In vitro interaction of voriconazole and anidulafungin against triazole-resistant Aspergillus fumigatus

Abstract

Voriconazole is the recommended drug of first choice to treat infections caused by Aspergillus fumigatus. The efficacy of voriconazole might be hampered by the emergence of azole resistance. However, the combination of voriconazole with anidulafungin could improve therapeutic outcomes in azole-resistant invasive aspergillosis (IA). The in vitro interaction between voriconazole and anidulafungin was determined against voriconazole-susceptible and voriconazole-resistant (substitutions in the cyp51A gene, including single point [M220I and G54W] and tandem repeat [34-bp tandem repeat in the promoter region of the cyp51A gene in combination with substitutions at codon L98 and 46-bp tandem repeat in the promoter region of the cyp51A gene in combination with mutation at codons Y121 and T289] mutations) clinical A. fumigatus isolates using a checkerboard microdilution method with spectrophotometric analysis and a viability-based XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} assay within 2 h of exposure after 24 and 48 h of incubation at 35 °C to 37 °C. Fractional inhibitory concentration (FIC) indexes (FICis) were determined using different MIC endpoints and Bliss independence analysis performed based on the response surface calculation of the no-drug interaction. Significant synergistic interactions obtained based on measuring the FIC index were dependent on the MIC endpoint, in which FICs were inversely related to voriconazole and anidulafungin MICs and were influenced by the CYP51A genotype. A statistically significant difference was observed between FIC indexes of isolates harboring tandem repeat mutations and wild-type controls (P = 0.006 by one-way analysis of variance [ANOVA]), indicating that synergy is decreased in azole-resistant strains. Our results indicated that a combination of voriconazole and anidulafungin might be effective against infections caused by both azole-susceptible and azole-resistant A. fumigatus isolates, but the combination could possibly be less effective in voriconazole-resistant strains with high MICs. Studies in vivo and in vitro-in vivo correlation investigations are required to validate the potential synergy of voriconazole and anidulafungin.

Fig 1

Graphical distribution of mean and standard error of the mean of FIC indexes determined at 10% and 25% growth endpoints for 25 A. fumigatus isolates. None of the data sets analyzed had ∑FIC_maxs higher than 1.25, indicating that antagonism was not observed.

Fig 2

Mean and standard errors of the mean of FICs with respect to cyp51 substitutions for 25 A. fumigatus isolates, indicating that the FIC indexes are dependent on the type of mutation. The vertical bars indicate that the mean FIC indexes did not differ significantly among VCZ-S isolates and those with M220I and G54W mutations; a statistically significant difference was observed between isolates harboring TR₃₄/L98H and TR₄₆/Y121F/T289A mutations and wild-type controls (P < 0.05).

Fig 3

Interaction surfaces obtained from response surface analysis of the Bliss independence no-interaction model for the in vitro combination of VCZ plus AFG against a VCZ-susceptible A. fumigatus isolate (MIC of VCZ, 0.25 mg/liter; MEC of AFG, 0.03 mg/liter) and a VCZ-resistant A. fumigatus isolate (MIC of VCZ, 4 mg/liter; MEC of AFG, 0.03 mg/liter). The x and y axes represent the efficacies of VCZ and AFG, respectively. The z axis is the percent ΔE. The zero plane represents Bliss-independent interactions, whereas the volumes above the zero plane represent statistically significantly synergistic (positive ΔE) interactions. The magnitude of interactions is directly related to ΔE. The different tones in three-dimensional plots represent different percentile bands of synergy. (A) Synergistic interaction. The mean ΔE ± standard error of the mean and sum ΔE were 3.23% ± 1.09% and 271%, respectively, after 48 h. (B) Antagonistic interaction. The mean ΔE ± standard error of the mean and sum ΔE were −2.47% ± 0.40% and −208%, respectively, after 48 h.

Fig 4

Interpretation of voriconazole and anidulafungin interactions for 25 A. fumigatus isolates utilizing two different definitions: the cutoff values proposed previously by Meletiadis et al. (46) (synergistic if the FIC index was ≤1, additive if the FIC index was >1 to ≤1.25, and antagonistic if the FIC index was >1.25) and the commonly used FIC index range of 0.5 to 4 proposed previously by Greco et al. (47) and which is generally recommended to define drug interactions in most combination studies of antifungal agents (synergistic if the FIC index was ≤0.5, indifferent if the FIC index was >0.5 to ≤4, and antagonistic if the FIC index was >4).