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Sequential exposure of Aspergillus fumigatus to itraconazole and caspofungin: evidence of enhanced in vitro activity
Diagnostic Microbiology and Infectious Disease 47 (2003) 415– 419
www.elsevier.com/locate/diagmicrobio
Mycology
Sequential exposure of Aspergillus fumigatus to itraconazole and caspofungin: evidence of enhanced in vitro activity Dimitrios P. Kontoyiannisa,c,*, Russell E. Lewisa,c, Michail S. Lionakisa, Nathaniel D. Alberta, Gregory S. Mayb, Issam I. Raada a
Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA b Laboratory Medicine, The University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA c University of Houston College of Pharmacy, Houston, TX, USA Received 17 January 2003; received in revised form 29 April 2003
Abstract We investigated the in vitro activity of sequential itraconazole and caspofungin against 10 isolates of Aspergillus fumigatus. Previous exposure of A. fumigatus to itraconazole resulted in dose-dependent enhanced effects of caspofungin and vice versa. Our finding suggests a preferential role for azole-caspofungin sequential combinations and merits further in vivo investigation. © 2003 Elsevier Inc. All rights reserved. Keywords: Caspofungin; Itraconazole; A. fumigatus
1. Introduction Invasive aspergillosis (IA) is the leading cause of infectious death in bone marrow transplant recipients and patients with hematologic malignancies (Kontoyiannis and Bodey, 2002). Itraconazole (ITZ), a broad-spectrum triazole, and caspofungin (CAS), a novel echinocandin that inhibits fungal cell wall biosynthesis, have activity against Aspergillus spp. and show promise for the treatment of IA (Groll et al., 1998). The availability of ITZ in formulations that result in reliable serum levels (i.e., i.v. and oral suspension) has led to its increasing use as prophylaxis in patients at high-risk for IA (Harousseau et al., 2000). Similarly, the potent activity of echinocandins against Candida spp. (including the azole-resistant non-albicans Candida spp) (Groll et al., 1998) and their activity against Aspergillus spp (Maertens et al., 2002) make CAS appealing for both empiric and prophylactic use in patients with febrile neutropenia who are at risk for invasive fungal infections Presented in part at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 2002 (Abstract #M-851). * Corresponding author. Tel.: ⫹1-713-792-6237; fax: ⫹1-713-7456839. E-mail address: [email protected] (D.P. Kontoyiannis). 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(03)00136-6
(Van Burik et al., 2002). Therefore, ITZ and CAS are expected to be frequently used in different sequences in the course of IA treatment (Kontoyiannis and Bodey, 2002). The clinical question is whether breakthrough IA that occurs despite the use of either drug would result in higher likelihood of subsequent failure of the other drug. There have been no preclinical studies of the staggered administration of these drugs. To that end, we studied whether prior exposure to ITZ or CAS affects the subsequent activity of CAS or ITZ respectively in Aspergillus fumigatus.
