In vitro activity of rezafungin against common and rare Candida species and Saccharomyces cerevisiae.

BACKGROUND: Rezafungin is a novel echinocandin with excellent activity against common Candida species; however, limited data are available regarding rare Candida species.
METHODS: We determined the in vitro susceptibility of 689 clinical isolates of 5 common and 19 rare Candida species, as well as Saccharomyces cerevisiae. The activity of rezafungin was compared with that of anidulafungin, caspofungin, micafungin, amphotericin B and fluconazole, using CLSI broth microdilution methodology (Fourth Edition: M27).
RESULTS: Rezafungin MIC90 values were 0.06 mg/L for Candida albicans (n=125), Candida tropicalis (n=51), Candida dubliniensis (n=22), Candida inconspicua (n=41), Candida sojae (n=10), Candida lipolytica (n=10) and Candida pulcherrima (n=10), 0.12 mg/L for Candida glabrata (n=81), Candida krusei (n=53), Candida kefyr (n=52) and Candida fabianii (n=15), 0.25 mg/L for Candida lusitaniae (n=46) and Candida auris (n=19), 0.5 mg/L for Candida metapsilosis (n=15) and S. cerevisiae (n=21), 1 mg/L for Candida orthopsilosis (n=15) and Candida guilliermondii (n=27) and 2 mg/L for Candida parapsilosis sensu stricto (n=59). Caspofungin MIC90 values were 0.25-2 mg/L for all species, while micafungin and anidulafungin MIC90 values were similar to those of rezafungin. Fluconazole resistance was found in C. albicans (5.6%) and C. glabrata (4.9%); rezafungin was effective against these isolates as well. Amphotericin B MIC values did not exceed 2 mg/L.
CONCLUSIONS: Rezafungin showed excellent in vitro activity against both WT and azole-resistant Candida species, as well as against S. cerevisiae. Rezafungin had similar activity to other echinocandins (excluding caspofungin) against common Candida species and, notably, against clinically relevant uncommon Candida species.

Introduction

Rezafungin is a novel, once-weekly echinocandin in development for treatment of candidaemia and invasive candidiasis, with a Phase 3 trial currently under way, and for prophylaxis against invasive fungal infections caused by Candida, Aspergillus and Pneumocystis spp. in patients undergoing blood or marrow transplantation., Rezafungin has excellent in vitro activity, comparable to that of other echinocandins, against the five most frequently isolated Candida species (Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis sensu stricto and Candida krusei) using either CLSI or EUCAST broth microdilution (BMD) methodologies. However, limited data are available regarding activity against less common Candida species. Therefore, our study aimed at determining the in vitro susceptibility of 668 clinical isolates of 5 common and 19 rare Candida species, as well as 21 clinical isolates of Saccharomyces cerevisiae. The activity of rezafungin was compared with that of five licensed systemic antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole and amphotericin B).

Materials and methods

Isolates

The vast majority of 689 non-duplicate clinical isolates were collected between January 2005 and December 2018 in our diagnostic laboratory. All C. albicans, C. glabrata, C. tropicalis, C. parapsilosis sensu stricto, C. krusei, Candida rugosa, Candida catenulata, Lodderomyces elongisporus and S. cerevisiae isolates were derived from normally sterile body sites (blood, cerebrospinal, pleural and peritoneal fluids, deep wounds, etc.). At least one-third of the isolates were cultured from normally sterile body sites in the case of the other Candida species as well. In addition, 14 ATCC and type strains were also tested (Table 1). Clinical isolates were obtained prior to antifungal administration and identified with MALDI Biotyper (Bruker, Bremen, Germany) and/or PCR ribotyping.

Table 1.

MIC values of rezafungin and comparator antifungal agents for Candida ATCC and type strains

