Fixed-Ratio Combination Testing of an Echinocandin, Anidulafungin, and an Azole, Voriconazole, against 1,467 Candida Species Isolates

Anidulafungin is a newer echinocandin antifungal agent, approved by the United States Food and Drug Administration (U.S. FDA) in 2006 (18), with well-documented potency against Candida spp. and a MIC₉₀ value of 0.06 μg/ml reported for a sample of 2,869 Candida albicans isolates across a 6-year collection (16). In common with other echinocandins, anidulafungin MIC₉₀ values for C. parapsilosis and C. guilliermondii can be elevated (1 to 4 μg/ml) (8, 10, 16, 17).

Voriconazole is a triazole antifungal that is structurally related to fluconazole (1). Voriconazole has broad-spectrum activity against Candida spp. and has been approved by the U.S. FDA for the treatment of esophageal candidiasis and invasive candidiasis in nonneutropenic patients (11, 19). Compared to fluconazole, voriconazole demonstrates enhanced activity against Candida spp. (5, 15). Voriconazole is active against the commonly isolated species of Candida, including C. albicans (MIC₉₀, 0.015 to 0.06 μg/ml), C. tropicalis (MIC₉₀, 0.12 to 0.25 μg/ml), and C. parapsilosis (MIC₉₀, 0.06 to 0.12 μg/ml) (14, 15). Notably, voriconazole is also active against C. krusei (MIC₉₀, 0.5 μg/ml), a species considered to be intrinsically resistant to fluconazole (MIC₉₀, 64 μg/ml) (14, 15). Although activity has also been demonstrated against C. glabrata, MICs are higher (MIC₉₀, 1 to 2 μg/ml) and resistance is more common than with other species (4, 14, 15).

This protocol was designed to (i) quantify the potency and spectrum of anidulafungin and voriconazole using CLSI reference methods (2, 3) and (ii) assess the activity of anidulafungin combined with voriconazole (2:1 ratio) against a large collection of Candida spp. (8). The results were analyzed by directly comparing the MIC values of each agent tested alone or as a component of the 2:1 ratio test. The ratio was selected to mimic the concurrent exposures (area under the curve [AUC] and maximum drug concentration in serum [C_max]) to each antifungal agent as listed in the U.S. FDA product package inserts (18-20).

A total of 1,467 Candida sp. fungemia isolates were submitted by participating medical centers in North America, Europe, Latin America, and the Asia-Pacific region to the central monitoring laboratory (JMI Laboratories, North Liberty, IA) for testing (8). All strains were tested by the reference broth microdilution methods for yeasts as recommended by the CLSI M27-A3 approved standard (2, 3). Interpretive criteria used for yeasts when testing voriconazole and anidulafungin were those of the CLSI (M27-S3, 2008) (2, 3, 17). The quality control (QC) strains utilized included C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 (2, 3, 7); all QC results were within published MIC limits (3, 7).

The activities of the two agents tested against the over 1,400 Candida spp. (MIC_50/90 and percent susceptible) were as follows: anidulafungin, 0.03/1 μg/ml and 99.0%; voriconazole, ≤0.06/0.25 μg/ml and 98.3%. The combination of anidulafungin and voriconazole demonstrated enhanced activity when tested at a 2:1 ratio against these Candida spp. (Table 1). Anidulafungin tested alone had a MIC₉₀ of 1 μg/ml, whereas that value was reduced to 0.06 μg/ml when it was combined with voriconazole. Similarly, the voriconazole MIC₉₀ of 0.25 μg/ml was decreased to ≤0.03 μg/ml when it was tested with anidulafungin. The combination was 8- to 16-fold more potent than each agent was alone (MIC₉₀ comparison), and all anidulafungin/voriconazole (2:1 ratio) MIC test values were ≤1/0.5 μg/ml, i.e., below the suggested susceptibility breakpoint for one or both agents (3, 17).

TABLE 1.

