In vitro Activity of Isavuconazole and Comparators Against Clinical Isolates of Molds from a Multicenter Study in China

Figures & data

Table 1 Molecular Description of CYP51 Homologous Genes Related to Azoles-Resistance in Mucor circinelloides

Genes	Gene ID	Location	Total Length (nt)	CDS Length (nt)	Total Length (aa)	A. fumigatus Homologues Genes	A. fumigatus Homologues Proteins	Functions
CYP51F1	HMPREF1544_03888	KE123937:33,460.35,232(+)	1773	1539	512	CYP51B (GenBank: AF338660.1)	CYP51B (Protein ID: AAK73660.1)	Lanosterol 14-alpha demethylase
CYP51F5	HMPREF1544_08704	KE124036:54,965.56,622(+)	1658	1533	510	CYP51A (GenBank: AF338659.1)	CYP51A (Protein ID: AAK73659.1)	Lanosterol 14-alpha demethylase

Table 2 MIC Distributions for Isavuconazole Against Aspergillus spp. and Species of Mucorales Order Using CLSI Broth Microdilution Methods

Species (No. Tested)	No. of Isolates at MIC (µg/mL)
	≤0.008	0.016	0.03	<0.06	0.06	0.12	0.25	0.5	1	2	4	8	>8
Aspergillus spp. (105)					1	5	1	26	56	15	1
A. fumigatus complex (75)								24	45	6
A. niger complex (11)									3	7	1
A. flavus complex(10)									8	2
A. terreus complex (9)					1	5	1	2
Mucorales (26)					2		3	9	8	2		1	1
Mucor spp. (2)								1				1
Mucor circinelloides (2)								1				1
Rhizopus spp. (13)					1		2	4	5	1
R. oryzae/R. arrhizus (11)					1		1	3	5	1
R. microsporus (1)								1
R. stolonifer (1)							1
Rhizomucor spp. (4)							1	1	2
Rhizomucor pusillus (3)							1		2
Rhizomucor miehei (1)								1
Syncephalastrum spp. (3)								2					1
Syncephalastrum racemosum (3)								2					1
Lichtheimia spp. (4)					1			1	1	1
Lichtheimia ramosa (4)					1			1	1	1

Note: Numbers in bold are median MIC values.

Abbreviations: CLSI, Clinical and Laboratory Standards Institute; MIC, minimum inhibitory concentration.

Table 3 Antifungal Activity of Isavuconazole and Comparator Antifungal Agents (Voriconazole and Amphotericin B) Against Aspergillus spp. and Species of Mucorales Order Collected in China Between 2017 and 2020

Antifungal Drugs	ISA (μg/mL)							VOR (μg/mL)							AMB (μg/mL)
Genus (No. of Isolates Tested)	MIC Range	mMIC	MIC	MIC50	MIC90	WT* (%)	Non-WT* (%)	MIC Range	mMIC	MIC	MIC50	MIC90	WT* (%)	Non-WT* (%)	MIC Range	mMIC	MIC	MIC50	MIC90	WT* (%)	Non-WT* (%)
Aspergillus spp. (105)	0.06–4	1	–	1	2	–	–	0.06–2	1	–	1	2	–	–	0.25–>8	4	–	4	8	–	–
A. fumigatus complex (75)	0.5–2	1	–	1	1	69 (92)	6 (8)	0.25–2	1	–	1	1	70 (93.3)	5 (6.7)	2–8	4	–	4	8	8 (10.7)	67 (89.3)
A. niger complex (11)	1–4	2	–	2	2	11 (100)	0	0.5–2	1	–	1	2	11 (100)	0	2–4	2	–	2	4	7 (63.6)	4 (36.4)
A. flavus complex (10)	1–2	1	–	1	2	8 (80)	2 (20)	1–2	1	–	1	2	10 (100)	0	4–8	4	–	4	8	6 (60)	4 (40)
A. terreus complex (9)	0.06–0.5	0.12	–	0.12	0.5	9 (100)	0	0.06–0.5	0.12	–	0.12	0.5	9 (100)	0	0.25–>8	2	–	2	>8	8 (88.9)	1 (11.1)
Mucorales (26)	0.06–>8	0.5	–	0.5	2	–	–	4–>8	>8	–	>8	>8	–	–	0.12–2	1	–	1	1	–	–
Rhizopus spp. (13)	0.06–2	0.5	–	0.5	1	–	–	4–>8	8	–	8	>8	–	–	0.25–2	1	–	1	2	–	–
R. oryzae/R. arrhizus (11)	0.06–2	1	–	1	1	–	–	4–>8	8	–	8	>8	–	–	0.25–2	1	–	1	2	–	–
R. microsporus (1)^#	–	–	0.5					–	–	>8					–	–	0.5
R. stolonifer (1) ^#	–	–	0.3					–	–	4					–	–	1
Rhizomucor spp. (4)^#	0.25–1	0.75	–					>8	>8	–					0.5–1	0.75	–
Rhizomucor pusillus (3)^#	0.25–1	1	–					>8	>8	–					0.5–1	0.5	–
Rhizomucor miehei (1)^#	–	–	0.5					–	–	8					–	–	1
Mucor spp. (2)^#	0.5–8	4.25	–					>8	>8	–					0.5–1	1	–
Mucor circinelloides (2)^#	0.5–8	4.25						>8	>8	–					0.5–1	1	–
Mucor circinelloides-M49	–	–	0.5					–	–	>8					–	–	0.5
Mucor circinelloides-M35	–	–	8					–	–	>8					–	–	1
Syncephalastrum spp. (3)^#	0.5–>8	0.5	–					>8	>8	–					0.25–1	0.5	–
Syncephalastrum racemosum (3)^#	0.5–>8	0.5	–					>8	>8	–					0.25–1	0.5	–
Lichtheimia spp. (4)^#	0.06–2	0.75	–					4–>8	>6	–					0.12–1	0.75	–
Lichtheimia ramosa (4)^#	0.06–2	0.75	–					4–>8	>6	–					0.12–1	0.75	–

