Comparative Effects of Micafungin, Caspofungin, and Anidulafunginagainst a Difficult-To-Treat Fungal Opportunistic Pathogen,Candida glabrata

Elisabetta Spreghini,a Fiorenza Orlando,b Maurizio Sanguinetti,c Brunella Posteraro,c Daniele Giannini,d Esther Manso,e

and Francesco Barchiesia

Department of Biomedical Sciences and Public Health, Clinic Infectious Disease, Universita Politecnica delle Marche, Ancona, Italya; Experimental Animal Models for AgingUnits, Scientific Technological Area N. Masera, I.N.R.C.A.-I.R.R.C.S., Ancona, Italyb; Institute of Microbiology, Universita Cattolica del Sacro Cuore, Rome, Italyc; Centro diGestione Presidenza Medicina e Chirurgia, Universita Politecnica delle Marche, Ancona, Italyd; and Laboratory of Microbiology, Azienda Ospedaliero-Universitaria,Ospedali Riuniti Umberto I-Lancisi-Salesi, Ancona, Italye

The aim of this study was to compare the in vitro and in vivo activities of micafungin, caspofungin, and anidulafungin againstCandida glabrata. The MICs against 28 clinical isolates showed that the overall susceptibilities to caspofungin and to micafun-gin were not statistically different in the absence of human serum, whereas the isolates were less susceptible to micafungin thanto caspofungin in its presence. Minimum fungicidal concentrations, as well as time-kill experiments, showed that caspofunginwas more active than anidulafungin, while micafungin was superior to either caspofungin or anidulafungin without serum; itsaddition rendered caspofungin and micafungin equally effective. A murine model of systemic candidiasis against a C. glabrata-susceptible isolate was performed to study the effects of all three echinocandins, and kidney burden counts showed that caspo-fungin, micafungin, and anidulafungin were active starting from 0.25, 1, and 5 mg/kg of body weight/day, respectively. Twoechinocandin-resistant strains of C. glabrata were selected: C. glabrata 30, a laboratory strain harboring the mutation Fks2p-P667T, and C. glabrata 51, a clinical isolate harboring the mutation Fks2p-D666G. Micafungin activity was shown to be as effec-tive as or more effective than that of caspofungin or anidulafungin in terms of MICs. In vivo studies against these resistantstrains showed that micafungin was active starting from 1 mg/kg/day, while caspofungin was effective only when administratedat higher doses of 5 or 10 mg/kg/day. Although a trend toward colony reduction was observed with the highest doses of anidula-fungin, a significant statistical difference was never reached.

Candida glabrata has recently been reported to be the secondmost common cause of invasive candidiasis, and there are

increasing amounts of data showing its important role in deter-mining either superficial or deep-seated infections (4, 18). Sys-temic infections due to C. glabrata are characterized by a highmortality rate, and they are difficult to treat due to reduced sus-ceptibility of the species to azole drugs, especially to fluconazole(26). According to the published guidelines, amphotericin B canbe used to treat infections due to C. glabrata, especially in pro-foundly immunocompromised hosts (22). Fortunately, the spe-cies also appears to be highly susceptible to the echinocandins (i.e.,caspofungin, anidulafungin, and micafungin), making theseagents valuable alternatives as first-line therapy against this Can-dida species (22). Interestingly, patients suffering from systemiccandidiasis due to C. glabrata showed a trend, although not asignificant one, to a better clinical outcome when treated withmicafungin (87%) rather than with caspofungin (67%) (23).

The three echinocandin antifungal agents anidulafungin,caspofungin, and micafungin have a unique mechanism of action,inhibiting �-1,3-D-glucan synthase, an enzyme that is necessaryfor the synthesis of �-1,3-D-glucan polymers, essential compo-nents of the cell walls of several fungi (8).

Although resistance to echinocandins remains relatively low, agrowing number of reports have been published about break-through infections involving high-MIC isolates in patients receiv-ing echinocandin therapy (29, 31).

The aim of this study was to compare the effects of all threeFDA-approved echinocandins against C. glabrata. In particular,

the potential fungicidal activities of these compounds were inves-tigated both in vitro and in a neutropenic murine model of sys-temic infection due to echinocandin-susceptible and -resistantstrains of C. glabrata.

