Methods: To establish a lethal C. albicans infection, larval stage 4 worms [n=30/group (gp)] were fed various doses of yeast (2.5x10ex2 – 2.5x10ex5 cells/gp) for 4h at 20°C or 30°C. The infection was evaluated by monitoring worms for mortality and determining fungal burden in worm homogenates by plating for colony forming units every 24h for 4d post-challenge. To examine the worm’s ability to ingest L-AmB and to determine drug toxicity, uninfected worms were fed L-AmB (6.3 - 25 ug/gp) for 4h at 30°C, and drug toxicity evaluated by survival with drug concentrations determined by bioassay of worm homogenates. Lack of toxicity allowed us to evaluate L-AmB activity in worms challenged for 4h at 30°C with 2.5 x 10ex5 yeast cells/gp and then treated with L-AmB (0.5 - 25 ug/gp) for 4h at 30°C, with survival rate and fungal burden to assess L-AmB treatment.
Results: Calbicans infection was established in worms challenged with all yeast doses, with optimum infection observed with 2.5x10ex5 yeast cells/gp at 30°C (13% survival in infected worms vs. 87% in uninfected worms). We observed that uninfected worms could take up L-AmB at doses of 6.3 - 25 ug/gp and yet was not toxic for the worms (93-95% survival). In worms exposed to yeast and treated with L-AmB, complete clearance of infection was achieved with the higher doses (6.3 to 25 ug/gp), while lower doses (1.6 to 3.1 ug/gp) significantly reduced the fungal burden (p≤0.05). Infected worms, not treated with L-AmB had only 10% survival, while L-AmB improved survival in a dose-dependent manner giving 40% survival for 0.5 ug L-AmBi/gp and 100% survival for doses of 6.3 ug/gp and higher.
Conclusion: By using fungal burden as a readout of efficacy, along with survival, we have established a quantitative, reproducible, flexible method for examining the activity of L-AmB in C. elegans which could be expanded for use in evaluating other antifungal drugs and different pathogenic fungi.
J. Adler-Moore, None