ATCC and type strains	MIC (mg/L)
	RZF	ANF	CAS	MCF	FLC	AMB
C. krusei ATCC 6258	0.03–0.06	0.03–0.06	0.5–1	0.12–0.25	8–32	0.5–1
C. parapsilosis ATCC 22019	0.5–1	0.5–2	0.5–1	1–2	0.5–1	0.25–1
C. albicans ATCC 10231	0.03	0.015	0.25	0.008	0.25	0.25
C. glabrata ATCC 90030	0.06	0.0015	0.5	0.03	>32	0.5
C. tropicalis ATCC 750	0.06	0.015	0.12	0.03	1	1
C. dubliniensis CD36	0.03	0.015	0.06	0.015	0.25	0.25
C. auris ATCC 21092	0.06	0.03	0.25	0.12	0.5	0.5
C. inconspicua ATCC 16783	0.06	0.008	0.12	0.03	32	0.5
C. orthopsilosis ATCC 96139	2	1	1	0.5	0.5	0.5
C. metapsilosis ATCC 96144	0.5	0.5	0.25	0.25	1	0.5
C. africana ATCC 2669	0.03	≤0.004	0.12	0.06	0.12	0.25
C. sojae CBS 7871	0.06	≤0.004	0.25	0.015	0.25	0.12
C. rugosa ATCC 2142	0.03	0.06	0.12	0.12	1	0.25
C. guilliermondii ATCC 6260	1	0.5	0.5	2	1	0.5

RZF, rezafungin; ANF, anidulafungin; CAS, caspofungin; MCF, micafungin; FLC, fluconazole; AMB, amphotericin B.

Antifungal susceptibility testing

All isolates were tested by BMD according to CLSI in RPMI-1640 (Sigma, Budapest, Hungary). MIC assays were conducted in U-bottom, tissue culture-treated microtitre test plates (TPP Techno Plastic Products AG, Switzerland; cat. no. 92097). Rezafungin pure powder was provided by Cidara Therapeutics, Inc. (San Diego, CA, USA). Caspofungin, micafungin and anidulafungin were obtained from Molcan Corporation (Richmond Hill, Ontario, Canada). Amphotericin B and fluconazole were purchased from Sigma (Budapest, Hungary). The concentration ranges tested were 0.004–2 mg/L for rezafungin, anidulafungin, caspofungin and micafungin, 0.015–8 mg/L for amphotericin B and 0.06–32 mg/L for fluconazole. In cases of the ‘psilosis’ group and Candida guilliermondii, the ranges for all four echinocandins were 0.015–8 mg/L.

Neither a clinical breakpoint nor an epidemiological cut-off value (ECV) has been published for rezafungin against Candida species. The revised species-specific CLSI clinical breakpoints were used in cases of C. albicans, C. glabrata, C. parapsilosis sensu stricto, C. tropicalis, C. krusei and C. guilliermondii for anidulafungin, caspofungin, micafungin and fluconazole (except C. guilliermondii for fluconazole, where an ECV of ≤8 mg/L was used)., The previously established ECVs were used for anidulafungin, caspofungin, micafungin and fluconazole for Candida kefyr, Candida lusitaniae, Candida dubliniensis and Candida orthopsilosis. For amphotericin B, an ECV of ≤2 mg/L was used for the previously mentioned Candida species., In the case of Candida auris for anidulafungin, caspofungin, micafungin, amphotericin B and fluconazole, we used the tentative MIC breakpoints as suggested by the CDC. For other Candida species and S. cerevisiae neither clinical breakpoints nor ECVs exist, thus only MIC50/90 values are shown.

Quality control strains C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were included on each day of testing (25 different days).

Results

MIC values for the 14 ATCC and type strains are shown in Table 1. MIC values of the six antifungal agents for the quality control strains were always within the accepted ranges., MIC values of rezafungin also fell always within the 24 h quality control ranges tentatively accepted by CLSI (January 2018 Subcommittee on Antifungal Susceptibility Tests meeting; C. krusei ATCC 6258, 0.015–0.12 mg/L; C. parapsilosis ATCC 22019, 0.25–1 mg/L).

Activity of echinocandins against Candida species and S. cerevisiae isolates

Rezafungin inhibited all C. albicans isolates at ≤0.12 mg/L, similarly to anidulafungin and micafungin, but only 70.4% were susceptible to caspofungin (Table 2). Susceptibility patterns were similar to the closely related C. dubliniensis and Candida africana isolates, as well as to C. tropicalis (Table 2).

Table 2.

