Abstract
E1210 is a first-in-class broad-spectrum antifungal that suppresses hyphal growth by inhibiting fungal glycophosphatidylinositol (GPI) biosynthesis. In the present study, we extend these findings by examining the activity of E1210 and comparator antifungal agents against Aspergillus spp. by using the methods of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) to test wild-type (WT) as well as amphotericin B (AMB)-resistant (-R) and azole-R strains (as determined by CLSI methods). Seventy-eight clinical isolates of Aspergillus were tested including 20 isolates of Aspergillus flavus species complex (SC), 22 of A. fumigatus SC, 13 of A. niger SC, and 23 of A. terreus SC. The collection included 15 AMB-R (MIC, ≥2 μg/ml) isolates of A. terreus SC and 10 itraconazole-R (MIC, ≥4 μg/ml) isolates of A. fumigatus SC (7 isolates), A. niger SC (2 isolates), and A. terreus SC (1 isolate). Comparator antifungal agents included anidulafungin, caspofungin, amphotericin B, itraconazole, posaconzole, and voriconazole. Both CLSI and EUCAST methods were highly concordant for E1210 and all comparators. The essential agreement (EA; ±2 log2 dilution steps) was 100% for all comparisons with the exception of posaconazole versus A. terreus SC (EA = 91.3%). The minimum effective concentration (MEC)/MIC90 values (μg/ml) for E1210, anidulafungin, caspofungin, itraconazole, posaconazole, and voriconazole, respectively, were as follows for each species: for A. flavus SC, 0.03, ≤0.008, 0.12, 1, 1, and 1; for A. fumigatus SC, 0.06, 0.015, 0.12, >8, 1, and 4; for A. niger SC, 0.015, 0.03, 0.12, 4, 1, and 2; and for A. terreus SC, 0.06, 0.015, 0.12, 1, 0.5, and 1. E1210 was very active against AMB-R strains of A. terreus SC (MEC range, 0.015 to 0.06 μg/ml) and itraconazole-R strains of A. fumigatus SC (MEC range, 0.03 to 0.12 μg/ml), A. niger SC (MEC, 0.008 μg/ml), and A. terreus SC (MEC, 0.015 μg/ml). In conclusion, E1210 was a very potent and broad-spectrum antifungal agent regardless of in vitro method applied, with excellent activity against AMB-R and itraconazole-R strains of Aspergillus spp.
INTRODUCTION
The introduction of mold-active antifungal agents of the triazole (itraconazole, posaconazole, voriconazole) and echinocandin (anidulafungin, caspofungin, micafungin) classes has dramatically increased the options for prophylaxis and empirical or directed therapy for the prevention and treatment of invasive aspergillosis (IA) (2, 4, 5, 11, 25, 31). Although resistance to these agents among clinical isolates of Aspergillus has been considered to be uncommon, both increased resistance and breakthrough infections have been reported increasingly among patients with long-term exposure to these agents (1, 3, 14, 16, 19, 20, 26, 30). These observations have prompted a call for an expanded search for new antifungal agents with novel mechanisms of action, as well as an expanded role for antifungal susceptibility testing of Aspergillus spp. (4, 8, 11, 12, 15, 24, 25, 30).
E1210 (Eisai Co., Tokyo, Japan) is a novel first-in-class broad-spectrum antifungal agent that inhibits the inositol acylation step in fungal glycophosphatidylinositol (GPI) biosynthesis, resulting in defects in various steps in cell wall biosynthesis with accompanying inhibition of cell wall growth, hyphal elongation, and attachment of fungal cells to biological substrates (27, 28, 32). Differences in the inositol acylation of GPI in fungi and in human cells suggest that this may prove to be a good target for drugs directed against fungi (yeasts and molds) that do not impair inositol acylation in human cells (27, 28).