MATERIALS AND METHODS

In vitro studies. (i) Isolates. A total of 30 isolates of C. glabrata werestudied: 21 were isolated from blood samples, while the other 7 wererecovered from the gastrointestinal, respiratory, and urinary tracts. Eachstrain represented a unique isolate from a patient. Additionally, twoechinocandin-resistant strains were evaluated: C. glabrata 51, a clinicalisolate (provided by M. Sanguinetti) that harbors a mutation in FKS2(A1997G; Fks2p-D666G), as well a laboratory strain, C. glabrata 30, whichharbors another mutation in FKS2 (C1999A; Fks2p-P667T). The latterstrain was selected in vitro by plating C. glabrata 11 (caspofungin MIC,0.03 �g/ml) on 20 �g/ml caspofungin-containing yeast extract-peptone-dextrose (YPD) agar plates. Yeast isolates were identified at the specieslevel by conventional morphological and biochemical methods and werestored at �70°C in 10% glycerol. Before the initiation of the study, yeast

Received 14 October 2011 Returned for modification 12 November 2011Accepted 13 December 2011

Published ahead of print 27 December 2011

Address correspondence to Elisabetta Spreghini, e.spreghini@univpm.it.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.05872-11

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isolates were subcultured on antimicrobial-agent-free medium to ensureviability and purity.

(ii) Antifungal drugs. Amphotericin B was used as pure powder(Sigma Chemical, Milan, Italy) for in vitro studies and as a commercialpreparation (Fungizone; Bristol-Myers Squibb S.p.A.) for in vivo studies.Anidulafungin (Pfizer, Inc.), caspofungin (Merck & Co., Inc.), and mica-fungin (Astellas Pharma Inc.) were supplied as pure powders and used forthe in vitro and in vivo experiments.

For in vitro studies, stock solutions of amphotericin B and anidulafun-gin were prepared in dimethyl sulfoxide (DMSO) (Sigma), while caspo-fungin and micafungin were prepared in sterile water. Further dilutionswere prepared in test medium. For in vivo studies, solutions of amphoter-icin B, anidulafungin, caspofungin, and micafungin were prepared fol-lowing the manufacturers’ instructions.

(iii) Broth dilution. Antifungal susceptibility testing was performedby the broth microdilution method in accordance with the Clinical andLaboratory Standards Institute (CLSI) document M27-A3 (7). The finalconcentrations of drugs ranged from 0.002 to 2.0 �g/ml for the suscepti-ble strains and from 0.03 to 16 �g/ml for the resistant strains. Plates wereincubated at 35°C for 24 h (48 h for amphotericin B), and readings wereperformed both visually and spectrophotometrically. Anidulafungin,

caspofungin, and micafungin MICs were considered to be the first con-centrations of the antifungal agent at which the turbidity in the well was50% less than that in the control well. Amphotericin B MICs were con-sidered to be the first concentration of the antifungal agent at which theturbidity in the well was 100% less than that in the control well. Each testwas run in quintuplicate and repeated on 2 different days. MIC values arethe median MIC of 5 replicate determinations on 2 different days.

After MIC readings, the whole suspension from each well above theMIC were withdrawn and plated in duplicate onto 150-mm Sabourauddextrose agar (SDA) plates. The inoculated plates were incubated at 35°C,and minimum fungicidal concentrations (MFCs) were recorded after 48to 72 h. The MFC was defined as the lowest concentration of drug yieldingno growth (27). Each isolate was tested in triplicate.

Antifungal susceptibility testing was performed as described abovewith and without 50% human serum in the medium (21, 33). The humanserum was prepared from the blood of healthy volunteers.

C. tropicalis ATCC 750 was utilized as an additional strain, and C.parapsilosis ATCC 22019 was included on each day of testing as a CLSI-recommended quality control strain.

(iv) Killing curves. Killing experiments were performed against twostrains (C. glabrata 11, and C. glabrata 30). Briefly, three to five colonies of

TABLE 1 In vitro susceptibilities of 28 clinical isolates of C. glabrata to amphotericin B, anidulafungin, caspofungin, and micafungina

