Activities of E1210 and Comparator Agents Tested by CLSI and EUCAST Broth Microdilution Methods against Fusarium and Scedosporium Species Identified Using Molecular Methods

INTRODUCTION

The Fusarium spp. and Scedosporium spp. are among the most antifungal drug-resistant organisms encountered in clinical practice (1, 2, 7, 21, 25, 32, 38). Optimal treatment against infection with these fungal pathogens has not been established, and anecdotal successes have been reported with various agents alone or in combination (4, 12, 14, 18, 20, 23, 26, 33, 34, 40).

Among more than 60 species of Fusarium associated with human infections (24, 35), Fusarium solani is the most frequent (∼50% of cases), followed by F. oxysporum (∼20%) and F. verticilloides (∼10%) (21, 22). F. solani is also the most common species in fusarial keratitis (5, 9, 24, 25). Molecular phylogenetic studies have shown that the more commonly reported fusarial pathogens represent species complexes and cannot be identified to the species level by traditional morphological methods alone (21, 24, 25, 38). DNA sequence-based molecular identification tools are increasingly used to enable accurate species determination (3, 25, 36).

The typical in vitro antifungal susceptibility profile of Fusarium spp. demonstrates relative resistance to most antifungal agents (1, 21, 25, 32). Notably, some species may exhibit different patterns of susceptibility: F. solani and F. verticilloides are usually resistant to azoles and exhibit higher amphotericin B MIC values than other species, whereas F. oxysporum may be susceptible to voriconazole and posaconazole (1, 21, 32). The echinocandins are not active against Fusarium spp. (25).

Fungi of the genus Scedosporium (teleomorph Pseudallescheria) include members of the Pseudallescheria boydii species complex (SC) (Scedosporium aurantiacum, S. apiospermum, and S. boydii) as well as S. prolificans (7, 11, 17). S. apiospermum is generally considered to be resistant to amphotericin B, the MIC values of which are elevated, and the clinical response is very poor despite the use of high-dose regimens (7). The extended-spectrum azoles are active in vitro against S. apiospermum, and both posaconazole and voriconazole have been used in the treatment of central nervous system (CNS) abscesses (18, 20). S. prolificans is considered to be resistant to virtually all of the systemically active antifungal agents, including the extended-spectrum azoles and the echinocandins (7, 32). Synergistic activity between azoles and terbinafine against S. prolificans has been demonstrated in vitro (7), and patients with invasive fungal infection (IFI) due to S. prolificans have been successfully managed with a combination of terbinafine and voriconazole in addition to aggressive surgical debridement and immune reconstitution (4, 12, 14, 34, 40). As with Fusarium spp., the identification of the various species of Scedosporium is best accomplished using molecular identification methods (3, 7, 11, 17, 35).

E1210 (Eisai, Ltd., Japan) is a novel first-in-class broad-spectrum antifungal agent (with activity against yeasts and molds) that was designed to specifically inhibit the function of the protein GWT1, which plays an important role in fungal cell wall assembly (19). E1210 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 (19, 36, 37, 39). Preliminary data using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) method (6) have demonstrated excellent potency and spectrum against various species of Fusarium and Scedosporium (19). These studies were limited by the small number of molecularly identified isolates tested, lack of antifungal comparators, and a lack of comparison of E1210 activity as determined by the reference BMD methods of the CLSI and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) (6, 10).

In the present study, we have used a panel of Fusarium spp. and Scedosporium spp., all of which have been identified by molecular methods, to examine the activities of E1210 and comparator agents as determined by both CLSI and EUCAST BMD methods.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

The molecular identification of 67 Fusarium isolates was performed by TEF sequencing, and analysis was performed using the FUSARIUM-ID and Fusarium MLST databases. ITS and 28S sequencing was performed in some instances to confirm TEF results. Isolates were identified to the SC level, as follows: Gibberella (Fusarium) fujikuroi SC (30 strains), Fusarium solani SC (15 strains), Fusarium oxysporum SC (15 strains), Fusarium dimerum SC (two strains), and Fusarium incarnatum-equisei SC (one strain). Four isolates could not be identified to the SC level and remained as Fusarium spp. “NOS” (not otherwise specified). TEF, ITS and 28S sequences for these four isolates had similarity below 98% to sequences deposited in the international databases.