Comparisons of anidulafungin and voriconazole tested alone or in a 2:1 ratio combination against 1,467 Candida spp. clinical isolates^d

Species (no. of isolates tested), antifungal agent,c and test	Cumulative % inhibited at MIC (μg/ml)a of:
	≤0.03	0.06	0.12	0.25	0.5	1	2	4	8
C. albicans (768)
ANF
Alone	88.9	99.6	100.0
Combination	81.1	99.9	100.0
VRC
Alone	—b	98.9	100.0
Combination	99.9	100.0
C. glabrata (204)
ANF
Alone	9.3	72.1	96.6	98.0	98.0	100.0
Combination	32.5	88.7	97.0	99.0	99.5	100.0
VRC
Alone	—	14.8	48.3	66.5	83.3	90.2	93.6	98.0	100.0
Combination	88.7	97.0	99.0	99.5	100.0
C. tropicalis (156)
ANF
Alone	73.3	92.4	98.7	98.7	100.0
Combination	24.4	81.4	98.7	100.0
VRC
Alone	—	95.5	97.4	97.4	100.0
Combination	81.4	98.7	100.0
C. krusei (30)
ANF
Alone	13.3	70.0	90.0	90.0	93.3	96.7	100.0
Combination	3.3	63.3	90.0	90.0	100.0
VRC
Alone	—	0.0	16.7	73.3	83.3	90.0	100.0
Combination	63.3	90.0	90.0	100.0
C. lusitaniae
ANF
Alone	0.0	0.0	14.3	78.6	100.0
Combination	92.9	92.9	100.0
VRC
Alone	—	92.9	92.9	100.0
Combination	92.9	100.0
C. parapsilosis (247)
ANF
Alone	0.4	1.6	1.6	2.0	12.2	47.4	93.9	100.0
Combination	55.4	82.9	90.0	95.8	99.2	100.0
VRC
Alone	—	88.3	94.6	98.3	99.6	99.6	100.0
Combination	82.9	90.0	95.8	99.2	100.0
Other Candida spp. (48)
ANF
Alone	18.8	39.6	50.0	52.1	54.2	64.6	83.3	100.0
Combination	56.8	67.6	86.5	100.0
VRC
Alone	—	62.2	83.8	89.2	94.6	97.3	100.0
Combination	67.6	86.5	100.0
Total (1,467)
ANF
Alone	57.4	76.1	81.3	82.3	84.5	90.9	99.0	100.0
Combination	61.7	91.9	97.2	99.0	99.8	100.0
VRC
Alone	—	82.0	89.4	94.0	97.1	98.3	99.1	99.7	100.0
Combination	91.9	97.2	99.0	99.8	100.0

^aMIC read at 24 h and 50% decrease in growth in accordance with CLSI criteria (2, 3). Anidulafungin/voriconazole (2:1 ratio) test consistent with in vivo ratios of C_maxand AUC.

^b—, untested concentration.

^cANF, anidulafungin; VRC, voriconazole.

^dSENTRY Program, 2006 and 2007.

The combination of anidulafungin and voriconazole resulted in improved activity against the putatively resistant species C. glabrata (voriconazole) and C. parapsilosis (anidulafungin) (Table 1). The MIC₉₀ for C. glabrata decreased from 1 μg/ml (90.2%) for voriconazole alone to 0.06 μg/ml (97.0%) when it was combined with anidulafungin. Likewise, the MIC₉₀ for C. parapsilosis decreased from 2 μg/ml (93.9%) for anidulafungin alone to 0.12 μg/ml (90.0%) when it was combined with voriconazole. Recently, Garcia-Effron et al. (6) suggested that the susceptibility breakpoint for the echinocandins be lowered from ≤2 μg/ml to ≤0.25 μg/ml to improve the detection of fks mutant strains of Candida. As shown in Table 1, the combination of anidulafungin and voriconazole would capture 99.0% of the isolates tested at an MIC of ≤0.25 μg/ml versus only 82.3% when anidulafungin was tested alone.

These in vitro results markedly expand upon what has been reported elsewhere (12) concerning the activity of anidulafungin and voriconazole in combination versus Candida and suggest a favorable interaction of these agents when they are used together against Candida spp. As resistances emerge among these fungal pathogens (6, 9, 13), combination treatments could assume a valuable therapeutic role (11).