In Vitro Susceptibilities of Clinical Isolates of Candida Species, Cryptococcus neoformans, and Aspergillus Species to Itraconazole: Global
http://www.100md.com
微生物临床杂志 2005年第8期
Departments of Pathology Medicine, Roy J. and Lucille A. Carver College of Medicine
Department of Epidemiology
College of Public Health, University of Iowa, Iowa City, Iowa 52242
ABSTRACT
The in vitro activity of itraconazole was determined against 7,299 isolates of Candida spp., 1,615 isolates of Cryptococcus neoformans, and 445 isolates of Aspergillus spp. obtained from over 200 medical centers worldwide. Itraconazole was active against all Candida spp. (96% of MICs were 1 μg/ml) with the exception of C. glabrata (77% of MICs were 1 μg/ml). Itraconazole inhibited 94% of C. krusei and 84% of other fluconazole-resistant Candida species, exclusive of C. glabrata, at a MIC of 1 μg/ml. Itraconazole was not active against fluconazole-resistant isolates of C. glabrata. Only modest activity was seen against C. neoformans (80% of MICs were 1 μg/ml); however, itraconazole showed excellent activity against Aspergillus spp. (94% of MICs were 1 μg/ml). These results provide an update on the antifungal activity of itraconazole against major opportunistic fungal pathogens. In light of the new intravenous formulation of itraconazole these data suggest that this agent remains a viable systemically active antifungal agent.
INTRODUCTION
Invasive fungal infections remain a major threat to the health of immunocompromised hosts worldwide (3, 4, 10, 15, 16, 26, 29, 36, 40). Among the myriad of opportunistic fungal pathogens, the three most important groups include Candida spp., Cryptococcus neoformans, and Aspergillus spp. (6, 9, 13, 15, 22, 24, 25, 28, 29). In recent years, several new developments in the area of antifungal therapy have improved the ability of physicians to prevent and treat these important mycoses (14, 23, 29, 31-33, 35, 37). Certainly a great deal of attention has been paid to the new triazoles (e.g., voriconazole, posaconazole, and ravuconazole), with their potency and improved spectrum of activity, including activity against fluconazole-resistant Candida spp., C. neoformans, and Aspergillus spp. (6, 7, 12, 13, 22, 24, 32, 33, 37, 38). Despite the well-founded enthusiasm for the new antifungal agents, it is important to continue to assess the activity of the older, more established agents, especially those, such as itraconazole, for which the development of new formulations has overcome some of the major deficiencies (i.e., bioavailability) of the drug when it was first introduced (2-5, 10, 16, 18, 19, 29, 36, 39, 40). The inherent potency and broad spectrum of itraconazole (1, 5, 13, 16, 17, 22, 24, 29, 38), coupled with the availability of an intravenous formulation (IVF), suggests that the use of itraconazole as first-line therapy for some of the opportunistic mycoses may well become an option (29).
The development of itraconazole as an important antifungal agent has lagged due to the lack of an IVF and the poor and erratic bioavailability of the oral capsule formulation (2, 5, 29, 39). Although bioavailability was much improved with the oral solution, it remains somewhat variable and is clearly inferior to that of fluconazole or voriconazole (2, 29, 36, 39). The introduction of the IVF of itraconazole in 1999 broadened the potential utility of itraconazole (29, 39). Whereas the suggested "target" concentration of 0.5 μg/ml in serum (29, 30) was difficult to achieve reliably with either oral formulation, it is now very easy to obtain peak concentrations of 2 to 4 μg/ml and troughs of 1 μg/ml using the IVF (4, 19, 29, 36, 39).
Although itraconazole has often been used as a comparator in surveys of the in vitro antifungal susceptibility of opportunistic fungal pathogens, it has rarely been the primary focus of such studies; thus, its activity against opportunistic fungi is generally underappreciated (5, 8, 9, 13, 22, 24, 25). In part, this may also be due to a perception of rather poor activity of itraconazole against Candida spp. given the very conservative breakpoints (susceptible MIC, 0.12 μg/ml; susceptible-dose dependent MIC, 0.25 to 0.5 μg/ml; resistant MIC, 1 μg/ml) assigned by the Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards) (30). These breakpoints were assigned based entirely on MICs for isolates of Candida spp. obtained from patients with oropharyngeal candidiasis who were treated with oral itraconazole (capsule and/or solution) and in whom serum concentrations of <0.5 μg/ml were common (30). The category of susceptible-dose dependent was applied to those isolates for which the MIC was determined to be 0.25 to 0.5 μg/ml on the basis of the recognition of improved clinical and microbiological response when serum concentrations were 0.5 μg/ml (30). Given the ability to reliably achieve serum concentrations of itraconazole of 1 μg/ml throughout the dosing interval with the IVF (4, 19, 36, 39), it is now reasonable to reassess the potency and spectrum of activity of itraconazole against the major opportunistic fungi. As such, we will discuss the data presented herein with a focus on the proportion of isolates tested for which the itraconazole MIC is 1 μg/ml. Such data are necessary to more accurately assess the "treatability" of the pathogens in light of the new formulation (IVF) of itraconazole and can be considered the first step towards a readjustment of the interpretive breakpoints.
In the present study we determined the in vitro activity of itraconazole against an international collection (representing over 200 medical centers) of more than 9,000 clinical isolates of Candida spp. (7,299 isolates), C. neoformans (1,615 isolates), and Aspergillus spp. (445 isolates). This report represents the largest experience with itraconazole tested by CLSI reference methods (20, 21). The results are presented as the cumulative percentage of isolates inhibited at each concentration throughout the dilution series (full-range MICs) to facilitate comparison with other studies using the CLSI methods.