2. Materials and methods We tested the susceptibility of 10 clinical isolates of A. fumigatus to CAS and ITZ. Aspergillus growth was examined in yeast extract glucose (YAG) plates prepared using standard methods. CAS and ITZ were obtained from Merck Research Laboratories (Merck & Co. Inc., Rathway, NJ) and Janssen Pharmaceutical (Titusville, NJ) respectively. CAS was dissolved in sterile water and ITZ in dimethyl sulfoxide. The concentration of the stock solutions of both drugs was 10 mg/ml. To test the susceptibility of A. fumigatus to ITZ following exposure to CAS, YAG-plates containing increasing
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concentrations of CAS (0, 0.0625, 0.125, 0.25, 0.5, 1, and 4 g/ml) were each swab inoculated with 200 L of a standardized suspension of Aspergillus conidia (106 conidia/ml) of each isolate. Stock conidial suspensions were prepared from 3-day-old cultures grown on YAG-plates. Conidia were counted using hemocytometry. After inoculation, the plates were incubated at 37°C for 12-16 h. The following day, a 1⁄4-inch-diameter paper disk (Schleicher and Schuell, Keene, N.H.) containing 10 g of ITZ (25 L of the ITZ stock solution) was applied on the agar surface and plates were incubated for another 48 h at 37°C. To test the susceptibility of A. fumigatus to CAS following exposure to ITZ, YAG-plates containing increasing concentrations of ITZ (0, 10, 50, 75, 100, 150, and 300 ng/ml) were similarly swab inoculated with 200 L of a standardized suspension of Aspergillus conidia (106 conidia/ml) of each isolate. After inoculation, the plates were incubated at 37°C for 12-16 h. The following day, a disk containing 20 g of CAS (50 L of the CAS stock solution) was applied on plates, which were then incubated for another 48 h at 37°C. To evaluate the effects of simultaneous exposure to both drugs, YAG-plates containing different concentrations of CAS (0, 0.25 g/ml, 0.5 g/ml and 1 g/ml) were each swab inoculated with 200 L of conidial suspensions (106 conidia/ml) of each isolate. A disk containing 10 g of ITZ was placed on the agar surface 15 min after isolates were plated and plates were incubated for 48 h at 37°C. The radius of the zone of growth inhibition was measured in millimeters at 48 h of incubation. All assays were conducted in triplicate in three different experiments. The Kruskal-Wallis test was used to compare measurements. Differences were further analyzed posthoc using the Tukeys Test for multiple comparisons. All comparisons were considered statistically significant for P values ⱕ0.05.
3. Results Previous exposure of A. fumigatus isolates to ITZ or CAS resulted in enhanced in vitro effects of CAS or ITZ, respectively (Table 1, Fig. 1A and 1B). This attenuated growth of Aspergillus isolates upon the sequential exposure to CAS and ITZ was not due to growth inhibition caused by the increasing concentrations of either drug previously added in the plate; in pilot experiments all 10 isolates grew in YAG-plates containing increasing concentrations of CAS (up to 4 g/ml) or ITZ (up to 300 ng/ml) (data not shown). The positive interaction of the two drugs increased over time, reaching its maximum effect at 48 h; this effect was sustained throughout 96 h (data not shown). Consistent enhancement of the activity of one drug following exposure to the other was seen in all 10 isolates, although strain-dependent differences
Table 1 Summary of the growth inhibition responses of 10 clinical isolates of A. fumigatus to sequential ITZ and CAS exposure using disk diffusion assay Sequence of drug administration
A. ITZ followed by CAS in disk (20g) ITZ concentration in plates (ng/mL) 0 (CAS alone) 10 75 100 150 300 B. CAS followed by ITZ in disk (10g) CAS concentrations in plates (g/mL) 0 (ITRA alone) 0.0625 0.125 0.25 0.5 1 4 C. Simultaneous CAS (in plates) and ITZ in disk (10g) CAS concentrations in plates (g/mL) 0 0.25 0.5 1
Mean radius of zone of growth inhibition (in mm) ⫾ SD
No zone 0.2 ⫾ 0.1 1.2 ⫾ 0.4a 3.9 ⫾ 0.7a 9.7 ⫾ 2a 13 ⫾ 3a
3.04 3.85 ⫾ 1,3 4.45 ⫾ 1,4 7.8 ⫾ 1.1a 8.4 ⫾ 1.3a 8.9 ⫾ 1.3a 8.2 ⫾ 1.5a 12.1 ⫾ 0.7 12.5 ⫾ 1.1 12.6 ⫾ 1.3 12.8 ⫾ 1.2
ITZ, itraconazole; CAS, Caspofungin. a P ⬍ 0.05 vs. control SD, standard deviation
were noted. In contrast, the simultaneous exposure of A. fumigatus isolates to CAS and ITZ did not result in enhanced in vitro effects (Table 1). The zone of growth inhibition caused by ITZ in disk was larger when A. fumigatus isolates were exposed to ITZ and CAS simultaneously (Table 1C), compared to the zone when ITZ in the disk was applied after prior growth of A. fumigatus in CAS-containing plates (Table 1B). This is consistent with our previous experience on the activity of ITZ when applied in freshly inoculated plated versus against pregrown lawns (Kontoyiannis et al., 2000).
