In Vitro Activities of Isavuconazole and Comparator Antifungal Agents Tested against a Global Collection of Opportunistic Yeasts and Molds

Abstract

Isavuconazole is a new broad-spectrum triazole with a favorable pharmacokinetic and safety profile. We report the MIC distributions for isavuconazole and 111 isolates of Candida (42 Candida albicans, 25 Candida glabrata, 22 Candida parapsilosis, 14 Candida tropicalis, and 8 Candida krusei isolates), as determined by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution (BMD) methods. Also, the relative activities of isavuconazole, itraconazole, fluconazole, posaconazole, voriconazole, and the three echinocandins were assessed against a recent (2011) global collection of 1,358 isolates of Candida spp., 101 of Aspergillus spp., 54 of non-Candida yeasts, and 21 of non-Aspergillus molds using CLSI BMD methods. The overall essential agreement (EA) (±2 log₂ dilutions) between the CLSI and EUCAST methods was 99.1% (EA at ±1 log₂ dilution, 90.1% [range, 80.0 to 100.0%]). The activities of isavuconazole against the larger collection of Candida spp. and Aspergillus spp. were comparable to those of posaconazole and voriconazole; the MIC₉₀ values for isavuconazole, posaconazole, and voriconazole against Candida spp. were 0.5, 1, and 0.25 μg/ml and against Aspergillus spp. were 2, 1, and 1 μg/ml, respectively. Isavuconazole showed good activities against Cryptococcus neoformans (MIC₉₀, 0.12 μg/ml) and other non-Candida yeasts (MIC₉₀, 1 μg/ml) but was less potent against non-Aspergillus molds (MIC₉₀, >8 μg/ml). Isavuconazole MIC values for three mucormycete isolates were 4, 1, and 2 μg/ml, whereas all three were inhibited by 1 μg/ml posaconazole. Isavuconazole demonstrates broad-spectrum activity against this global collection of opportunistic fungi, and the CLSI and EUCAST methods can be used to test this agent against Candida, with highly comparable results.

INTRODUCTION

Isavuconazole (formerly BAL4815) is a new triazole antifungal agent with broad in vitro activity against clinically relevant fungi (1–3). This compound can be administered either orally or intravenously as a prodrug, which then is converted by plasma esterases into the active component (3–6). Previous in vitro studies have demonstrated potent antifungal activity against both common and uncommon fungal pathogens, including Candida, Aspergillus, non-Candida yeasts, and non-Aspergillus molds (7–15). Furthermore, isavuconazole significantly reduced the organism burden in kidneys of mice infected with Candida tropicalis and the organism burden in both kidneys and brains of neutropenic mice infected with Candida krusei (16). It was as effective as voriconazole and much more effective than fluconazole in reducing the brain burden in mice infected with C. krusei (16). Presently, isavuconazole is in late-stage clinical development for the treatment of invasive candidiasis, invasive aspergillosis, and non-Aspergillus molds (3).

The newer triazoles (posaconazole and voriconazole) and the echinocandins (anidulafungin, caspofungin, and micafungin) have markedly expanded the options for prophylactic, empirical, and directed therapy for fungal infections (17, 18). Although resistance to these agents among clinical isolates of opportunistic fungi is considered uncommon, increased resistance and breakthrough infections have been observed among patients with long-term exposure to even the newest antifungal agents (19–25). These observations have prompted a call for enhanced surveillance efforts and an expanded role for antifungal susceptibility testing, especially for Candida and Aspergillus spp. (13, 20, 22, 23, 26, 27).

The in vitro MIC profile of isavuconazole against yeasts and molds has been determined in various studies using the broth microdilution (BMD) method of either the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (7–13, 15); however, only one study provided a “head-to-head” comparison of these two methods for testing isavuconazole and the comparison was limited to testing isavuconazole against Aspergillus flavus (10). To date, there are no studies comparing the activity of isavuconazole against Candida as determined by both CLSI and EUCAST BMD methods. Given the important roles that the two methods play in resistance surveillance for Candida, it is important to demonstrate the comparability of isavuconazole MIC values for Candida spp. determined by the two methods.

