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In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Candida spp. as determined by CLSI broth microdilution method
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Diagnostic Microbiology and Infectious Disease 71 (2011) 167 – 170 www.elsevier.com/locate/diagmicrobio
In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Candida spp. as determined by CLSI broth microdilution method☆ Michael A. Pfallera , Katsura Hatab , Ronald N. Jonesa,c , Shawn A. Messera , Gary J. Moeta , Mariana Castanheiraa,⁎ a
JMI Laboratories, North Liberty, IA 52317, USA b Eisai, Tsukuba 300-2635, Japan c Tufts University School of Medicine, Boston, MA 02111, USA Received 26 April 2011; accepted 2 May 2011
Abstract The in vitro activity of the novel antifungal agent E1210 and four comparators (caspofungin, fluconazole, posaconazole, and voriconazole) was determined against 90 clinical isolates of Candida using Clinical and Laboratory Standards Institute methods. The collection was composed of 21 Candida albicans, 20 C. glabrata, 25 C. parapsilosis, and 24 C. tropicals, and also included 21 fluconazole-resistant and 15 caspofungin-resistant strains. E1210 was highly active against all the species tested and was more potent than all comparators. The MIC90 results (μg/mL) for E1210, caspofungin, fluconazole, posaconazole, and voriconazole, respectively, were as follows by species: C. albicans (0.06, 4, ≥64, 0.5, 0.5), C. glabrata (0.06, 2, 32, 1, 1), C. parapsilosis (0.06, 4, 16, 0.12, 0.25), and C. tropicalis (0.06, 4, ≥64, 0.5, 2). E1210 was also the most active agent against fluconazole-resistant strains of C. albicans (MIC range, 0.015–0.12 μg/mL), C. glabrata (0.06 μg/mL), C. parapsilosis (MIC range, 0.06–0.05 μg/mL), and C. tropicalis (MIC range, 0.008–0.06 μg/mL), and was the most potent agent tested against caspofungin-resistant strains of C. albicans (MIC range, 0.008–0.12 μg/mL), C. glabrata (MIC range, 0.03–0.06 μg/mL), and C. tropicalis (MIC range, 0.015–0.06 μg/mL). © 2011 Elsevier Inc. All rights reserved. Keywords: E1210; Candida; Resistance; GPI inhibitor
1. Introduction New formulations and classes of antifungal agents have dramatically expanded the options for the treatment of invasive candidiasis (Chen et al., 2010). Despite these advances, the steady emergence of strains with intrinsic and acquired resistance to both new and established antifungal agents continues to be a concern prompting an expanded search for new antifungal agents with novel mechanisms of action (Fridkin, 2005; Sipsas et al., 2009; Spellberg et al., 2006). ☆ This study was sponsored by an educational/research grant from Eisai Co. (Japan). ⁎ Corresponding author. Tel.: +1 319 665 3370; fax: +1 319 665 3371. E-mail address: [email protected] (M. Castanheira).
0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2011.05.001
E1210 (Eisai, Japan) is a novel first-in-class broadspectrum antifungal agent that inhibits the inositol acylation step in fungal glycophosphatidylinositol (GPI) biosynthesis resulting in defects in various steps in cell wall biosynthesis leading to the inhibition of cell growth, hyphal elongation, and attachment of fungal cells to biological substrates (Tsukahara et al., 2003; Umemura et al., 2003; Watanabe et al., 2010). Differences in the inositol acylation of GPI in yeast and human cells suggest that this could be a good target for drugs directed against yeasts that do not impair inositol acylation in human cells (Tsukahara et al., 2003; Umemura et al., 2003). Preliminary data using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) method have demonstrated the excellent potency and spectrum of E1210 against Candida spp. (Miyazaki et al., 2010). In the present study, we extend these findings by
