Mapping the Madurella mycetomatis pathogen and host responses during and after antifungal treatment to identify prognostic therapeutic markers for mycetoma

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During FY2022 we completed two major tasks.First, the library preparations and sequencing for transcriptomics analysis was successfully completed.Second, pharmacokinetics of antifungal agents in Galleria mellonella larvae. For the trsanscriptomics analysis, total RNA was extracted from six type of samples at 4 timepoints (6hr,30hr,72hr, and 168hr) in triplicates (4x3 =12) the type of samples are (Healthy G. mellonella larvae,infected G. mellonella larvae with M. mycetomatis, Healthy G. mellonella larvae treated with itraconazole,infected G. mellonella larvae with M. mycetomatis treated with itraconazole (n=12),Healthy G. mellonella larvae treated with ravuconazole and infected Galleria mellonella larvae with M. mycetomatis treated with ravuconazole.Total of 72 RNA-seq libraries were prepared and sequenced. Since we have extra RNA aliquot reaming, we prepared and sequenced LQ-ssCAGE (low quantity single strand CAGE) libraries from the same samples (n=72).Raw sequenced reads mapped against the G. mellonella larvae (host) genome and then against the pathogen M. mycetomatis genome. We obtained a high mapping ratio according to RNA-seq standards.As for the pharmacokinetics(PK) of antifungal agents in G. mellonella larvae,The PK of itraconazole was determined after the administration of a single dose to uninfected G. mellonella larvae. Concentrations of antifungal agents were present during 24hr after injection and above the MIC. The AUC24h for itraconazole (61.9% of human AUC) was lower in G. mellonella larvae than in humans.