Preliminary data obtained using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) method (6) have demonstrated the excellent E1210 spectrum and potency against both yeasts and molds, including Aspergillus spp. (18). With regard to Aspergillus spp., these studies are limited by the small numbers of isolates tested, the lack of antifungal comparators, and the lack of a comparison of the E1210 potency as determined by the reference BMD methods of the CLSI and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) (6, 9, 10). Currently there are two independent reference standards for BMD susceptibility testing of antifungal agents against Aspergillus spp. (6, 9, 10). These two methods are similar in that both use BMD, RPMI 1640 broth, incubation at 35 to 37°C for 48 h, and a complete (100%) inhibition (triazoles) or minimum effective concentration (MEC; lowest concentration of drug that produces stubby aberrant hyphal growth) MIC/MEC endpoint. They differ, however, in inoculum concentration (0.4 × 104 to 5 × 104 CFU/ml [CLSI] versus 2 × 105 to 5 × 105 CFU/ml [EUCAST]), glucose content of the medium (0.2% [CLSI] versus 2.0% [EUCAST]), and rounded (CLSI) versus flat-bottom (EUCAST) microdilution wells (6, 9, 10). Although we have shown previously that both methods provide concordant results when testing Aspergillus spp. against itraconazole, posaconazole, and voriconazole (21), similar data are not available for E1210 or the echinocandins anidulafungin and caspofungin. Given the important role that both methods currently play in antifungal resistance surveillance and regulatory evaluations of new agents, it is important to demonstrate the comparability of their results in the preclinical development of new antifungal agents. In the present study, we have used a collection of Aspergillus spp. isolates selected to represent both polyene- and triazole-resistant strains to examine the activity of E1210 and comparator agents as determined by both CLSI and EUCAST reference BMD methods.
MATERIALS AND METHODS
Organisms.
A total of 78 clinical isolates of Aspergillus spp. obtained from centers participating in the 2008-2009 ARTEMIS and SENTRY Antimicrobial Surveillance Programs (22, 23) were tested against E1210, amphotericin B, anidulafungin, caspofungin, itraconazole, posaconazole, and voriconazole. The collection included 20 isolates of A. flavus species complex (SC), 22 of A. fumigatus SC, 13 of A. niger SC, and of 23 A. terreus SC. The phenotypically resistant isolates (as determined by CLSI methods) included 15 amphotericin B-resistant (MIC, ≥2 μg/ml) isolates of A. terreus SC and 10 itraconazole-resistant (MIC, ≥4 μg/ml) isolates of A. fumigatus SC (7 isolates), A. niger SC (2 isolates), and A. terreus SC (1 isolate). The isolates were obtained from a variety of sources, including sputum, bronchoscopy, and tissue biopsy specimens, and represented individual infection episodes. The isolates were collected at individual study sites and sent to JMI Laboratories (North Liberty, IA) for identification and susceptibility testing as described previously (17, 23). All isolates were identified by standard microscopic morphology (29) and were stored as spore suspensions in sterile distilled water at room temperature until used in the study. Before testing, each isolate was subcultured at least twice on potato dextrose agar (Remel, Lenexa, KS) to ensure viability and purity.
Antifungal agents.
Reference powders of E1210, amphotericin B, anidulafungin, caspofungin, itraconazole, posaconazole, and voriconazole were obtained from their respective manufacturers. Stock solutions were prepared in dimethyl sulfoxide, and serial doubling dilutions in RPMI 1640 medium (Sigma, St. Louis, MO) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma) were made.
Antifungal susceptibility testing.
All isolates were tested for in vitro susceptibility to E1210 and comparators using the CLSI and EUCAST BMD methods (6, 9, 10). CLSI BMD testing was performed as outlined in document M38-A2 (6) by using RPMI 1640 medium with 0.2% glucose, round-bottom microdilution trays, inocula of 0.4 × 104 to 5 × 104 CFU/ml, and incubation at 35°C for 48 h. MIC values for the triazoles were determined visually as the lowest concentration of drug that caused complete inhibition of growth (first clear well) relative to that of the growth control. MEC values for E1210, anidulafungin, and caspofungin were determined as described previously (18, 22) and by the CLSI (6). The MEC endpoint was chosen for E1210 due to the fact that, similar to the echinocandins, E1210 inhibits cell wall synthesis and hyphal extension of Aspergillus spp., resulting in aberrant hyphal growth (short, stubby, highly branched hyphae) but not complete growth inhibition. Preliminary data generated by the sponsor and in experiments at JMI Laboratories (unpublished) found that as with the echinocandins, when an MIC for E1210 is determined (complete growth inhibition), an endpoint is not achieved for Aspergillus spp.