168
M.A. Pfaller et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 167–170
Table 1 In vitro activity of a novel broad-spectrum antifungal, E1210, and comparator agents tested against Candida spp. as determined by CLSI broth microdilution methods Species (no. tested)
C. albicans (21)
C. glabrata (20)
C. parapsilosis (25)
C. tropicalis (24)
Antifungal agent
E1210 Caspofungin Fluconazole Posaconazole Voriconazole E1210 Caspofungin Fluconazole Posaconazole Voriconazole E1210 Caspofungin Fluconazole Posaconazole Voriconazole E1210 Caspofungin Fluconazole Posaconzole Voriconazole
% by categorya,b
MIC (μg/mL) Range
50%
90%
S
SDD/I
R
0.008–0.12 0.12–N16 0.12–N128 0.01–1 0.008–2 ≤0.008–0.25 0.12–4 0.5–128 0.06–2 ≤0.008–1 ≤0.008–0.5 0.5–4 0.5–≥64 0.03–0.5 0.008–1 0.008–0.06 0.12–4 0.25–≥64 0.015–≥8 0.015–≥8
0.015 0.25 0.25 0.03 0.015 0.03 0.25 8 0.5 0.12 0.03 1 1 0.12 0.015 0.03 0.25 0.5 0.06 0.03
0.06 4 64 0.5 0.5 0.06 2 32 1 1 0.06 4 16 0.12 0.25 0.06 4 ≥64 0.5 2
NA 71.4 57.1 NA 76.2 NA 60.0 NA NA 60.0 NA 87.5 75.0 NA 87.5 NA 73.9 73.9 NA 73.9
NA 0.0 4.8 NA 14.3 NA 15.0 94.4 NA NA NA 12.5 4.2 NA 8.3 NA 4.8 0.0 NA 4.4
NA 23.8 38.1 NA 9.5 NA 25.0 5.6 NA 20.0 NA 0.0 21.7 NA 4.2 NA 33.8 26.1 NA 21.7
a
S = Susceptible; SDD = susceptible dose dependent; I = intermediate; R = resistant; NA = not available. Breakpoints for each antifungal and species: fluconazole and C. albicans, C. parapsilosis, and C. tropicalis (S, ≤2 μg/mL; SDD, 4 μg/mL; R ≥8 μg/mL); fluconazole and C. glabrata (SDD, ≤32 μg/mL; R, ≥64 μg/mL); caspofungin and C. albicans and C. tropicalis (S, ≤0.25 μg/mL; I, 0.5 μg/mL; R, ≥1 μg/mL); caspofungin and C. glabrata (S, ≤0.12 μg/mL; I, 0.25 μg/mL; R, ≥0.5 μg/mL); caspofungin and C. parapsilosis (S, ≤2 μg/mL; I, 4 μg/mL; R, ≥8 μg/mL); voriconazole and C. albicans, C. tropicalis, and C. parapsilosis (S, ≤0.12 μg/mL; I, 0.25–0.5 μg/mL; R, ≥1 μg/mL); voriconazole and C. glabrata (S, ≤0.5 μg/ mL; R, N0.5 μg/mL). b
examining the activity of E1210 and comparator agents against recent clinical isolates of Candida spp., including azole- and echinocandin-resistant strains. A total of 90 clinical isolates of Candida spp. were tested including 21 isolates of C. albicans, 20 of C. glabrata, 25 of C. parapsilosis, and 24 of C. tropicalis. The collection included 20 fluconazole-resistant strains (8 C. albicans [MIC, ≥8 μg/mL], 1 C. glabrata [MIC, 64 μg/mL], 5 C. parapsilosis [MIC, ≥8 μg/mL], and 6 C. tropicalis [MIC, ≥8 μg/mL]) and 15 caspofungin-resistant (MIC, ≥1 μg/mL) strains (5 each of C. albicans, C. glabrata, and C. tropicalis) (Pfaller et al., 2010a, 2010d). The caspofungin-resistant strains all contained mutations in the fks1 or fks2 hotspot regions (Castanheira et al., 2010; Pfaller et al., 2010b). All isolates were sent to JMI Laboratories (North Liberty, IA) for identification and susceptibility testing as described previously (Pfaller et al., 2010c). The isolates were identified by standard methods and stored as water suspensions until used in the study. Prior to testing, each isolate was passaged at least twice on Sabouraud dextrose agar (Remel, Lenexa, KS) and CHROMagar Candida (Becton Dickinson and Company, Sparks, MD) to ensure purity and viability. Reference powders of E1210, caspofungin, fluconazole, posaconazole, and voriconazole were obtained from their respective manufacturers. Stock solutions were prepared in dimethyl sulfoxide, and serial 2-fold dilutions in RPMI 1620 medium (Sigma, St. Louis, MO) buffered to pH 7.0 with
