Ex Vivo Plasmodium malariae Culture Method for Antimalarial Drugs Screen in the Field

Abstract

In vitro and ex vivo cultivation of Plasmodium (P) falciparum has facilitated active research into the malaria parasite toward the quest for basic knowledge and the discovery of effective drug treatments. Such a drug discovery program is currently difficult for P. malariae simply because of the absence of in vitro and ex vivo cultivation system for its asexual blood stages supporting antimalarial evaluation. Despite availability of artemisinin combination therapies effective on P. falciparum, P. malariae is being increasingly detected in malaria endemic countries. P. malariae is responsible for chronic infections and is associated with a high burden of anemia and morbidity. Here, we optimized and adapted ex vivo conditions under which P. malariae can be cultured and used for screening antimalarial drugs. Subsequently, this enabled us to test compounds such as artemether, chloroquine, lumefantrine, and quinine for ex vivo antimalarial activity against P. malariae.

Keywords: Plasmodium malariae; drug discovery; ex vivo culture; malaria; nonfalciparum.

Figure 1

Microscopy and molecular detection of Human malaria species. (A) Giemsa-stained asexual blood stages of Human malaria parasites and (B) molecular detection of human malaria parasites species in test samples 1–10 along with control for Pf = Plasmodium falciparum, Pm = Plasmodium malariae, Poc = Plasmodium ovale curtisi, Pow = Plasmodium ovale wallikeri, Pv = Plasmodium vivax. W1 = Control 1, W2 = Control 2, bp = Base pairs, and MW = Molecular weight.

Figure 2

P. malariae maturation and growth monitoring in ex vivo culture using flow cytometry. (a) Time 0 h freshly collected P. malariae asexual stages, (b) time 24 h cultured P. malariae asexual blood stages, (c) time 48 h cultured P. malariae asexual blood stages, and (d) time 72 h cultured P. malariae asexual blood stages, (e) culture starting 0 h time point negative control of parasite free red blood cells, (f) culture time 72 h end point negative control of parasite free red blood cells. iRBC = infected red blood cells.

Figure 3

Plasmodium malariae maturation and growth. (a) Giemsa-stained P. malariae parasites at defined time points of the culture. (b) Five P. malariae isolates parasitemia at defined time points of the culture measured using light microscopy. (c) Nineteen field isolates of P. malariae comparative growth measured using flow cytometry. At time 0 h, 0.5% parasitemia of P. malariae asexual blood stages are cultured for 24, 48, and 72 h, and times respective parasitemia of viable parasites were evaluated using flow cytometry. Parasites were detected with Sybr Green while viability was measured using deep red mito-tracker.

Figure 4

P. malariae sensitivity to references antimalarial drugs. (A) Dose response curves of artemether against P. malariae asexual blood stages. (B) Dose response curves of lumefantrine against P. malariae asexual blood stages. (C) Dose response curves of chloroquine against P. malariae asexual blood stages. (D) Dose response curves of quinine against P. malariae asexual blood stages.