In the present study, we examine the in vitro activities of isavuconazole and comparator antifungal agents against 1,534 clinical fungal isolates (1,358 isolates of Candida spp., 101 of Aspergillus spp., 54 of non-Candida yeasts, and 21 of non-Aspergillus molds) collected in 2011 from bloodstream infections, normally sterile sites, and respiratory tract specimens, as part of the global SENTRY Antimicrobial Surveillance Program. This study is unique in several respects. First, we provide a direct comparison of isavuconazole MIC values determined by the CLSI and EUCAST BMD methods for a subset of 111 Candida isolates. Second, we used molecular methods to confirm the identification of the less common species of Candida, as well as the non-Candida yeasts and all of the filamentous fungi. Finally, we applied the newly revised clinical breakpoints (CBPs) for the echinocandins, fluconazole, and voriconazole to determine the resistance profiles of various Candida species (27) and used the epidemiological cutoff values (ECVs) for these agents, as well as itraconazole and posaconazole, to detect emerging resistance among less common species of Candida (27) and among Aspergillus isolates (28).

MATERIALS AND METHODS

Organisms and sources.

A total of 1,573 nonduplicate fungal strains were collected prospectively from 75 medical centers located in North America (30 sites, 733 strains), Europe (24 sites, 498 strains), Latin America (10 sites, 176 strains), and the Asia-Pacific region (11 sites, 166 strains). These strains were recovered consecutively from patients with bloodstream infections (1,042 strains), from normally sterile body fluids, tissues, or abscesses (163 strains), from respiratory tract specimens (215 strains), or from unspecified infection sites (153 strains).

Isolates were identified at participant institutions using methods routinely employed at the submitting laboratory, including the use of Vitek, MicroScan, API strips, and AuxaColor systems supplemented by classic methods for yeast and mold identification (29, 30). Isolates were submitted to JMI Laboratories (North Liberty, IA), where the identification was confirmed by morphological, biochemical, and molecular methods (31–34). Yeast isolates were subcultured and screened using CHROMagar Candida (Becton, Dickinson, Sparks, MD) to ensure purity and to differentiate Candida albicans/Candida dubliniensis, Candida tropicalis, and Candida krusei. Biochemical tests including Vitek 2 (bioMérieux, Hazelwood, MO) testing, trehalose assimilation (Candida glabrata), and growth at 45°C (C. albicans/C. dubliniensis) also were used to establish the identification of common Candida species. Molecular methods were used for common species of Candida that could not be definitively identified using phenotypic methods or that presented unusual phenotypic or biochemical profiles, as well as all uncommon species of Candida, non-Candida yeasts, and all molds. Candida spp. and other yeasts were identified using sequence-based methods for the internal transcribed spacer (ITS) region, 28S ribosomal subunit (D1/D2), and intergenic spacer (IGS) (Debaryomyces spp.) and IGS1 (Trichosporon spp.) (31, 34). All mold isolates were subcultured and analyzed by ITS sequencing followed by specific molecular species identification within genera, i.e., β-tubulin for Aspergillus spp., translation elongation factor (TEF) for Fusarium spp., and 28S subunit for all other genera of molds (34). Nucleotide sequences were examined using Lasergene software (DNA Star, Madison, WI) and then compared to database sequences using BLAST (http://blast.ncbi.nlm.nih.gov). Fusarium isolates were analyzed for TEF sequences using the Fusarium-ID database (http://www.isolate.fusariumdb.org/index.php) and the Fusarium multilocus sequence typing (MLST) database (http://www.cbs.knaw.nl/fusarium/BiolomicsInfo.aspx) (34).