0.165 mL morpholinepropane-sulforic acid buffer (Sigma) were made. BMD testing was performed in accordance with the guidelines in CLSI (2008a) document M27-A3 by using RPMI 1640 medium with 0.2% glucose, and inoculums of 0.5 × 103 to 2.5 × 103 cells/mL and incubation at 35 °C. MIC values for all five antifungal agents were determined visually after 24 h of incubation, as the lowest concentration of drug that caused a significant diminution (≥50% inhibition) of growth below that of the drug-free control (CLSI, 2008a). We used the recently revised CLSI breakpoints to identify strains as susceptible (S), susceptible dose dependent (SDD), intermediate (I), or resistant (R) to caspofungin, fluconazole, and voriconazole (Pfaller et al., 2010a, 2010d). Caspofungin MIC values of ≤0.25 μg/mL (S), 0.5 μg/mL (I), and ≥0.5 μg/mL (R) were used for C. albicans and C. tropicalis; MIC values of ≤2 μg/mL (S), 4 μg/mL (I), and ≥8 μg/mL (R) were used for C. parapsilosis; and MIC values of ≤0.12 μg/ mL (S), 0.25 μg/mL (I), and ≥0.5 μg/mL (R) were used for C. glabrata; fluconazole MIC results of ≤2 μg/mL (S), 4 μg/ mL (SDD), and ≥8 μg/mL (R) were used to classify isolates of C. albicans, C. tropicalis, and C. parapsilosis; and MIC values of ≤32 μg/mL (SDD) and ≥64 μg/mL (R) were used for C. glabrata; voriconazole MIC results of ≤0.12 μg/mL (S), 0.25–0.5 μg/mL (I), and ≥1 μg/mL (R) were used for C. albicans, C. tropicalis, and C. parapsilosis; and MIC values of ≤0.5 μg/mL (wild-type [WT]) and N0.5 μg/mL
M.A. Pfaller et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 167–170
(non-WT) were used for C. glabrata. Quality control was performed by testing the CLSI-recommended strains C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (CLSI, 2008b). Table 1 shows the in vitro activity of E1210 and the four antifungal comparators tested against the 4 major species of Candida. E1210 was highly active against all species tested (MIC90 of 0.06 μg/mL for all four species) and was more potent than all of the comparators against all Candida species. E1210 was not active against C. krusei (MIC50, N16 μg/mL; data not shown). The challenging nature of this organism collection is evident in the high levels of resistance to caspofungin (0.0% to 33.8%), fluconazole (5.6% to 26.1%), and voriconazole (4.2% to 22.2%; see Table 1). E1210 was the most active agent against fluconazoleresistant strains (Table 2) of C. albicans (MIC range, 0.015–0.12 μg/mL), C. glabrata (0.06 μg/mL), C. parapsilosis (MIC range, 0.06–0.5 μg/mL), and C. tropicalis (MIC range, 0.008–0.06 μg/mL). Cross-resistance between fluconazole and voriconazole, and to a lesser extent posaconazole, was evident among isolates of C. albicans (25% voriconazole-resistant), C. glabrata (100%), and C. tropicalis (83%). E1210 was also the most potent agent against caspofungin-resistant strains (Table 3) of C. albicans (MIC range, 0.008–0.12 μg/mL), C. glabrata (MIC range, 0.03–0.06 μg/ mL), and C. tropicalis (MIC range, 0.015–0.06 μg/mL). Although cross-resistance between echinocandins and azoles is unusual, it is notable that this collection included three strains of C. albicans with resistance to both caspofungin
169
Table 3 In vitro activity of E1210 and comparator agents tested against caspofungin-resistant Candida spp. as determined by the CLSI broth microdilution method Species
C. albicans
C. glabrata
C. tropicalis
Isolate no.
