Authors

Discussion

Visceral Leishmaniasis (VL) is caused by the protozoan parasites Leishmania donovani and L. infantum which are transmitted by female phlebotomine sandlflies. VL causes >50,000 deaths annually, new drugs are urgently required. Our in vitro screening cascade to identify new phenotypic hits against L. donovani which could progress to new clinical candidates starts with an axenic amastigote luminescence assay, followed by a high-content image-based intracellular assay (LdBob/THP1). However, to identify compounds that are effective against clinically relevant strains from different geographical locations development of a strain panel was undertaken.

Low passage metacyclic parasites were used to infect CD14+ M-CSF differentiated human PBMC which were subsequently dispensed onto compound containing 96 well plates for 96h. These were stained with Sytox green and imaged by the Operetta to generate potency and toxicity data. (Buffy coat was obtained ethically and its research use was in accordance with the terms of donor informed consent).

High levels of PBMC infection was observed for all Leishmania strains. However, replication rates varied and strain-specific variation in potency profiles was reported for some compounds. In addition, the potency data generated in this assay was a better predictor of pharmacokinetic/ pharmacodynamic relationships reported in our in vivo animal model relative to the LdBob/THP1 assay.

Incorporation of a Leishmania strain panel using primary macrophages and clinically relevant strains has proved essential in building confidence in hits coming through our VL screening cascade and has improved predictability of in vivo efficacy results.