Activity of Anidulafungin in a Murine Model of Candida krusei Infection: Evaluation of Mortality and Disease Burden by Quantitative Tissue Cultures and Measurement of Serum (1,3)-{beta}-D-Glucan Levels

Experience with anidulafungin against Candida krusei is limited.Immunosuppressed mice were injected with 1.3 x 10⁷ to 1.5 x10⁷ CFU of C. krusei. Animals were treated with saline, 40 mg/kgfluconazole, 1 mg/kg amphotericin B, or 10 and 20 mg/kg anidulafunginfor 5 days. Anidulafungin improved survival and significantlyreduced the number of CFU/g in kidneys and serum β-glucanlevels.

INTRODUCTION

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INTRODUCTION

Non-albicans Candida species are increasing in incidence ascauses of invasive candidiasis in the United States and worldwide(11). This is particularly important as Candida krusei has intrinsicresistance to azoles, and there are reports of reduced susceptibilityto amphotericin B (AMB) (9, 16, 17). Recent research has shownthat even minor delays in appropriate antifungal therapy areassociated with increased morbidity and mortality and that itis difficult to predict non-albicans Candida species infectionreliably (2, 3).

Echinocandins are novel antifungals that have activity in vitroactivity against C. krusei and other non-albicans Candida species(19, 21). Proof in an animal model that C. krusei can be treatedwith anidulafungin (AFG) would be a valuable contribution tothe understanding of the drug, especially since clinical trialshave limited numbers of patients with infection by these species.Our objective was to measure the efficacy of AFG in terms ofsurvival and organism burden as measured by quantitative tissuecultures and serum (1,3)-β-D-glucan (BG) levels in a murinemodel of C. krusei infection. BG levels are emerging as possiblesurrogate marker to evaluate the response to treatment of fungalinfections (12).

(This study was presented as abstract M-1840 at the 47th InterscienceConference on Antimicrobial Agents and Chemotherapy, Chicago,IL, 2007 [14].)

Animals.

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Animals.

Male CF-1 mice (Harlan Sprague-Dawley) weighing 20 to 30 g wereused in this study. This study was reviewed and approved bythe University of Texas Health Science Center Animal WelfareCommittee.

Isolate and inoculum.

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Isolate and inoculum.

C. krusei strain LMR 39-14, a bloodstream isolate from a clinicalisolate collection, was used for all experiments. The isolatespecies was confirmed by the API 32 method (Biomerieux), andantifungal susceptibility testing was carried out by the CLSIM27-A3 microdilution method (1). The MICs for fluconazole (FLU),AMB, and AFG were >64, 1.0, and 0.06 µg/ml, respectively.Animals were inoculated by intravenous tail injection with 1.3x 10⁷ to 1.5 x 107 CFU per animal. Inocula were confirmed byserial plating after each experiment.

Immunosuppression.

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Immunosuppression.

Animals were immunosuppressed by a single dose of 200 mg/kgof body weight fluorouracil (5-FU) intraperitoneally (i.p.)3 days prior to inoculation and a single dose of 4 mg/kg ofbody weight dexamethasone subcutaneously on the day of inoculation.

Experimental interventions.

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Experimental interventions.

Twenty-five animals per group were assigned to the followingtreatment regimens: (i) i.p. normal saline every 24 h (q24h)(placebo) for 5 days, (ii) 10 mg/kg i.p. AFG q24h for 5 days,(iii) 20 mg/kg i.p. AFG q24h for 5 days, (iv) 40 mg/kg i.p.FLU q24h for 5 days, and (v) 1 mg/kg i.p. AMB q24h for 5 days.AFG doses were based on previous pharmacodynamic studies (4,20). The experiment was run in duplicate.

Evaluations.

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Evaluations.

Fifteen animals from each group were observed for survival.Five animals from each group were sacrificed on day 5 postinoculationand five animals on day 10 postinoculation to harvest the kidneysand blood. Quantitative tissue cultures were performed by homogenizationof both kidneys, serial dilutions, and plating on Sabourauddextrose agar. Plates were incubated for 48 h, and the numbersof CFU per gram of tissue were recorded and calculated. Thelevel of BG in serum was measured using the Fungitell kit followingthe manufacturer's instructions (Fungitell package insert, 2007;Associates of Cape Cod, Falmouth, MA). The detection thresholdswere 40 CFU/g of tissue for quantitative cultures and 31 pg/mlfor BG (according to the Fungitell package insert).