8647 8649 8650 8651 8667 8653 8655 8658 8659 8661 8662 8663 8664 8684 8685
MIC (μg/mL) CSF
FLU
PSC
VRC
E1210
8 4 1 1 N8 4 4 2 2 1 2 4 1 4 4
0.25 0.12 N64 32 64 8 32 1 16 32 0.25 0.25 0.5 0.5 0.25
0.03 0.03 0.12 0.12 0.25 0.5 1 0.25 1 1 0.03 0.03 0.03 0.03 0.03
0.015 ≤0.008 2 0.06 0.5 0.25 0.5 0.03 0.5 1 0.015 0.03 0.03 0.015 0.015
0.03 ≤0.008 0.06 0.015 0.12 0.03 0.03 0.03 0.03 0.06 0.015 0.015 0.03 0.03 0.06
and fluconazole, all of which were inhibited by very low concentrations (0.015–0.12 μg/mL) of E1210. In conclusion, E1210 (Miyazaki et al., 2010; Tsukahara et al., 2003; Umemura et al., 2003; Watanabe et al., 2010) was identified as a potent, novel antifungal agent with impressive activity against both azole- and echinocandinresistant strains of Candida. Further development of this new antifungal agent appears warranted. Acknowledgments
Table 2 In vitro activity of E1210 and comparator agents tested against fluconazoleresistant Candida spp. as determined by CLSI broth microdilution methods Species
C. ablicans
C. glabrata C. parapsilosis
C. tropicalis
Isolate no.
MIC (μg/mL)a FLU
PSC
VRC
CSF
E1210
373 28 8676 8677 8679 8650 8651 8667 3472 2626 666 147 676 730 10873 16245 410 540 1628 699
64 32 32 32 32 N64 32 64 N64 16 8 8 32 64 8 N64 64 N64 N64 16
0.25 0.12 1 0.5 1 0.12 0.12 0.25 2 0.12 0.12 0.12 0.12 0.5 0.5 4 0.25 0.25 N8 0.5
0.12 0.06 2 0.5 0.5 2 0.06 0.5 1 0.12 0.25 0.06 0.5 1 0.25 8 2 2 N8 1
0.12 0.25 0.25 0.25 0.25 1 1 N8 0.25 2 2 1 1 4 0.12 0.25 0.12 0.12 0.5 0.25
0.015 0.03 0.03 0.06 0.03 0.06 0.015 0.12 0.06 0.06 0.06 0.06 0.06 0.5 0.06 ≤0.008 0.06 0.03 ≤0.008 0.06
a FLU = Fluconazole; PSC = posaconazole; VRC = voriconazole; CSF = caspofungin.
The authors would like to thank Drs. D.S. Perlin (Public Health Research Institute, New Jersey Medical School, UMDNJ) and D.J. Diekema (University of Iowa) for kindly supplying some resistance phenotypes of Candida spp. References Castanheira M, Woosley LN, Pfaller MA, Diekema DJ, Messer SA, Jones RN (2010) Low prevalence of fks1 hotspot 1 mutations in a worldwide collection of Candida spp. Antimicrob Agents Chemother 54:2655–2659. Chen SC, Playford EG, Sorrell TC (2010) Antifungal therapy in invasive fungal infections. Curr Opin Pharmacol 10:522–530. Clinical and Laboratory Standards Institute (CLSI) (2008a) Reference method for broth dilution antifungal susceptibility testing of yeasts. 3rd ed, M27-A3. Wayne, PA: CLSI. Clinical and Laboratory Standards Institute (CLSI) (2008b) Reference method for broth dilution antifungal susceptibility testing of yeasts: 3rd informational supplement, (M27-S3). Wayne, PA: CLSI. Fridkin SK (2005) The changing face of fungal infections in health care settings. Clin Infect Dis 41:1455–1460. Miyazaki M, Horii T, Hata K, Watanabe M (2010) In vitro antifungal activity of E1210: A novel antifungal with activity against clinically important yeasts and moulds. 50th ICAAC, September 12–15, 2010. pp. Abstr. F1-840. Boston, Massachusetts, USA. Pfaller MA, Andes D, Diekema DJ, Espinel-Ingroff A, Sheehan D, The Subcommittee for Antifungal Susceptibility Testing (2010a) Wild-type
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MIC distributions, epidemiological cutoff values and species-specific clinical breakpoints for fluconazole and Candida: time for harmonization of CLSI and EUCAST broth microdilution methods. Drug Resist Updat 13:180–195. Pfaller MA, Castanheira M, Diekema DJ, Messer SA, Moet GJ, Jones RN (2010b) Comparison of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Etest methods with the CLSI broth microdilution method for echinocandin susceptibility testing of Candida species. J Clin Microbiol 48:1592–1599. Pfaller MA, Castanheira M, Messer SA, Moet GJ, Jones RN (2010c) Variation in Candida spp. distribution and antifungal resistance rates among bloodstream infection isolates by patient age: report from the SENTRY Antimicrobial Surveillance Program (2008–2009). Diagn Microbiol Infect Dis 68:278–283. Pfaller MA, Diekema DJ, Andes D, Arendrup MC, Brown SD, Motyl M, Perlin DS, The Subcommittee for Antifungal Susceptibility Testing (2010d) Clinical breakpoints for the echinocandins and Candida revisited: integration of molecular, clinical, and microbiological data to arrive at species-specific interpretive criteria. Drug Resist Updat (in press).
