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

于2022财政年度，我们完成了两项主要工作。第一，成功完成转录组学分析的文库制备和测序。第二，抗真菌剂在大蜡螟幼虫中的药代动力学。对于转录组学分析，在4个时间点（6小时、30小时、72小时和168小时）从六种类型的样品中提取总RNA，一式三份（4 × 3 =12）。大蜡螟幼虫感染G. Mellonella幼虫与M. mycetomatis、健康G.用伊曲康唑处理的大蜡螟幼虫感染G. Mellonella幼虫与M.伊曲康唑治疗组12例，健康G.用雷武康唑处理大蜡螟幼虫，用M.共制备72个RNA-seq文库并测序。由于我们有额外的RNA等分试样，我们从相同的样品（n=72）制备并测序了LQ-ssCAGE（低量单链CAGE）文库。大蜡螟幼虫（寄主）基因组，然后对病原菌M.霉菌基因组。 根据RNA-seq标准，我们获得了较高的作图率。对未感染的大蜡螟幼虫单次给药后测定伊曲康唑的药代动力学。大蜡螟幼虫注射后24小时内存在抗真菌剂浓度，且高于MIC。伊曲康唑的AUC 24 h（人AUC的61.9%）在G.大蜡螟幼虫比人类多。

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.