4. Discussion We found that pre-exposure of A. fumigatus to ITZ or CAS results in enhanced in vitro effects of subsequent CAS or ITZ use respectively. This finding contrasts with the apparently antagonistic effect observed when amphotericin B is used after ITZ, both in vitro and in animal models of IA (Kontoyiannis et al., 2000; Lewis et al., 2002). The exact
D.P. Kontoyiannis et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 415– 419
417
Fig. 1A. A representative isolate of A. fumigatus (isolate AF 34) has been pre-grown for 16 h in YAG plates containing ITZ at concentrations of 0, 10, 75, 100, 150 and 300 (ng/mL). A disk containing 20 g of CAS was applied on the agar surface the following day. Plates were incubated at 37°C for 48 h and the zone of growth inhibition caused by CAS was then assessed.
mechanism of this positive in vitro triazole-echinocandin interaction is unknown. ITZ is an ergosterol-depleting agent and CAS inhibits cell wall biosynthesis of the growing hyphal tips (Groll et al., 1998; Bowman et al., 2002). The enhanced effect of each drug following staggered administration of the other would suggest a feedback regulation between ergosterol and cell wall biosynthesis. It is possible that exposure of A. fumigatus to an ergosteroldepleting agent (ITZ) could result in downregulation of cell wall remodeling, thus augmenting the effects of subsequent use of an echinocandin. The influence of ergosterol depletion to cell wall remodeling has been
previously suggested in Saccharomyces cerevisiae and C. albicans (Pfaller and Riley, 1992; Kontoyiannis, 1999; Bammert and Fostel, 2000). Conversely, CAS-induced fungal cell wall alterations could affect ITZ entry or efflux. The relevance of this observation needs to be validated in animal models of IA. For example, it would be interesting to determine whether breakthrough IA following ITZ prophylaxis responds better to CAS or to amphotericin B-based regimens. Our finding should stimulate further investigations for the identification of effective sequences and combinations of antifungal therapy for treatment of breakthrough IA.
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Fig. 1B. A representative isolate of A. fumigatus (isolate AF 34) has been pre-grown for 16 h in YAG plates containing CAS at concentrations of 0, 0.0625, 0.125, 0.25, 0.5 and 1 (g/mL). A disk containing 10 g of ITZ was applied on the agar surface the following day. Plates were incubated at 37°C for 48 h and the zone of growth inhibition caused by ITZ was then assessed.
References Bammert, G. F., & Fostel, J. M. (2000). Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergostetrol. Antimicrob Agents Chemother 44, 1225–1263. Bowman, J. C., Hicks, P. S., Kurtz, M. B., Rosen, H., Schmatz, D. M., Liberator, P., et al (2002). The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. Antimicrob Agents Chemother 46, 3001–3012. De Backer, M. D., Ilyina, T., Ma, X. J., Vandoninck, S., Luyten, W. H., & Vanden Bossche, H. (2001). Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray. Antimicrob Agents Chemother 45, 1660 –1670. Groll, A. H., Piscitelli, S. C., & Walsh, T. H. (1998). Clinical pharmacology of systemic antifungal agents: A comprehensive review of agents in clinical use, current investigational compounds, and pu-
tative targets for antifungal drug development. Adv Pharmacol 44, 343–500. Harousseau, J. L., Dekker, A. W., Stamatoulas-Bastard, A., Fassas, A., Linkesch, W., Gouveia, J., et al (2000). Itraconazole oral solution for primary prophylaxis of fungal infections in patients with hematological malignancy and profound neutropenia: a randomized, double-blind, double-placebo, multicenter trial comparing itraconazole to amphotericin B. Antimicrob Agents Chemother 44, 1887–1893. Kontoyiannis, D. P. (1999). Genetic analysis of azole resistance by transposon mutagenesis in Saccharomyces cerevisiae. Antimicrob Agents Chemother 43, 2731–2735. Kontoyiannis, D. P., & Bodey, G. P. (2002). Invasive aspergillosis in 2002: An update. Eur J Clin Microbiol Infect Dis 21, 161–172. Kontoyiannis, D. P., Lewis, R. E., Sagar, N., May, G., Prince, R. A., & Rolston, K. V. (2000). Itraconazole-amphotericin B antagonism in Aspergillus fumigatus: an E-test-based strategy. Antimicrob Agents Chemother 44, 2915–2918.
