Experience with anidulafungin against Candida krusei is limited. Immunosuppressed mice were injected with 1.3 x 10(7) to 1.5 x 10(7) CFU of C. krusei. Animals were treated with saline, 40 mg/kg fluconazole, 1 mg/kg amphotericin B, or 10 and 20 mg/kg anidulafungin for 5 days. Anidulafungin improved survival and significantly reduced the number of CFU/g in kidneys and serum beta-glucan levels

Chen, Enuo

Ostrosky-Zeichner, Luis

Paetznick, Victor L

Rodriguez, Jose

Sheehan, Daniel J

Name not available

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.

DigitalCommons@The Texas Medical Center

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2009, p. 1639–1641 Vol. 53, No. 40066-4804/09/$08.000 doi:10.1128/AAC.00955-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.Activity of Anidulafungin in a Murine Model of Candida krusei Infection:Evaluation of Mortality and Disease Burden by Quantitative TissueCultures and Measurement of Serum (1,3)--D-Glucan LevelsLuis Ostrosky-Zeichner,1* Victor L. Paetznick,1 Jose Rodriguez,1 Enuo Chen,1 and Daniel J. Sheehan2Laboratory of Mycology Research, University of Texas Medical School at Houston, Houston, Texas,1 andPfizer, Inc., New York, New York2Received 18 July 2008/Returned for modification 17 October 2008/Accepted 6 January 2009Experience with anidulafungin against Candida krusei is limited. Immunosuppressed mice were injected with1.3  107 to 1.5  107 CFU of C. krusei. Animals were treated with saline, 40 mg/kg fluconazole, 1 mg/kgamphotericin B, or 10 and 20 mg/kg anidulafungin for 5 days. Anidulafungin improved survival and signifi-cantly reduced the number of CFU/g in kidneys and serum -glucan levels.Non-albicans Candida species are increasing in incidence ascauses of invasive candidiasis in the United States and world-wide (11). This is particularly important as Candida krusei hasintrinsic resistance to azoles, and there are reports of reducedsusceptibility to amphotericin B (AMB) (9, 16, 17). Recentresearch has shown that even minor delays in appropriateantifungal therapy are associated with increased morbidity andmortality and that it is difficult to predict non-albicans Candidaspecies infection reliably (2, 3).Echinocandins are novel antifungals that have activity invitro activity against C. krusei and other non-albicans Candidaspecies (19, 21). Proof in an animal model that C. krusei can betreated with anidulafungin (AFG) would be a valuable contri-bution to the understanding of the drug, especially since clin-ical trials have limited numbers of patients with infection bythese species. Our objective was to measure the efficacy ofAFG in terms of survival and organism burden as measured byquantitative tissue cultures and serum (1,3)--D-glucan (BG)levels in a murine model of C. krusei infection. BG levels areemerging as possible surrogate marker to evaluate the re-sponse to treatment of fungal infections (12).(This study was presented as abstract M-1840 at the 47thInterscience Conference on Antimicrobial Agents and Chemo-therapy, Chicago, IL, 2007 [14].)Animals. Male CF-1 mice (Harlan Sprague-Dawley) weigh-ing 20 to 30 g were used in this study. This study was reviewedand approved by the University of Texas Health Science Cen-ter Animal Welfare Committee.Isolate and inoculum. C. krusei strain LMR 39-14, a blood-stream isolate from a clinical isolate collection, was used for allexperiments. The isolate species was confirmed by the API 32method (Biomerieux), and antifungal susceptibility testing wascarried out by the CLSI M27-A3 microdilution method (1).The MICs for fluconazole (FLU), AMB, and AFG were 64,1.0, and 0.06 g/ml, respectively. Animals were inoculated byintravenous tail injection with 1.3  107 to 1.5  107 CFU peranimal. Inocula were confirmed by serial plating after eachexperiment.Immunosuppression. Animals were immunosuppressed by asingle dose of 200 mg/kg of body weight fluorouracil (5-FU)intraperitoneally (i.p.) 3 days prior to inoculation and a singledose of 4 mg/kg of body weight dexamethasone subcutaneouslyon the day of inoculation.Experimental interventions. Twenty-five animals per groupwere assigned to the following treatment regimens: (i) i.p.normal saline every 24 h (q24h) (placebo) for 5 days, (ii) 10mg/kg i.p. AFG q24h for 5 days, (iii) 20 mg/kg i.p. AFG q24hfor 5 days, (iv) 40 mg/kg i.p. FLU q24h for 5 days, and (v) 1mg/kg i.p. AMB q24h for 5 days. AFG doses were based onprevious pharmacodynamic studies (4, 20). The experimentwas run in duplicate.Evaluations. Fifteen animals from each group were ob-served for survival. Five animals from each group were sacri-ficed on day 5 postinoculation and five animals on day 10postinoculation to harvest the kidneys and blood. Quantitativetissue cultures were performed by homogenization of bothkidneys, serial dilutions, and plating on Sabouraud dextroseagar. Plates were incubated for 48 h, and the numbers of CFUper gram of tissue were recorded and calculated. The level ofBG in serum was measured using the Fungitell kit followingthe manufacturer’s instructions (Fungitell package insert,2007; Associates of Cape Cod, Falmouth, MA). The detectionthresholds were 40 CFU/g of tissue for quantitative culturesand 31 pg/ml for BG (according to the Fungitell package in-sert).Statistical analysis. As results were similar between the du-plicate runs, data were pooled for the final analysis. Survivalcurves were estimated using the Kaplan-Meier product limitmethod and compared using the log-rank test. Log-trans-formed CFU counts and BG levels were compared by analysisof variance and the Kruskal-Wallis method, respectively. Find-ings were considered statistically significant if P was 0.05.As shown in Fig. 1, survival was statistically improved forboth AFG groups compared to the saline, FLU, or AMBgroups. There were no significant differences among the twoAFG doses, and likewise there were no significant differences* Corresponding author. Mailing address: Division of InfectiousDiseases, University of Texas Medical School at Houston, 6431 Fan-nin, MSB 2.112, Houston, TX 77030. Phone: (713) 500-6733. Fax:(713) 500-5495. E-mail: Luis.Ostrosky-Zeichner@uth.tmc.edu. Published ahead of print on 12 January 2009.1639between FLU and saline. AMB significantly prolonged survivalcompared to saline and FLU.As shown in Table 1, both doses of AFG produced a statis-tically significant 2-log reduction of kidney CFU/g comparedto saline and FLU on day 5 and an4-log reduction by day 10.No statistically significant reductions were seen for FLU orAMB compared to saline, and no differences were seen be-tween the two doses of AFG either. BG levels significantlydecreased with both AFG doses compared to saline, FLU, andAMB on days 5 and 10. There were no significant differencesbetween the two doses or between the levels found for eachdose on day 5 and day 10.AFG showed antifungal activity in this murine model of C.krusei infection, as evidenced by improved survival and re-duced organism burden by quantitative tissue cultures andserum BG.AFG shows good in vitro activity against C. krusei. ARTEMIS,a global surveillance program (17), reported a MIC90 of 0.06g/ml for 121 isolates of this species. As stated in the intro-duction, there are no reports of animal models exploring theefficacy of this agent in disease caused by this particular spe-cies. Clinical experience has anecdotally shown variable re-sponse rates of C. krusei to AFG. In the first open-label expe-rience with AFG and candidemia, Krause et al. (7) reportedfive cases of disease caused by C. krusei infection, one of whichwas considered a microbiological failure at the end of therapy.The largest randomized trial of AFG for invasive candidiasissystematically excluded patients with infection by C. kruseisince the comparator was FLU (18). Development of resis-tance while under treatment appears to be rare. Marr et al.FIG. 1. Kaplan-Meier plot of survival estimates for immunocompromised CF-1 mice infected with 1.3  107 to 1.5  107 CFU of C. krusei byintravenous 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 statisticallyimproved survival (P  0.05) compared to placebo, AMB, or FLU. AMB statistically improved survival (P  0.05) compared to placebo and FLU.TABLE 1. Tissue burden in kidneys and BG levels in serumTreatmentgroupaDay 5b Day 10bMean 	 SDtissue burden(log10 CFU/g)Mean 	 SDserum BGlevel (pg/ml)Mean 	 SDtissue burden(log10 CFU/g)Mean 	 SDserum BGlevel (pg/ml)Uninfected ND 52 	 30 ND 44 	 72Saline 6.66 	 0.30 1,942 	 574 5.22 	 2.17 1,670 	 962FLU 7.01 	 0.56 2,714 	 2,636 4.77 	 1.74 1,292 	 734AMB 5.95 	 0.47 2,559 	 2,872 4.96 	 0.66 1,742 	 1,108AFG10 4.06 	 0.38*†‡ 256 	 144*†‡ 0.96 	 1.54*†‡ 624 	 497*†‡AFG20 3.97 	 0.34*†‡ 351 	 264*†‡ 1.59 	 1.68*†‡ 693 	 816*‡a The treatment regimens were as follows: Saline, normal saline solution q24h(placebo) for 5 days; FLU, 40 mg/kg i.p. FLU q24h for 5 days; AMB, 1 mg/kg i.p.AMB q24h for 5 days; AFG10, 10 mg/kg i.p. AFG q24h for 5 days; and AFG20,20 mg/kg i.p. q24h for 5 days.b Log10-transformed CFU were compared by analysis of variance, and BGlevels were compared by the Kruskal-Wallis method. ND, not determined. , P0.05 compared to saline; †, P  0.05 compared to FLU; ‡, P  0.05 compared tosaline.1640 OSTROSKY-ZEICHNER ET AL. ANTIMICROB. AGENTS CHEMOTHER.(5, 6) reported a C. krusei strain from a patient with leukemiathat displayed reduced susceptibility to echinocandin drugs:the strain contained a heterozygous mutation of FKS1. Otherechinocandins have consistently shown good activity againstC. krusei in a limited 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 evidenced byimproved survival and reduced organism burden compared toplacebo and other antifungals. Further clinical experience iswarranted.This study was funded by an Independent Investigator Researchgrant from Pfizer, Inc.REFERENCES1. Clinical Laboratory Standards Institute. 2008. 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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

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.

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