Isolate

Median MIC (�g/ml)b Median MFC (�g/ml)c

RPMI RPMI plus 50% serum RPMI RPMI plus 50% serum

AMB ANID CAS MICA AMB ANID CAS MICA AMB ANID CAS MICA AMB ANID CAS MICA

C. tropicalis ATCC 750 0.5 0.06 0.06 0.06 0.5 0.25 0.125 1.0 1.0 0.06 0.06 0.125 0.5 0.5 0.125 1.0C. parapsilosis ATCC

220190.5 0.25 0.25 0.5 0.5 �2.0 1.0 �2.0 1.0 0.5 0.5 0.5 0.5 �2.0 1.0 �2.0

C. glabrata1 1.0 0.25 0.03 0.016 0.5 2.0 0.25 1.0 2.0 2.0 1.0 0.03 2.0 �2.0 0.5 2.02 0.5 0.06 0.008 0.008 0.5 1.0 1.0 0.5 1.0 1.0 �2.0 0.06 1.0 �2.0 2.0 1.03 0.5 0.06 0.03 0.016 0.5 1.0 0.5 0.5 1.0 1.0 0.5 0.125 1.0 �2.0 1.0 1.04 2.0 0.50 0.03 0.03 0.5 2.0 0.25 1.0 1.0 2.0 1.0 0.03 2.0 �2.0 0.5 2.05 1.0 0.06 0.03 0.016 0.5 2.0 0.25 1.0 2.0 2.0 0.5 0.03 2.0 �2.0 0.5 1.06 1.0 0.06 0.06 0.016 0.5 2.0 0.25 1.0 2.0 2.0 1.0 0.06 1.0 �2.0 1.0 2.07 1.0 0.06 0.06 0.016 0.5 1.0 0.25 0.5 2.0 1.0 1.0 0.125 1.0 �2.0 1.0 2.08 1.0 0.03 0.06 0.03 0.5 2.0 0.25 1.0 2.0 �2.0 0.5 0.125 2.0 �2.0 0.5 2.09 2.0 0.06 0.03 0.016 0.5 1.0 0.25 0.5 2.0 2.0 1.0 0.03 1.0 �2.0 2.0 1.010 1.0 0.06 0.06 0.016 0.5 1.0 0.25 0.5 2.0 1.0 1.0 0.06 1.0 �2.0 2.0 1.011 2.0 0.06 0.03 0.03 0.5 0.5 0.25 0.5 2.0 1.0 0.5 0.06 1.0 �2.0 2.0 0.512 1.0 0.125 0.03 0.016 0.25 1.0 0.25 0.5 2.0 1.0 0.5 0.03 0.5 �2.0 0.5 2.013 1.0 0.125 0.016 0.03 0.5 1.0 0.25 0.5 2.0 2.0 1.0 0.06 1.0 �2.0 0.5 1.014 1.0 0.125 0.03 0.03 0.5 1.0 0.25 0.5 2.0 �2.0 1.0 0.06 2.0 �2.0 0.5 1.015 1.0 0.125 0.03 0.03 0.5 1.0 0.5 0.5 2.0 1.0 0.5 0.06 2.0 �2.0 0.5 1.016 1.0 0.125 0.06 0.03 0.5 1.0 0.5 0.5 2.0 2.0 1.0 0.125 1.0 �2.0 1.0 1.017 1.0 0.125 0.03 0.03 1.0 1.0 0.5 0.5 2.0 �2.0 0.125 0.06 1.0 �2.0 0.5 1.018 2.0 0.125 0.03 0.03 2.0 1.0 0.25 1.0 2.0 1.0 0.5 0.06 2.0 �2.0 0.5 1.019 2.0 0.125 0.03 0.03 2.0 1.0 0.25 1.0 2.0 1.0 0.5 0.06 2.0 �2.0 0.5 1.020 2.0 0.06 0.03 0.03 2.0 1.0 0.25 1.0 2.0 2.0 0.5 0.06 2.0 �2.0 0.5 1.021 1.0 0.06 0.06 0.03 1.0 1.0 0.25 0.5 2.0 1.0 0.5 0.06 1.0 �2.0 0.5 1.022 1.0 0.125 0.06 0.03 1.0 1.0 0.25 0.5 2.0 1.0 0.5 0.06 1.0 �2.0 0.5 1.023 1.0 0.06 0.06 0.016 1.0 1.0 0.5 1.0 2.0 �2.0 0.5 0.06 1.0 �2.0 0.5 0.524 1.0 1.0 0.03 0.008 0.5 1.0 0.25 0.5 2.0 1.0 0.06 0.016 1.0 �2.0 0.5 1.026 1.0 0.125 0.06 0.008 1.0 0.5 0.25 0.25 2.0 0.5 1.0 0.016 1.0 �2.0 1.0 0.527 1.0 0.125 0.125 0.03 0.5 1.0 0.5 1.0 2.0 2.0 2.0 0.125 1.0 �2.0 1.0 1.028 0.5 0.125 0.06 0.03 0.5 1.0 0.25 0.5 2.0 1.0 1.0 0.125 1.0 �2.0 1.0 0.529 0.5 0.25 0.125 0.016 0.5 1.0 0.5 0.5 1.0 1.0 2.0 0.06 0.25 �2.0 1.0 0.5

a AMB, amphotericin B; ANID, anidulafungin; CAS, caspofungin; MICA, micafungin.b The AMB MIC was defined as the lowest drug concentration at which there was complete inhibition of growth after 48 h of incubation; the ANID, CAS, and MICA MICs weredefined as the lowest concentration at which there was a visually prominent reduction in growth (approximately 50%) relative to the drug-free growth control after 24 h ofincubation.c The MFC was defined as the lowest concentration of antifungal compound yielding no growth.