Sipsas NV, Lewis RE, Tarrand J, Hachem R, Rolston KV, Raad II, Kontoyiannis DP (2009) Candidemia in patients with hematologic malignancies in the era of new antifungal agents (2001–2007): stable incidence but changing epidemiology of a still frequently lethal infection. Cancer 115:4745–4752. Spellberg BJ, Filler SG, Edwards Jr JE (2006) Current treatment strategies for disseminated candidiasis. Clin Infect Dis 42:244–251. Tsukahara K, Hata K, Nakamoto K, Sagane K, Watanabe NA, Kuromitsu J, Kai J, Tsuchiya M, Ohba F, Jigami Y, Yoshimatsu K, Nagasu T (2003) Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly. Mol Microbiol 48:1029–1042. Umemura M, Okamoto M, Nakayama K, Sagane K, Tsukahara K, Hata K, Jigami Y (2003) GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast. J Biol Chem 278: 23639–23647. Watanabe N, Horii T, Miyazaki M, Hata K (2010) E1210, a new broadspectrum antifungal, inhibits glycosylphosphatidylinositl (GPI) biosynthesis and effects Candida albicans cell characteristics. 50th ICAAC, September 12–15, 2010. pp. Abstr. F1-841. Boston, Massachusetts, USA.
Diagnostic Microbiology and Infectious Disease 71 (2011) 167 – 170 www.elsevier.com/locate/diagmicrobio
In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Candida spp. as determined by CLSI broth microdilution method☆ Michael A. Pfallera , Katsura Hatab , Ronald N. Jonesa,c , Shawn A. Messera , Gary J. Moeta , Mariana Castanheiraa,⁎ a
JMI Laboratories, North Liberty, IA 52317, USA b Eisai, Tsukuba 300-2635, Japan c Tufts University School of Medicine, Boston, MA 02111, USA Received 26 April 2011; accepted 2 May 2011
Abstract The in vitro activity of the novel antifungal agent E1210 and four comparators (caspofungin, fluconazole, posaconazole, and voriconazole) was determined against 90 clinical isolates of Candida using Clinical and Laboratory Standards Institute methods. The collection was composed of 21 Candida albicans, 20 C. glabrata, 25 C. parapsilosis, and 24 C. tropicals, and also included 21 fluconazole-resistant and 15 caspofungin-resistant strains. E1210 was highly active against all the species tested and was more potent than all comparators. The MIC90 results (μg/mL) for E1210, caspofungin, fluconazole, posaconazole, and voriconazole, respectively, were as follows by species: C. albicans (0.06, 4, ≥64, 0.5, 0.5), C. glabrata (0.06, 2, 32, 1, 1), C. parapsilosis (0.06, 4, 16, 0.12, 0.25), and C. tropicalis (0.06, 4, ≥64, 0.5, 2). E1210 was also the most active agent against fluconazole-resistant strains of C. albicans (MIC range, 0.015–0.12 μg/mL), C. glabrata (0.06 μg/mL), C. parapsilosis (MIC range, 0.06–0.05 μg/mL), and C. tropicalis (MIC range, 0.008–0.06 μg/mL), and was the most potent agent tested against caspofungin-resistant strains of C. albicans (MIC range, 0.008–0.12 μg/mL), C. glabrata (MIC range, 0.03–0.06 μg/mL), and C. tropicalis (MIC range, 0.015–0.06 μg/mL). © 2011 Elsevier Inc. All rights reserved. Keywords: E1210; Candida; Resistance; GPI inhibitor
1. Introduction New formulations and classes of antifungal agents have dramatically expanded the options for the treatment of invasive candidiasis (Chen et al., 2010). Despite these advances, the steady emergence of strains with intrinsic and acquired resistance to both new and established antifungal agents continues to be a concern prompting an expanded search for new antifungal agents with novel mechanisms of action (Fridkin, 2005; Sipsas et al., 2009; Spellberg et al., 2006). ☆ This study was sponsored by an educational/research grant from Eisai Co. (Japan). ⁎ Corresponding author. Tel.: +1 319 665 3370; fax: +1 319 665 3371. E-mail address: [email protected] (M. Castanheira).
