Abstract
We examined the in vitro activity of caspofungin, posaconazole, voriconazole, ravuconazole, itraconazole, and amphotericin B against 448 recent clinical mold isolates. The endpoint for reading caspofungin was the minimum effective concentration (MEC). Among the triazoles, posaconazole was most active, inhibiting 95% of isolates at ≤1 μg/ml, followed by ravuconazole (91%), voriconazole (90%), and itraconazole (79%). Caspofungin and amphotericin B inhibited 93% and 89% of isolates at ≤1 μg/ml, respectively, with caspofungin demonstrating an MEC 90 of 0.12 μg/ml. All three new triazoles and caspofungin inhibited >95% of Aspergillus spp. at ≤1 μg/ml compared to 83% for itraconazole and 91% for amphotericin B. Amphotericin B inhibited only 38% of Aspergillus terreus isolates at ≤1 μg/ml, whereas the three new triazoles and caspofungin inhibited all A. terreus at ≤0.5 μg/ml. The new triazoles and caspofungin have excellent in vitro activity against a very large collection of recent clinical isolates of Aspergillus spp., and some in vitro activity against selected other filamentous fungi.
Invasive infections due to Aspergillus spp. and other filamentous fungi have emerged as prominent causes of infectious morbidity and mortality in the United States and worldwide (6, 7, 20). Treatment of these infections with available antifungal agents still results in an unacceptably high associated mortality (18).
Two new antifungal agents recently have been introduced for treatment of invasive aspergillosis or other invasive mold infections. Voriconazole and caspofungin offer new alternatives for therapy of these difficult infections (10, 11). In addition, the investigational triazoles ravuconazole and posaconazole have been demonstrated to have in vitro potency against Aspergillus spp. and selected other filamentous fungi (8, 9, 24).
Since the availability of these agents represents an exciting opportunity for improving the outcome of invasive infections due to filamentous fungi, their in vitro activity against contemporary clinical isolates is of great interest. We performed a 2-year, 20-center survey of filamentous fungal infections from January 2000 through December 2001. We previously reported preliminary results of the activity of new triazoles against molds collected during the first year of this survey (24). We now report final 2-year results, including the in vitro activity of caspofungin, the new triazoles, amphotericin B, and itraconazole against over 400 recent clinical isolates of filamentous fungi collected from January 2000 through December of 2001. In the process, we present the largest collection of clinical mold isolates tested against caspofungin yet reported.
MATERIALS AND METHODS
Organisms.
A total of 448 unique clinical isolates (one per patient, duplicate patient isolates excluded) of filamentous fungi were obtained from 20 different medical centers in the United States and Canada between January 2000 and December 2001. These centers were participants in the SENTRY Antimicrobial Surveillance Program. The isolates were obtained from sputum, bronchoscopy, and tissue biopsy specimens. The collection of isolates included 372 isolates of Aspergillus spp., including 256 Aspergillus fumigatus, 30 A. flavus, 29 A. niger, 20 A. versicolor, 16 A. terreus, 4 A. nidulans, and 3 A. ustus isolates, 1 A. oryzae isolate, 1 A. glaucus isolate, and 12 isolates of unspecified Aspergillus spp. The collection included 76 other filamentous fungi, including 35 Penicillium spp., 11 Fusarium spp., 6 Paecilomyces spp., 5 Rhizopus spp., 3 Mucor spp., and 2 Scedosporium apiospermum isolates and one isolate each of Absidia sp., Acremonium sp., Bipolaris sp., Chrysosporium sp., Cladophialophora bantiana, Curvularia sp., Exophiala dermatitidis, Fonsecaeae pedrosoi, Geotrichum sp., Pithomyces sp., Trichophyton mentagrophytes, Trichophyton sp., Ulocladium sp., and Wangiella sp. All isolates were sent on Amies charcoal transport media to the central laboratory at the University of Iowa for reference identification and susceptibility testing. They were stored as spore suspensions in sterile distilled water at room temperature. Before being tested, each isolate was subcultured at least twice on potato dextrose agar (Remel, Lenexa, Kans.) to ensure viability and purity.