Notes: *Based on epidemiological cut-off values published by CLSI;²² values reported using WT and non-WT for the mentioned target drug because no breakpoints available on Aspergillus species from CLSI.^{22 #}The number of the remaining isolates for Mucorales order was too low to calculate the MIC₅₀ and MIC₉₀, and ECVs are also unavailable for Mucorales order from CLSI,²² hence other isolates for Mucorales order are not presented.

Abbreviations: ISA, isavuconazole; VOR, voriconazole; AMB, amphotericin B; MIC, minimum inhibitory concentration; mMIC, median MIC; MIC₅₀, 50% minimum inhibitory concentration; MIC₉₀, 90% minimum inhibitory concentration; WT, wild-type.

Table 4 MIC Distributions for Voriconazole Against Aspergillus spp. and Species of Mucorales Order Using CLSI Broth Microdilution Methods

Species (No. Tested)	No. of Isolates at MIC (µg/mL)
	≤0.008	0.016	0.03	<0.06	0.06	0.12	0.25	0.5	1	2	4	8	>8
Aspergillus spp. (105)					2	4	4	33	52	10
A. fumigatus complex (75)							2	31	37	5
A. niger complex(11)								1	7	3
A. flavus complex (10)									8	2
A. terreus complex (9)					2	4	2	1
Mucorales (26)											5	5	16
Mucor spp. (2)													2
Mucor circinelloides (2)													2
Rhizopus spp. (13)											3	4	6
R. oryzae/R. arrhizus (11)											2	4	5
R. microsporus (1)													1
R. stolonifer (1)											1
Rhizomucor spp. (4)												1	3
Rhizomucor pusillus (3)													3
Rhizomucor miehei (1)												1
Syncephalastrum spp. (3)													3
Syncephalastrum racemosum (3)													3
Lichtheimia spp. (4)											2		2
Lichtheimia ramosa (4)											2		2

Note: Numbers in bold are median MIC values.

Abbreviations: CLSI, Clinical and Laboratory Standards Institute; MIC, minimum inhibitory concentration.