Scedosporium isolates (63 strains) were identified using ITS and/or 28S sequencing, and among 63 isolates, 28 were Scedosporium apiospermum, 28 were S. prolificans, and 7 were S. aurantiacum. Table 1 summarizes the in vitro susceptibilities of the 130 isolates of Fusarium spp. and Scedosporium spp. to E1210 and six comparators as determined by CLSI and EUCAST BMD methods. Overall, E1210 was highly active against all Fusarium spp. tested (67 strains): MEC_50/90 of 0.06/0.12 μg/ml for the CLSI method and 0.06/0.25 for the EUCAST method (Table 1). The activity of this antifungal agent was higher than the activities noted for anidulafungin, caspofungin, itraconazole, posaconazole, voriconazole, and amphotericin B (MEC/MIC₉₀ values [CLSI and EUCAST] of >8, >8, >8, >8, >8, and 4 μg/ml, respectively).

F. solani SC strains (MEC_50/90 [CLSI], 0.03/0.06 μg/ml) were slightly more susceptible to E1210 than isolates identified as Gibberella fujikuroi SC and F. oxysporum SC (MEC_50/90 [CLSI], 0.03/0.12 and 0.06/0.25 μg/ml, respectively) (Table 1). The highest CLSI E1210 MEC value for F. solani SC was only 0.06 μg/ml, whereas for G. fujikuroi SC and F. oxysporum SC, the values were 0.25 and 0.5 μg/ml, respectively (Table 1). F. dimerum SC and F. incarnatum-equiseti SC had E1210 MEC results of 0.06 μg/ml by the CLSI method and from ≤0.008 to 0.12 μg/ml by the EUCAST method (data not shown).

E1210 was very potent against the Scedosporium species tested (Table 1). E1210 CLSI MEC results ranged from 0.03 to 0.25 μg/ml (MEC₉₀, 0.12 μg/ml) for S. apiospermum (Table 1). With the exception of voriconazole (MIC₉₀, 1 μg/ml), the comparator agents showed limited activity against this species: MIC/MEC₉₀ values were >8 (caspofungin), 4 (anidulafungin), 8 (itraconazole), 4 (posaconazole), and >4 μg/ml (amphotericin B). S. prolificans strains had E1210 CLSI MEC results of 0.03 to 0.12 μg/ml (MEC₉₀, 0.12 μg/ml) (Table 1). Comparator agents displayed limited activity against S. prolificans strains, and MIC/MEC₉₀ values were ≥4 μg/ml for amphotericin B and anidulafungin and >8 μg/ml for caspofungin, itraconazole, posaconazole, and voriconazole (Table 1). The MEC₅₀ for E1210 against S. aurantiacum was 0.06 μg/ml versus 0.5 to >8 μg/ml for the comparators (data not shown). The MIC/MEC₉₀ was not calculated for this species due to the low number of strains tested.

The comparison of CLSI and EUCAST testing conditions demonstrates for the first time that these methods were highly concordant when used to test new and established antifungal agents against Fusarium and Scedosporium spp. (Table 1). The %EA (±2 log₂ dilutions) was >93% for all comparisons, with the exception of posaconazole and F. oxysporum SC (60%) and posaconazole and voriconazole against S. aurantiacum (85.7%; data not shown). Overall, when testing Fusarium spp. (67 isolates), the %EA between CLSI and EUCAST MIC/MEC results were 88.1% for posaconazole, 94.0% for E1210, 95.5% for itraconazole, 98.5% for voriconazole, and 100.0% for caspofungin, anidulafungin, and amphotericin B. Likewise, when testing Scedosporium spp. (63 isolates), the %EA were 96.8% for posaconazole and caspofungin, 98.4% for E1210, itraconazole, and voriconazole, and 100.0% for anidulafungin and amphotericin B (Table 1).

There are several important findings that can be derived from this study. First, we have documented the excellent spectrum and potency of E1210 against molecularly identified strains of Fusarium and Scedosporium species, including polyene-, azole-, and echinocandin-resistant strains. E1210 was the most active of the seven antifungal agents tested against these uncommon, but clinically important opportunistic fungal pathogens. These findings are especially notable for S. prolificans, a species that appears completely resistant to the antifungal agents currently available for the treatment of invasive fungal infections (Table 1). Second, we document for the first time the high level of intermethod agreement between the CLSI and EUCAST reference tests for testing E1210, the echinocandins, the triazoles, and amphotericin B against species of Fusarium and Scedosporium.

E1210 is a potent, novel antifungal agent with impressive in vitro activity against Fusarium and Scedosporium spp. as well as against Aspergillus (19, 29) and Candida spp. (19, 31, 39). In vitro susceptibility testing of E1210 and other mold-active antifungal agents may be accomplished by either of the CLSI or EUCAST broth microdilution methods with comparable results. Further development of this new antifungal agent (E1210) appears warranted.