Susceptibility testing.
Posaconazole (Schering-Plough Research Institute, Kenilworth, N.J.), ravuconazole (Bristol-Myers Squibb, Wallingford, Conn.), voriconazole (Pfizer Pharmaceutical Group, New York, N.Y.), caspofungin (Merck Research Laboratories, Rahway, N.J.), and amphotericin B (Sigma Chemical Co., St. Louis, Mo.) were all obtained as reagent-grade powders from their respective manufacturers. The broth microdilution method was performed according to NCCLS guidelines (21). Stock solutions of voriconazole, ravuconazole, and amphotericin B were prepared in dimethyl sulfoxide (Sigma), itraconazole and posaconazole were prepared in polyethylene glycol, and caspofungin was prepared in water; all were diluted to 100 times their final concentrations, further diluted in RPMI 1640 medium buffered to pH 7.0 with morpholinepropanesulfonic acid buffer, and dispensed into 96-well microdilution trays. Trays containing a 0.1-ml aliquot in each well of the appropriate drug solution (2× final concentration) were subjected to quality control and then sealed and stored at −70°C until used. The final concentrations of the drugs in the wells ranged from 0.008 to 8.0 μg/ml. The stock conidial suspension (106 spores/ml) was diluted to a final inoculum concentration of 0.4 × 104 to 5 × 104 CFU/ml and dispensed into the microdilution wells. The inoculated microdilution trays were incubated at 35°C and read at 48 h. The MIC endpoint for the azoles and amphotericin B was defined as the lowest concentration that produced complete inhibition of growth, whereas the minimum effective concentration (MEC) endpoint for caspofungin was defined according to published methods (1, 15).
Quality control.
Quality control was ensured by testing the following strains (4, 21): A. flavus ATCC 204304, Candida parapsilosis ATCC 22019, and Candida krusei ATCC 6258. All results were within the recommended limits of the NCCLS (21) or other published limits if NCCLS recommended limits were not available (e.g., for caspofungin) (4).
Statistical analysis.
MIC distributions for different triazole antifungal agents were compared by using the Wilcoxin signed-rank test. The alpha value was set at 0.05, and all P values were two tailed.
RESULTS AND DISCUSSION
Table 1 presents the in vitro activities of each antifungal agent against the Aspergillus spp., including the percentage of isolates of each species that were inhibited at an MIC or MEC of ≤1 μg of the antifungal agents/ml. Each of three new and investigational triazoles (posaconazole, ravuconazole, and voriconazole) had greater in vitro activity than itraconazole against the Aspergillus isolates tested (P < 0.01 for difference in MIC distribution of new triazoles compared to itraconazole against A. fumigatus, A. niger, A. versicolor, and all Aspergillus spp. combined). These data confirm previous reports of the in vitro activity of the new and investigational triazoles against Aspergillus spp. (8, 9, 16, 17, 22). The clinical promise of one new triazole, voriconazole, was demonstrated in a recent report of its superiority to amphotericin B as primary therapy of invasive aspergillosis (10).
TABLE 1.
