Abstract
BACKGROUND: Talaromyces marneffei causes talaromycosis, a life-threatening fungal disease with limited treatment options. The standard treatment of amphotericin B (AmB) induction followed by itraconazole consolidation still results in 15% to 30% mortality. This study aimed to investigate the potential of AmB and flucytosine (5FC) combination therapy to enhance antifungal activity. METHODS: The in vitro antifungal activity of AmB and 5FC alone and in combination against 60 T. marneffei clinical isolates was evaluated using a validated colorimetric antifungal susceptibility assay and the checkerboard method. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration inhibiting ≥ 95% fungal growth (MIC(95)) for both AmB and 5FC. The combination effect between AmB and 5FC against T. marneffei was determined using fractional inhibitory concentration index. Combination effects were further tested using a time-kill assay. RESULTS: The MIC(95) was 0.25 - 2 μg/mL (geometric mean [GM] 0.68 μg/mL) for AmB, and 0.03 - 0.5 μg/mL (GM 0.28 μg/mL) for 5FC. Full synergy was observed in 4 isolates (7%), and indifference was observed in the remaining 56 isolates (93%). The time-kill experiments revealed a concentration-dependent fungicidal activity of AmB, and concentration-independent fungistatic effect of 5FC. Synergy between AmB and 5FC was confirmed, showing greater than 2-log(10) reduction in colony forming units when used in combination. No antagonism was observed. CONCLUSIONS: Our study demonstrated in vitro evidence of synergistic activity between AmB and 5FC against T. marneffei, providing the evidence to support in vivo and clinical trial testing of AmB and 5FC combination therapy, and dosing reduction strategies of 5FC. LAY SUMMARY: This study demonstrated in vitro synergy between amphotericin B and flucytosine against Talaromyces marneffei, providing the proof of concept to test this antifungal combination in a clinical trial of talaromycosis.