In Vitro Activities of Anidulafungin against More than 2,500 Clinical Isolates of Candida spp., Including 315 Isolates Resistant to Fluconazole

Anidulafungin is an echinocandin antifungal agent with potentactivity against Candida spp. We assessed the in vitro activityof anidulafungin against 2,235 clinical isolates of Candidaspp. using the CLSI broth microdilution method. Anidulafunginwas very active against Candida spp. (the MIC at which 90% ofstrains are inhibited [MIC₉₀] was 2 µg/ml when MIC endpointcriteria of partial inhibition [MIC-2] were used). Candida albicans,C. glabrata, C. tropicalis, C. krusei, and C. kefyr were themost susceptible species of Candida (MIC₉₀, 0.06 to 0.12 µg/ml),and C. parapsilosis, C. lusitaniae, and C. guilliermondii werethe least susceptible (MIC₉₀, 0.5 to 2 µg/ml). In addition,315 fluconazole-resistant isolates were tested, and 99% wereinhibited by $≤$ 1 µg/ml of anidulafungin. These results providefurther evidence for the spectrum and potency of anidulafunginactivity against a large and geographically diverse collectionof clinically important isolates of Candida spp.

INTRODUCTION

Top

Introduction

Anidulafungin is an investigational echinocandin with potentfungicidal activity against many species of Candida (1, 4, 5,6, 11, 12, 13, 14, 17, 18). Anidulafungin is now in phase IIIclinical trials and has been shown to be safe and efficaciousin treating invasive candidiasis (8). Although several studiesdocumenting the in vitro activity of anidulafungin against Candidaspp. have been published, these studies employed test methodsthat utilize either a more conservative MIC endpoint criterion(100% inhibition or MIC-0), an extended incubation time (48h), or both and are either limited in the number of isolatesof the various species of Candida tested or are restricted inthe geographical distribution of the tested strains (1, 4, 11,14, 17, 18).

In the present study, we determined the in vitro activity ofanidulafungin against an international collection of more than2,000 clinical isolates of Candida representing predominatelybloodstream infection and other invasive forms of candidiasis.We also provide an evaluation of anidulafungin activity against315 isolates with resistance (MIC, $≥$ 64 µg/ml) to fluconazole.Because there is some controversy over the optimal method forperforming in vitro susceptibility testing of the echinocandins(3), there is currently no standardized method for testing theseagents against Candida. We have elected to use the method recommendedby the Clinical and Laboratory Standards Institute (CLSI, formerlyNCCLS), which employs RPMI 1640 broth, 24 h of incubation, anda partial inhibition (MIC-2, or $≥$ 50% inhibition relative to control)MIC endpoint (3, 9, 10, 15, 16). The results are presented asthe cumulative percentage of isolates inhibited at each concentrationthroughout the dilution series (full-range MICs) to facilitatecomparison with other studies using the CLSI method.

MATERIALS AND METHODS

Top

Materials and Methods

Organisms. A total of 2,235 clinical isolates of Candida spp. obtainedfrom 91 different medical centers internationally were tested.These isolates were contributed as part of an ongoing prospectivesurveillance program and represent the incident isolate obtainedfrom a given infectious episode. The collection included thefollowing numbers of isolates: C. albicans, 1,181; C. glabrata,265; C. parapsilosis, 328; C. tropicalis, 278; C. krusei, 59;C. lusitaniae, 34; C. guilliermondii, 57; C. kefyr, 15; andmiscellaneous Candida spp., 18. The isolates represented theAsia-Pacific region (354 isolates from 16 study sites), LatinAmerica (542 isolates from 15 study sites), Europe (668 isolatesfrom 32 study sites), and North America (671 isolates from 28study sites) (Table 1). In addition, a collection of 315 isolatesof Candida spp. previously characterized as resistant to fluconazole(MIC, $≥$ 64 µg/ml) (15) was tested in order to determinethe activity of anidulafungin against these clinically importantstrains: C. albicans (41 isolates), C. glabrata (110 isolates),C. krusei (146 isolates), and Candida spp. (18 isolates). Theisolates were all recent clinical isolates (2001 to 2004) andwere from blood or normally sterile body fluids (cerebrospinalfluid, pleural fluid, or peritoneal fluid) and tissue. The isolateswere identified by standard methods (7) and were stored as watersuspensions until they were used in the study.

View this table:
[in this window]
[in a new window]
TABLE 1. Species distribution of Candida isolates by geographic region

Antifungal agents. Standard antifungal powder of anidulafungin (Vicuron, Inc.,King of Prussia, PA) was obtained from the manufacturer. Stocksolutions were prepared in dimethyl sulfoxide. Serial twofolddilutions were prepared exactly as outlined in CLSI documentM27-A2 (9). Final dilutions were made in RPMI 1640 medium (Sigma,St. Louis, MO) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonicacid (MOPS) buffer (Sigma). Aliquots (0.1 ml) of the antifungalagent at twice the final concentration were dispensed into wellsof plastic microdilution trays. The trays were sealed and frozenat –70°C until they were used.