In vitro susceptibilities of filamentous fungi isolated during 2000 and 2001
| Species (no. of isolates tested) | Antifungal agent | MIC or MECa (μg/ml)
|
MIC or MECa range (μg/ml) | % Total at ≤1 μg/ml | |
|---|---|---|---|---|---|
| 50% | 90% | ||||
| A. fumigatus (256) | Amphotericin | 1 | 1 | 0.5->8 | 96.1 |
| Caspofungin | 0.03 | 0.06 | 0.015-2 | 99.2 | |
| Itraconazole | 1 | 2 | 0.12-2 | 87.1 | |
| Posaconazole | 0.25 | 0.5 | 0.03-2 | 99.6 | |
| Ravuconazole | 0.25 | 0.5 | 0.06-2 | 98.8 | |
| Voriconazole | 0.25 | 0.5 | 0.06-4 | 98.8 | |
| A. flavus (30) | Amphotericin | 1 | 2 | 1-2 | 70.0 |
| Caspofungin | 0.03 | 0.06 | 0.015-0.12 | 100 | |
| Itraconazole | 0.5 | 1 | 0.25-2 | 96.7 | |
| Posaconazole | 0.25 | 0.5 | 0.12-1 | 100 | |
| Ravuconazole | 0.5 | 1 | 0.06-1 | 100 | |
| Voriconazole | 0.5 | 1 | 0.12-1 | 100 | |
| A. niger (29) | Amphotericin | 1 | 1 | 0.25-1 | 100 |
| Caspofungin | 0.03 | 0.06 | 0.015-0.06 | 100 | |
| Itraconazole | 2 | 2 | 0.5-4 | 44.8 | |
| Posaconazole | 0.5 | 1 | 0.25-1 | 100 | |
| Ravuconazole | 0.5 | 2 | 0.25-2 | 86.2 | |
| Voriconazole | 1 | 2 | 0.12-4 | 65.5 | |
| A. versicolor (20) | Amphotericin | 1 | 2 | 1-2 | 80.0 |
| Caspofungin | 0.03 | 0.12 | 0.015-4 | 90.5 | |
| Itraconazole | 1 | 2 | 0.12-2 | 65.0 | |
| Posaconazole | 0.5 | 1 | 0.06-2 | 90.0 | |
| Ravuconazole | 0.25 | 1 | 0.03-2 | 95.2 | |
| Voriconazole | 0.5 | 1 | 0.06-2 | 95.0 | |
| A. terreus (16) | Amphotericin | 2 | 2 | 1-4 | 37.5 |
| Caspofungin | 0.03 | 0.06 | 0.015-0.12 | 100 | |
| Itraconazole | 0.5 | 0.5 | 0.25-1 | 100 | |
| Posaconazole | 0.12 | 0.25 | 0.06-0.25 | 100 | |
| Ravuconazole | 0.25 | 0.5 | 0.03-0.5 | 100 | |
| Voriconazole | 0.25 | 1 | 0.06-0.5 | 100 | |
| Penicillium spp. (35) | Amphotericin | 1 | 2 | 0.12-2 | 85.7 |
| Caspofungin | 0.03 | 0.12 | 0.015->8 | 97.4 | |
| Itraconazole | 1 | 2 | 0.25-2 | 77.1 | |
| Posaconazole | 0.5 | 1 | 0.06-2 | 97.1 | |
| Ravuconazole | 0.5 | 4 | 0.015-8 | 80.0 | |
| Voriconazole | 0.5 | 2 | 0.03->8 | 80.0 | |
| Fusarium spp. (11) | Amphotericin | 1 | 2 | 1-2 | 81.8 |
| Caspofungin | >8 | >8 | >8->8 | 0 | |
| Itraconazole | >8 | >8 | 2->8 | 0 | |
| Posaconazole | >8 | >8 | 0.5->8 | 18.2 | |
| Ravuconazole | 8 | >8 | 0.25->8 | 18.2 | |
| Voriconazole | 4 | >8 | 0.25->8 | 18.2 | |
| Paecilomyces spp. (6) | Amphotericin | 0.5 | 0.06->8 | 66.7 | |
| Caspofungin | 0.06 | 0.03-8 | 83.3 | ||
| Itraconazole | 0.25 | 0.06-2 | 83.3 | ||
| Posaconazole | 0.12 | 0.03-0.5 | 100 | ||
| Ravuconazole | 0.25 | 0.03-4 | 83.3 | ||
| Voriconazole | 0.25 | 0.03-2 | 83.3 | ||
| Rhizopus spp. (5) | Amphotericin | 1 | 0.5-1 | 100 | |
| Caspofungin | >8 | >8->8 | 0 | ||
| Itraconazole | 4 | 1->8 | 20 | ||
| Posaconazole | 2 | 1-4 | 40 | ||
| Ravuconazole | >8 | 0.5->8 | 20 | ||
| Voriconazole | 2 | 1->8 | 40 | ||
| Mucor spp. (3) | Amphotericin | 0.5 | 0.5-1 | 100 | |
| Caspofungin | >8 | >8->8 | 0 | ||
| Itraconazole | 2 | 2->8 | 0 | ||
| Posaconazole | 1 | 0.5->8 | 66.7 | ||
| Ravuconazole | >8 | 1->8 | 33.3 | ||
| Voriconazole | 2 | 1->8 | 33.3 | ||
| All Aspergillus (372) | Amphotericin | 1 | 1 | 0.12->8 | 90.6 |
| Caspofungin | 0.03 | 0.06 | 0.015-4 | 98.4 | |
| Itraconazole | 1 | 2 | 0.12->8 | 83.1 | |
| Posaconazole | 0.25 | 0.5 | 0.03->8 | 98.1 | |
| Ravuconazole | 0.25 | 1 | 0.03->8 | 96.8 | |
| Voriconazole | 0.5 | 1 | 0.03->8 | 95.2 | |
| All filamentous fungi (448) | Amphotericin | 1.0 | 2.0 | 0.06->8 | 89.3 |
