssanthou{at}kus.se


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Klepser, M. E., Wolfe, E. J., Jones, R. N. et al. (1997). Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B tested against Candida albicans. Antimicrobial Agents and Chemotherapy 41, 1392–5.[Abstract]

2 . Minguez, F., Lima, J. E., Garcia, M. T. et al. (1996). Influence of human serum on the postantifungal effect of four antifungal agents on Candida albicans. Chemotherapy 42, 273–9.[CrossRef][ISI][Medline]

3 . Turnidge, J. D., Gudmundsson, S., Vogelman, B. et al. (1994). The postantibiotic effect of antifungal agents against common pathogenic yeasts. Journal of Antimicrobial Chemotherapy 34, 83–92.[Abstract]

4 . Vitale, R. G., Mouton, J. W., Afeltra, J. et al. (2002). Method for measuring postantifungal effect in Aspergillus species. Antimicrobial Agents and Chemotherapy 46, 1960–5.[Abstract/Free Full Text]

5 . Manavathu, E. K., Ramesh, M. S., Baskaran, I. et al. (2004). A comparative study of the post-antifungal effect (PAFE) of amphotericin B, triazoles and echinocandins on Aspergillus fumigatus and Candida albicans. Journal of Antimicrobial Chemotherapy 53, 386–9.[Abstract/Free Full Text]

6 . Chryssanthou, E., Cars, O. & Sjölin, J. (2002). New automated method for determining postantifungal effect of amphotericin B against Candida species: Effects of concentration, exposure time, and area under the curve. Antimicrobial Agents and Chemotherapy 46, 4016–8.[Abstract/Free Full Text]

7 . National Committee for Clinical Laboratory Standards. (1998). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi: Approved Standard M38-A. NCCLS, Wayne, PA, USA.

8 . Manavathu, E. K., Cutright, J. L. & Chandrasekar, P. H. (1998). Organism-dependent fungicidal activities of azoles. Antimicrobial Agents and Chemotherapy 42, 3018–21.[Abstract/Free Full Text]

9 . Andes, D., Stamsted, T. & Conklin, R. (2001). Pharmacodynamics of amphotericin B in a neutropenic-mouse disseminated candidiasis model. Antimicrobial Agents and Chemotherapy 45, 922–6.[Abstract/Free Full Text]





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