MATERIALS AND METHODS
Organisms. A total of 9,359 clinical isolates of Candida spp. (7,299 isolates), Cryptococcus neoformans (1,615 isolates), and Aspergillus spp. (445 isolates) obtained from more than 200 medical centers worldwide were tested. These were all clinical isolates collected between 1992 and 2004. The Candida and C. neoformans isolates were obtained from blood and normally sterile body fluids, and the Aspergillus isolates were from respiratory specimens. Isolates were identified by standard methods (11, 34) and stored as water suspensions until they were used. Prior to testing, each isolate was subcultured on potato dextrose agar (Remel, Lenexa, Kans.) to ensure viability and purity.
Susceptibility testing. Standard antifungal powder of itraconazole (Janssen, Beerse, Belguim) was obtained from the manufacturer. Broth microdilution trays were prepared in house at the University of Iowa as described previously (7, 24, 25, 28). Broth microdilution testing of Candida spp. and C. neoformans was performed in the Molecular Epidemiology and Fungus Testing Laboratory (Iowa City) exactly as outlined in CLSI document M27-A2 (20), and testing of Aspergillus was performed as described in CLSI document M38-A (21). The concentrations of itraconazole tested ranged from 0.007 to 8 μg/ml. MIC endpoints for Candida and Aspergillus spp. were determined after 48 h of incubation and for C. neoformans after 72 h of incubation. The MICs of itraconazole were defined as the lowest concentrations that produced either a prominent (approximately 50%) decrease (Candida and Cryptococcus spp.) or a complete (100%; Aspergillus spp.) inhibition of growth relative to that of the drug-free control well (20, 21).
Quality control. Quality control was ensured by testing the following strains (20, 31): A. flavus ATCC 204304, C. parapsilosis ATCC 22019, and C. krusei ATCC 6258. All results were within the recommended limits of the CLSI (20, 21).
RESULTS AND DISCUSSION
Table 1 summarizes the in vitro susceptibility of 7,299 isolates of Candida spp. and 1,615 isolates of C. neoformans to itraconazole. Overall, itraconazole was quite active against all species of Candida (96% of isolates were inhibited by 1 μg/ml) with the exception of C. glabrata (77% inhibited by 1 μg/ml). This level of activity was unchanged over time and when stratified by geographic regions of the world (data not shown). Itraconazole had little meaningful activity against fluconazole-resistant isolates of C. glabrata (Table 2); however, 86% of the fluconazole-resistant isolates of C. albicans, C. parapsilosis, C. tropicalis, and C. krusei were inhibited by 1 μg of itraconazole per ml. Overall, 94% of all isolates of C. krusei (those for which fluconazole MICs were either 64 or 64 μg/ml) were susceptible to itraconazole at concentrations of 1 μg/ml (Table 1).
Itraconazole was less active against C. neoformans (Table 1). Although 80% of the isolates tested were inhibited by 1 μg of itraconazole per ml, the poor penetration of itraconazole into the central nervous system makes this drug a less attractive option for treating cryptococcosis than other azoles (39).
In similarity to the results recently reported by Verweij et al. (38), itraconazole showed good activity (94% inhibited by 1 μg/ml) against a large collection of Aspergillus spp. (Table 3). Notably, there were no isolates of A. fumigatus for which the MICs of itraconazole were greater than 2 μg/ml. Likewise, Verweij et al. (38) reported only three isolates of A. fumigatus for which itraconazole MICs were >2 μg/ml.
These findings provide a contemporary look at the spectrum and potency of itraconazole against three major groups of opportunistic fungal pathogens as determined by standardized reference broth microdilution methods. Given the changes in formulation of itraconazole and the ability to reliably maintain serum concentrations above 1 μg/ml, one can now begin to assess the data presented in Table 1 to Table 3 in terms of those species that may be covered by this agent (i.e., those for which the MIC is 1 μg/ml). In this regard, more than 90% of invasive isolates of Candida and Aspergillus spp. from throughout the world may be considered "susceptible" to itraconazole at concentrations of 1 μg/ml. Indeed, elevated itraconazole MICs (>2 μg/ml) were extremely uncommon among isolates of Candida and Aspergillus spp. (Table 1 to Table 3). Aside from C. glabrata, even strains of fluconazole-resistant Candida spp. show itraconazole MICs of 1 μg/ml.
As seen with fluconazole and Candida spp. (27), and as reported by Verweij et al. (38) for Aspergillus, we have found no evidence that the in vitro activity of itraconazole has decreased over time (data not shown). Thus, primary resistance to itraconazole among clinical isolates of Candida (except for C. glabrata) and Aspergillus spp. appears to be very low. The fact that the spectrum and potency of the drug has been sustained over a 13-year period (1992 to 2004) despite the use of itraconazole for treatment and prophylaxis indicates that acquired resistance is low as well.
The availability of more readily bioavailable oral and parenteral formulations of itraconazole has given this established antifungal agent "new life" (29, 39). We report the MIC data herein as a continuous distribution of results throughout the range of concentrations tested to allow one to better evaluate the activity of the drug against readily achievable serum and tissue concentrations. Such data should prove useful in reevaluating the MIC interpretive breakpoints for itraconazole and serve as an aid in guiding the application of this drug clinically.
ACKNOWLEDGMENTS
We thank Linda Elliott for excellent secretarial assistance in the preparation of the manuscript.
This study was supported in part by grants by Bristol-Myers Squibb, Pfizer Pharmaceuticals, and Schering-Plough Research Institute.
REFERENCES
Barchiesi, F., A. L. Colombo, D. A. McGough, A. W. Fothergill, and M. G. Rinaldi. 1994. In vitro activity of itraconazole against fluconazole-susceptible and -resistant Candida albicans isolates from oral cavities of patients infected with human immunodeficiency virus. Antimicrob. Agents Chemother. 38:1530-1533.