Statistical analysis.

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Statistical analysis.

As results were similar between the duplicate runs, data werepooled for the final analysis. Survival curves were estimatedusing the Kaplan-Meier product limit method and compared usingthe log-rank test. Log-transformed CFU counts and BG levelswere compared by analysis of variance and the Kruskal-Wallismethod, respectively. Findings were considered statisticallysignificant if P was <0.05.

As shown in Fig. 1, survival was statistically improved forboth AFG groups compared to the saline, FLU, or AMB groups.There were no significant differences among the two AFG doses,and likewise there were no significant differences between FLUand saline. AMB significantly prolonged survival compared tosaline and FLU.

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FIG. 1. Kaplan-Meier plot of survival estimates for immunocompromised CF-1 mice infected with 1.3 x 10⁷ to 1.5 x 10⁷ CFU of C. krusei by intravenous tail injection. Treatment group abbreviations (n = 30): Saline, i.p. normal aline solution; Anidula 10 MKD, 10 mg/kg/day i.p. AFG; Anidula 20 MKD, 20 mg/kg/day i.p. AFG; Flu 40 MKD, 40 mg/kg/day i.p. FLU; AMB 1 MKD, 1 mg/kg/day i.p. AMB. Both AFG doses statistically improved survival (P < 0.05) compared to placebo, AMB, or FLU. AMB statistically improved survival (P < 0.05) compared to placebo and FLU.

As shown in Table 1, both doses of AFG produced a statisticallysignificant $~$ 2-log reduction of kidney CFU/g compared to salineand FLU on day 5 and an $~$ 4-log reduction by day 10. No statisticallysignificant reductions were seen for FLU or AMB compared tosaline, and no differences were seen between the two doses ofAFG either. BG levels significantly decreased with both AFGdoses compared to saline, FLU, and AMB on days 5 and 10. Therewere no significant differences between the two doses or betweenthe levels found for each dose on day 5 and day 10.

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TABLE 1. Tissue burden in kidneys and BG levels in serum

AFG showed antifungal activity in this murine model of C. kruseiinfection, as evidenced by improved survival and reduced organismburden by quantitative tissue cultures and serum BG.

AFG shows good in vitro activity against C. krusei. ARTEMIS,a global surveillance program (17), reported a MIC₉₀ of 0.06µg/ml for 121 isolates of this species. As stated in theintroduction, there are no reports of animal models exploringthe efficacy of this agent in disease caused by this particularspecies. Clinical experience has anecdotally shown variableresponse rates of C. krusei to AFG. In the first open-labelexperience with AFG and candidemia, Krause et al. (7) reportedfive cases of disease caused by C. krusei infection, one ofwhich was considered a microbiological failure at the end oftherapy. The largest randomized trial of AFG for invasive candidiasissystematically excluded patients with infection by C. kruseisince the comparator was FLU (18). Development of resistancewhile under treatment appears to be rare. Marr et al. (5, 6)reported a C. krusei strain from a patient with leukemia thatdisplayed reduced susceptibility to echinocandin drugs: thestrain contained a heterozygous mutation of FKS1. Other echinocandinshave consistently shown good activity against C. krusei in alimited number of patients in clinical trials (8, 10, 13, 15).

This study provides information regarding the in vivo activityof AFG against C. krusei. AFG seemed effective in controllingthe infection in this murine model of disease, as evidencedby improved survival and reduced organism burden compared toplacebo and other antifungals. Further clinical experience iswarranted.

ACKNOWLEDGMENTS

This study was funded by an Independent Investigator Researchgrant from Pfizer, Inc.

FOOTNOTES

* Corresponding author. Mailing address: Division of Infectious Diseases, University of Texas Medical School at Houston, 6431 Fannin, MSB 2.112, Houston, TX 77030. Phone: (713) 500-6733. Fax: (713) 500-5495. E-mail: Luis.Ostrosky-Zeichner{at}uth.tmc.edu

Published ahead of print on 12 January 2009.

REFERENCES

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