2.7. Antifungal Drug Synergy Assay

Synergistic interactions were evaluated by the checkerboard method according to EUCAST guidelines [47]. S. aurantiacum conidia (1 × 10⁵/mL) were grown in 96-well plates containing supplemented RPMI in the presence of selected compounds (0.156–10 μm) combined with fluconazole (5–320 μm), voriconazole (0.47–30 μm), or caspofungin (0.625–40 μm). After incubation for 72 h at 37 °C, MIC was evaluated at 600 nm and cell viability was assessed by the XTT-reduction assay at 490 nm using a spectrophotometer (Bio-Rad, Hercules, CA, USA). An inhibition of at least 80% was defined as a cut-off for minimum inhibitory concentration (MIC). Minimum effective concentration (MEC) was used to assess caspofungin activity and its interaction with auranofin and iodoquinol, since MEC values are considered more suitable for echinocandins analysis [48]. Interactions were determined by two different methods, the fractional inhibitory concentration index (FICI) and the Bliss independence model.

Fractional inhibitory concentration index was calculated using the following formula: (MIC combined/MIC drug A alone) + (MIC combined/MIC drug B alone). The results were classified as: synergistic effect, FICI of ≤0.5; no effect, FICI of >0.5–4.0; antagonistic effect, FICI of >4.0 [49].

Bliss independence model was performed according to Meletiadis and colleagues and Zhao and colleagues [50,51]. The following formula was used to assess the drug interaction: E_exp = E_a + E_b − E_a × E_b, in which E_exp is the expected efficacy of drug combination, E_a is the efficacy of drug A (auranofin or iodoquinol), and E_b is the efficacy of drug B (fluconazole, voriconazole or caspofungin). The results were classified as: synergistic effect, E_obs > E_exp; indifference, E_obs = E_exp; antagonistic effect, E_obs < E_exp.