Barone, J. A., B. L. Moskovitz, J. Guarnieri, A. E. Hassell, J. L. Colaizzi, R. H. Bierman, and L. Jessen. 1998. Enhanced bioavailability of itraconazole in hyroxypropyl--cyclodextrin solution versus capsules in healthy volunteers. Antimicrob. Agents Chemother. 42:1862-1865.
Boogaerts, M., D. J. Winston, E. J. Bow, G. Garber, A. C. Reboli, A. P. Schwarer, N. Novitzky, A. Boehme, E. Chwetzoff, K. De Beule, and the Itraconazole Neutropenia Study Group. 2001. Intravenous and oral itraconazole versus intravenous amphotericin B deoxycholate as empirical antifungal therapy for persistent fever in neutropenic patients with cancer who are receiving broad spectrum antibacterial therapy: a randomized controlled trial. Ann. Intern. Med. 135:412-422.
Boogaerts, M. A., J. Maertens, R. van der Geest, A. Bosly, J.-L. Michaux, A. van Hoof, M. Cleeren, R. Wostenborghs, and K. DeBeule. 2001. Pharmacokinetics and safety of a 7-day administration of intravenous itraconazole followed by a 14-day administration of itraconazole oral solution in patients with hematologic malignancy. Antimicrob. Agents Chemother. 45:981-985.
Carteledge, J. D., J. Midgely, and B. G. Gazzard. 1997. Itraconazole solution: higher serum drug concentrations and better clinical response rates than the capsule formulation in acquired immunodeficiency syndrome patients with candidosis. J. Clin. Pathol. 50:477-480.
Cuenca-Estrella, M., D. Rodriguez, B. Almirante, J. Morgan, A. M. Planes, M. Almela, J. Mensa, F. Sachez, J. Ayatis, M. Gimenexz, M. Salvado, D. W. Warnock, A. Pahissa, and J. L. Rodriguez-Tudela. 2005. In vitro susceptibilities of bloodstream isolates of Candida species to six antifungal agents: results from a population-based active surveillance programme, Barcelona, Spain, 2002-2003. J. Antimicrob. Chemother. 55:194-199.
Diekema, D. J., S. A. Messer, R. J. Hollis, R. N. Jones, and M. A. Pfaller. 2003. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi. J. Clin. Microbiol. 41:3623-3626.
Espinel-Ingroff, A., K. Boyle, and D. J. Sheehan. 2001. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: review of the literature. Mycopathologia 150:101-115.
Hajjeh, R. A., A. M. Sofair, L. H. Harrison, G. M. Lyon, B. A. Arthington-Skaggs, S. A. Mirza, M. Phelan, J. Morgan, W. Lee-Yang, M. A. Ciblak, L. E. Benjamin, L. T. Sanza, S. Huie, S. F. Yeo, M. E. Brandt, and D. W. Warnock. 2004. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J. Clin. Microbiol. 42:1519-1527.
Harousseau, J. L., A. W. Dekker, A. Stamatoullas-Bastard, A. Fassas, W. Linkesch, J. Gouveia, R. DeBock, M. Rovira, W. F. Seifert, H. Joosen, M. Peeters, and K. De Beule. 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 and amphotericin B. Antimicrob. Agents Chemother. 44:1887-1893.
Hazen, K. C., and S. A. Howell. 2003. Candida, Cryptococcus, and other yeasts of medical importance, p. 1693-1711. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8th ed., vol. 2. American Society for Microbiology, Washington, D.C.
Hoffman, H. L., E. J. Ernst, and M. E. Klepser. 2000. Novel triazole antifungal agents. Expert Opin. Investig. Drugs 9:593-605.
Hsueh, P.-R., Y.-J. Lau, Y.-C. Chuang, J.-H. Wan, W.-K. Huang, J.-M. Shyr, J.-J. Yan, K.-W. Yu, W.-C. Ko, Y.-C. Yang, Y.-C. Liu, L.-J. Teng, C.-Y. Liu, and K.-T. Luh. 2005. Antifungal susceptibilities of clinical isolates of Candida species, Cryptococcus neoformans, and Aspergillus species from Taiwan: surveillance of multicenter antimicrobial resistance in Taiwan program data from 2003. Antimicrob. Agents Chemother. 49:512-517.
Maertens, J., and M. Boogaerts. 2003. Caspofungin in the treatment of candidosis and aspergillosis. Int. J. Infect. Dis. 7:94-101.
Marr, K. A., K. Seidel, T. C. White, and R. A. Bowden. 2000. Candidemia in allogeneic blood and marrow transplant recipients: evolution of risk factors after the adoption of prophylactic fluconazole. J. Infect. Dis. 181:309-316.
Marr, K. A., F. Crippa, W. Leisenring, M. Hoyle, M. Boeckh, S. A. Balajee, W. G. Nichols, B. Musher, and L. Corey. 2004. Itraconazole versus fluconazole for prevention of fungal infections in patients receiving allogeneic stem cell transplants. Transplantation 103:1527-1533.
Martinez-Suarez, J. V., and J. L. Rodriguez-Tudela. 1995. Patterns of in vitro activity of itraconazole and imidazole antifungal agents against Candida albicans with decreased susceptibility to fluconazole from Spain. Antimicrob. Agents Chemother. 39:1512-1516.
Mattiuzzi, G. N., H. Kantarjian, S. O'Brien, D. P. Kontoyiannis, F. Giles, X. Zhou, J. Lim, B. N. Bekele, S. Faderi, J. Cortes, S. Peirce, G. J. Leitz, I. Raad, and E. Estey. 2004. Intravenous itraconazole for prophylaxis of systemic fungal infections in patients with acute myelogenous leukemia and high-risk myelodysplastic syndrome undergoing induction chemotherapy. Cancer 100:568-573.