Among the 1,364 isolates of Candida, there were 578 isolates of C. albicans, 266 of C. glabrata, 262 of C. parapsilosis, 130 of C. tropicalis, 45 of C. krusei, and 83 of miscellaneous Candida spp. (28 Candida lusitaniae, 22 C. dubliniensis, 11 Candida kefyr, eight Candida guilliermondii, five Candida fermentati, three Candida intermedia/pseudointermedia, two Candida lipolytica, and one each of Candida catenulata, Candida haemulonii, Candida inconspicua, and Candida pararugosa). The collection also included Cryptococcus neoformans (46 isolates), Trichosporon spp. (eight isolates, including three Trichosporon asahii isolates, one Trichosporon ovoides isolate, and four Trichosporon isolates not identified at the species level), Lodderomyces elongisporus (two isolates), Saccharomyces cerevisiae (two isolates), and one isolate each of Debaryomyces fabryi, Dipodascus capitatus, Kodamaea ohmeri, Rhodotorula mucilaginosa, Rhodotorula sp., and a yeast not identified at the species level. Molds included 71 isolates of Aspergillus fumigatus, 33 miscellaneous Aspergillus spp. (11 Aspergillus niger species complex [SC], 10 Aspergillus flavus SC, six Aspergillus terreus SC, three Aspergillus sydowii, one Aspergillus foetidus, and two Aspergillus not identified at the species level), and 41 other molds (eight Penicillium spp., four Paecilomyces spp., three Gibberella fujikuroi SC, three Sarocladium spp., three Scedosporium apiospermum, two Fusarium solani SC, two Rhizopus microsporus group, two Alternaria spp., two Cladosporium spp., two Coprinellus radians, and one each of Cochliobolus lunatus, Coprinopsis cinerea, Curvularia trifolii, Exophiala dermatitidis, Geosmithia argillacea, Phanerochaete chrysosporium, Rhizomucor pusillus, Curvularia sp., Trichoderma sp., and an unidentified mold).

Antifungal susceptibility testing.

All yeast isolates were tested for in vitro susceptibility to isavuconazole (11 dilutions; range, 0.008 to 8 μg/ml), posaconazole (11 dilutions; range, 0.008 to 8 μg/ml), voriconazole (11 dilutions; range, 0.008 to 8 μg/ml), fluconazole (12 dilutions; range, 0.06 to 128 μg/ml), anidulafungin (12 dilutions; range, 0.008 to 16 μg/ml), caspofungin (12 dilutions; range, 0.008 to 16 μg/ml), and micafungin (12 dilutions; range, 0.008 to 16 μg/ml) using CLSI BMD methods (35). MIC results for all agents were read following 24 h of incubation at 35°C in testing against Candida spp., whereas MIC results were read after 48 h in testing against non-Candida yeasts. In all instances, the MIC values were determined visually as the lowest concentrations of drug that caused significant (≥50%) growth inhibition (35, 36).

A subset of 111 isolates of Candida spp. (randomly selected to represent the five major species, i.e., C. albicans [42 isolates], C. glabrata [25 isolates], C. parapsilosis [22 isolates], C. tropicalis [14 isolates], and C. krusei [8 isolates]) were tested against isavuconazole using EUCAST BMD methods as outlined in EUCAST document EDef 7.1 (37), using RPMI 1640 medium with 2.0% glucose, inocula of 0.5 × 10⁵ to 2.5 × 10⁵ cells/ml, and incubation at 35°C. Isavuconazole MIC values were determined using a spectrophotometer (at 530 nm) after 24 h of incubation, as the lowest concentrations of drug that resulted in ≥50% inhibition of growth relative to the growth control. The MIC results for isavuconazole obtained with the EUCAST method were compared with those obtained with the CLSI method. High off-scale MIC results were converted to the next highest concentration, and low off-scale MIC results were left unchanged. Discrepancies of more than ±1 log₂ dilution and more than ±2 log₂ dilutions were used to calculate essential agreement (EA) between the two BMD methods for testing isavuconazole.

In vitro susceptibility testing of Aspergillus spp. and other molds against isavuconazole, the other mold-active triazoles (itraconazole, posaconazole, and voriconazole), and the echinocandins (anidulafungin, caspofungin, and micafungin) was performed with BMD methods as described in CLSI document M38-A2 (38). The triazole MICs and the echinocandin minimum effective concentrations (MECs) were determined as described in the CLSI reference method (38).

