In vivo bioluminescence imaging in Galleria mellonella to non-invasively quantify fungal infection over time

Longitudinal imaging of bioluminescent fungi in Galleria mellonella can complement standard survival and health scoring data by quantifying the fungal burden over time and monitoring early infection before clinical symptoms arise, and by more sensitively and more early detection of treatment effects. By using bioluminescence flux as a readout of fungal burden instead of relying only on secondary health effects, drug doses can be linked to a quantitative reduction in fungal burden which is valuable towards further experimenting in e.g. mice. It also provides a more standardized readout that is less prone to inter-lab variability in e.g. rearing of larvae, possibly leading to intrinsic differences in health. We validated and published this method for aspergillosis (azole-resistant and susceptible) and cryptococcosis, and have shown translatability of this method to Galleria mellonella models of Candida, Fusarium and Mucorales spp. In short, larvae are injected with the bioluminescent fungus of interest, and after injection of 40 µg/g D-luciferin, they are placed individually in a black 12-well plate inside of the IVIS Spectrum (or other detection system) and photon flux from each individual larva can be quantified. Daily imaging was performed. Longitudinal follow-up has been tested up to 1 week, depending on the virulence and inoculum size of the infectious agent. Treatment was injected daily starting from 1h post-infection. Between imaging timepoints, larvae were kept individually in 12-well plates at 37°C in the dark with food during the whole experiment.