0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2011.05.001
E1210 (Eisai, Japan) is a novel first-in-class broadspectrum antifungal agent that inhibits the inositol acylation step in fungal glycophosphatidylinositol (GPI) biosynthesis resulting in defects in various steps in cell wall biosynthesis leading to the inhibition of cell growth, hyphal elongation, and attachment of fungal cells to biological substrates (Tsukahara et al., 2003; Umemura et al., 2003; Watanabe et al., 2010). Differences in the inositol acylation of GPI in yeast and human cells suggest that this could be a good target for drugs directed against yeasts that do not impair inositol acylation in human cells (Tsukahara et al., 2003; Umemura et al., 2003). Preliminary data using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) method have demonstrated the excellent potency and spectrum of E1210 against Candida spp. (Miyazaki et al., 2010). In the present study, we extend these findings by
168
M.A. Pfaller et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 167–170
Table 1 In vitro activity of a novel broad-spectrum antifungal, E1210, and comparator agents tested against Candida spp. as determined by CLSI broth microdilution methods Species (no. tested)
C. albicans (21)
C. glabrata (20)
C. parapsilosis (25)
C. tropicalis (24)
Antifungal agent
E1210 Caspofungin Fluconazole Posaconazole Voriconazole E1210 Caspofungin Fluconazole Posaconazole Voriconazole E1210 Caspofungin Fluconazole Posaconazole Voriconazole E1210 Caspofungin Fluconazole Posaconzole Voriconazole
% by categorya,b
MIC (μg/mL) Range
50%
90%
S
SDD/I
R
0.008–0.12 0.12–N16 0.12–N128 0.01–1 0.008–2 ≤0.008–0.25 0.12–4 0.5–128 0.06–2 ≤0.008–1 ≤0.008–0.5 0.5–4 0.5–≥64 0.03–0.5 0.008–1 0.008–0.06 0.12–4 0.25–≥64 0.015–≥8 0.015–≥8
0.015 0.25 0.25 0.03 0.015 0.03 0.25 8 0.5 0.12 0.03 1 1 0.12 0.015 0.03 0.25 0.5 0.06 0.03
0.06 4 64 0.5 0.5 0.06 2 32 1 1 0.06 4 16 0.12 0.25 0.06 4 ≥64 0.5 2
NA 71.4 57.1 NA 76.2 NA 60.0 NA NA 60.0 NA 87.5 75.0 NA 87.5 NA 73.9 73.9 NA 73.9
NA 0.0 4.8 NA 14.3 NA 15.0 94.4 NA NA NA 12.5 4.2 NA 8.3 NA 4.8 0.0 NA 4.4
NA 23.8 38.1 NA 9.5 NA 25.0 5.6 NA 20.0 NA 0.0 21.7 NA 4.2 NA 33.8 26.1 NA 21.7
a
S = Susceptible; SDD = susceptible dose dependent; I = intermediate; R = resistant; NA = not available. Breakpoints for each antifungal and species: fluconazole and C. albicans, C. parapsilosis, and C. tropicalis (S, ≤2 μg/mL; SDD, 4 μg/mL; R ≥8 μg/mL); fluconazole and C. glabrata (SDD, ≤32 μg/mL; R, ≥64 μg/mL); caspofungin and C. albicans and C. tropicalis (S, ≤0.25 μg/mL; I, 0.5 μg/mL; R, ≥1 μg/mL); caspofungin and C. glabrata (S, ≤0.12 μg/mL; I, 0.25 μg/mL; R, ≥0.5 μg/mL); caspofungin and C. parapsilosis (S, ≤2 μg/mL; I, 4 μg/mL; R, ≥8 μg/mL); voriconazole and C. albicans, C. tropicalis, and C. parapsilosis (S, ≤0.12 μg/mL; I, 0.25–0.5 μg/mL; R, ≥1 μg/mL); voriconazole and C. glabrata (S, ≤0.5 μg/ mL; R, N0.5 μg/mL). b