We used the recently revised CLSI CBP values to identify strains of the five most common species of Candida (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei) resistant to the echinocandins, fluconazole, and voriconazole (27). Anidulafungin, caspofungin, and micafungin MIC values of >0.5 μg/ml were considered to indicate resistance to C. albicans, C. tropicalis, and C. krusei, and MIC results of >4 μg/ml were categorized as resistant for C. parapsilosis; anidulafungin and caspofungin MIC values of >0.25 μg/ml and micafungin MIC values of >0.12 μg/ml were considered resistant for C. glabrata. Fluconazole MIC results of >4 μg/ml were defined as resistant for C. albicans, C. parapsilosis, and C. tropicalis, and MICs of >32 μg/ml were considered resistant for C. glabrata. All isolates of C. krusei were defined as resistant to fluconazole. The CLSI resistance breakpoint for voriconazole is >0.5 μg/ml for C. albicans, C. tropicalis, and C. parapsilosis, and MIC results of >1 μg/ml were categorized as resistant for C. krusei; CLSI has not assigned CBPs for voriconazole and C. glabrata and recommends the ECV of 0.5 μg/ml be used to differentiate wild-type (WT) from non-WT strains of this species (27).

CBPs have not been established for any antifungal agent and the less common species of Candida, non-Candida yeasts, Aspergillus spp., or the non-Aspergillus molds; however, ECVs have been established for echinocandins and triazoles and six species of Candida that are encountered less frequently (C. lusitaniae, C. guilliermondii, C. dubliniensis, C. kefyr, C. orthopsilosis, and C. pelliculosa) (27). ECVs also have been developed for A. fumigatus, A. flavus, A. terreus, and A. niger and itraconazole, posaconazole, and voriconazole (28); itraconazole and voriconazole MIC values of >1 μg/ml were considered non-WT for A. fumigatus, A. flavus, and A. terreus, and itraconazole and voriconazole MIC values of >2 μg/ml were considered non-WT for A. niger. Posaconazole MIC values of >0.5 μg/ml were considered non-WT for A. fumigatus, A. terreus, and A. niger, and MIC results of >0.25 μg/ml were considered non-WT for A. flavus. Isolates of these Aspergillus spp. for which triazole MIC results exceed the ECV are considered to be non-WT and may harbor acquired mutations in the cyp51A gene (39). Quality control was performed as recommended in CLSI documents M27-A3 (35) and M38-A2 (38), using C. krusei ATCC 6258, C. parapsilosis ATCC 22019, A. flavus ATCC 204304, and A. fumigatus MYA-3626.

RESULTS AND DISCUSSION

Comparison of CLSI and EUCAST BMD methods for testing isavuconazole against Candida spp.

Table 1 summarizes the in vitro susceptibilities of 111 Candida isolates to isavuconazole determined with the CLSI and EUCAST BMD methods. The MIC₉₀ values determined with the two methods for each species were comparable to those published previously and demonstrated excellent coverage of these common species by isavuconazole (3). The EA (±2 log₂ dilutions) between the two reference methods was 99.1% for isavuconazole across all five Candida spp. (range, 96.0 to 100.0%) (Table 1). Good agreement between the two methods also was observed using the more-restrictive criterion (±1 log₂ dilution) for EA (overall rate, 90.1% [range, 80.0 to 100.0%]) (Table 1). The MIC values generated with the EUCAST method tended to be slightly higher than those obtained with the CLSI method for most species. These results demonstrate a high level of concordance between the isavuconazole MIC results produced with the two reference methods in testing of Candida spp.

Table 1.