Mohr, J. F., K. W. Finkel, J. H. Rex, J. R. Rodriguez, G. J. Leitz, and L. Ostrosky-Zeichner. 2004. Pharmacokinetics of intravenous itraconazole in stable hemodialysis patients. Antimicrob. Agents Chemother. 48:3151-3153.
National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.
National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Ostrosky-Zeichner, L., J. H. Rex, P. G. Pappas, R. J. Hamill, R. A. Larsen, H. W. Horowitz, W. G. Powderly, N. Hyslop, C. A. Kauffman, J. Cleary, J. E. Mangino, and J. Lee. 2003. Antifungal susceptibility survey of 2,000 bloodstream Candida isolates in the United States. Antimicrob. Agents Chemother. 47:3149-3154.
Pappas, P. G., J. H. Rex, J. D. Sobel, S. G. Filler, W. E. Dismukes, T. J. Walsh, and J. E. Edwards. 2004. Guidelines for treatment of candidiasis. Clin. Infect. Dis. 38:161-189.
Pfaller, M. A., S. A. Messer, R. J. Hollis, R. N. Jones, and D. J. Diekema. 2002. In vitro activities of ravuconazole and voriconazole compared with those of four approved systemic antifungal agents against 6,970 clinical isolates of Candida spp. Antimicrob. Agents Chemother. 46:1723-1727.
Pfaller, M. A., D. J. Diekema, S. A. Messer, R. J. Hollis, and R. N. Jones. 2003. In vitro activities of caspofungin compared with those of fluconazole and itraconazole against 3,959 clinical isolates of Candida spp., including 157 fluconazole-resistant isolates. Antimicrob. Agents Chemother. 47:1068-1071.
Pfaller, M. A., and D. J. Diekema. 2004. Rare and emerging opportunistic fungal pathogens: concerns for resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 42:4419-4431.
Pfaller, M. A., and D. J. Diekema. 2004. Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconazole susceptibility of bloodstream isolates of Candida. Clin. Microbiol. Infect. 10(Suppl. 1):11-23.
Pfaller, M. A., S. A. Messer, L. Boyken, C. Rice, S. Tendolkar, R. J. Hollis, and D. J. Diekema. 2005. Global trends in the antifungal susceptibility of Cryptococcus neoformans: 1990 to 2004. J. Clin. Microbiol. 43:2163-2167.
Pierard, G.-E., M. Kharfi, M.-D. Salomon-Neiera, M.-R. Kamoun, and C. Pierard-Franchimont. 2004. Itraconazole in human aspergillosis revisited. J. Mycol. Med. 14:192-200.
Rex, J. H., M. A. Pfaller, J. N. Galgiani, M. S. Bartlett, A. Espinel-Ingroff, M. A. Ghannoum, M. Lancaster, F. C. Odds, M. G. Rinaldi, T. J. Walsh, and A. L. Barry. 1997. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and Candida infections. Clin. Infect. Des. 24:235-247.
Saag, M. S., R. J. Graybill, R. A. Larson, P. G. Pappas, J. R. Perfect, W. G. Powderly, J. D. Sobel, and W. E. Dismukes. 2000. Practice guidelines for the management of cryptococcal disease. Clin. Infect. Dis. 30:710-718.
Sabo, J. A., and S. M. Abdel-Rahman. 2000. Voriconazole: a new triazole antifungal. Ann. Pharmacother. 34:1032-1043.
Sheehan, D. J., C. A. Hitchcock, and C. M. Sibley. 1999. Current and emerging azole antifungal agents. Clin. Microbiol. Rev. 12:40-79.
Sigler, L., and P. E. Verweij. 2003. Aspergillus, Fusarium, and other opportunistic moniliaceous fungi, p. 1726-1760. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed., vol. 2. American Society for Microbiology, Washington, D.C.
Stevens, D. A., V. L. Kau, M. A. Judson, V. A. Morrison, S. Dummer, D. W. Denning, J. E. Bennett, T. J. Walsh, T. F. Patterson, and G. A. Pankey. 2000. Practice guidelines for diseases caused by Aspergillus. Clin. Infect. Dis. 30:696-709.
Vandewoude, K., D. Vogelaers, J. Decruyenaere, P. Jaqmin, K. DeBeule, A. van Peter, R. Woestenborghs, K. Groen, and F. Colardyn. 1997. Concentrations in plasma and safety of 7 days of intravenous itraconazole followed by 2 weeks of oral itraconazole solution in patients in intensive care units. Antimicrob. Agents Chemother. 41:2714-2718.
Verweij, P. E., B. E. de Pauw, and J. F. Meis. 1999. Voriconazole. Curr. Opin. Anti-Infect. Investig. Drugs 1:361-372.
Verweij, P. E., D. T. A. Te Dorsthorst, A. J. M. M. Rijs, H. G. De Vries-Hospers, J. F. G. M. Meis, and the Dutch Interuniversity Working Party for Invasive Mycoses. 2002. Nationwide survey of in vitro activities of itraconazole and voriconazole against clinical Aspergillus fumigatus isolates cultured between 1945 and 1998. J. Clin. Microbiol. 40:2648-2650.
Willems, L., R. van der Geest, and K. De Beule. 2001. Itraconazole oral solution and intravenous formulations: a review of pharmacokinetics and pharmacodynamics. J. Clin. Pharm. Ther. 26:159-169.