D.P. Kontoyiannis et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 415– 419 Lewis, R. E., Prince, R. A., & Kontoyiannis, D. P. (2002). Pretreatment with itraconazole attenuates the efficacy of increasing amphotericin B dosages in a murine model of invasive pulmonary aspergillosis. Antimicrob Agents Chemother 46, 3208 –3214. Maertens, J., Raad, I. I., Petrikkos, D., Selleslag, D., Petersen, F., Sable,C., Kartsonis, N., Ngai, A., Taylor, A., Patterson, T., Denning, D., Walsh, T. (2002). Update of the multicenter noncomparative study of caspofungin (CAS) in adults with invasive Aspergillus (IA) refractory or intolerant to other antifungal agents: Analysis of 90 patients. 42nd Interscience Con-
419
ference on Antimicrobial Agents and Chemotherapy; September 2002; San Diego, CA. Abstract 388:M-868. Pfaller, M., & Riley, J. (1992). Effects of fluconazole on the sterol and carbohydrate composition of four species of Candida. Eur J Clin Microbiol Infect Dis 11, 152–156. Van Burik, J., Ratanatharathorn, V., Lipton, J., Miller, C., Bunin, N., Walsh, T. J. (2002). Randomized, double-blind trial of micafungin versus fluconazole for prophylaxis of invasive fungal infections in patients undergoing hematopoietic stem cell transplant. Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 2002.
www.elsevier.com/locate/diagmicrobio
Mycology
Sequential exposure of Aspergillus fumigatus to itraconazole and caspofungin: evidence of enhanced in vitro activity Dimitrios P. Kontoyiannisa,c,*, Russell E. Lewisa,c, Michail S. Lionakisa, Nathaniel D. Alberta, Gregory S. Mayb, Issam I. Raada a
Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA b Laboratory Medicine, The University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA c University of Houston College of Pharmacy, Houston, TX, USA Received 17 January 2003; received in revised form 29 April 2003
Abstract We investigated the in vitro activity of sequential itraconazole and caspofungin against 10 isolates of Aspergillus fumigatus. Previous exposure of A. fumigatus to itraconazole resulted in dose-dependent enhanced effects of caspofungin and vice versa. Our finding suggests a preferential role for azole-caspofungin sequential combinations and merits further in vivo investigation. © 2003 Elsevier Inc. All rights reserved. Keywords: Caspofungin; Itraconazole; A. fumigatus
1. Introduction Invasive aspergillosis (IA) is the leading cause of infectious death in bone marrow transplant recipients and patients with hematologic malignancies (Kontoyiannis and Bodey, 2002). Itraconazole (ITZ), a broad-spectrum triazole, and caspofungin (CAS), a novel echinocandin that inhibits fungal cell wall biosynthesis, have activity against Aspergillus spp. and show promise for the treatment of IA (Groll et al., 1998). The availability of ITZ in formulations that result in reliable serum levels (i.e., i.v. and oral suspension) has led to its increasing use as prophylaxis in patients at high-risk for IA (Harousseau et al., 2000). Similarly, the potent activity of echinocandins against Candida spp. (including the azole-resistant non-albicans Candida spp) (Groll et al., 1998) and their activity against Aspergillus spp (Maertens et al., 2002) make CAS appealing for both empiric and prophylactic use in patients with febrile neutropenia who are at risk for invasive fungal infections Presented in part at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 2002 (Abstract #M-851). * Corresponding author. Tel.: ⫹1-713-792-6237; fax: ⫹1-713-7456839. E-mail address: [email protected] (D.P. Kontoyiannis). 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(03)00136-6
(Van Burik et al., 2002). Therefore, ITZ and CAS are expected to be frequently used in different sequences in the course of IA treatment (Kontoyiannis and Bodey, 2002). The clinical question is whether breakthrough IA that occurs despite the use of either drug would result in higher likelihood of subsequent failure of the other drug. There have been no preclinical studies of the staggered administration of these drugs. To that end, we studied whether prior exposure to ITZ or CAS affects the subsequent activity of CAS or ITZ respectively in Aspergillus fumigatus.