In vitro susceptibilities of Candida spp. to isavuconazole, as determined with CLSI and EUCAST broth microdilution methods

Species	BMD methoda	MIC (μg/ml)b			EA (%) atc:
		Range	50%	90%	±1 dilution	±2 dilutions
C. albicans (n = 42)	CLSI	≤0.008–0.03	0.015	0.015	100.0	100.0
	EUCAST	≤0.008–0.015	≤0.008	0.015
C. glabrata (n = 25)	CLSI	0.12–8	0.5	1	80.0	96.0
	EUCAST	0.12–4	1	2
C. parapsilosis (n = 22)	CLSI	≤0.008–0.06	0.03	0.06	86.4	100.0
	EUCAST	≤0.008–0.12	0.06	0.06
C. tropicalis (n = 14)	CLSI	0.03–0.5	0.06	0.5	85.7	100.0
	EUCAST	0.015–1	0.06	0.5
C. krusei (n = 8)	CLSI	0.25–0.5	0.25	NDd	87.5	100.0
	EUCAST	0.25–1	0.5	ND
Total (n = 111)	CLSI	≤0.008–8	0.03	0.5	90.1	99.1
	EUCAST	≤0.008–4	0.03	1

^aCLSI, Clinical and Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing.

^b50% and 90%, MIC values that encompass 50% and 90% of the isolates tested, respectively.

^cEA, essential agreement of MIC values within 1 log₂ or 2 log₂ dilutions.

^dND, not determined due to the number of isolates (<10 isolates).

In vitro activities of isavuconazole and comparators tested against Candida spp.

Table 2 shows the MIC distributions for isavuconazole and six comparator antifungal agents against 1,358 isolates of Candida encompassing 11 different species, as determined with 24-h CLSI BMD methods. Results for species with two or fewer isolates in the surveillance program are not shown. The vast majority of isolates for each species of Candida, with the exception of C. glabrata, were inhibited by ≤1 μg/ml isavuconazole, posaconazole, and voriconazole. The overall MIC₉₀ values for these 3 azoles were 0.5, 1, and 0.25 μg/ml, respectively. Similarly, most species except C. parapsilosis and C. guilliermondii were inhibited by ≤0.5-μg/ml levels of each of the echinocandins.

Table 2.

MIC distributions for isavuconazole and six comparator antifungal agents tested against Candida spp. using CLSI broth microdilution methods

Species (n)	Antifungal agenta	No. of isolates with an MIC (μg/ml) of:
		≤0.008	0.015	0.03	0.06	0.12	0.25	0.5	1	2	4	8	16	32	64	128
C. albicans (578)	ISA	222	312	40	2	1							1
	PSC	10	95	286	163	21	2						1
	VRC	519	51	6		1							1
	FLC				20	351	187	12	6	1						1
	ANF	70	170	188	123	23	1	3
	CSF	5	131	342	89	7	2		1	1
	MCF	54	376	144	2				2
C. glabrata (266)	ISA			3	11	24	57	91	50	12	10	8
	PSC				1	1	7	26	119	85	11	2	14
	VRC	1		5	34	94	82	20	6	10	9	5
	FLC							2	3	25	57	112	36	6	5	20
	ANF		1	34	166	58	4	1	1	1
	CSF		4	186	60	11	3	1	1
	MCF	43	201	17	2	1		1	1
C. parapsilosis (262)	ISA		9	55	107	70	11	4	4				2
	PSC		3	45	151	54	8	1
	VRC	85	143	17	10	4	2	1
	FLC					4	53	158	28	12	2	2	2			1
	ANF			1		1	4	22	67	137	30
	CSF			1	6	27	153	66	9
	MCF				1	4	6	39	166	46
C. tropicalis (130)	ISA	1	10	42	47	19	4	4	2		1
	PSC	1	5	46	57	13	6	2
	VRC	14	41	49	8	11	4	1	1
	FLC					17	60	35	5	6	4	1		1	1
	ANF	14	41	56	13	4	2
	CSF	2	26	77	20	3	2
	MCF	4	37	65	23	1
C. krusei (45)	ISA				2	3	12	19	9
	PSC					2	14	25	4
	VRC					8	29	7		1
	FLC												11	27	6	1
	ANF			16	24	5
	CSF				16	12	17
	MCF		1		15	29
C. lusitaniae (28)	ISA		9	12	5	2
	PSC		6	3	14	4	1
	VRC	18	8	2
	FLC				1	4	12	6	3	2
	ANF			1	1	1	7	14	3	1
	CSF		1	1	2	6	15	3
	MCF			3	4	6	11	3	1
C. dubliniensis (22)	ISA	8	9	4	1
	PSC	1	8	11	2
	VRC	19	2			1
	FLC				7	10	4						1
	ANF		1	6	8	6	1
	CSF			7	13	2
	MCF	1	3	13	5
C. kefyr (11)	ISA	2	8	1
	PSC		1	2	5	3
	VRC	7	4
	FLC					1	6	4
	ANF			2	5	1	1	1	1
	CSF	2	6	2	1
	MCF			6	2	1		1	1
C. guilliermondii (8)	ISA						3	3	1		1
	PSC					3	3	2
	VRC				6		1		1
	FLC									5	1		1		1
	ANF						1		1	5	1
	CSF				2	1	1	4
	MCF					1	2	1	3	1
C. fermentati (5)	ISA					2	2		1
	PSC			1	2	2
	VRC			3	1	1
	FLC								3	2
	ANF							3	1	1
	CSF				1	3	1
	MCF						3	2
C. intermedia/pseudointermedia (3)	ISA	2			1
	PSC		2	1
	VRC	2		1
	FLC					2		1
	ANF	1		2
	CSF			3
	MCF		2		1