Winston, D. J., R. T. Maziarz, P. H. Chandrasekar, H. M. Lazarus, M. Goldman, J. Blumer, G. Leitz, and M. C. Territo. 2003. Intravenous and oral itraconazole versus intravenous and oral fluconazole for long-term antifungal prophylaxis in allogeneic hematopoietic stem-cell transplant recipients. Ann. Intern. Med. 138:705-713.(M. A. Pfaller, L. Boyken,)
Department of Epidemiology
College of Public Health, University of Iowa, Iowa City, Iowa 52242
ABSTRACT
The in vitro activity of itraconazole was determined against 7,299 isolates of Candida spp., 1,615 isolates of Cryptococcus neoformans, and 445 isolates of Aspergillus spp. obtained from over 200 medical centers worldwide. Itraconazole was active against all Candida spp. (96% of MICs were 1 μg/ml) with the exception of C. glabrata (77% of MICs were 1 μg/ml). Itraconazole inhibited 94% of C. krusei and 84% of other fluconazole-resistant Candida species, exclusive of C. glabrata, at a MIC of 1 μg/ml. Itraconazole was not active against fluconazole-resistant isolates of C. glabrata. Only modest activity was seen against C. neoformans (80% of MICs were 1 μg/ml); however, itraconazole showed excellent activity against Aspergillus spp. (94% of MICs were 1 μg/ml). These results provide an update on the antifungal activity of itraconazole against major opportunistic fungal pathogens. In light of the new intravenous formulation of itraconazole these data suggest that this agent remains a viable systemically active antifungal agent.
INTRODUCTION
Invasive fungal infections remain a major threat to the health of immunocompromised hosts worldwide (3, 4, 10, 15, 16, 26, 29, 36, 40). Among the myriad of opportunistic fungal pathogens, the three most important groups include Candida spp., Cryptococcus neoformans, and Aspergillus spp. (6, 9, 13, 15, 22, 24, 25, 28, 29). In recent years, several new developments in the area of antifungal therapy have improved the ability of physicians to prevent and treat these important mycoses (14, 23, 29, 31-33, 35, 37). Certainly a great deal of attention has been paid to the new triazoles (e.g., voriconazole, posaconazole, and ravuconazole), with their potency and improved spectrum of activity, including activity against fluconazole-resistant Candida spp., C. neoformans, and Aspergillus spp. (6, 7, 12, 13, 22, 24, 32, 33, 37, 38). Despite the well-founded enthusiasm for the new antifungal agents, it is important to continue to assess the activity of the older, more established agents, especially those, such as itraconazole, for which the development of new formulations has overcome some of the major deficiencies (i.e., bioavailability) of the drug when it was first introduced (2-5, 10, 16, 18, 19, 29, 36, 39, 40). The inherent potency and broad spectrum of itraconazole (1, 5, 13, 16, 17, 22, 24, 29, 38), coupled with the availability of an intravenous formulation (IVF), suggests that the use of itraconazole as first-line therapy for some of the opportunistic mycoses may well become an option (29).
The development of itraconazole as an important antifungal agent has lagged due to the lack of an IVF and the poor and erratic bioavailability of the oral capsule formulation (2, 5, 29, 39). Although bioavailability was much improved with the oral solution, it remains somewhat variable and is clearly inferior to that of fluconazole or voriconazole (2, 29, 36, 39). The introduction of the IVF of itraconazole in 1999 broadened the potential utility of itraconazole (29, 39). Whereas the suggested "target" concentration of 0.5 μg/ml in serum (29, 30) was difficult to achieve reliably with either oral formulation, it is now very easy to obtain peak concentrations of 2 to 4 μg/ml and troughs of 1 μg/ml using the IVF (4, 19, 29, 36, 39).
Although itraconazole has often been used as a comparator in surveys of the in vitro antifungal susceptibility of opportunistic fungal pathogens, it has rarely been the primary focus of such studies; thus, its activity against opportunistic fungi is generally underappreciated (5, 8, 9, 13, 22, 24, 25). In part, this may also be due to a perception of rather poor activity of itraconazole against Candida spp. given the very conservative breakpoints (susceptible MIC, 0.12 μg/ml; susceptible-dose dependent MIC, 0.25 to 0.5 μg/ml; resistant MIC, 1 μg/ml) assigned by the Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards) (30). These breakpoints were assigned based entirely on MICs for isolates of Candida spp. obtained from patients with oropharyngeal candidiasis who were treated with oral itraconazole (capsule and/or solution) and in whom serum concentrations of <0.5 μg/ml were common (30). The category of susceptible-dose dependent was applied to those isolates for which the MIC was determined to be 0.25 to 0.5 μg/ml on the basis of the recognition of improved clinical and microbiological response when serum concentrations were 0.5 μg/ml (30). Given the ability to reliably achieve serum concentrations of itraconazole of 1 μg/ml throughout the dosing interval with the IVF (4, 19, 36, 39), it is now reasonable to reassess the potency and spectrum of activity of itraconazole against the major opportunistic fungi. As such, we will discuss the data presented herein with a focus on the proportion of isolates tested for which the itraconazole MIC is 1 μg/ml. Such data are necessary to more accurately assess the "treatability" of the pathogens in light of the new formulation (IVF) of itraconazole and can be considered the first step towards a readjustment of the interpretive breakpoints.
In the present study we determined the in vitro activity of itraconazole against an international collection (representing over 200 medical centers) of more than 9,000 clinical isolates of Candida spp. (7,299 isolates), C. neoformans (1,615 isolates), and Aspergillus spp. (445 isolates). This report represents the largest experience with itraconazole tested by CLSI reference methods (20, 21). The results are presented as the cumulative percentage of isolates inhibited at each concentration throughout the dilution series (full-range MICs) to facilitate comparison with other studies using the CLSI methods.