examining the activity of E1210 and comparator agents against recent clinical isolates of Candida spp., including azole- and echinocandin-resistant strains. A total of 90 clinical isolates of Candida spp. were tested including 21 isolates of C. albicans, 20 of C. glabrata, 25 of C. parapsilosis, and 24 of C. tropicalis. The collection included 20 fluconazole-resistant strains (8 C. albicans [MIC, ≥8 μg/mL], 1 C. glabrata [MIC, 64 μg/mL], 5 C. parapsilosis [MIC, ≥8 μg/mL], and 6 C. tropicalis [MIC, ≥8 μg/mL]) and 15 caspofungin-resistant (MIC, ≥1 μg/mL) strains (5 each of C. albicans, C. glabrata, and C. tropicalis) (Pfaller et al., 2010a, 2010d). The caspofungin-resistant strains all contained mutations in the fks1 or fks2 hotspot regions (Castanheira et al., 2010; Pfaller et al., 2010b). All isolates were sent to JMI Laboratories (North Liberty, IA) for identification and susceptibility testing as described previously (Pfaller et al., 2010c). The isolates were identified by standard methods and stored as water suspensions until used in the study. Prior to testing, each isolate was passaged at least twice on Sabouraud dextrose agar (Remel, Lenexa, KS) and CHROMagar Candida (Becton Dickinson and Company, Sparks, MD) to ensure purity and viability. Reference powders of E1210, caspofungin, fluconazole, posaconazole, and voriconazole were obtained from their respective manufacturers. Stock solutions were prepared in dimethyl sulfoxide, and serial 2-fold dilutions in RPMI 1620 medium (Sigma, St. Louis, MO) buffered to pH 7.0 with
0.165 mL morpholinepropane-sulforic acid buffer (Sigma) were made. BMD testing was performed in accordance with the guidelines in CLSI (2008a) document M27-A3 by using RPMI 1640 medium with 0.2% glucose, and inoculums of 0.5 × 103 to 2.5 × 103 cells/mL and incubation at 35 °C. MIC values for all five antifungal agents were determined visually after 24 h of incubation, as the lowest concentration of drug that caused a significant diminution (≥50% inhibition) of growth below that of the drug-free control (CLSI, 2008a). We used the recently revised CLSI breakpoints to identify strains as susceptible (S), susceptible dose dependent (SDD), intermediate (I), or resistant (R) to caspofungin, fluconazole, and voriconazole (Pfaller et al., 2010a, 2010d). Caspofungin MIC values of ≤0.25 μg/mL (S), 0.5 μg/mL (I), and ≥0.5 μg/mL (R) were used for C. albicans and C. tropicalis; MIC values of ≤2 μg/mL (S), 4 μg/mL (I), and ≥8 μg/mL (R) were used for C. parapsilosis; and MIC values of ≤0.12 μg/ mL (S), 0.25 μg/mL (I), and ≥0.5 μg/mL (R) were used for C. glabrata; fluconazole MIC results of ≤2 μg/mL (S), 4 μg/ mL (SDD), and ≥8 μg/mL (R) were used to classify isolates of C. albicans, C. tropicalis, and C. parapsilosis; and MIC values of ≤32 μg/mL (SDD) and ≥64 μg/mL (R) were used for C. glabrata; voriconazole MIC results of ≤0.12 μg/mL (S), 0.25–0.5 μg/mL (I), and ≥1 μg/mL (R) were used for C. albicans, C. tropicalis, and C. parapsilosis; and MIC values of ≤0.5 μg/mL (wild-type [WT]) and N0.5 μg/mL