^aISA, isavuconazole; PSC, posaconazole; VRC, voriconazole; FLC, fluconazole; ANF, anidulafungin; CSF, caspofungin; MCF, micafungin.

Resistance to fluconazole was uncommon among these incident isolates of Candida, ranging from 0.3% (C. albicans) to 9.4% (C. glabrata) (Table 2). Cross-resistance between isavuconazole and one or more of the other triazoles was noted for one isolate each of C. albicans, C. parapsilosis, C. tropicalis, and C. guilliermondii. Isolates of C. glabrata that were resistant to fluconazole generally showed non-WT MIC values for voriconazole (MIC, >0.5 μg/ml) and posaconazole (MIC, >2 μg/ml) and MIC results of >1 μg/ml for isavuconazole. The intrinsically fluconazole-resistant species C. krusei was inhibited by ≤1-μg/ml levels of isavuconazole, posaconazole, and voriconazole. Among the less common species of Candida shown in Table 2, all except C. guilliermondii and C. fermentati were inhibited by ≤0.12 μg/ml isavuconazole. In general, isavuconazole was slightly less potent than voriconazole but more potent than posaconazole against this contemporary (2011) collection of Candida spp.

The MIC profiles for the echinocandins were representative of susceptible or WT distributions for the species of Candida shown in Table 2. Among the 578 isolates of C. albicans, 99.5% were susceptible to anidulafungin (MIC, ≤0.25 μg/ml) and 99.7% were susceptible to both caspofungin (MIC, ≤0.25 μg/ml) and micafungin (MIC, ≤0.25 μg/ml). Two isolates of C. albicans were resistant to both caspofungin and micafungin and showed intermediate resistance to anidulafungin (MIC, 0.5 μg/ml); both isolates were shown to have an fks resistance mutation (data not shown). Isolates of C. glabrata also were quite susceptible to the echinocandins (range, 97.3 to 98.9%); however, three isolates were resistant to anidulafungin (MIC, >0.25 μg/ml) and either showed intermediate resistance (one isolate; micafungin MIC, 0.12 μg/ml; caspofungin MIC, 0.25 μg/ml) or were resistant (two isolates; micafungin MIC, >0.12 μg/ml; caspofungin MIC, >0.25 μg/ml) to micafungin and caspofungin. All three isolates contained an fks resistance mutation (data not shown). Echinocandin resistance was not detected among the remaining species of Candida with the exception of C. kefyr, for which two isolates were non-WT for both anidulafungin (MIC, >0.25 μg/ml) and micafungin (MIC, >0.12 μg/ml). Both isolates contained a mutation in fks (data not shown). These results confirm the excellent spectrum and potency of the echinocandins versus Candida spp. and also illustrate that in vitro susceptibility test results are predictive of fks resistance mutations using either CLSI CBPs or ECVs.

In vitro activities of isavuconazole and comparators tested against Aspergillus spp.

