Viability and Burden of Leishmania in Extralesional Sites during Human Dermal Leishmaniasis

Understanding of the dynamics and distribution of Leishmania in the human host is fundamental to the targeting of control measures and their evaluation. Amplification of parasite gene sequences in clinical samples from cutaneous leishmaniasis patients has provided evidence of Leishmania in blood, other tissues and sites distinct from the lesion and of persistence of infection after clinical resolution of disease. However, there is uncertainty about the interpretation of the presence of Leishmania DNA as indicative of viable parasites. Because RNA is short-lived and labile, its presence provides an indicator of viability. We amplified Leishmania 7SLRNA, a molecule involved in intracellular protein translocation, to establish viability and estimate parasite load in blood monocytes, tonsil swab samples, and tissue fluid from healthy skin of patients with dermal leishmaniasis. Results showed that during active dermal leishmaniasis, viable Leishmania are present in blood monocytes, tonsils and normal skin in quantities similar to that in lesions, demonstrating widespread dissemination of infection and subclinical involvement of tissues beyond the lesion site. Leishmania 7SLRNA will be useful in deciphering the role of human infection in transmission

Integration of multiple epigenomic marks improves prediction of variant impact in saturation mutagenesis reporter assay

The integrative analysis of highâ throughput reporter assays, machine learning, and profiles of epigenomic chromatin state in a broad array of cells and tissues has the potential to significantly improve our understanding of noncoding regulatory element function and its contribution to human disease. Here, we report results from the CAGI 5 regulation saturation challenge where participants were asked to predict the impact of nucleotide substitution at every base pair within five diseaseâ associated human enhancers and nine diseaseâ associated promoters. A library of mutations covering all bases was generated by saturation mutagenesis and altered activity was assessed in a massively parallel reporter assay (MPRA) in relevant cell lines. Reporter expression was measured relative to plasmid DNA to determine the impact of variants. The challenge was to predict the functional effects of variants on reporter expression. Comparative analysis of the full range of submitted prediction results identifies the most successful models of transcription factor binding sites, machine learning algorithms, and ways to choose among or incorporate diverse datatypes and cellâ types for training computational models. These results have the potential to improve the design of future studies on more diverse sets of regulatory elements and aid the interpretation of diseaseâ associated genetic variation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151884/1/humu23797_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151884/2/humu23797.pd