| Caspofungin | 0.03 | 0.12 | 0.015->8 | 92.8 | |
| Itraconazole | 1.0 | 2.0 | 0.06->8 | 79.0 | |
| Posaconazole | 0.25 | 1.0 | 0.03->8 | 94.6 | |
| Ravuconazole | 0.25 | 1.0 | 0.015->8 | 91.3 | |
| Voriconazole | 0.5 | 1.0 | 0.03->8 | 90.2 | |
Caspofungin, using MEC as the in vitro susceptibility testing endpoint (1, 15), also had excellent in vitro activity against all species of Aspergillus tested, inhibiting 90% of isolates at an MEC of 0.06 μg/ml and >98% at an MEC of ≤1 μg/ml. Caspofungin has been approved by the U.S. Food and Drug Administration for use in refractory cases of invasive aspergillosis and may hold promise for treatment of amphotericin B-resistant aspergillosis or as part of combination regimens with triazoles or amphotericin (2, 13, 14, 26). Importantly, direct comparisons of MIC and MEC values should not be made, since they represent different inhibition endpoints for different antifungal classes. In particular, the MEC endpoint for echinocandins recognizes that they do not produce complete macroscopic growth inhibition of Aspergillus spp. but rather partial inhibition associated with the development of short, stubby, highly branched, and abnormal hyphae (1, 15).
Of particular note, the triazoles and caspofungin all had excellent in vitro activity against A. terreus (100% inhibited at an MIC or MEC of ≤1 μg/ml), a species against which amphotericin B demonstrated poor in vitro activity (38% inhibited at an MIC of ≤1 μg/ml) and against which amphotericin B has poor clinical efficacy (12).
Table 1 also shows the activity of each agent against other species of filamentous fungi that were isolated in sufficient numbers to merit examining individually. All tested agents were less active against these filamentous fungi than against Aspergillus spp. Caspofungin, which is not generally considered to be active against molds other than Aspergillus spp., demonstrated some in vitro activity against Penicillium spp. (MEC90, 0.12 μg/ml; 97% inhibited at an MEC of ≤1 μg/ml) and Paecilomyces spp. (five of six isolates inhibited at an MEC of ≤1 μg/ml) but no activity against Fusarium spp. or the zygomycetes. It remains to be seen whether caspofungin or other echinocandins will have any role, either alone or in combination with other agents (2, 3, 27), in the treatment of infections due to molds other than Aspergillus spp.
The new and investigational triazoles also demonstrated some activity against miscellaneous molds, particularly Penicillium and Paecilomyces spp. None of the agents except amphotericin B had good in vitro activity against Fusarium spp. (90% MIC [MIC90] or MEC90 of >8 μg/ml for all triazoles and caspofungin). These data are consistent with previously published in vitro data (8, 9, 15, 17, 19, 24, 25). Despite poor in vitro activity, the clinical response to voriconazole and posaconazole has been described in some cases of Fusarium spp. infection (23; R. Y. Hachem, I. I. Raad, C. M. Afif, et al., 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1009, 2000). We report in vitro results for only eight zygomycetes (five Rhizopus and three Mucor spp.). The zygomycetes have been demonstrated to be a heterogeneous group with regard to in vitro antifungal susceptibility (5), and more data are needed in order to determine which new or investigational agents are active against individual species.
