In Vitro Evaluation of Olorofim and Amphotericin B Combination Therapy Against Talaromyces marneffei

The dimorphic fungus Talaromyces marneffei causes talaromycosis, a life-threatening fungal disease with limited treatment options. Olorofim, a first-in-class orotomide antifungal that targets pyrimidine synthesis essential for fungal growth, has low minimum inhibitory concentration (MIC) against T. marneffei and clinical efficacy against other invasive fungal diseases. Here, we tested the hypothesis that olorofim synergistically enhances amphotericin B (AmB), a potent membrane-targeting antifungal, against T. marneffei in 55 clinical isolates using a validated colorimetric checkerboard assay. The MIC was defined as the lowest drug concentration inhibiting ≥ 95% of fungal growth. Drug interactions were assessed using the fractional inhibitory concentration index (FICI), which defines ≤0.5 as synergy, 0.5 < FICI ≤ 4.0 as indifference, and FICI > 4 as antagonism. We found that interactions between AmB and olorofim were indifferent across all 55 isolates (0.5 < FICI ≤ 1.03). Time-kill assays showed an expected concentration-dependent fungicidal activity for AmB, but a concentration-independent fungistatic activity for olorofim against T. marneffei. Combinations of AmB and olorofim were also indifferent in time-kill experiments. Although synergy was not observed, and olorofim is unlikely to enhance AmB induction therapy, olorofim may have a role in the consolidation and maintenance therapy of talaromycosis.