The MIC distributions for isavuconazole, the other mold-active triazoles, and the echinocandins for five species of Aspergillus are shown in Table 3. In general, the MIC distributions for agents in both classes conformed to the WT distributions described previously (28, 40). Isavuconazole MIC results were ≤2 μg/ml for all species except A. niger SC, for which MIC values of ≥4 μg/ml were determined for 27% of the isolates tested, similar to findings reported by Guinea and colleagues (7). One isolate of A. fumigatus for which the itraconazole MIC was 2 μg/ml (non-WT) also was found to exhibit an elevated MIC for isavuconazole (4 μg/ml), suggesting cross-resistance. Overall, the in vitro activity of isavuconazole against Aspergillus spp. (MIC_50/90, 1/2 μg/ml) was most comparable to that of itraconazole (MIC_50/90, 1/1 μg/ml), and isavuconazole was less active than either posaconazole (MIC_50/90, 0.5/1 μg/ml) or voriconazole (MIC_50/90, 0.5/1 μg/ml).

Table 3.

MIC/MEC distributions for isavuconazole and six comparator antifungal agents tested against Aspergillus spp. using CLSI broth microdilution methods

Species (n)	Antifungal agenta	No. of isolates with an MIC/MEC (μg/ml) of:
		≤0.008	0.015	0.03	0.06	0.12	0.25	0.5	1	2	4	≥8
A. fumigatus (71)	ISA							6	48	16	1
	ITR							7	63	1
	PSC					2	21	40	8
	VRC						20	46	5
	ANF	1	9	48	12	1
	CSF		4	44	23
	MCF	10	39	21	1
A. niger (11)	ISA									8	2	1
	ITR								6	5
	PSC							5	6
	VRC							5	4	2
	ANF	6	3	2
	CSF		3	4	4
	MCF	5	3	3
A. flavus (10)	ISA								1	8	1
	ITR								10
	PSC						3	5	1
	VRC							3	7
	ANF	6	1	3
	CSF		6	4
	MCF	4	4	2
A. terreus SC (6)b	ISA					1		3	2
	ITR							2	4
	PSC						3	3
	VRC							5	1
	ANF		3	2		1
	CSF		4	1			1
	MCF	4	1			1
A. sydowii (3)	ISA						1		1	1
	ITR								1	2
	PSC							1	2
	VRC								2	1
	ANF		1	1	1
	CSF		1	2
	MCF		1	2

^aISA, isavuconazole; ITR, itraconazole; PSC, posaconazole; VRC, voriconazole; ANF, anidulafungin; CSF, caspofungin; MCF, micafungin.

^bSC, species complex.

In vitro activities of isavuconazole and comparators tested against non-Candida yeasts and non-Aspergillus molds.

The MIC distributions for isavuconazole and the triazole and echinocandin comparators for the non-Candida yeasts and non-Aspergillus molds for which identification was confirmed by molecular methods are shown in Table 4. Given that these species are uncommon in most regions of the world and data concerning their in vitro susceptibilities to most antifungals remain limited, we elected to present these MIC distributions despite the small number of isolates tested. The 46 isolates of C. neoformans all exhibited WT susceptibilities to fluconazole (MIC, ≤8 μg/ml), posaconazole (MIC, ≤0.25 μg/ml), and voriconazole (MIC, ≤0.12 μg/ml) (Table 4) (41). As expected, the echinocandins were inactive against both C. neoformans and Trichosporon spp. encountered in the 2011 SENTRY Program. In contrast, the triazoles, including isavuconazole, showed potent activity against both Trichosporon and Cryptococcus yeasts.

Table 4.