MATERIALS AND METHODS
Organisms. A total of 9,359 clinical isolates of Candida spp. (7,299 isolates), Cryptococcus neoformans (1,615 isolates), and Aspergillus spp. (445 isolates) obtained from more than 200 medical centers worldwide were tested. These were all clinical isolates collected between 1992 and 2004. The Candida and C. neoformans isolates were obtained from blood and normally sterile body fluids, and the Aspergillus isolates were from respiratory specimens. Isolates were identified by standard methods (11, 34) and stored as water suspensions until they were used. Prior to testing, each isolate was subcultured on potato dextrose agar (Remel, Lenexa, Kans.) to ensure viability and purity.
Susceptibility testing. Standard antifungal powder of itraconazole (Janssen, Beerse, Belguim) was obtained from the manufacturer. Broth microdilution trays were prepared in house at the University of Iowa as described previously (7, 24, 25, 28). Broth microdilution testing of Candida spp. and C. neoformans was performed in the Molecular Epidemiology and Fungus Testing Laboratory (Iowa City) exactly as outlined in CLSI document M27-A2 (20), and testing of Aspergillus was performed as described in CLSI document M38-A (21). The concentrations of itraconazole tested ranged from 0.007 to 8 μg/ml. MIC endpoints for Candida and Aspergillus spp. were determined after 48 h of incubation and for C. neoformans after 72 h of incubation. The MICs of itraconazole were defined as the lowest concentrations that produced either a prominent (approximately 50%) decrease (Candida and Cryptococcus spp.) or a complete (100%; Aspergillus spp.) inhibition of growth relative to that of the drug-free control well (20, 21).
Quality control. Quality control was ensured by testing the following strains (20, 31): A. flavus ATCC 204304, C. parapsilosis ATCC 22019, and C. krusei ATCC 6258. All results were within the recommended limits of the CLSI (20, 21).
RESULTS AND DISCUSSION
Table 1 summarizes the in vitro susceptibility of 7,299 isolates of Candida spp. and 1,615 isolates of C. neoformans to itraconazole. Overall, itraconazole was quite active against all species of Candida (96% of isolates were inhibited by 1 μg/ml) with the exception of C. glabrata (77% inhibited by 1 μg/ml). This level of activity was unchanged over time and when stratified by geographic regions of the world (data not shown). Itraconazole had little meaningful activity against fluconazole-resistant isolates of C. glabrata (Table 2); however, 86% of the fluconazole-resistant isolates of C. albicans, C. parapsilosis, C. tropicalis, and C. krusei were inhibited by 1 μg of itraconazole per ml. Overall, 94% of all isolates of C. krusei (those for which fluconazole MICs were either 64 or 64 μg/ml) were susceptible to itraconazole at concentrations of 1 μg/ml (Table 1).
Itraconazole was less active against C. neoformans (Table 1). Although 80% of the isolates tested were inhibited by 1 μg of itraconazole per ml, the poor penetration of itraconazole into the central nervous system makes this drug a less attractive option for treating cryptococcosis than other azoles (39).
In similarity to the results recently reported by Verweij et al. (38), itraconazole showed good activity (94% inhibited by 1 μg/ml) against a large collection of Aspergillus spp. (Table 3). Notably, there were no isolates of A. fumigatus for which the MICs of itraconazole were greater than 2 μg/ml. Likewise, Verweij et al. (38) reported only three isolates of A. fumigatus for which itraconazole MICs were >2 μg/ml.
These findings provide a contemporary look at the spectrum and potency of itraconazole against three major groups of opportunistic fungal pathogens as determined by standardized reference broth microdilution methods. Given the changes in formulation of itraconazole and the ability to reliably maintain serum concentrations above 1 μg/ml, one can now begin to assess the data presented in Table 1 to Table 3 in terms of those species that may be covered by this agent (i.e., those for which the MIC is 1 μg/ml). In this regard, more than 90% of invasive isolates of Candida and Aspergillus spp. from throughout the world may be considered "susceptible" to itraconazole at concentrations of 1 μg/ml. Indeed, elevated itraconazole MICs (>2 μg/ml) were extremely uncommon among isolates of Candida and Aspergillus spp. (Table 1 to Table 3). Aside from C. glabrata, even strains of fluconazole-resistant Candida spp. show itraconazole MICs of 1 μg/ml.
As seen with fluconazole and Candida spp. (27), and as reported by Verweij et al. (38) for Aspergillus, we have found no evidence that the in vitro activity of itraconazole has decreased over time (data not shown). Thus, primary resistance to itraconazole among clinical isolates of Candida (except for C. glabrata) and Aspergillus spp. appears to be very low. The fact that the spectrum and potency of the drug has been sustained over a 13-year period (1992 to 2004) despite the use of itraconazole for treatment and prophylaxis indicates that acquired resistance is low as well.
The availability of more readily bioavailable oral and parenteral formulations of itraconazole has given this established antifungal agent "new life" (29, 39). We report the MIC data herein as a continuous distribution of results throughout the range of concentrations tested to allow one to better evaluate the activity of the drug against readily achievable serum and tissue concentrations. Such data should prove useful in reevaluating the MIC interpretive breakpoints for itraconazole and serve as an aid in guiding the application of this drug clinically.
ACKNOWLEDGMENTS
We thank Linda Elliott for excellent secretarial assistance in the preparation of the manuscript.
This study was supported in part by grants by Bristol-Myers Squibb, Pfizer Pharmaceuticals, and Schering-Plough Research Institute.
REFERENCES
Barchiesi, F., A. L. Colombo, D. A. McGough, A. W. Fothergill, and M. G. Rinaldi. 1994. In vitro activity of itraconazole against fluconazole-susceptible and -resistant Candida albicans isolates from oral cavities of patients infected with human immunodeficiency virus. Antimicrob. Agents Chemother. 38:1530-1533.