Activity of rezafungin and comparator antifungal agents against Candida species and S. cerevisiae clinical isolates

Species (n)	Drug	MIC (mg/L)				Susceptibility (%)			Percentage of MICs above ECV
		range	mode	MIC50	MIC90	S	I/SDD	R
C. albicans (125)	RZF	0.008–0.12	0.03	0.03	0.06
	ANF	≤0.004–0.06	≤0.004	0.008	0.03	100
	CAS	0.06–0.5	0.25	0.25	0.5	70.4	29.6
	MCF	≤0.004–0.25	0.015	0.015	0.06	100
	FLC	0.12 to >32	0.12	0.12	0.25	94.4		5.6
	AMB	0.12–2	0.5	0.5	1
C. glabrata (81)	RZF	0.06–0.25	0.06	0.06	0.12
	ANF	0.008–0.12	0.03	0.03	0.06	100
	CAS	0.12–1	0.5	0.5	0.5	4.9	32.1	63
	MCF	0.008–0.06	0.015	0.03	0.06	100
	FLC	0.25 to >32	4	2	16		95.1	4.9
	AMB	0.25–2	0.5	0.5	1
C. parapsilosis (59)	RZF	0.5–2	1	1	2
	ANF	0.25–2	1	1	2	100
	CAS	0.25–2	1	1	2	100
	MCF	0.5–2	2	2	2	100
	FLC	0.12–4	0.5	0.5	1	96.6	3.4
	AMB	0.12–1	0.5	0.5	1
C. tropicalis (51)	RZF	0.015–0.12	0.06	0.06	0.06
	ANF	≤0.004–0.06	0.03	0.015	0.03	100
	CAS	0.03–0.5	0.25	0.25	0.5	70.6	29.4
	MCF	0.015–0.12	0.03	0.03	0.06	100
	FLC	0.06–0–5	0.25	0.25	0.5	100
	AMB	0.25–1	0.5	0.5	1
C. krusei (53)	RZF	0.06–0.12	0.06	0.06	0.12
	ANF	0.015–0.25	0.06	0.06	0.12	100
	CAS	0.12–1	1	1	1	11.3	22.6	66.1
	MCF	0.03–0.25	0.25	0.25	0.25	100
	FLC	8 to >32	32	32	>32
	AMB	0.5–2	1	1	1
C. kefyr (52)	RZF	0.015–0.25	0.06	0.06	0.12
	ANF	0.008–0.12	0.03	0.03	0.06
	CAS	0.25–1	0.25	0.25	0.5				100
	MCF	0.008–0.12	0.06	0.06	0.12
	FLC	0.12–4	0.12	0.12	0.5				1.9
	AMB	0.25–1	0.5	0.5	1
C. lusitaniae (46)	RZF	0.015–0.5	0.12	0.12	0.25
	ANF	0.008–0.25	0.03	0.03	0.06
	CAS	0.12–1	0.5–1	0.5	1
	MCF	0.015–0.5	0.12	0.12	0.25
	FLC	0.06–32	0.25	0.25	4				10.9
	AMB	0.12–1	0.5	0.5	1
C. guilliermondii (27)	RZF	0.5–2	1	1	1
	ANF	0.25–2	1	1	2	100
	CAS	0.25–1	0.5	0.5	1	100
	MCF	0.5–2	1	1	2	100
	FLC	1–32	2	2	4				7.4
	AMB	0.25–1	0.5	0.5	1
C. dubliniensis (22)	RZF	0.015–0.06	0.06	0.06	0.06
	ANF	≤0.004–0.03	0.015	0.015	0.03
	CAS	0.03–0.5	0.25	0.12	0.25				50
	MCF	0.008–0.12	0.03	0.03	0.03
	FLC	0.06–0.25	0.12	0.12	0.25
	AMB	0.06–0.5	0.12	0.25	0.5
C. auris (19)	RZF	0.03–0.25	0.12–0.25	0.12	0.25
	ANF	0.03–0.5	0.03	0.06	0.25
	CAS	0.25–1	0.5	0.5	1
	MCF	0.06–2	0.25	0.25	0.5
	FLC	0.5 to >32	>32	>32	>32				68.4
	AMB	0.12–1	1	0.5	1
C. orthopsilosis (15)	RZF	0.12–1	1	0.5	1
	ANF	0.12–1	1	1	1
	CAS	0.5–1	1	0.5	1				53.3
	MCF	0.25–1	0.5	0.5	1
	FLC	0.12–0.5	0.25	0.25	0.5
	AMB	0.12–0.5	0.25	0.25	0.5
C. metapsilosis (15)	RZF	0.25–0.5	0.5	0.5	0.5
	ANF	0.12–0.5	0.25	0.25	0.5
	CAS	0.12–1	0.25	0.25	0.5
	MCF	0.06–0.5	0.25	0.25	0.5
	FLC	0.5–16	0.5–1	1	1
	AMB	0.25–1	0.25–0.5	0.25	0.5
S. cerevisiae (21)	RZF	0.03–0.5	0.5	0.25	0.5
	ANF	0.015–0.5	0.12	0.12	0.5
	CAS	0.5–1	1	1	1
	MCF	0.12–0.5	0.25	0.25	0.25
	FLC	2–8	4	4	8
	AMB	0.25–1	0.5	0.5	1
C. fabianii (15)	RZF	0.03–0.12	0.06	0.06	0.12
	ANF	0.015–0.25	0.06	0.06	0.12
	CAS	0.5–1	1	1	1
	MCF	0.06–0.5	0.06	0.06	0.12
	FLC	0.12–2	0.5	0.5	2
	AMB	0.25–1	0.5	0.5	1
C. inconspicua (41)	RZF	0.015–0.06	0.06	0.06	0.06
	ANF	≤0.004–0.015	0.008	0.008	0.015
	CAS	0.03–0.5	0.25	0.25	0.5
	MCF	≤0.004–0.12	0.03	0.03	0.06
	FLC	8 to >32	16	32	>32
	AMB	0.06–1	0.5	0.5	1
C. sojae (10)	RZF	0.03–0.06	0.06	0.06	0.06
	ANF	≤0.004–0.03	0.03	0.015	0.03
	CAS	0.12–1	0.5	0.25	0.5
	MCF	0.015–0.12	0.06	0.03	0.06
	FLC	0.12–0.25	0.25	0.25	0.25
	AMB	0.12–1	0.5	0.5	0.5
C. lipolytica (10)	RZF	0.03–0.06	0.06	0.06	0.06
	ANF	0.03–0.12	0.12	0.06	0.12
	CAS	0.12–1	0.25	0.25	0.5
	MCF	0.06–1	1	0.25	1
	FLC	0.5–2	0.5	0.5	1
	AMB	0.12–0.5	0.25	0.25	0.5
C. pulcherrima (10)	RZF	0.015–0.06	0.03	0.03	0.06
	ANF	0.015–0.06	0.015	0.015	0.06
	CAS	0.12–1	1	0.5	1
	MCF	0.008–0.5	0.06	0.06	0.25
	FLC	0.12–0.5	0.25	0.25	0.25
	AMB	0.12–1	0.5	0.5	1
Other yeast spp. (17)a	RZF	≤0.004–0.5	0.03, 0.06	0.06	0.25
	ANF	≤0.004–1	0.03	0.03	0.25
	CAS	0.06–1	0.12	0.25	0.5
	MCF	≤0.004–0.5	0.03	0.03	0.25
	FLC	0.06–8	0.12, 0.25	0.25	4
	AMB	0.06–1	0.25	0.25	1

