Activities of Caspofungin, Itraconazole, Posaconazole, Ravuconazole, Voriconazole, and Amphotericin B against 448 Recent Clinical Isolates of Filamentous Fungi

We examined the in vitro activity of caspofungin, posaconazole,voriconazole, ravuconazole, itraconazole, and amphotericin Bagainst 448 recent clinical mold isolates. The endpoint forreading caspofungin was the minimum effective concentration(MEC). Among the triazoles, posaconazole was most active, inhibiting95% of isolates at $<=$ 1 µg/ml, followed by ravuconazole (91%),voriconazole (90%), and itraconazole (79%). Caspofungin andamphotericin B inhibited 93% and 89% of isolates at $<=$ 1 µg/ml,respectively, with caspofungin demonstrating an MEC 90 of 0.12µg/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 Binhibited only 38% of Aspergillus terreus isolates at $<=$ 1 µg/ml,whereas the three new triazoles and caspofungin inhibited allA. terreus at $<=$ 0.5 µg/ml. The new triazoles and caspofunginhave excellent in vitro activity against a very large collectionof recent clinical isolates of Aspergillus spp., and some invitro activity against selected other filamentous fungi.

INTRODUCTION

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Introduction

Invasive infections due to Aspergillus spp. and other filamentousfungi have emerged as prominent causes of infectious morbidityand mortality in the United States and worldwide (6, 7, 20).Treatment of these infections with available antifungal agentsstill results in an unacceptably high associated mortality (18).

Two new antifungal agents recently have been introduced fortreatment of invasive aspergillosis or other invasive mold infections.Voriconazole and caspofungin offer new alternatives for therapyof these difficult infections (10, 11). In addition, the investigationaltriazoles ravuconazole and posaconazole have been demonstratedto have in vitro potency against Aspergillus spp. and selectedother filamentous fungi (8, 9, 24).

Since the availability of these agents represents an excitingopportunity for improving the outcome of invasive infectionsdue to filamentous fungi, their in vitro activity against contemporaryclinical isolates is of great interest. We performed a 2-year,20-center survey of filamentous fungal infections from January2000 through December 2001. We previously reported preliminaryresults of the activity of new triazoles against molds collectedduring the first year of this survey (24). We now report final2-year results, including the in vitro activity of caspofungin,the new triazoles, amphotericin B, and itraconazole againstover 400 recent clinical isolates of filamentous fungi collectedfrom January 2000 through December of 2001. In the process,we present the largest collection of clinical mold isolatestested against caspofungin yet reported.

MATERIALS AND METHODS

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Materials and Methods

Organisms. A total of 448 unique clinical isolates (one per patient, duplicatepatient isolates excluded) of filamentous fungi were obtainedfrom 20 different medical centers in the United States and Canadabetween January 2000 and December 2001. These centers were participantsin the SENTRY Antimicrobial Surveillance Program. The isolateswere obtained from sputum, bronchoscopy, and tissue biopsy specimens.The collection of isolates included 372 isolates of Aspergillusspp., including 256 Aspergillus fumigatus, 30 A. flavus, 29A. niger, 20 A. versicolor, 16 A. terreus, 4 A. nidulans, and3 A. ustus isolates, 1 A. oryzae isolate, 1 A. glaucus isolate,and 12 isolates of unspecified Aspergillus spp. The collectionincluded 76 other filamentous fungi, including 35 Penicilliumspp., 11 Fusarium spp., 6 Paecilomyces spp., 5 Rhizopus spp.,3 Mucor spp., and 2 Scedosporium apiospermum isolates and oneisolate 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 onAmies charcoal transport media to the central laboratory atthe University of Iowa for reference identification and susceptibilitytesting. They were stored as spore suspensions in sterile distilledwater at room temperature. Before being tested, each isolatewas 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.), andamphotericin B (Sigma Chemical Co., St. Louis, Mo.) were allobtained as reagent-grade powders from their respective manufacturers.The broth microdilution method was performed according to NCCLSguidelines (21). Stock solutions of voriconazole, ravuconazole,and amphotericin B were prepared in dimethyl sulfoxide (Sigma),itraconazole and posaconazole were prepared in polyethyleneglycol, and caspofungin was prepared in water; all were dilutedto 100 times their final concentrations, further diluted inRPMI 1640 medium buffered to pH 7.0 with morpholinepropanesulfonicacid buffer, and dispensed into 96-well microdilution trays.Trays containing a 0.1-ml aliquot in each well of the appropriatedrug solution (2x final concentration) were subjected to qualitycontrol and then sealed and stored at -70°C until used.The final concentrations of the drugs in the wells ranged from0.008 to 8.0 µg/ml. The stock conidial suspension (10⁶spores/ml) was diluted to a final inoculum concentration of0.4 x 10⁴ to 5 x 10⁴ CFU/ml and dispensed into the microdilutionwells. The inoculated microdilution trays were incubated at35°C and read at 48 h. The MIC endpoint for the azoles andamphotericin B was defined as the lowest concentration thatproduced complete inhibition of growth, whereas the minimumeffective concentration (MEC) endpoint for caspofungin was definedaccording 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 recommendedlimits of the NCCLS (21) or other published limits if NCCLSrecommended limits were not available (e.g., for caspofungin)(4).