MIC distributions for isavuconazole and comparator antifungal agents tested against non-Candida yeasts and non-Aspergillus molds using CLSI broth microdilution methods

Species (n)	Antifungal agenta	No. of isolates with an MIC/MEC (μg/ml) of:
		≤0.008	0.015	0.03	0.06	0.12	0.25	0.5	1	2	4	8	16	32	64	128
Cryptococcus neoformans (46)	ISA		4	21	17	4
	PSC			4	17	22	2	1
	VRC	2	13	17	13	1
	FLC						1	3	13	16	11	2
	ANF											1		45
	CSF											13	31	2
	MCF													46
Trichosporon spp. (8)b	ISA				2	1	2	2	1
	PSC				2	1	3	2
	VRC			2	4	1		1
	FLC							1	2							5
	ANF												1	7
	CSF											3	5
	MCF													8
Penicillium spp. (8)c	ISA		1		1	1	2	1	2
	ITR						1	4	2	1
	PSC					1	4	2	1
	VRC							5	1	1			1
	ANF	2	1	2	2	1
	CSF		3	1	2	1			1
	MCF	1	2	2	1
Paecilomyces spp. (4)d	ISA								3	1
	PCS						1	1	2
	VRC						1	2		1
	ANF			1	1	2
	CSF			1	1	2
	MCF		1	1	2
Gibberella fujikuroi SC (3)	ISA			1								1	1
	PSC						1				1		1
	VRC		1							1			1
	ANF										1			2
	CSF												1	2
	MCF												3
Sarocladium spp. (3)e	ISA										1	1	1
	PSC									2	1
	VRC								1	1	1
	ANF	1	1							1
	CSF		2									1
	MCF	1		1									1
Scedosporium apiospermum (3)	ISA						1			2
	PSC							1	1	1
	VRC					1		2
	ANF								1	1	1
	CSF						1	1					1
	MCF							2						1

^aISA, isavuconazole; ITR, itraconazole; PSC, posaconazole; VRC, voriconazole; FLC, fluconazole; ANF, anidulafungin; CSF, caspofungin; MCF, micafungin.

^bIncludes T. asahii (three isolates), T. ovoides (one isolate), and Trichosporon not identified at the species level (four isolates).

^cIncludes Penicillium subgenus Aspergilloides (three isolates) and one isolate each of Penicillium citrinum, Penicillium roqueforti, Penicillium subgenus Furcatum, Penicillium subgenus Terverticillata, and Penicillium not identified at the species level.

^dIncludes Paecilomyces lilacinus (three isolates) and Paecilomyces variotii (one isolate).

^eIncludes Sarocladium kiliense (two isolates) and Sarocladium bacillisporum (one isolate).

Among the non-Aspergillus molds, isavuconazole, posaconazole, and voriconazole all were moderately active (MIC, ≤2 μg/ml) against Penicillium spp., Paecilomyces spp., and Scedosporium apiospermum and were less active against Gibberella fujikuroi SC and Sarocladium spp. (Table 4). Isavuconazole MIC results for three mucormycete isolates (Rhizomucor pusillus [one isolate] and Rhizopus microsporus group [two isolates]) were 4, 1, and 2 μg/ml, whereas all three isolates were inhibited by 1 μg/ml posaconazole (data not shown). The echinocandins showed good activity (MEC, ≤0.06 μg/ml) against isolates of Penicillium spp., Paecilomyces spp., and Sarocladium spp.

Conclusions.

Several important observations can be made from this global survey. First, we have documented excellent agreement between the CLSI and EUCAST BMD methods for isavuconazole tested against Candida spp. The EA (±2 log₂ dilutions) of 99.1% (EA at ±1 log₂ dilution, 90.1% [range, 80.0 to 100.0%]) compares favorably with that observed for isavuconazole with these two methods in testing against Aspergillus flavus (98.9%), as reported by Rudramurthy and colleagues (10). Second, we have used the new CBPs and ECVs for the triazole and echinocandin antifungal agents to demonstrate the generally low levels of resistance in a large collection of molecularly characterized isolates of Candida and Aspergillus. Furthermore, the isolates of Candida for which the echinocandin MIC values exceeded the thresholds defined by the CBPs or ECVs possessed resistance mutations in hot spot regions of the fks genes. Finally, we both confirm and extend the literature documenting the potency and spectrum of the newest triazole, isavuconazole, tested against a global collection of opportunistic fungal pathogens. This broad spectrum of activity suggests that isavuconazole might be a useful addition to the antifungal armamentarium.