# In vitro evidence to support amphotericin B and flucytosine combination therapy for talaromycosis

## TL;DR

This study shows that combining amphotericin B and flucytosine can more effectively fight Talaromyces marneffei, a deadly fungus, compared to using either drug alone.

## Contribution

The study provides in vitro evidence for synergistic antifungal activity of amphotericin B and flucytosine against Talaromyces marneffei.

## Key findings

- Amphotericin B and flucytosine combination showed synergy in 7% of T. marneffei isolates.
- The drug combination caused a greater than 2-log10 reduction in colony forming units.
- No antagonism was observed between the two drugs in the tested isolates.

## Abstract

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.

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 (MIC95) 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.

The MIC95 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-log10 reduction in colony forming units when used in combination. No antagonism was observed.

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.

Talaromycosis is an emerging, life-threatening, invasive fungal infection affecting people with weakened immune systems and is caused by the fungus Talaromyces marneffei, which is endemic to Southeast Asia. The mortality despite antifungal treatment is up to 30%, yet treatment options are limited to just two drugs: amphotericin B, which is associated with substantial drug toxicity, and itraconazole, which has poor absorption and limited effectiveness. In this study, we investigated whether combining amphotericin B with the existing antifungal drug flucytosine could improve antifungal activity against Talaromyces marneffei, compared to amphotericin B or flucytosine alone. These drugs inhibit fungal replication through different mechanisms, and their combination has been shown to improve tissue penetration and treatment outcomes in other fungal infections. Using checkerboard and time-kill assays on 60 clinical isolates obtained from talaromycosis patients, we found that the two-drug combination inhibited the growth of Talaromyces marneffei more effectively compared to the individual drugs. Our findings support the potential of combining amphotericin B and flucytosine as a more effective treatment strategy for talaromycosis and warrant further clinical evaluation. By strengthening the evidence for improved use of existing antifungal agents, our study contributes to advancing treatment options for this neglected fungal disease.