Antifungal susceptibility studies. Broth microdilution testing of all 2,550 isolates was performedas described previously (16) in accordance with the guidelinesof CLSI document M27-A2 (9), using a final inoculum concentrationof (1.5 ± 1.0) x 10³ cells/ml, RPMI 1640 medium, andincubation at 35°C for 24 h. MIC endpoints for anidulafunginwere defined as the lowest concentration that produced a prominentdecrease in turbidity ( $≥$ 50% or MIC-2) relative to that of thedrug-free control well (2, 9, 16).

RESULTS AND DISCUSSION

Top

Results and Discussion

The species distribution of the isolates tested, stratifiedby the geographic region of origin, is shown in Table 1. Allof the major species were represented, including less commonand "emerging" species, such as C. guilliermondii. It is notablethat the species distributions of Candida bloodstream infectionisolates contributed by study sites in the Asia-Pacific andLatin American regions were considerably different than thatseen in North America.

Whereas C. glabrata was much more frequently isolated than eitherC. parapsilosis or C. tropicalis in North America, it was lesscommon than either of these two species in the Asia-Pacificregion and in Latin America. Similarly, C. parapsilosis wasmore common than C. glabrata among isolates contributed fromEurope. Among the less frequently isolated species of Candida,C. krusei was more common among European isolates and C. guilliermondiiwas more common in Latin America, where it ranked above bothC. glabrata and C. krusei among all bloodstream infection isolates.The species diversity, the number of contributing study sites,and the broad (worldwide) geographic representation are strengthsof this database.

Table 2 summarizes the in vitro susceptibilities of 2,235 isolatesof Candida spp. to anidulafungin using the MIC-2 endpoint methoddescribed above. Overall, anidulafungin was quite active againstthis broad range of Candida species (MIC at which 50% of thestrains are inhibited [MIC₅₀], 0.06 µg/ml; MIC₉₀, 2 µg/ml).C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyrwere the species most susceptible to anidulafungin (MIC₉₀, 0.06to 0.12 µg/ml), and C. parapsilosis (MIC₉₀, 2 µg/ml),C. lusitaniae (MIC₉₀, 0.5 µg/ml), and C. guilliermondii(MIC₅₀, 2 µg/ml) were the least susceptible. Notably,100% of C. glabrata and C. krusei isolates were inhibited by $≤$ 0.25 µg/ml of anidulafungin. Despite differences in thespecies distribution across the four geographic regions, therewas no difference in the activity of anidulafungin, overallor by species, when stratified by region.

View this table:
[in this window]
[in a new window]
TABLE 2. In vitro susceptibilities of 2,235 clinical isolates of Candida spp. to anidulafungin^a

The activity of anidulafungin against the 315 fluconazole-resistantisolates (Table 3) was equal to or better than its activityagainst the larger group of more azole-susceptible strains (Table2). Notably, 100% of fluconazole-resistant isolates of C. glabrataand C. krusei were inhibited by $≤$ 0.5 µg/ml of anidulafungin(Table 3).

View this table:
[in this window]
[in a new window]
TABLE 3. In vitro activity of anidulafungin against 315 fluconazole-resistant clinical isolates of Candida spp.^a

These findings confirm and extend those reported previouslyregarding the anticandidal activity of anidulafungin (1, 4,11, 15, 17, 18). Anidulafungin exhibited potent activity againstvirtually all species of Candida, including those with resistanceto fluconazole. As seen with caspofungin (15, 16), there appearto be two broad groups of Candida species that can be differentiatedby the degree of susceptibility to anidulafungin. One groupincludes the common species C. albicans, C. glabrata, C. tropicalis,and C. krusei (as well as the less common C. kefyr) and is highlysusceptible to anidulafungin (MIC₉₀, $≤$ 0.12 µg/ml), whereasthe second group includes C. parapsilosis and less common species,such as C. lusitaniae and C. guilliermondii, and is 4- to 16-foldless susceptible to anidulafungin (MIC₉₀, 0.5 to 2 µg/ml)(Table 2). Preliminary data suggest that all of these speciesmay respond clinically in a similar fashion to anidulafungintreatment (8). The MICs for 99% of isolates in both groups are $≤$ 2 µg/ml when tested using the partial inhibition endpointcriteria, a concentration that is exceeded throughout the dosinginterval following the administration of anidulafungin at standarddoses of 100 mg/day (8, 13).

In summary, we have demonstrated that anidulafungin has potentin vitro activity against a broad range of Candida species fromthroughout the world. The emerging in vivo data from animalmodels and from clinical trials support the efficacy of anidulafunginin the treatment of invasive candidiasis. The fungicidal natureof anidulafungin coupled with sustained serum concentrationsthat exceed the MIC₉₀ of virtually all Candida species makesit a very promising systemic antifungal agent.

ACKNOWLEDGMENTS

We thank Linda Elliott for excellent assistance in the preparationof the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Medical Microbiology Division, C606 GH, Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242. Phone: (319) 384-9566. Fax: (319) 356-4916. E-mail: michael-pfaller{at}uiowa.edu.

REFERENCES

Top