2. Materials and methods We tested the susceptibility of 10 clinical isolates of A. fumigatus to CAS and ITZ. Aspergillus growth was examined in yeast extract glucose (YAG) plates prepared using standard methods. CAS and ITZ were obtained from Merck Research Laboratories (Merck & Co. Inc., Rathway, NJ) and Janssen Pharmaceutical (Titusville, NJ) respectively. CAS was dissolved in sterile water and ITZ in dimethyl sulfoxide. The concentration of the stock solutions of both drugs was 10 mg/ml. To test the susceptibility of A. fumigatus to ITZ following exposure to CAS, YAG-plates containing increasing
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concentrations of CAS (0, 0.0625, 0.125, 0.25, 0.5, 1, and 4 g/ml) were each swab inoculated with 200 L of a standardized suspension of Aspergillus conidia (106 conidia/ml) of each isolate. Stock conidial suspensions were prepared from 3-day-old cultures grown on YAG-plates. Conidia were counted using hemocytometry. After inoculation, the plates were incubated at 37°C for 12-16 h. The following day, a 1⁄4-inch-diameter paper disk (Schleicher and Schuell, Keene, N.H.) containing 10 g of ITZ (25 L of the ITZ stock solution) was applied on the agar surface and plates were incubated for another 48 h at 37°C. To test the susceptibility of A. fumigatus to CAS following exposure to ITZ, YAG-plates containing increasing concentrations of ITZ (0, 10, 50, 75, 100, 150, and 300 ng/ml) were similarly swab inoculated with 200 L of a standardized suspension of Aspergillus conidia (106 conidia/ml) of each isolate. After inoculation, the plates were incubated at 37°C for 12-16 h. The following day, a disk containing 20 g of CAS (50 L of the CAS stock solution) was applied on plates, which were then incubated for another 48 h at 37°C. To evaluate the effects of simultaneous exposure to both drugs, YAG-plates containing different concentrations of CAS (0, 0.25 g/ml, 0.5 g/ml and 1 g/ml) were each swab inoculated with 200 L of conidial suspensions (106 conidia/ml) of each isolate. A disk containing 10 g of ITZ was placed on the agar surface 15 min after isolates were plated and plates were incubated for 48 h at 37°C. The radius of the zone of growth inhibition was measured in millimeters at 48 h of incubation. All assays were conducted in triplicate in three different experiments. The Kruskal-Wallis test was used to compare measurements. Differences were further analyzed posthoc using the Tukeys Test for multiple comparisons. All comparisons were considered statistically significant for P values ⱕ0.05.
