Malaria and visceral leishmaniasis are major killer parasitic diseases. These diseases though occur primarily in tropical countries; economic burden and overall health impacts of malaria and leishmaniasis are global. The emergence of drug-resistant and more-virulent strains of the pathogens has further amplified the problems. New drug discovery approaches primarily rely on in vitro and in vivo models of the disease. The pathogens causing leishmaniasis and malaria are intracellular. Leishmania parasite grows as amastigotes in macrophages cells, and malaria parasite grows within the hepatocytes or erythrocytes. The intracellular forms of the pathogens are responsible for the pathophysiology of the diseases. New phenotypic cell-based models have been developed for leishmaniasis and malaria, those have been employed for in vitro/in vivo screening for new drug discovery. A parasite-rescue and transformation assay was developed for macrophage-internalized Leishmania donovani amastigotes. The assay has been applied for high-throughput screening of a library of plants’ fractions. Two fluorescent transgenic cell lines of L. donovani were developed with mCherry and Citrine reporter genes by stable transfection approach. The transgenic cell lines have shown stable and constitutive expression of the fluorescent reporter proteins. The in vitro screening methods were developed with the transgenic leishmania cells employing flow-cytometric and fluorescent microscopy analyses. Analysis of parasitemia and intra-erythrocytic growth of the parasite are hallmarks of malaria research. A flow-cytometric assay, based on staining of the malaria parasites with LDS-751, a fluorescent cell-permeant nucleic acid stain, was developed for parasitemia analysis. Staining of malaria-infected RBCs may be performed directly without additional processing. Selective staining of malaria-infected erythrocytes by LDS-751 was confirmed with fluorescent microscopy. The method has been applied for flow-cytometric analysis of parasitemia in mice blood infected with Plasmodium berghei and human blood infected with P. falciparum. The utility of this developed method was established for both in vitro and in vivo antimalarial drug screenings. Establishment of the new phenotypic assay will expedite the process of new drug discovery against the tropical parasitic diseases. These assays would also have utility for understanding biology, virulence, and pathogenesis of malaria and leishmania pathogens
