Activities of Micafungin against 315 Invasive Clinical Isolates of Fluconazole-Resistant Candida spp.

Micafungin is a new echinocandin exhibiting broad-spectrum activityagainst Candida spp. The activity of the echinocandins againstCandida species known to express intrinsic or acquired resistanceto fluconazole is of interest. We determined the MICs of micafunginand caspofungin against 315 invasive clinical (bloodstream andother sterile-site) isolates of fluconazole-resistant Candidaspecies obtained from geographically diverse medical centersbetween 2001 and 2004. MICs were determined using broth microdilutionaccording to the CLSI reference method M27-A2. RPMI 1640 wasused as the test medium, and we used the MIC endpoint of prominentgrowth reduction at 24 h. Among the 315 fluconazole-resistantCandida isolates, 146 (46%) were C. krusei, 110 (35%) were C.glabrata, 41 (13%) were C. albicans, and 18 (6%) were less frequentlyisolated species. Micafungin had good in vitro activity againstall fluconazole-resistant Candida spp. tested; the MICs at which50% (MIC₅₀) and 90% (MIC₉₀) of isolates were inhibited were0.03 µg/ml and 0.06 µg/ml, respectively. All thefluconazole-resistant Candida spp. were inhibited at a micafunginMIC that was $≤$ 1 µg/ml. Among the most common fluconazole-resistantCandida spp. tested in the collection, C. glabrata exhibitedthe lowest micafungin MICs (MIC₉₀, $≤$ 0.015 µg/ml), followedby C. albicans (MIC₉₀, 0.03 µg/ml) and C. krusei (MIC₉₀,0.06 µg/ml). The new echinocandin micafungin has excellentin vitro activity against 315 invasive clinical isolates offluconazole-resistant Candida, which represents the largestcollection to date of fluconazole-resistant Candida isolatestested against micafungin. Micafungin may prove useful in thetreatment of infections due to azole-resistant Candida.

INTRODUCTION

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Introduction

In the United States, Candida spp. are the fourth most commoncause of nosocomial bloodstream infection (13, 14). These infectionsresult in increased mortality (5) and longer hospital stays(3, 18) with associated higher health care costs. Of specialconcern has been the emergence of non-Candida albicans speciesexhibiting resistance to the current spectrum of azoles (6,8, 12-14). Treatment of azole-resistant infections has remaineda challenge for clinicians and has driven the need for alternativeagents having novel mechanisms of action.

The development of the echinocandin class of systemic antifungalagents has added to the limited number of drugs used againstCandida spp. In contrast to the azoles, which inhibit ergosterolsynthesis, echinocandins interrupt the synthesis of 1,3-ß-Dglucan, an important component of the fungal cell wall (2).The echinocandins have been shown to exhibit in vitro activityagainst both Candida spp. and Aspergillus spp. (4, 10, 11, 15,17, 19).

Micafungin is the most recently available echinocandin, currentlyFDA approved for use in esophageal candidiasis and for the prophylaxisof invasive fungal infections in hematopoietic stem cell transplantpatients. The activity of micafungin against clinical isolatesof azole-resistant Candida spp. is of interest. We examinedthe in vitro activities of micafungin against 315 invasive clinicalisolates of fluconazole-resistant Candida spp. from medicalcenters worldwide, using Clinical and Laboratory Standards Institute(CLSI, formerly the NCCLS) broth microdilution methods withRPMI 1640 broth, incubation for 24 h, and an MIC endpoint definedas a prominent reduction in growth ( $≥$ 50% inhibition relativeto control growth) (9). Caspofungin was also tested as a comparatorthat has FDA approval for the treatment of invasive candidiasis.

MATERIALS AND METHODS

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Materials and Methods

Organisms. A total of 315 isolates of Candida spp. from blood or normallysterile body fluid were collected from diverse medical centersworldwide and sent to the University of Iowa for testing. Thecollection included 146 C. krusei, 110 C. glabrata, 41 C. albicans,6 C. parapsilosis, 3 C. tropicalis, 3 C. guillermondii, and2 yeast species isolates and 1 isolate each of C. inconspicua,C. kefyr, C. lipolytica, and C. norvegensis. Isolates were identifiedby standard methods (20) and stored in water vials at ambientroom temperature until used in the study. At the time of testing,the isolates were subcultured twice on potato dextrose agar(Remel, Inc., Lenexa, KS) to achieve exponential growth andto ensure purity.