EUCAST BMD testing was performed exactly as detailed by EUCAST (10) by using RPMI 1640 medium with 2.0% glucose, flat-bottom microdilution trays, inocula of 2 × 105 to 5 × 105 CFU/ml, and incubation at 35°C. MICs (triazoles) and MECs (E1210, echinocandins) were determined after 48 h as described above and by both CLSI (6) and EUCAST (10). Quality control was assured by testing the following strains recommended by CLSI and by EUCAST (7): Candida parapsilosis ATCC 22019, C. krusei ATCC 6258, and A. flavus ATCC 204304 (6, 9).
Analysis.
The MIC/MEC results for each antifungal agent obtained with the EUCAST method (9) were compared to those of the CLSI BMD method (6). High off-scale MIC/MEC results were converted to the next highest concentration and low off-scale MIC/MEC results were left unchanged. Discrepancies of more than ±2 log2 dilution steps among MIC results were used to calculate the level of essential agreement (EA) between the two methods with a target of ≥95% EA at ≤2 doubling dilutions.
RESULTS AND DISCUSSION
Table 1 summarizes the in vitro susceptibilities of tested Aspergillus spp. to E1210 and five comparators as determined by the CLSI and EUCAST BMD methods (6, 9, 10). All Aspergillus spp. were inhibited by ≤0.06 μg/ml of E1210 as determined by both methods. The echinocandins (anidulafungin and caspofungin) displayed the lowest MEC50/MIC50 values among comparator agents (MEC50, ≤0.008 μg/ml for anidulafungin for all species; MEC50 range from 0.06 to 0.12 μg/ml for caspofungin) followed by posaconazole (MIC50s of 0.5 μg/ml for all Aspergillus spp.).
Table 1.
In vitro susceptibilities of Aspergillus spp. to E1210 and comparators as determined by CLSI and EUCAST broth microdilution methods
Species (no. tested) | Antifungal agent | Test method | No. of isolates at indicated MIC/MEC (μg/ml) |
% EAa | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
≤0.008 | 0.015 | 0.03 | 0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | ≥8 | ||||
A. flavus SC b (20) | E1210 | EUCAST | 4 | 14 | 2 | 100.0 | ||||||||
CLSI | 1 | 14 | 4 | 1 | ||||||||||
Anidulafungin | EUCAST | 19 | 1 | 100.0 | ||||||||||
CLSI | 20 | |||||||||||||
Caspofungin | EUCAST | 4 | 16 | 100.0 | ||||||||||
CLSI | 1 | 15 | 4 | |||||||||||
Itraconazole | EUCAST | 7 | 13 | 100.0 | ||||||||||
CLSI | 7 | 13 | ||||||||||||
Posaconazole | EUCAST | 2 | 17 | 1 | 100.0 | |||||||||
CLSI | 1 | 16 | 3 | |||||||||||
Voriconazole | EUCAST | 1 | 12 | 7 | 100.0 | |||||||||
CLSI | 4 | 14 | 1 | 1 | ||||||||||
A. fumigatus SC (22) | E1210 | EUCAST | 5 | 13 | 4 | 100.0 | ||||||||
CLSI | 1 | 1 | 16 | 4 | ||||||||||
Anidulafungin | EUCAST | 17 | 5 | 100.0 | ||||||||||
CLSI | 14 | 8 | ||||||||||||
Caspofungin | EUCAST | 3 | 15 | 4 | 100.0 | |||||||||
CLSI | 4 | 17 | 1 | |||||||||||
Itraconazole | EUCAST | 2 | 13 | 7 | 100.0 | |||||||||
CLSI | 1 | 14 | 1 | 6 | ||||||||||
Posaconazole | EUCAST | 13 | 1 | 2 | 5 | 1 | 100.0 | |||||||
CLSI | 8 | 7 | 6 | 1 | ||||||||||
Voriconazole | EUCAST | 1 | 12 | 4 | 4 | 1 | 100.0 | |||||||
CLSI | 4 | 10 | 3 | 4 | 1 | |||||||||
A. niger SC (13) | E1210 | EUCAST | 5 | 6 | 2 | 100.0 | ||||||||
CLSI | 8 | 5 | ||||||||||||
Anidulafungin | EUCAST | 13 | 100.0 | |||||||||||
