IDENTIFICATION AND CLASSIFICATION OF THE MALARIA PARASITE BLOOD DEVELOPMENTAL STAGES, USING IMAGING FLOW CYTOMETRY

Malaria is the most devastating parasitic disease caused by the unicellular protozoa of the Plasmodium genus, and is responsible up to a million deaths each year. Plasmodium falciparum (Pf) and P. vivax are responsible for most clinical cases of malaria in humans. Pf life cycle is complex, and the transmission of the parasite between humans via mosquitos involves a remarkable series of morphological transformations. The human blood stage is responsible for all disease pathology; the parasites undergo asexual multiplication inside the red blood cell (RBC) where the parasites mature through the ring, trophozoite and schizont stages. During the asexual cycle a small portion of the parasite population develop into sexual forms (gametocytogenesis), thus ensuring parasite transfer to the mosquito host.

Flow cytometry (FC) methods were previously shown to detect Pf-iRBCs, in live or fixed cells, using DNA stain Hoechst (HO), and RNA stain Thiazole Orange (TO). Here, by using Imaging Flow Cyotmetry (IFC) we demonstrate a clear detection of each of the Pf-infected RBC life stages. This technique allows multi-channel, high resolution imaging of individual parasites, as well as detailed morphological quantification of Pf-iRBCs cultures. We developed an improved classification probing of the blood stages, both asexual and sexual. This method provides an accurate quantification and robust measurement of parasitemia profile and can serve as a valuable tool in malaria research and drugs screen assays.