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each isolate from a 48-h growth plate were suspended in 10 ml of steriledistilled water, and the turbidity was adjusted using spectrophotometricmethods to 0.5 McFarland standard (approximately 1 � 106 to 5 � 106

CFU/ml). The solution was further diluted at 1:50 and 1:10 with RPMI1640 medium buffered with morpholinepropanesulfonic acid (MOPS),and 250 �l of the adjusted fungal suspension was added to 250 �l of eitherRPMI 1640 medium buffered with MOPS free of drug or the growthmedium plus an appropriate amount of drug. Drugs were used at finalconcentrations of 0.25�, 1�, 4�, 32�, 64�, 128�, and 256� the MIC.Test solutions were placed on a shaker and incubated at 35°C. At 0, 4, and24 h following the introduction of the test isolate into the system, 50-�laliquots were removed from each test solution. After 10-fold serial dilu-tions, 50-�l aliquots of undiluted and diluted samples were streaked ontoSDA plates for colony count determination. Following incubation at 35°Cfor 48 to 72 h, the number of CFU on each plate was determined. Fungi-cidal activity was considered to be achieved when the number of CFU permilliliter was �99.9% of the initial inoculum size (27). The limit of de-tection was 19 CFU. Experiments were performed in triplicate.

FKS gene sequence analysis. Sequencing of partial portions of theFKS1 and FKS2 genes was performed using previously described primers(30). DNA from yeast cultures grown at 37°C overnight in YPD (2% yeastextract, 4% Bacto Peptone, 4% dextrose) broth medium was extractedusing the QIAamp DNA Mini Kit (Qiagen, Milan, Italy) according to themanufacturer’s instructions. PCR products were sequenced, the se-quences of both strands were analyzed using a 3130 Genetic Analyzer(Applied Biosystems, Foster City, CA), and multiple-amino-acid align-ments were derived by using Vector NTI Advance (version 11.5; Invitro-gen Inc., Milan, Italy).

In vivo studies. CD1 female mice (Charles River, Calco, Italy) weigh-ing 25 g were rendered neutropenic by intraperitoneal (i.p.) administra-tion of cyclophosphamide (200 mg/kg of body weight/day) on day �4 anddays 1 and 4 postinfection.

The mice were infected intravenously with 1.35 � 108 CFU/mouseof C. glabrata 11, with 1.6 � 108 CFU/mouse of C. glabrata 30, and with1.2 � 108 CFU/mouse of C. glabrata 51 given in a 0.2-ml volume. Alldrugs were administered i.p. for 6 consecutive days in a 0.2-ml volume,starting 24 h postchallenge. Each echinocandin was given at 0.25, 1, 5,and 10 mg/kg/day, while amphotericin B (the control drug) was givenat 1 mg/kg/day. Drug efficacy was assessed by determining the numberof CFU per kidney pair. Briefly, the mice were sacrificed, the kidneyswere homogenized, and diluted or undiluted aliquots, including theentire organ, were grown on SDA plates for colony count determina-tion. Tissue burden experiments were performed on days 3, 5, and 7postinfection, which corresponded to a total of 2, 4, and 6 days oftherapy, respectively. The susceptibilities of C. glabrata 30 to the echi-nocandins were determined after 7 days of animal treatments. Briefly,colonies of the tissue burden counts obtained either from control ortreatment groups were analyzed, and the MIC values did not show anydifference from the initial strain. There were from 6 to 9 animals ineach group. Animal experiments were conducted with the approval ofthe University of Ancona Ethics Committee.

Statistical analysis. In vitro and in vivo studies were compared amongthe different groups using the Kruskal-Wallis test and Dunn’s test for posthoc comparisons between each treatment group and the control group(Prism 5; GraphPad Software). Two-sided P values of �0.05 were consid-ered statistically significant.