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(non-WT) were used for C. glabrata. Quality control was performed by testing the CLSI-recommended strains C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (CLSI, 2008b). Table 1 shows the in vitro activity of E1210 and the four antifungal comparators tested against the 4 major species of Candida. E1210 was highly active against all species tested (MIC90 of 0.06 μg/mL for all four species) and was more potent than all of the comparators against all Candida species. E1210 was not active against C. krusei (MIC50, N16 μg/mL; data not shown). The challenging nature of this organism collection is evident in the high levels of resistance to caspofungin (0.0% to 33.8%), fluconazole (5.6% to 26.1%), and voriconazole (4.2% to 22.2%; see Table 1). E1210 was the most active agent against fluconazoleresistant strains (Table 2) of C. albicans (MIC range, 0.015–0.12 μg/mL), C. glabrata (0.06 μg/mL), C. parapsilosis (MIC range, 0.06–0.5 μg/mL), and C. tropicalis (MIC range, 0.008–0.06 μg/mL). Cross-resistance between fluconazole and voriconazole, and to a lesser extent posaconazole, was evident among isolates of C. albicans (25% voriconazole-resistant), C. glabrata (100%), and C. tropicalis (83%). E1210 was also the most potent agent against caspofungin-resistant strains (Table 3) of C. albicans (MIC range, 0.008–0.12 μg/mL), C. glabrata (MIC range, 0.03–0.06 μg/ mL), and C. tropicalis (MIC range, 0.015–0.06 μg/mL). Although cross-resistance between echinocandins and azoles is unusual, it is notable that this collection included three strains of C. albicans with resistance to both caspofungin
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Table 3 In vitro activity of E1210 and comparator agents tested against caspofungin-resistant Candida spp. as determined by the CLSI broth microdilution method Species
C. albicans
C. glabrata
C. tropicalis
Isolate no.
8647 8649 8650 8651 8667 8653 8655 8658 8659 8661 8662 8663 8664 8684 8685
MIC (μg/mL) CSF
FLU
PSC
VRC
E1210
8 4 1 1 N8 4 4 2 2 1 2 4 1 4 4
0.25 0.12 N64 32 64 8 32 1 16 32 0.25 0.25 0.5 0.5 0.25
0.03 0.03 0.12 0.12 0.25 0.5 1 0.25 1 1 0.03 0.03 0.03 0.03 0.03
0.015 ≤0.008 2 0.06 0.5 0.25 0.5 0.03 0.5 1 0.015 0.03 0.03 0.015 0.015
0.03 ≤0.008 0.06 0.015 0.12 0.03 0.03 0.03 0.03 0.06 0.015 0.015 0.03 0.03 0.06
and fluconazole, all of which were inhibited by very low concentrations (0.015–0.12 μg/mL) of E1210. In conclusion, E1210 (Miyazaki et al., 2010; Tsukahara et al., 2003; Umemura et al., 2003; Watanabe et al., 2010) was identified as a potent, novel antifungal agent with impressive activity against both azole- and echinocandinresistant strains of Candida. Further development of this new antifungal agent appears warranted. Acknowledgments
Table 2 In vitro activity of E1210 and comparator agents tested against fluconazoleresistant Candida spp. as determined by CLSI broth microdilution methods Species
C. ablicans
C. glabrata C. parapsilosis
C. tropicalis
Isolate no.
MIC (μg/mL)a FLU
PSC
VRC
CSF
E1210
373 28 8676 8677 8679 8650 8651 8667 3472 2626 666 147 676 730 10873 16245 410 540 1628 699
64 32 32 32 32 N64 32 64 N64 16 8 8 32 64 8 N64 64 N64 N64 16
0.25 0.12 1 0.5 1 0.12 0.12 0.25 2 0.12 0.12 0.12 0.12 0.5 0.5 4 0.25 0.25 N8 0.5
0.12 0.06 2 0.5 0.5 2 0.06 0.5 1 0.12 0.25 0.06 0.5 1 0.25 8 2 2 N8 1
0.12 0.25 0.25 0.25 0.25 1 1 N8 0.25 2 2 1 1 4 0.12 0.25 0.12 0.12 0.5 0.25
0.015 0.03 0.03 0.06 0.03 0.06 0.015 0.12 0.06 0.06 0.06 0.06 0.06 0.5 0.06 ≤0.008 0.06 0.03 ≤0.008 0.06
a FLU = Fluconazole; PSC = posaconazole; VRC = voriconazole; CSF = caspofungin.
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