Barone, J. A., B. L. Moskovitz, J. Guarnieri, A. E. Hassell, J. L. Colaizzi, R. H. Bierman, and L. Jessen. 1998. Enhanced bioavailability of itraconazole in hyroxypropyl--cyclodextrin solution versus capsules in healthy volunteers. Antimicrob. Agents Chemother. 42:1862-1865.
Boogaerts, M., D. J. Winston, E. J. Bow, G. Garber, A. C. Reboli, A. P. Schwarer, N. Novitzky, A. Boehme, E. Chwetzoff, K. De Beule, and the Itraconazole Neutropenia Study Group. 2001. Intravenous and oral itraconazole versus intravenous amphotericin B deoxycholate as empirical antifungal therapy for persistent fever in neutropenic patients with cancer who are receiving broad spectrum antibacterial therapy: a randomized controlled trial. Ann. Intern. Med. 135:412-422.
Boogaerts, M. A., J. Maertens, R. van der Geest, A. Bosly, J.-L. Michaux, A. van Hoof, M. Cleeren, R. Wostenborghs, and K. DeBeule. 2001. Pharmacokinetics and safety of a 7-day administration of intravenous itraconazole followed by a 14-day administration of itraconazole oral solution in patients with hematologic malignancy. Antimicrob. Agents Chemother. 45:981-985.
Carteledge, J. D., J. Midgely, and B. G. Gazzard. 1997. Itraconazole solution: higher serum drug concentrations and better clinical response rates than the capsule formulation in acquired immunodeficiency syndrome patients with candidosis. J. Clin. Pathol. 50:477-480.
Cuenca-Estrella, M., D. Rodriguez, B. Almirante, J. Morgan, A. M. Planes, M. Almela, J. Mensa, F. Sachez, J. Ayatis, M. Gimenexz, M. Salvado, D. W. Warnock, A. Pahissa, and J. L. Rodriguez-Tudela. 2005. In vitro susceptibilities of bloodstream isolates of Candida species to six antifungal agents: results from a population-based active surveillance programme, Barcelona, Spain, 2002-2003. J. Antimicrob. Chemother. 55:194-199.
Diekema, D. J., S. A. Messer, R. J. Hollis, R. N. Jones, and M. A. Pfaller. 2003. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi. J. Clin. Microbiol. 41:3623-3626.
Espinel-Ingroff, A., K. Boyle, and D. J. Sheehan. 2001. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: review of the literature. Mycopathologia 150:101-115.
Hajjeh, R. A., A. M. Sofair, L. H. Harrison, G. M. Lyon, B. A. Arthington-Skaggs, S. A. Mirza, M. Phelan, J. Morgan, W. Lee-Yang, M. A. Ciblak, L. E. Benjamin, L. T. Sanza, S. Huie, S. F. Yeo, M. E. Brandt, and D. W. Warnock. 2004. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J. Clin. Microbiol. 42:1519-1527.
Harousseau, J. L., A. W. Dekker, A. Stamatoullas-Bastard, A. Fassas, W. Linkesch, J. Gouveia, R. DeBock, M. Rovira, W. F. Seifert, H. Joosen, M. Peeters, and K. De Beule. 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 and amphotericin B. Antimicrob. Agents Chemother. 44:1887-1893.
Hazen, K. C., and S. A. Howell. 2003. Candida, Cryptococcus, and other yeasts of medical importance, p. 1693-1711. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8th ed., vol. 2. American Society for Microbiology, Washington, D.C.
Hoffman, H. L., E. J. Ernst, and M. E. Klepser. 2000. Novel triazole antifungal agents. Expert Opin. Investig. Drugs 9:593-605.
Hsueh, P.-R., Y.-J. Lau, Y.-C. Chuang, J.-H. Wan, W.-K. Huang, J.-M. Shyr, J.-J. Yan, K.-W. Yu, W.-C. Ko, Y.-C. Yang, Y.-C. Liu, L.-J. Teng, C.-Y. Liu, and K.-T. Luh. 2005. Antifungal susceptibilities of clinical isolates of Candida species, Cryptococcus neoformans, and Aspergillus species from Taiwan: surveillance of multicenter antimicrobial resistance in Taiwan program data from 2003. Antimicrob. Agents Chemother. 49:512-517.
Maertens, J., and M. Boogaerts. 2003. Caspofungin in the treatment of candidosis and aspergillosis. Int. J. Infect. Dis. 7:94-101.
Marr, K. A., K. Seidel, T. C. White, and R. A. Bowden. 2000. Candidemia in allogeneic blood and marrow transplant recipients: evolution of risk factors after the adoption of prophylactic fluconazole. J. Infect. Dis. 181:309-316.
Marr, K. A., F. Crippa, W. Leisenring, M. Hoyle, M. Boeckh, S. A. Balajee, W. G. Nichols, B. Musher, and L. Corey. 2004. Itraconazole versus fluconazole for prevention of fungal infections in patients receiving allogeneic stem cell transplants. Transplantation 103:1527-1533.
Martinez-Suarez, J. V., and J. L. Rodriguez-Tudela. 1995. Patterns of in vitro activity of itraconazole and imidazole antifungal agents against Candida albicans with decreased susceptibility to fluconazole from Spain. Antimicrob. Agents Chemother. 39:1512-1516.
Mattiuzzi, G. N., H. Kantarjian, S. O'Brien, D. P. Kontoyiannis, F. Giles, X. Zhou, J. Lim, B. N. Bekele, S. Faderi, J. Cortes, S. Peirce, G. J. Leitz, I. Raad, and E. Estey. 2004. Intravenous itraconazole for prophylaxis of systemic fungal infections in patients with acute myelogenous leukemia and high-risk myelodysplastic syndrome undergoing induction chemotherapy. Cancer 100:568-573.