In summary, caspofungin and the new triazoles posaconazole, ravuconazole, and voriconazole have excellent in vitro activity against Aspergillus spp. and variable activity against selected other filamentous fungi. In addition, an in vitro-in vivo correlation is needed for both new and established antifungal agents against the filamentous fungi.
Acknowledgments
This study was supported in part by research and educational grants from Bristol-Myers Squibb Company (SENTRY), Pfizer Pharmaceuticals, and Schering-Plough Research Institute.
We thank all participating SENTRY centers.
REFERENCES
- 1.Arikan, S., M. Lozano-Chiu, V. Paetznick, and J. H. Rex. 2001. In vitro susceptibility testing methods for caspofungin against Aspergillus and Fusarium isolates. Antimicrob. Agents Chemother. 45:327-330. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Arikin, S., M. Lozano-Chiu, V. Paetznick, and J. H. Rex. 2002. In vitro synergy of caspofungin and amphotericin B against Aspergillus and Fusarium spp. Antimicrob. Agents Chemother. 46:245-247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Arikan, S., V. Paetznick, and J. H. Rex. 2002. Comparative evaluation of disk diffusion with microdilution assay in susceptibility testing of caspofungin against Aspergillus and Fusarium isolates. Antimicrob. Agents Chemother. 46:3084-3087. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Barry, A. L., M. A. Pfaller, S. D. Brown, A. Espinel-Ingroff, M. A. Ghannoum, C. Knapp, R. P. Rennie, J. H. Rex, and M. G. Rinaldi. 2000. Quality control limits for broth microdilution susceptibility tests of ten antifungal agents. J. Clin. Microbiol. 38:3457-3459. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Dannaoui, E., J. Meletiadis, J. W. Mouton, J. F. Meis, and P. E. Verweij. 2003. In vitro susceptibilities of zygomycetes to conventional and new antifungals. J. Antimicrob. Chemother. 51:45-52. [DOI] [PubMed] [Google Scholar]
- 6.Dasbach, E. J., G. M. Davies, and S. M. Teutsch. 2000. Burden of aspergillus-related hospitalizations in the United States. Clin. Infect. Dis. 31:1524-1528. [DOI] [PubMed] [Google Scholar]
- 7.Denning, D. W., A. Marinas, and J. Cohen. 1998. An EORTC multicentre prospective survey of invasive aspergillosis in haematological patients: diagnosis and therapeutic outcome. J. Infect. 37:173-180. [DOI] [PubMed] [Google Scholar]
- 8.Espinel-Ingroff, A. 1998. Comparison of in vitro activities of the new triazole SCH 56592 and the echinocandins MK-0991 and LY-303366 against opportunistic filamentous and dimorphic fungi and yeasts. J. Clin. Microbiol. 36:2950-2956. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Espinel-Ingroff, A. 2001. In vitro fungicidal activities of voriconazole, itraconazole, and amphotericin B against opportunistic moniliaceous and dematiaceous fungi. J. Clin. Microbiol. 39:954-958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Herbrecht, R., D. W. Denning, T. F. Patterson, et al. 2002. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N. Engl. J. Med. 347:408-415. [DOI] [PubMed] [Google Scholar]
- 11.Hoang, A. 2001. Caspofungin acetate: an antifungal agent. Am. J. Health Syst. Pharm. 58:1206-1214. [DOI] [PubMed] [Google Scholar]
- 12.Iwen, R. C., M. E. Rupp, A. N. Langnas, E. C. Reed, and S. H. Hinrichs. 1998. Invasive pulmonary aspergillosis due to Aspergillus terreus: 12-year experience and review of the literature. Clin. Infect. Dis. 26:1092-1097. [DOI] [PubMed] [Google Scholar]