RESULTS

The in vitro susceptibilities of 28 clinical isolates of C. glabrataagainst amphotericin B, anidulafungin, caspofungin, and mica-fungin, either with or without 50% human serum, are reported inTable 1 and Fig. 1. The geometric mean MICs of anidulafungin,caspofungin, and micafungin were 0.10, 0.04, and 0.02 �g/ml,respectively. In the presence of human serum, the geometric meanMICs of anidulafungin, caspofungin, and micafungin increased to

1.08, 0.32, and 0.62 �g/ml, respectively. Multiple-comparisonanalysis of MIC values showed that the overall susceptibilities tocaspofungin and to micafungin were not statistically different inthe absence of human serum, whereas the isolates were less sus-ceptible to micafungin than to caspofungin in its presence (P �0.05). In general, anidulafungin was the less active echinocandinagainst the C. glabrata isolates. The MFCs obtained without serumshowed that caspofungin was more active than anidulafungin,while micafungin was superior to either caspofungin or anidula-fungin. However, the addition of serum rendered caspofunginand micafungin equally effective. Again, both drugs were moreeffective than anidulafungin (Fig. 1).

Then, C. glabrata 11 was selected to compare the fungicidalactivities of all the drugs (Fig. 2). In these experiments, drugs wereutilized at concentrations of 0.25�, 1�, 4�, and 32� the MICwith or without human serum. After 24 h of incubation, ampho-tericin B yielded killing activity at a concentration of 32� theMIC, regardless of the absence or presence of serum. In the ab-sence of serum, micafungin exerted fungicidal activity starting

FIG 1 Geometric means of the MICs (top) and MFCs (bottom) of C. glabrataclinical isolates (n � 28) with or without serum (S). The error bars representthe 95% confidence Intervals. �, P � 0.05 for caspofungin (CAS) versusanidulafungin (ANID); ¶, P � 0.05 for micafungin (MICA) versus CAS; §, P �0.05 for MICA versus ANID.

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from 1� the MIC after 24 h of incubation. At the same timeinterval, caspofungin was fungicidal at doses of 4� and 32� theMIC, while anidulafungin yielded a fungicidal effect at 32� theMIC. In the presence of serum, micafungin, and caspofungin werefungicidal at 4� and 32� MIC, while anidulafungin did not reachfungicidal activity.

C. glabrata 11 was also utilized for in vivo studies. Mice wereinfected with 1.35 � 108 CFU/mouse, and the drug activities werestudied on days 3, 5, and 7 postinfection. The results for kidneytissue burden obtained with this strain are reported in Fig. 3. After2 days of treatment, caspofungin was active starting at a dose of 1mg/kg/day, while micafungin was active at doses of 5 and 10 mg/kg/day. On day 5 postinfection, all doses of caspofungin were ef-fective at reducing the burden. Micafungin was active starting at adose of 1 mg/kg/day. Although, anidulafungin showed a trend inreduction, the agent did not significantly decrease the fungal bur-den with respect to the control after 2 and 4 days of treatment atany tested doses. On day 7 postinfection, all three echinocandinsat 5 and 10 mg/kg/day were effective at reducing the counts againstthe controls. At 1 mg/kg/day, either caspofungin or micafungin,but not anidulafungin, was active. At the lowest dose tested (0.25mg/kg/day), only caspofungin was active.

In order to investigate the effects of anidulafungin, caspofun-gin, and micafungin against echinocandin-resistant isolates of C.glabrata, two additional strains were selected for further in vitroand in vivo studies. C. glabrata 30 is a laboratory strain harboringa mutation in FKS2 (C1999A; Fks2p-P667T), while C. glabrata 51is a clinical isolate harboring another FKS2 mutation (A1997G;Fks2p-D666G). In vitro susceptibility tests of these two strains areshown in Table 2. Time-kill studies were performed against C.glabrata 30, and the results are shown in Fig. 4. In these experi-ments, drugs were utilized at concentrations of 0.25�, 1�, 4�,32�, 64�, 128�, and 256� the MIC with or without humanserum. Amphotericin B yielded killing activity against this isolateafter 24 h of incubation at a concentration of 32� the MIC either

with or without serum. Anidulafungin, caspofungin, and mica-fungin exerted fungicidal activity at 128� the MIC in the absenceof serum after 24 h of incubation; in the presence of serum, allthree echinocandins were not active against this isolate of C.glabrata.

Both resistant strains of C. glabrata were utilized to comparethe efficacies of the echinocandins in vivo, and the results areshown in Fig. 5. Against C. glabrata 30, micafungin was effective atdoses of 1, 5, and 10 mg/kg/day and caspofungin was active at 5and 10 mg/kg/day. Similarly, against C. glabrata 51, micafunginwas effective at doses of 1, 5, and 10 mg/kg/day, while caspofunginwas active only at a dose of 10 mg/kg/day. Although for bothstrains anidulafungin at 5 and 10 mg/kg/day showed a trend to-ward reduction of CFU with respect to the controls, a statisticallysignificant difference was never reached by using the multiple-comparison analyses.

