Figure 2. Assays available for determining the stage of activity and potency of potential antimalarial compounds.

To detect likely prophylactic activity, P. berghei or P. yoelli sporozoites are seeded onto human hepatoma cells and then the infection rate is imaged⁴¹ or detected enzymatically¹²³. To detect prophylactic in vivo activity, rodent malaria sporozoites are injected into a mouse shortly before or after the mouse has been treated with the compound. The infection can be visualized with luciferase (with genetically modified parasites) or by measuring reduction in blood stage parasitemia and/or improved survival. The radical cure potential of a compound is tested using the hypnozoite-forming monkey model, P. cynolmolgi¹¹. In the in vitro assay, P. cynolmolgi sporozoites are seeded onto primary monkey hepatocytes and imaged to determine the ratio between large, rapidly developing schizonts and dormant “small forms” (thought to be hypnozoites). Radical cure agents will eliminate all parasites including hypnozoites, whereas prophylactic compounds will act against growing schizonts only. In the in vivo model, monkeys are infected with P. cynolmolgi sporozoites followed by treatment with a compound that eliminates all blood stage parasites (e.g. chloroquine) and then by a potential radical cure compound¹²⁴. The monkeys are then monitored over several months to measure reductions in the frequency of hypnozoite-caused relapses. The IC₅₀ of P. falciparum blood stages is typically measured in a parasite proliferation assay²⁰. P. vivax blood stage sensitivity is determined using a schizont maturation assay using parasites taken directly from infected patients¹²⁵. The in vivo efficacy of blood-stage compounds is typically measured in mice infected with P. berghei or P. yoelli, although SCID mice can be infected with P. falciparum¹²⁶. Transmission-blocking activity can be assayed in vitro by looking either at the viability¹²⁷ or the development¹²⁸ of purified P. falciparum gametocytes or ookinetes⁵². The ability of gametocytes to infect mosquitoes is measured using a standard membrane feeding assay (with P. falciparum)^129,130 or by direct feeding from an infected mouse(with P. berghei or P. yoelli)⁵³. After feeding the number of oocysts per mosquito midgut is counted to determine drug efficacy.

Notes: Pf, P. falciparum; Pv, P. vivax; Pb, P. berghei; Py, P. yoelli; Pc, P. cynomolgi.