- 13.Kirkpatrick, W. R., S. Perea, B. J. Coco, and T. F. Patterson. 2002. Efficacy of caspofungin alone and in combination with voriconazole in a guinea pig model of invasive aspergillosis. Antimicrob. Agents Chemother. 46:2564-2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Koss, T., B. Bagheri, C. Zeana, M. F. Romagnoli, and M. E. Grossman. 2002. Amphotericin B-resistant Aspergillus flavus infection successfully treated with caspofungin, a novel antifungal agent. J. Am. Acad. Dermatol. 46:945-947. [DOI] [PubMed]
- 15.Kurtz, M. B., I. B. Heath, J. Marrinan, et al. 1994. Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-β-d-glucan synthase. Antimicrob. Agents Chemother. 38:1480-1489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Marco, F., M. A. Pfaller, S. A. Messer, and R. N. Jones. 1998. Antifungal activity of a new triazole, voriconazole, compared with three other antifungal agents tested against clinical isolates of filamentous fungi. Med. Mycol. 36:433-436. [PubMed] [Google Scholar]
- 17.Marco, F., M. A. Pfaller, S. A. Messer, and R. N. Jones. 1998. In vitro activity of a new triazole antifungal agent, Sch 56592, against clinical isolates of filamentous fungi. Mycopathologia 141:73-77. [DOI] [PubMed] [Google Scholar]
- 18.Marr, K. A., R. A. Carter, F. Crippa, A. Wald, and L. Corey. 2002. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin. Infect. Dis. 34:909-917. [DOI] [PubMed] [Google Scholar]
- 19.McGinnis, M. R., L. Pasarell, D. A. Sutton, A. W. Fothergill, C. R. Cooper, and M. G. Rinaldi. 1997. In vitro evaluation of voriconazole against some clinically important fungi. Antimicrob. Agents Chemother. 41:1832-1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.McNeil, M. M., S. L. Nash, R. A. Hajjeh, et al. 2001. Trends in mortality due to invasive mycotic diseases in the United States, 1980-1997. Clin. Infect. Dis. 33:641-647. [DOI] [PubMed] [Google Scholar]
- 21.National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Approved standard M38-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
- 22.Oakley, K. L., C. B. Moore, and D. W. Denning. 1997. In vitro activity of SCH 56592 and comparison with activities of amphotericin B and itraconazole against Aspergillus spp. Antimicrob. Agents Chemother. 41:1124-1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Perfect, J. R., K. A. Marr, T. J. Walsh, R. N. Greenberg, et al. 2003. Voriconazole treatment of less common, emerging or refractory fungal infections. Clin. Infect. Dis. 36:1122-1131. [DOI] [PubMed] [Google Scholar]
- 24.Pfaller, M. A., S. A. Messer, R. J. Hollis, et al. 2002. Antifungal activities of posaconazole, ravuconazole and voriconazole compared to those of itraconazole and amphotericin B against 239 clinical isolates of filamentous fungi: report from the SENTRY Antimicrobial Surveillance Program, 2000. Antimicrob. Agents Chemother. 46:1032-1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Radford, S. A., E. M. Johnson, and D. W. Warnock. 1997. In vitro studies of activity of voriconazole, a new triazole antifungal agent, against emerging and less common mold pathogens. Antimicrob. Agents Chemother. 41:841-843. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Rubin, M. A., K. C. Carroll, and B. C. Cahill. 2002. Caspofungin in combination with itraconazole for the treatment of invasive aspergillosis in humans. Clin. Infect. Dis. 34:1160-1161. [DOI] [PubMed] [Google Scholar]
- 27.Safdar, A. 2002. Progressive cutaneous hyalohyphomycosis due to Paecilomyces lilacinus: rapid response to treatment with caspofungin and itraconazole. Clin. Infect. Dis. 34:1415-1417. [DOI] [PubMed] [Google Scholar]