DISCUSSION

Our findings showed that all 28 clinical isolates recovered frompatients hospitalized in our department presented anidulafungin,caspofungin, and micafungin MICs within the previously re-ported ranges for wild-type strains of C. glabrata (3, 5, 24, 25).Also, our results showed that the MICs were within the suscepti-bility ranges for all three echinocandins, with the exception of twoisolates showing an intermediate MIC value for anidulafungin(0.25 �g/ml). In fact, the recently proposed CLSI clinical interpre-tive MIC breakpoints (CBPs) for anidulafungin and caspofunginagainst C. glabrata are �0.12 �g/ml for susceptible isolates (S),0.25 �g/ml for intermediate isolates (I), and �0.5 �g/ml for re-sistant isolates (R), while the CPBs for micafungin are �0.06�g/ml for S isolates, 0.12 �g/ml for I isolates, and �0.25 �g/ml forR isolates (24).

It has been reported that echinocandins exert fungicidal activ-ity against yeasts (10, 11). Therefore, we investigated this charac-teristic by determining either the MFCs or the killing curves. In

FIG 2 Time-kill plots for amphotericin B, anidulafungin, caspofungin, and micafungin against C. glabrata isolate 11. Each data point represents the meanresult � SEM (standard error of the mean). The dotted lines represent a 99.9% growth reduction compared with that of the initial inoculum (fungicidal effect).�, control; �, 0.25� MIC; o, 1� MIC; p, 4� MIC; �, 32� MIC.

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general, our MFCs were within the reported ranges for all threeechinocandins (11), with a rank order of activity of micafungin �caspofungin � anidulafungin.

Interestingly, a similar rank order was maintained when the

“cidal” activity was investigated by killing experiments. In general,all three echinocandins exerted fungicidal activities against thesusceptible isolate of C. glabrata. Our results are in agreement withthose previously reported for caspofungin by Nagappan et al. (19).These authors assessed the in vitro activity of caspofungin againstfluconazole-susceptible and -resistant isolates and observed thatthe drug was fungicidal at concentrations of 1 �g/ml and 4 �g/ml.A previous study reported that micafungin was fungicidal at con-centrations ranging from 4 to 16 times the MIC80 against C.glabrata isolates with MIC80s ranging from 0.0039 to 0.25 �g/ml(9). Similar to this study, our data showed that micafungin exertedfungicidal activity starting from 1 to 32 times the MIC (0.06/2.0�g/ml). In our hands, anidulafungin was fungicidal at concentra-tions of 32 times the MIC against the susceptible isolate, whereasprevious data reported “cidal” activity starting from 4 times theMIC (16, 20). It can be hypothesized that this difference might bedue to a slight modification of the experimental procedure (i.e.,drug preparation, drug lot, subcultured volumes, “cidal” defini-tion, etc).

It is known that echinocandins bind serum proteins at veryhigh levels (i.e., �99% to human plasma proteins for anidulafun-gin and approximately 97% to albumin for caspofungin) (15, 21).

FIG 3 Kidney tissue burdens of neutropenic CD1 mice infected intravenously with C. glabrata 11. The mice were treated daily with amphotericin B at 1mg/kg/day (�) and anidulafungin (o), caspofungin (�), or micafungin (Œ) at doses of 0.25, 1, 5, and 10 mg/kg/day. C, control. Tissue burden experiments wereperformed on days 3, 5, and 7 postinfection. There were from 6 to 9 animals in each group. The horizontal lines represent the median fungal burdens. *, P � 0.05versus the results for the control.

TABLE 2 In vitro susceptibility to amphotericin B, anidulafungin,caspofungin, and micafungin of two echinocandin-resistant strains ofC. glabrata

C. glabrataisolatea

Median MIC (�g/ml)b

RPMI RPMI plus 50% serum

AMB ANID CAS MICA AMB ANID CAS MICA

30 1.0 0.5 2.0 0.5 1.0 1.0 0.5 0.551 1.0 1.0 2.0 0.25 �16 2.0 2.0 0.5a C. glabrata 30 is a laboratory strain selected in vitro by plating the isolate C. glabrata11 on 20 �g/ml caspofungin-containing YPD agar plates (Fks2p-P667T); C. glabrata 51is a clinical isolate bearing a mutation in the FKS2 gene (Fks2p-D666G).b AMB, amphotericin B; ANID, anidulafungin; CAS, caspofungin; MICA, micafungin.TheAMB MIC was defined as the lowest drug concentration at which there was completeinhibition of growth after 48 h of incubation; the ANID, CAS, and MICA MICs weredefined as the lowest concentration at which there was a visually prominent reduction ingrowth (approximately 50%) relative to the drug-free growth control after 24 h ofincubation.