RZF, rezafungin; ANF, anidulafungin; CAS, caspofungin; MCF, micafungin; FLC, fluconazole; AMB, amphotericin B; S, susceptible; I, intermediate; SDD, susceptible-dose dependent; R, resistant.

Includes C. pararugosa (n=6), C. africana (n=3), C. catenulata (n=2), C. rugosa (n=2), L. elongisporus (n=2), C. intermedia (n=1) and C. carpophila (n=1).

Activity of rezafungin (MIC50/90, 0.06/0.12 mg/L) against C. glabrata was comparable to that of anidulafungin and micafungin. Susceptibility rates were 100%, 100% and 4.9% for anidulafungin, micafungin and caspofungin, respectively (Table 2).

All four echinocandins showed similar activities against the ‘psilosis’ group and C. guilliermondii; the most susceptible was Candida metapsilosis (MIC90 was 0.5 mg/L) (Table 2). In the case of 53.3% of C. orthopsilosis isolates, MICs were higher than the ECV (0.5 mg/L) for caspofungin (Table 2).

C. krusei isolates were inhibited by ≤0.12 mg/L rezafungin. All isolates were susceptible to anidulafungin and micafungin, but only 11.3% were susceptible to caspofungin (Table 2). Rezafungin, anidulafungin and micafungin, but not caspofungin, were highly active against the other fluconazole-resistant Candida species, Candida inconspicua (Table 2).

Rezafungin MIC50/90 values were 0.06/0.12 and 0.12/0.25 mg/L for C. kefyr and C. lusitaniae, respectively. Against these two species, anidulafungin was the most active. In the case of C. kefyr, MICs for all isolates were higher than the ECV (0.03 mg/L) for caspofungin (Table 2).

