Discussion

Visceral Leishmaniasis (VL) is caused by protozoan parasites Leishmania donovani and L. infantum. These are transmitted by the bite of infected female sandlflies. VL is fatal if untreated with an estimated 310 million people at risk of infection and >50,000 deaths annually. 90% of VL cases are recorded in Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan. The current drugs have issues with toxicity, painful injections, cost, and long treatment regimen, therefore, new treatments are urgently required. We have developed an in vitro screening cascade to identify new phenotypic hits against L. donovani which could progress to new clinical candidates for VL. Our cascade starts with an axenic amastigote luminescence assay, followed by a high-content image-based intracellular assay which uses L. donovani LdBob strain and PMA differentiated THP1 host cells. However, to identify compounds that are effective against clinically relevant strains from different geographical locations development of a L. donovani/ L. infantum strain panel was undertaken. This assay employs human peripheral blood monocytes (M-CSF differentiated) infected with low passage metacyclic parasites which are dispensed into 96 well plates for 96h. A single dye staining and analysis protocol was developed to generate potency and host cell toxicity data.