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Odabasi et al. (17) evaluated the effects of protein binding on theactivities of caspofungin, anidulafungin, and micafungin againstCandida and Aspergillus species. They observed that adding hu-man serum sharply increased the MICs of micafungin and anidu-lafungin and modestly affected the MIC of caspofungin. However,they also found that the increase in MICs does not appear to beconsistent with the rates of protein binding for the three com-pounds. Therefore, we performed in vitro studies by adding 50%human serum to RPMI 1640. Similar to what was observed byothers (15, 17, 21), the addition of serum to the medium increasedthe MICs of all three drugs. We also performed the experiments byadding 50% fetal bovine serum to the medium, and we obtainedsimilar results (data not shown).

In our hands, the ratios of geometric mean MICs (MIC valueswith/without serum) were 10.8, 8.0, and 31 for anidulafungin,caspofungin, and micafungin, respectively, while the ratios of geo-metric mean MFCs (MFCs values with/without serum) were 0.9and 17 for caspofungin and micafungin, respectively (the ratio foranidulafungin was not determined because the tested concentra-tions were too low with respect to the fungicidal range).

In general, our results are in agreement with previous in vitrostudies showing an increased echinocandin MIC when 50% serumor bovine serum albumin was added to RPMI 1640 (3, 12, 17).

Killing experiments conducted in the presence of serumshowed that both caspofungin and micafungin started to be fun-gicidal at 4 times the MIC and that the addition of serum did notmodify the fungicidal activity of caspofungin while it decreasedthat of micafungin. These results are in line with the higher serumbinding levels of micafungin compared to caspofungin (21).Time-kill plots of anidulafungin in the presence of serum neverreached the fungicidal effect, and additional studies should beperformed by using various drug lots and by increasing the anti-fungal agent concentration.

Since in vitro/in vivo correlation is not yet understood, we com-pared the in vivo activities of all three echinocandins in a neutro-penic murine model of candidiasis. In our hands, caspofungin

proved to be the most active drug (in terms of either time or doseeffectiveness) against this susceptible isolate. In fact, caspofunginstarted to be effective after only 2 days of treatment, while after 6days, the lowest effective doses were 0.25, 1, and 5 mg/kg/day forcaspofungin, micafungin, and anidulafungin, respectively.

Recently, Andes et al. (1, 2) investigated the in vivo activitiesof all three echinocandins against Candida spp., including C.glabrata, in a neutropenic murine model of disseminated candi-diasis. To compare the potencies of antifungal agents, they calcu-lated the 24-h static dose of each echinocandin and the doses re-quired to achieve a 1-log-unit reduction in colony counts (1).They observed that caspofungin required less drug on a mg/kgbasis for efficacy against all organisms than did the other twodrugs. Actually, the mean static doses were 21.1, 2.47, and 0.33mg/kg/24 h for anidulafungin, micafungin, and caspofungin, re-spectively, while mean doses to achieve 1-log-unit reduction were39, 5.88, and 1.16 mg/kg/24 h for anidulafungin, micafungin, andcaspofungin, respectively. In agreement with our results against C.glabrata, the echinocandins showed the following rank order ofactivity: caspofungin � micafungin � anidulafungin (1).

Our in vivo data on anidulafungin are similar to those observedby Gumbo et al. (14). These authors studied the activity of thedrug in a neutropenic murine model of disseminated candidiasisdue to a fluconazole-susceptible C. glabrata isolate. They foundthat doses of 8 and 10 mg/kg resulted in progressive declines inkidney fungal density, while data for mice that received 2 and 3mg/kg did not differ significantly from the controls.

When therapeutic options are limited (i.e., azole resistance,renal insufficiency, drug intolerance, etc.), an important clinicalquestion is whether an infection due to a yeast isolate with reducedsusceptibility to a given echinocandin might be treated by an in-creased dose of the same drug or by selecting a new drug belongingto the same family. Recently, Brzankalski et al. (6) showed thatcaspofungin dose escalation may overcome the in vitro resistanceof C. glabrata and be effective in vivo against resistant isolates.Additionally, the same authors suggested that aminocandin, an

FIG 4 Time-kill plots for amphotericin B, anidulafungin, caspofungin, and micafungin against C. glabrata isolate 30. Each data point represents the meanresult � SEM. The dotted lines represent a 99.9% growth reduction compared with that of the initial inoculum (fungicidal effect). �, control; �, 0.25� MIC;o, 1� MIC; p, 4� MIC; �, 32� MIC; �, 64� MIC; *, 128� MIC; 196 , 256� MIC.

