Infection and Treatment Protocols in Galleria mellonella for In Vivo Anti-Candida Drug Screening

Background/Objectives: Galleria mellonella (G. mellonella) larvae have emerged as a valuable in vivo model for antifungal drug screening. This study aimed to determine the optimal inoculum concentrations of Candida albicans (C. albicans) in G. mellonella, as well as the appropriate fluconazole concentrations, in order to standardize a preliminary screening method for compounds with antifungal activity. Methods: Larvae were infected with four C. albicans strains, including two reference strains (ATCC® 10231 and ATCC® 90028) and two oral isolates (A1 and A2). Fluconazole toxicity was evaluated at doses of 20, 40, and 80 mg/kg over a 72 h period. In the treatment assays, larvae were infected via the left pro-leg and treated with fluconazole, administered as a single or two doses, one hour after infection. Larval viability was monitored over five days based on movement, cocoon formation, and melanization, and survival data were analyzed using Kaplan–Meier curves and the log-rank test. Results: Fluconazole showed no toxicity at the tested concentrations. Infection with up to 2 × 107 cells/mL was non-lethal for most strains, except for A2, which exhibited 50% mortality within 48 h but it was effectively controlled with a single 20 mg/kg dose of fluconazole. Infection with 2 × 108 cells/mL resulted in complete mortality within 48 h; however, a single 80 mg/kg dose significantly improved survival. Conclusions: The G. mellonella model proved to be a suitable and reproducible in vivo system for the preliminary screening of antifungal compounds. The standardized experimental conditions established in this study support its applicability for evaluating antifungal activity in early research stages. Future studies should expand this approach to different fungal species and antifungal agents, as well as explore its applicability in combination therapies.