3. Results Previous exposure of A. fumigatus isolates to ITZ or CAS resulted in enhanced in vitro effects of CAS or ITZ, respectively (Table 1, Fig. 1A and 1B). This attenuated growth of Aspergillus isolates upon the sequential exposure to CAS and ITZ was not due to growth inhibition caused by the increasing concentrations of either drug previously added in the plate; in pilot experiments all 10 isolates grew in YAG-plates containing increasing concentrations of CAS (up to 4 g/ml) or ITZ (up to 300 ng/ml) (data not shown). The positive interaction of the two drugs increased over time, reaching its maximum effect at 48 h; this effect was sustained throughout 96 h (data not shown). Consistent enhancement of the activity of one drug following exposure to the other was seen in all 10 isolates, although strain-dependent differences
Table 1 Summary of the growth inhibition responses of 10 clinical isolates of A. fumigatus to sequential ITZ and CAS exposure using disk diffusion assay Sequence of drug administration
A. ITZ followed by CAS in disk (20g) ITZ concentration in plates (ng/mL) 0 (CAS alone) 10 75 100 150 300 B. CAS followed by ITZ in disk (10g) CAS concentrations in plates (g/mL) 0 (ITRA alone) 0.0625 0.125 0.25 0.5 1 4 C. Simultaneous CAS (in plates) and ITZ in disk (10g) CAS concentrations in plates (g/mL) 0 0.25 0.5 1
Mean radius of zone of growth inhibition (in mm) ⫾ SD
No zone 0.2 ⫾ 0.1 1.2 ⫾ 0.4a 3.9 ⫾ 0.7a 9.7 ⫾ 2a 13 ⫾ 3a
3.04 3.85 ⫾ 1,3 4.45 ⫾ 1,4 7.8 ⫾ 1.1a 8.4 ⫾ 1.3a 8.9 ⫾ 1.3a 8.2 ⫾ 1.5a 12.1 ⫾ 0.7 12.5 ⫾ 1.1 12.6 ⫾ 1.3 12.8 ⫾ 1.2
ITZ, itraconazole; CAS, Caspofungin. a P ⬍ 0.05 vs. control SD, standard deviation
were noted. In contrast, the simultaneous exposure of A. fumigatus isolates to CAS and ITZ did not result in enhanced in vitro effects (Table 1). The zone of growth inhibition caused by ITZ in disk was larger when A. fumigatus isolates were exposed to ITZ and CAS simultaneously (Table 1C), compared to the zone when ITZ in the disk was applied after prior growth of A. fumigatus in CAS-containing plates (Table 1B). This is consistent with our previous experience on the activity of ITZ when applied in freshly inoculated plated versus against pregrown lawns (Kontoyiannis et al., 2000).
4. Discussion We found that pre-exposure of A. fumigatus to ITZ or CAS results in enhanced in vitro effects of subsequent CAS or ITZ use respectively. This finding contrasts with the apparently antagonistic effect observed when amphotericin B is used after ITZ, both in vitro and in animal models of IA (Kontoyiannis et al., 2000; Lewis et al., 2002). The exact
D.P. Kontoyiannis et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 415– 419
417
Fig. 1A. A representative isolate of A. fumigatus (isolate AF 34) has been pre-grown for 16 h in YAG plates containing ITZ at concentrations of 0, 10, 75, 100, 150 and 300 (ng/mL). A disk containing 20 g of CAS was applied on the agar surface the following day. Plates were incubated at 37°C for 48 h and the zone of growth inhibition caused by CAS was then assessed.
mechanism of this positive in vitro triazole-echinocandin interaction is unknown. ITZ is an ergosterol-depleting agent and CAS inhibits cell wall biosynthesis of the growing hyphal tips (Groll et al., 1998; Bowman et al., 2002). The enhanced effect of each drug following staggered administration of the other would suggest a feedback regulation between ergosterol and cell wall biosynthesis. It is possible that exposure of A. fumigatus to an ergosteroldepleting agent (ITZ) could result in downregulation of cell wall remodeling, thus augmenting the effects of subsequent use of an echinocandin. The influence of ergosterol depletion to cell wall remodeling has been
previously suggested in Saccharomyces cerevisiae and C. albicans (Pfaller and Riley, 1992; Kontoyiannis, 1999; Bammert and Fostel, 2000). Conversely, CAS-induced fungal cell wall alterations could affect ITZ entry or efflux. The relevance of this observation needs to be validated in animal models of IA. For example, it would be interesting to determine whether breakthrough IA following ITZ prophylaxis responds better to CAS or to amphotericin B-based regimens. Our finding should stimulate further investigations for the identification of effective sequences and combinations of antifungal therapy for treatment of breakthrough IA.