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investigational echinocandin, has some potential in the treatmentof C. glabrata infections due to caspofungin-susceptible isolatesand that higher doses may be required against isolates with re-duced susceptibility to caspofungin (6). Also, Garcia-Effron et al.(13) performed a genetic analysis of FKS1 and FKS2 genes from 13echinocandin-resistant C. glabrata isolates. They demonstratedthat FKS mutations influenced the �-1,3-D-glucan synthase kinet-ics and the FKS gene expression and that the mutations werelinked to an echinocandin reduced-susceptibility phenotype. Inthe current study, we investigated the in vivo effects of the availableechinocandins against two echinocandin-resistant C. glabrata iso-lates, one harboring the mutation Fks2p-P667T and the other themutation Fks2p-D666G.

In our in vivo experiments, a fungicidal effect (i.e., organ ster-ilization) was never observed regardless of the drug or straintested, the dosages, or the duration of therapy. In general, all threeechinocandins at the active doses showed lower killing ratesagainst the resistant strains than the susceptible strain.

Interestingly, we observed that micafungin retained its efficacyagainst both fks2 mutant strains, being effective at doses as low as1 mg/kg/day. In C. albicans, Slater et al. (28) investigated the ef-fects of three doses of micafungin (5 h, 29 h, and 53 h postinfec-tion) in a murine model of disseminated candidiasis due to C.albicans fks1 heterozygous and homozygous mutants at Ser645.They observed that fungicidal activity in animals infected with anFKS1/fks1 heterozygote was reached only with doses as high as 20mg/kg, while animals infected with the homozygous fks1 mutantfailed to respond to any dosage.

In our study, we also demonstrated that caspofungin dose es-calation may overcome in vitro resistance. In fact, caspofungin wasstill active at 5 or 10 mg/kg against the two resistant strains. Anidu-lafungin showed a trend toward reduction of CFU with respect tothe controls at 5 and 10 mg/kg/day, but statistically significantdifferences were never reached. Our results are partially compara-ble to those reported by Wiederhold et al. (33). They comparedcaspofungin and anidulafungin in vitro and in vivo against twoclinical isolates of C. glabrata with caspofungin MICs of �1 �g/mland found that, despite enhanced in vitro potency of anidulafun-gin, treatment with the echinocandin did not result in reductionsin tissue burdens greater than those achieved by treatment withcaspofungin.

Recently, Wiederhold et al. (32) demonstrated that higherdoses of caspofungin (5 and 10 mg/kg) did improve survivalagainst an fks1p-S645P C. albicans-resistant isolate, but notagainst another isolate bearing the mutation fks1p-F641S. Theauthors concluded that the caspofungin effect against resistant C.albicans isolates may be associated with the virulence of the strain.Overall, these results suggest that there might be a linkage betweenthe increased echinocandin MICs, the specific FKS mutations, andthe potential for a successful clinical outcome.

In conclusion, we compared in vitro and in vivo effects ofanidulafungin, caspofungin, and micafungin against the difficult-to-treat fungal opportunistic pathogen C. glabrata. While all threedrugs were often fungicidal in vitro, they were not able to com-pletely eradicate the infection in this murine neutropenic model ofcandidiasis. Caspofungin, followed by micafungin, was the mostactive drug at reducing the kidney burden of mice infected withan echinocandin-susceptible strain. Interestingly, micafunginshowed the best in vivo antifungal activity against two resistantmutants of C. glabrata bearing specific mutations in the FKS2 hotspot region. A limitation of this in vivo study is that the mutantisolates showed a low level of resistance to echinocandins, andextrapolations to other mutants with more dominant mutationscannot be made. Further studies with a larger number of strainsshowing various levels of echinocandin resistance, as well as sev-eral schemes for therapies, should be done to better predict treat-ment success in clinical practice.

ACKNOWLEDGMENTS

We thank Pfizer for providing pure anidulafungin, Merck for providingpure caspofungin, and Astellas for providing pure micafungin.

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FIG 5 Kidney tissue burdens of neutropenic CD1 mice infected intravenouslywith C. glabrata 30 and C. glabrata 51. The mice were treated daily with anidu-lafungin (o), caspofungin (�), or micafungin (Œ) at doses of 0.25, 1, 5, and 10mg/kg/day. C, control. Tissue burden experiments were performed on day 7postinfection. There were 8 or 9 animals in each group. The horizontal linesrepresent the median fungal burdens. *, P � 0.05 versus the results for thecontrol.

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