Antifungal agents. Standard antifungal powders of micafungin (Astellas Pharma,Osaka, Japan), caspofungin (Merck and Co., Whitehouse Station,PA), and fluconazole (Pfizer, Inc., New York, NY) were receivedfrom the respective manufacturers and stored according to instructionsuntil stock solutions were prepared. Stock solutions were preparedin sterile water (micafungin, caspofungin) or dimethyl sulfoxide(fluconazole) and diluted in twofold increments as describedin the CLSI document M27-A2 (7). Final dilutions were preparedin RPMI 1640 medium (Sigma, St. Louis, MO) buffered to pH 7.0with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma). Eachantifungal agent was dispensed (0.1-ml aliquots, 2x final concentration)into 96-well round-bottomed microdilution panels (Sartedt, Inc.,Newton, NC) by using a QuickSpense II system (Dynatech Laboratories,Chantilly, VA). The panels were sealed and stored at –70°Cuntil thawed for use at the time of testing.

Inoculum preparation. Inocula were prepared by a spectrophotometric method as describedin the CLSI M27-A2 guidelines. At least five isolated yeastcolonies from a potato dextrose agar plate were sampled usinga sterile applicator stick and diluted to a concentration of1.0 x 10³ to 5.0 x 10³ cells/ml in RPMI 1640 medium. A 0.1-mlyeast inoculum was added to each well of the thawed panel volumesof a 2x-concentration antifungal agent to bring the final inoculumto 0.5 x 10³ to 2.5 x 10³ cells/ml and the final drug concentrationsto 1x. Final concentrations were 0.007 to 8.0 µg/ml formicafungin and caspofungin and 0.12 to 128.0 µg/ml forfluconazole.

Reading endpoints. The panels were incubated at 35°C, and MICs were determinedfor both 24 h and 48 h. MICs were determined by the visual methodwith the aid of a reading mirror, and the growth in each wellwas compared to that in drug-free growth control wells. TheMIC was defined as the lowest concentration at which a prominentdecrease in growth (approximately 50%) relative to the growthof the control occurred after 24 h of incubation (micafunginand caspofungin) or after 48 h of incubation (fluconazole).Interpretive criteria for fluconazole were those published byRex et al. (16) and the CLSI (7): isolates for which fluconazoleMICs are $≤$ 8.0 µg/ml are susceptible, isolates for whichMICs are 16 to 32 µg/ml are susceptible dose dependent,and isolates for which MICs are $≥$ 64 µg/ml are resistant.The fluconazole breakpoints apply to all Candida species exceptC. krusei, which is considered intrinsically resistant regardlessof the MIC recorded (7). Interpretive criteria for micafunginand caspofungin have not yet been established.

Quality control. Quality control was performed using C. krusei ATCC 6258 andC. parapsilosis ATCC 22019 for each batch of isolates tested,as recommended in CLSI document M27-A2 (1, 7).

RESULTS AND DISCUSSION

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Results and Discussion

All 146 isolates of C. krusei were considered resistant to fluconazole,including 3 isolates for which fluconazole MICs were $≤$ 8.0 µg/ml,35 isolates for which the MICs were 16 to 32 µg/ml, and108 isolates for which the MICs were $≥$ 64 µg/ml. Table 1summarizes the in vitro susceptibilities of 315 fluconazole-resistantCandida spp. Micafungin exhibited good in vitro activity againstall fluconazole-resistant Candida isolates tested; the MICsat which 50% (MIC₅₀) and 90% (MIC₉₀) of the isolates were inhibitedwere 0.03 µg/ml and 0.06 µg/ml, respectively. Allisolates were inhibited at $≤$ 1.0 µg/ml. Among the most commonfluconazole-resistant Candida encountered in the collection,C. glabrata was most susceptible (MIC₉₀ $≤$ 0.015 µg/ml),followed by C. albicans (MIC₉₀, 0.03 µg/ml) and C. krusei(MIC₉₀, 0.06 µg/ml).

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TABLE 1. Activities of micafungin and caspofungin against 315 invasive clinical isolates of Candida spp. that are resistant to fluconazole^a

Caspofungin was included in this study as a comparator thathas already been FDA approved for the treatment of invasivecandidiasis. The excellent in vitro activity of caspofunginagainst these clinically significant azole-resistant speciesconfirms that reported previously (10, 15). Although micafunginappears to be somewhat more active than caspofungin againstC. krusei (MIC₉₀, 0.06 µg/ml versus 0.25 µg/ml,respectively), all isolates of this species were inhibited by0.25 to 0.5 µg/ml of both agents.

These findings support those of Ostrosky-Zeichner et al. (10),who reported similar activity of micafungin against Candidaspp. despite using a more prolonged incubation time of 48 h.Micafungin exhibited potent activity against a large and geographicallydiverse collection of azole-resistant Candida species. The spectrumand potency of micafungin against these clinically importantisolates compare favorably with those of caspofungin, an echinocandinthat is licensed for the treatment of invasive candidiasis.The fungicidal nature of micafungin coupled with the maximumconcentrations of the drug in serum (8 µg/ml after a 75-mgdose [19]), which exceed the MIC₉₀s for all of the azole-resistantisolates, makes it a promising systemic antifungal agent.

ACKNOWLEDGMENTS

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

This study was supported in part by a grant from Astellas.

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

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