Statistical analysis. MIC distributions for different triazole antifungal agents werecompared by using the Wilcoxin signed-rank test. The alpha valuewas set at 0.05, and all P values were two tailed.

RESULTS AND DISCUSSION

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Results and Discussion

Table 1 presents the in vitro activities of each antifungalagent against the Aspergillus spp., including the percentageof isolates of each species that were inhibited at an MIC orMEC of $<=$ 1 µg of the antifungal agents/ml. Each of threenew and investigational triazoles (posaconazole, ravuconazole,and voriconazole) had greater in vitro activity than itraconazoleagainst the Aspergillus isolates tested (P < 0.01 for differencein MIC distribution of new triazoles compared to itraconazoleagainst A. fumigatus, A. niger, A. versicolor, and all Aspergillusspp. combined). These data confirm previous reports of the invitro activity of the new and investigational triazoles againstAspergillus spp. (8, 9, 16, 17, 22). The clinical promise ofone new triazole, voriconazole, was demonstrated in a recentreport of its superiority to amphotericin B as primary therapyof invasive aspergillosis (10).

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TABLE 1. In vitro susceptibilities of filamentous fungi isolated during 2000 and 2001

Caspofungin, using MEC as the in vitro susceptibility testingendpoint (1, 15), also had excellent in vitro activity againstall species of Aspergillus tested, inhibiting 90% of isolatesat 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 Administrationfor use in refractory cases of invasive aspergillosis and mayhold promise for treatment of amphotericin B-resistant aspergillosisor as part of combination regimens with triazoles or amphotericin(2, 13, 14, 26). Importantly, direct comparisons of MIC andMEC values should not be made, since they represent differentinhibition endpoints for different antifungal classes. In particular,the MEC endpoint for echinocandins recognizes that they do notproduce complete macroscopic growth inhibition of Aspergillusspp. but rather partial inhibition associated with the developmentof short, stubby, highly branched, and abnormal hyphae (1, 15).

Of particular note, the triazoles and caspofungin all had excellentin vitro activity against A. terreus (100% inhibited at an MICor MEC of $<=$ 1 µg/ml), a species against which amphotericinB demonstrated poor in vitro activity (38% inhibited at an MICof $<=$ 1 µg/ml) and against which amphotericin B has poorclinical efficacy (12).

Table 1 also shows the activity of each agent against otherspecies of filamentous fungi that were isolated in sufficientnumbers to merit examining individually. All tested agents wereless active against these filamentous fungi than against Aspergillusspp. Caspofungin, which is not generally considered to be activeagainst molds other than Aspergillus spp., demonstrated somein 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. Itremains to be seen whether caspofungin or other echinocandinswill have any role, either alone or in combination with otheragents (2, 3, 27), in the treatment of infections due to moldsother than Aspergillus spp.

The new and investigational triazoles also demonstrated someactivity against miscellaneous molds, particularly Penicilliumand Paecilomyces spp. None of the agents except amphotericinB had good in vitro activity against Fusarium spp. (90% MIC[MIC₉₀] or MEC₉₀ of >8 µg/ml for all triazoles andcaspofungin). These data are consistent with previously publishedin vitro data (8, 9, 15, 17, 19, 24, 25). Despite poor in vitroactivity, the clinical response to voriconazole and posaconazolehas 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). Wereport in vitro results for only eight zygomycetes (five Rhizopusand three Mucor spp.). The zygomycetes have been demonstratedto be a heterogeneous group with regard to in vitro antifungalsusceptibility (5), and more data are needed in order to determinewhich new or investigational agents are active against individualspecies.

In summary, caspofungin and the new triazoles posaconazole,ravuconazole, and voriconazole have excellent in vitro activityagainst Aspergillus spp. and variable activity against selectedother filamentous fungi. In addition, an in vitro-in vivo correlationis needed for both new and established antifungal agents againstthe filamentous fungi.

ACKNOWLEDGMENTS

This study was supported in part by research and educationalgrants from Bristol-Myers Squibb Company (SENTRY), Pfizer Pharmaceuticals,and Schering-Plough Research Institute.

We thank all participating SENTRY centers.

FOOTNOTES

* Corresponding author. Mailing address: C606 GH, University of Iowa College of Medicine, 200 Hawkins Dr., Iowa City, IA 52242. Phone: (319) 356-8615. Fax: (319) 356-4916. E-mail: daniel-diekema{at}uiowa.edu.

REFERENCES

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