CLSI | 12 | 1 | ||||||||||||
Caspofungin | EUCAST | 13 | 100.0 | |||||||||||
CLSI | 4 | 9 | ||||||||||||
Itraconazole | EUCAST | 1 | 3 | 6 | 3 | 100.0 | ||||||||
CLSI | 6 | 5 | 2 | |||||||||||
Posaconazole | EUCAST | 2 | 4 | 7 | 100.0 | |||||||||
CLSI | 1 | 8 | 4 | |||||||||||
Voriconazole | EUCAST | 4 | 5 | 4 | 100.0 | |||||||||
CLSI | 4 | 5 | 4 | |||||||||||
A. terreus SC (23) | E1210 | EUCAST | 2 | 17 | 4 | 100.0 | ||||||||
CLSI | 1 | 11 | 8 | 3 | ||||||||||
Anidulafungin | EUCAST | 20 | 3 | 100.0 | ||||||||||
CLSI | 15 | 8 | ||||||||||||
Caspofungin | EUCAST | 1 | 18 | 4 | 100.0 | |||||||||
CLSI | 21 | 2 | ||||||||||||
Itraconazole | EUCAST | 15 | 7 | 1 | 100.0 | |||||||||
CLSI | 1 | 15 | 6 | 1 | ||||||||||
Posaconazole | EUCAST | 1 | 14 | 7 | 1 | 91.3 | ||||||||
CLSI | 7 | 14 | 1 | 1 | ||||||||||
Voriconazole | EUCAST | 11 | 11 | 1 | 100.0 | |||||||||
CLSI | 4 | 13 | 5 | 1 |
EA, essential agreement.
SC, species complex.
The EA between the two reference methods was 100.0% for E1210 across all four Aspergillus spp. (Table 1). The MEC values generated by the EUCAST method (9, 10) tended to be 1 log2 dilution higher than those obtained by the CLSI method (6). Likewise, the overall EA between EUCAST and CLSI results was 100.0% for anidulafungin, caspofungin, itraconazole, and voriconazole but was 97.4% (76 of 78 results) for posaconazole. These results confirm the high level of concordance between the MIC results produced by both methods for the triazoles when testing Aspergillus spp. (21) and demonstrates for the first time that both reference methods generate comparable MEC values for E1210, anidulafungin, and caspofungin.
E1210 and the echinocandins were the most active agents against amphotericin B-resistant strains of A. terreus SC as determined by the CLSI method (Table 2). Anidulafungin was the most potent agent (MEC90, 0.015 μg/ml) followed by E1210 (MEC90, 0.06 μg/ml), caspofungin (MEC90, 0.25 μg/ml), posaconazole (MIC90, 0.5 μg/ml), itraconazole and voriconazole (MIC90, 1 μg/ml), and amphotericin B (4 μg/ml).
Table 2.
In vitro activities of E1210 and comparators against amphotericin B-resistant A. terreus SC as determined by CLSI broth microdilution methodsa
Isolate no. | MIC/MEC (μg/ml) forb,c: |
||||||
---|---|---|---|---|---|---|---|
AMB | ITR | PSC | VRC | ANF | CSF | E1210 | |
446 | 4 | 0.5 | 0.5 | 0.5 | 0.008 | 0.12 | 0.015 |
618 | 4 | 0.5 | 0.25 | 0.25 | 0.008 | 0.12 | 0.03 |
8691 | 2 | 1 | 0.5 | 1 | 0.015 | 0.25 | 0.06 |
8693 | 2 | 1 | 0.5 | 0.5 | 0.015 | 0.25 | 0.06 |
8694 | 2 | 0.5 | 0.25 | 0.5 | 0.008 | 0.12 | 0.03 |
8695 | 2 | 1 | 0.5 | 1 | 0.008 | 0.12 | 0.06 |
8696 | 2 | 0.25 | 0.25 | 0.25 | 0.008 | 0.12 | 0.015 |
8722 | 2 | 0.5 | 0.5 | 1 | 0.015 | 0.12 | 0.03 |
8723 | 2 | 0.5 | 0.25 | 0.5 | 0.008 | 0.12 | 0.03 |
8727 | 2 | 0.5 | 0.5 | 1 | 0.008 | 0.12 | 0.03 |
8728 | 2 | 0.5 | 0.5 | 0.5 | 0.008 | 0.12 | 0.03 |
8729 | 2 | 1 | 0.5 | 1 | 0.008 | 0.12 | 0.03 |
8730 | 2 | 0.5 | 0.5 | 1 | 0.008 | 0.12 | 0.03 |
8731 | 2 | 4 | 2 | >8 | 0.008 | 0.12 | 0.015 |
8733 | 2 | 0.5 | 0.5 | 1 | 0.015 | 0.12 | 0.015 |
The MICs and minimum effective concentrations (MECs) were determined after 48 h of incubation.