No MICs for C. auris exceeded the tentative MIC breakpoints against the three licensed echinocandins (Table 2). Rezafungin inhibited all isolates at ≤0.25 mg/L (MIC50/90, 0.12/0.25 mg/L). Activity of rezafungin (MIC50/90, 0.25/0.5 mg/L) against S. cerevisiae was comparable to anidulafungin, micafungin and caspofungin (Table 2).

Rezafungin MIC90 values for Candida fabianii, Candida sojae, Candida lipolytica and Candida pulcherrima were 0.12, 0.06, 0.06 and 0.06 mg/L, respectively. Anidulafungin and micafungin MIC90 values for C. fabianii, C. sojae and C. pulcherrima were similar to those of rezafungin. Micafungin MICs for C. lipolytica were higher compared with rezafungin and anidulafungin; MICs for 5 of 10 isolates were 0.5–1 mg/L (Table 2).

Against other yeast species represented by <10 isolates (Candida pararugosa, C. africana, C. catenulata, C. rugosa, L. elongisporus, Candida intermedia and Candida carpophila), rezafungin was active at ≤0.25 mg/L with the exception of C. carpophila (Table 2).

Activity of fluconazole and amphotericin B against Candida species and S. cerevisiae isolates

Fluconazole resistance was found in C. albicans (5.6%) and C. glabrata (4.9%). Two (3.4%) C. parapsilosis sensu stricto isolates showed dose-dependent susceptibility to fluconazole. In cases of C. guilliermondii, C. kefyr, C. lusitaniae and C. auris isolates, 7.4%, 1.9%, 10.9% and 68.4% of isolate MICs, respectively, were higher than their ECVs (Table 2).

Amphotericin B MICs with the exception of two, three and three C. albicans, C. glabrata and C. krusei isolates, respectively, (with 2 mg/L MICs for all cases) were ≤1 mg/L for all tested isolates (Table 2).

Discussion

Results of this study, consistent with other published data, demonstrate that rezafungin MIC values for the five most common Candida species, including fluconazole-resistant isolates, were comparable to those of anidulafungin and micafungin. Activity of rezafungin was comparable to anidulafungin and micafungin against C. kefyr, C. lusitaniae and C. dubliniensis, as well as against the emerging fluconazole-resistant C. auris and C. inconspicua. As expected, against C. guilliermondii, C. orthopsilosis, C. metapsilosis and C. carpophila all echinocandins including rezafungin showed higher MIC values, similarly to C. parapsilosis sensu stricto.

Rezafungin at ≤0.12 mg/L was active against very rare Candida species (C. lipolytica, C. fabianii, C. sojae, C. pulcherrima, C. pararugosa and C. rugosa) and showed activity against S. cerevisiae comparable to anidulafungin or micafungin. C. sojae has not been isolated from clinical specimens previously; our 10 isolates, including 1 bloodstream isolate, draw attention to this primarily plant pathogen frequently misidentified as Candida sake using traditional identification methods.

Although rezafungin MIC values were similar to those of micafungin and of anidulafungin, as might be expected given the latter’s structural similarities to rezafungin, MIC values of caspofungin were markedly higher for almost all Candida species studied. Our caspofungin MIC distributions were in line with the previously well-documented limitation that caspofungin susceptibility testing suffers from significant interlaboratory variability and is therefore not recommended. As all three echinocandin comparators (anidulafungin, caspofungin and micafungin) originated from the same supplier, drug source does not explain this difference in antifungal efficacy. However, the quality of the microtitre plate (treated versus untreated polystyrene trays) may have influenced differentially our MIC values obtained with the different echinocandins, as previously demonstrated by Fothergill et al. This may also be a factor in MIC differences found between laboratories testing rezafungin susceptibility. Although our MIC data derived from a single centre and we used tissue culture-treated microtitre test plates from the same batch, their impact on rezafungin MIC distribution could not be entirely ruled out.

We have provided a ‘head-to-head’ comparison between the three clinically available echinocandins and rezafungin using CLSI reference BMD methodology. Although the vast majority of clinical strains were isolated from a single centre, our anidulafungin, micafungin, fluconazole and amphotericin B MIC data for the tested Candida species were comparable to data reported by others. The investigational echinocandin rezafungin showed excellent in vitro activity against Candida species, including the emerging potentially MDR C. auris, as well as against S. cerevisiae. Rezafungin had similar activity to other echinocandins (excluding caspofungin) against common and, notably, against clinically relevant uncommon Candida species.