418
D.P. Kontoyiannis et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 415– 419
Fig. 1B. A representative isolate of A. fumigatus (isolate AF 34) has been pre-grown for 16 h in YAG plates containing CAS at concentrations of 0, 0.0625, 0.125, 0.25, 0.5 and 1 (g/mL). A disk containing 10 g of ITZ was applied on the agar surface the following day. Plates were incubated at 37°C for 48 h and the zone of growth inhibition caused by ITZ was then assessed.
References Bammert, G. F., & Fostel, J. M. (2000). Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergostetrol. Antimicrob Agents Chemother 44, 1225–1263. Bowman, J. C., Hicks, P. S., Kurtz, M. B., Rosen, H., Schmatz, D. M., Liberator, P., et al (2002). The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. Antimicrob Agents Chemother 46, 3001–3012. De Backer, M. D., Ilyina, T., Ma, X. J., Vandoninck, S., Luyten, W. H., & Vanden Bossche, H. (2001). Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray. Antimicrob Agents Chemother 45, 1660 –1670. Groll, A. H., Piscitelli, S. C., & Walsh, T. H. (1998). Clinical pharmacology of systemic antifungal agents: A comprehensive review of agents in clinical use, current investigational compounds, and pu-
tative targets for antifungal drug development. Adv Pharmacol 44, 343–500. Harousseau, J. L., Dekker, A. W., Stamatoulas-Bastard, A., Fassas, A., Linkesch, W., Gouveia, J., et al (2000). Itraconazole oral solution for primary prophylaxis of fungal infections in patients with hematological malignancy and profound neutropenia: a randomized, double-blind, double-placebo, multicenter trial comparing itraconazole to amphotericin B. Antimicrob Agents Chemother 44, 1887–1893. Kontoyiannis, D. P. (1999). Genetic analysis of azole resistance by transposon mutagenesis in Saccharomyces cerevisiae. Antimicrob Agents Chemother 43, 2731–2735. Kontoyiannis, D. P., & Bodey, G. P. (2002). Invasive aspergillosis in 2002: An update. Eur J Clin Microbiol Infect Dis 21, 161–172. Kontoyiannis, D. P., Lewis, R. E., Sagar, N., May, G., Prince, R. A., & Rolston, K. V. (2000). Itraconazole-amphotericin B antagonism in Aspergillus fumigatus: an E-test-based strategy. Antimicrob Agents Chemother 44, 2915–2918.
D.P. Kontoyiannis et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 415– 419 Lewis, R. E., Prince, R. A., & Kontoyiannis, D. P. (2002). Pretreatment with itraconazole attenuates the efficacy of increasing amphotericin B dosages in a murine model of invasive pulmonary aspergillosis. Antimicrob Agents Chemother 46, 3208 –3214. Maertens, J., Raad, I. I., Petrikkos, D., Selleslag, D., Petersen, F., Sable,C., Kartsonis, N., Ngai, A., Taylor, A., Patterson, T., Denning, D., Walsh, T. (2002). Update of the multicenter noncomparative study of caspofungin (CAS) in adults with invasive Aspergillus (IA) refractory or intolerant to other antifungal agents: Analysis of 90 patients. 42nd Interscience Con-
419
ference on Antimicrobial Agents and Chemotherapy; September 2002; San Diego, CA. Abstract 388:M-868. Pfaller, M., & Riley, J. (1992). Effects of fluconazole on the sterol and carbohydrate composition of four species of Candida. Eur J Clin Microbiol Infect Dis 11, 152–156. Van Burik, J., Ratanatharathorn, V., Lipton, J., Miller, C., Bunin, N., Walsh, T. J. (2002). Randomized, double-blind trial of micafungin versus fluconazole for prophylaxis of invasive fungal infections in patients undergoing hematopoietic stem cell transplant. Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 2002.