Mohr, J. F., K. W. Finkel, J. H. Rex, J. R. Rodriguez, G. J. Leitz, and L. Ostrosky-Zeichner. 2004. Pharmacokinetics of intravenous itraconazole in stable hemodialysis patients. Antimicrob. Agents Chemother. 48:3151-3153.
National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.
National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Ostrosky-Zeichner, L., J. H. Rex, P. G. Pappas, R. J. Hamill, R. A. Larsen, H. W. Horowitz, W. G. Powderly, N. Hyslop, C. A. Kauffman, J. Cleary, J. E. Mangino, and J. Lee. 2003. Antifungal susceptibility survey of 2,000 bloodstream Candida isolates in the United States. Antimicrob. Agents Chemother. 47:3149-3154.
Pappas, P. G., J. H. Rex, J. D. Sobel, S. G. Filler, W. E. Dismukes, T. J. Walsh, and J. E. Edwards. 2004. Guidelines for treatment of candidiasis. Clin. Infect. Dis. 38:161-189.
Pfaller, M. A., S. A. Messer, R. J. Hollis, R. N. Jones, and D. J. Diekema. 2002. In vitro activities of ravuconazole and voriconazole compared with those of four approved systemic antifungal agents against 6,970 clinical isolates of Candida spp. Antimicrob. Agents Chemother. 46:1723-1727.
Pfaller, M. A., D. J. Diekema, S. A. Messer, R. J. Hollis, and R. N. Jones. 2003. In vitro activities of caspofungin compared with those of fluconazole and itraconazole against 3,959 clinical isolates of Candida spp., including 157 fluconazole-resistant isolates. Antimicrob. Agents Chemother. 47:1068-1071.
Pfaller, M. A., and D. J. Diekema. 2004. Rare and emerging opportunistic fungal pathogens: concerns for resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 42:4419-4431.
Pfaller, M. A., and D. J. Diekema. 2004. Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconazole susceptibility of bloodstream isolates of Candida. Clin. Microbiol. Infect. 10(Suppl. 1):11-23.
Pfaller, M. A., S. A. Messer, L. Boyken, C. Rice, S. Tendolkar, R. J. Hollis, and D. J. Diekema. 2005. Global trends in the antifungal susceptibility of Cryptococcus neoformans: 1990 to 2004. J. Clin. Microbiol. 43:2163-2167.
Pierard, G.-E., M. Kharfi, M.-D. Salomon-Neiera, M.-R. Kamoun, and C. Pierard-Franchimont. 2004. Itraconazole in human aspergillosis revisited. J. Mycol. Med. 14:192-200.
Rex, J. H., M. A. Pfaller, J. N. Galgiani, M. S. Bartlett, A. Espinel-Ingroff, M. A. Ghannoum, M. Lancaster, F. C. Odds, M. G. Rinaldi, T. J. Walsh, and A. L. Barry. 1997. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and Candida infections. Clin. Infect. Des. 24:235-247.
Saag, M. S., R. J. Graybill, R. A. Larson, P. G. Pappas, J. R. Perfect, W. G. Powderly, J. D. Sobel, and W. E. Dismukes. 2000. Practice guidelines for the management of cryptococcal disease. Clin. Infect. Dis. 30:710-718.
Sabo, J. A., and S. M. Abdel-Rahman. 2000. Voriconazole: a new triazole antifungal. Ann. Pharmacother. 34:1032-1043.
Sheehan, D. J., C. A. Hitchcock, and C. M. Sibley. 1999. Current and emerging azole antifungal agents. Clin. Microbiol. Rev. 12:40-79.
Sigler, L., and P. E. Verweij. 2003. Aspergillus, Fusarium, and other opportunistic moniliaceous fungi, p. 1726-1760. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed., vol. 2. American Society for Microbiology, Washington, D.C.
Stevens, D. A., V. L. Kau, M. A. Judson, V. A. Morrison, S. Dummer, D. W. Denning, J. E. Bennett, T. J. Walsh, T. F. Patterson, and G. A. Pankey. 2000. Practice guidelines for diseases caused by Aspergillus. Clin. Infect. Dis. 30:696-709.
Vandewoude, K., D. Vogelaers, J. Decruyenaere, P. Jaqmin, K. DeBeule, A. van Peter, R. Woestenborghs, K. Groen, and F. Colardyn. 1997. Concentrations in plasma and safety of 7 days of intravenous itraconazole followed by 2 weeks of oral itraconazole solution in patients in intensive care units. Antimicrob. Agents Chemother. 41:2714-2718.
Verweij, P. E., B. E. de Pauw, and J. F. Meis. 1999. Voriconazole. Curr. Opin. Anti-Infect. Investig. Drugs 1:361-372.
Verweij, P. E., D. T. A. Te Dorsthorst, A. J. M. M. Rijs, H. G. De Vries-Hospers, J. F. G. M. Meis, and the Dutch Interuniversity Working Party for Invasive Mycoses. 2002. Nationwide survey of in vitro activities of itraconazole and voriconazole against clinical Aspergillus fumigatus isolates cultured between 1945 and 1998. J. Clin. Microbiol. 40:2648-2650.
Willems, L., R. van der Geest, and K. De Beule. 2001. Itraconazole oral solution and intravenous formulations: a review of pharmacokinetics and pharmacodynamics. J. Clin. Pharm. Ther. 26:159-169.
Winston, D. J., R. T. Maziarz, P. H. Chandrasekar, H. M. Lazarus, M. Goldman, J. Blumer, G. Leitz, and M. C. Territo. 2003. Intravenous and oral itraconazole versus intravenous and oral fluconazole for long-term antifungal prophylaxis in allogeneic hematopoietic stem-cell transplant recipients. Ann. Intern. Med. 138:705-713.(M. A. Pfaller, L. Boyken,)