The MICs for AMB (amphotericin B), ITR (itraconazole), PSC (posaconazole), and VRC (voriconazole) were read at complete (100%) inhibition.
The MECs for ANF (anidulafungin), CSF (caspofungin), and E1210 were read as described in CLSI document M38-A2 (6).
A similar rank order of activity was determined with the itraconazole-resistant isolates: anidulafungin (MEC90, 0.015 μg/ml) > E1210 (MEC90, 0.06 μg/ml) > caspofungin (MEC90, 0.12 μg/ml) > amphotericin B (MIC90, 1 μg/ml) > posaconazole (MIC90, 2 μg/ml) > voriconazole (MIC90, 8 μg/ml) > itraconazole (MIC90, >8 μg/ml) (Table 3). Whereas cross-resistance was apparent between itraconazole, posaconazole, and voriconazole, the distinctly different mechanisms of action represented by E1210, amphotericin B, and the echinocandins result in activity of these antifungal agents against this subset of Aspergillus species isolates that are of great clinical concern worldwide (3, 12, 13, 15, 30).
Table 3.
In vitro activities of E1210 and comparators against itraconazole-resistant Aspergillus spp. as determined by CLSI broth microdilution methods
Species | Isolate no. | MIC/MEC (μg/ml) fora: |
||||||
---|---|---|---|---|---|---|---|---|
ITR | PSC | VRC | AMB | ANF | CSF | E1210 | ||
A. fumigatus SC | 8686 | >8 | 1 | 2 | 1 | 0.015 | 0.12 | 0.12 |
8687 | >8 | 1 | 2 | 1 | 0.015 | 0.12 | 0.06 | |
8688 | >8 | 1 | 1 | 1 | 0.008 | 0.12 | 0.03 | |
8689 | >8 | 1 | 2 | 1 | 0.015 | 0.12 | 0.06 | |
8690 | >8 | 2 | 8 | 1 | 0.008 | 0.12 | 0.06 | |
8737 | 4 | 1 | 2 | 1 | 0.015 | 0.12 | 0.03 | |
8737 | >8 | 1 | 0.25 | 1 | 0.008 | 0.12 | 0.03 | |
A. niger SC | 8698 | 4 | 1 | 2 | 1 | 0.008 | 0.12 | 0.008 |
301 | 4 | 1 | 2 | 0.5 | 0.008 | 0.12 | 0.008 | |
A. terreus SC | 8731 | 4 | 2 | >8 | 2 | 0.008 | 0.12 | 0.015 |
There were several important findings derived from this investigation. First, we have documented the excellent in vitro potency for E1210 tested against contemporary isolates of Aspergillus spp., including polyene- and triazole-resistant strains. Second, we have confirmed previous observations (21) regarding the comparability of the CLSI and EUCAST BMD methods (6, 9, 10) when testing the triazoles against Aspergillus spp. Finally, we document the high level of agreement between the CLSI and EUCAST methods for testing E1210 and the echinocandins against Aspergillus spp. In conclusion, E1210 is a potent, novel antifungal agent with impressive activity against both wild-type and antifungal-resistant strains of Aspergillus species. Further development of this new antifungal agent appears warranted.
ACKNOWLEDGMENTS
We thank P. A. Clark and J. D. Feuerbach for their excellent data analysis support for the present study.
This study was sponsored by an educational/research grant from the Eisai Co. (Japan).
Footnotes
Published ahead of print on 15 August 2011.
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