Table 5 MIC Distributions for Amphotericin B Against Aspergillus spp. and Species of Mucorales Order Using CLSI Broth Microdilution Methods

Species (No. Tested)	No. of Isolates at MIC (µg/mL)
	≤0.008	0.016	0.03	<0.06	0.06	0.12	0.25	0.5	1	2	4	8	>8
Aspergillus spp. (105)							1		1	18	62	22	1
A. fumigatus complex (75)										8	49		18
A. niger complex (11)										7	4
A. flavus complex (10)											6	4
A. terreus complex (9)							1		1	3	3		1
Mucorales (26)						1	2	7	14	2
Mucor spp. (2)								1	1
Mucor circinelloides (2)								1	1
Rhizopus spp. (13)							1	2	8	2
R. oryzae/R. arrhizus (11)							1	1	7	2
R. microsporus (1)								1
R. stolonifer (1)									1
Rhizomucor spp. (4)								2	2
Rhizomucor pusillus (3)								2	1
Rhizomucor miehei (1)									1
Syncephalastrum spp. (3)							1	1	1
Syncephalastrum racemosum (3)							1	1	1
Lichtheimia spp. (4)						1		1	2
Lichtheimia ramosa (4)						1		1	2

Note: Numbers in bold are median MIC values.

Abbreviations: CLSI, Clinical and Laboratory Standards Institute; MIC, minimum inhibitory concentration.

Figure 1 Amino acid sequence alignment of lanosterol 14-α demethylase between Saccharomyces cerevisiae S288C and tested Mucor circinelloides CYP51s. Alignment using Saccharomyces cerevisiae S288C Erg11 (CYP51) as reference, Y140 (a tyrosine residue) is marked in bold, the previously confirmed mutation as correlation to resistance to voriconazole in different fungi species (analogous to Y140F in S. cerevisiae), and the change to phenylalanine (129F) is seen in both M. circinelloides CYP51F5s, displayed in bold and red. Another amino acid mutation of V (valine) to A (alanine) (position V311 in S. cerevisiae, and mutated to 293A for M. circinelloides numbering) potentially related to resistance to voriconazole is also observed in both M. circinelloides CYP51F5s, displayed in bold and red.

Table 6 Review of MICs of Isavuconazole and Comparator Antifungal Agents (Voriconazole and Amphotericin B) MICs of Major Mucorales Order Using CLSI Broth Microdilution Methods Reported in Different Countries

Species	Country	No. of Isolates Tested	MIC Range (or MIC) µg/mL
			ISA	VOR	AMB
Mucor circinelloides	Netherlands/India^Citation42	5	1–8	8–16	0.03–0.125
	USA^Citation30	67	2–>16	–	≤0.03–8
	Denmark^Citation40	13	1–8	1–>16	≤0.03–0.125
	China*	2	0.5–8	>8	0.5–1
Rhizopus arrhizus^#	Netherlands/India^Citation42	25	1–8	4–16	0.03–0.5
	USA^3028	114	0.125–4	–	≤0.03–1
	China*	11	0.06–2	4–>8	0.25–2
Rhizopus oryzae^#	Denmark^Citation40	6	0.125–2	4–8	≤0.03–0.25
Rhizopus microsporus	Netherlands/India^Citation42	23	0.5–8	1–16	0.03–1
	USA^3028	121	0.125–>16	–	≤0.03–1
	Denmark^Citation40	26	0.125–1	2–16	≤0.03–0.25
	China*	1	0.5	>8	0.5
Rhizopus stolonifer	Netherlands/India^Citation42	4	0.25–1	4–8	0.03–0.06
	China*	1	0.25	4	1
Rhizomucor pusillus	Netherlands/India^Citation42	7	1–8	8–16	0.06–1
	Denmark^Citation40	8	0.125–1	1–16	≤0.03–0.25
	China*	3	0.25–1	>8	0.5–1
Rhizomucor miehei	Netherlands/India^Citation42	1	8	16	1
	China*	1	0.5	8	1
Syncephalastrum racemosum	Netherlands/India^Citation42	15	0.125–8	4–16	0.03–0.5
	USA^3028	19	0.5–>16	–	≤0.03–1
	China*	3	0.5–>8	>8	0.25–1
Lichtheimia ramosa	Netherlands/India^Citation42	7	0.5–8	8–16	0.06–1
	USA^Citation30	22	≤0.03–1	–	0.125–2
	Denmark^Citation40	4	0.5–2	2–>16	≤0.03
	China*	4	0.06–2	4–>8	0.12–1

Notes: For Europe also CLSI data is presented. *Data from China reported in our study. ^#R. oryzae/R. arrhizus strains were difficult to distinguish by MALDI-TOF MS, hence combined data are presented for China.

Abbreviations: ISA, isavuconazole; VOR, voriconazole; AMB, amphotericin B; MIC, minimum inhibitory concentration; MALDI-TOF MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry.

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