The zoonotic malaria parasite Plasmodium knowlesi has recently been established in continuous in vitro culture. Here, the Plasmodium falciparum [ 3 H]hypoxanthine uptake assay was adapted for P. knowlesi and used to determine the sensitivity of this parasite to chloroquine, cycloguanil, and clindamycin. The data demonstrate that P. knowlesi is sensitive to all drugs, with 50% inhibitory concentrations (IC 50 s) consistent with those obtained with P. falciparum. This assay provides a platform to use P. knowlesi in vitro for drug discovery.
In 2015, there were an estimated 214 million clinical cases of malaria which resulted in ϳ438,000 deaths (1). Substantial funds have been invested in producing a malaria vaccine; however, the efficacy of experimental vaccines has been poor (2, 3), and as a result, vector control and drugs remain the mainstays for the prevention and treatment of malaria. While there has been a significant reduction in malaria-associated mortality and morbidity in recent years (1), there is concern that lack of sustained funding, together with insecticide and antimalarial drug resistance, will affect this progress (4). To prevent backward momentum in disease control and push toward the endgame strategy of malaria elimination, new chemotherapeutics with novel modes of action and activity against multiple species and life cycle stages are needed (5).The ability to easily and rapidly assess the activity of new lead compounds against multiple Plasmodium species has been limited to date, as only Plasmodium falciparum has been amenable to routine, long-term, continuous in vitro culture (6). While there have been some recent improvements to in vitro culture techniques for Plasmodium vivax, the culture of this parasite is still limited by the requirement of reticulocytes, and the parasite density over longterm culture is low (7). However, the recent adaptation of the zoonotic malaria species Plasmodium knowlesi (8) to continuous in vitro culture in human erythrocytes (9-11) has changed this position. Although a routine drug sensitivity assay for P. knowlesi has not yet been established, the ability to culture this parasite species in vitro provides researchers with an unprecedented opportunity to rapidly test new drug leads against two human-infecting Plasmodium species.In vitro assays for assessing malaria parasite growth inhibition are indispensable tools for the screening and evaluation of potential new drug leads and, also, for the surveillance of parasite drug resistance. A "gold standard" approach for assessing P. falciparum growth inhibition is the incorporation of [ 3 H]hypoxanthine into parasite nucleic acids (12). As Plasmodium parasites are unable to synthesize purines de novo, they must scavenge these metabolic precursors for growth. Thus, supplementation of parasite cultures with [3 H]hypoxanthine results in the incorporation of this radiolabeled purine into nucleic acids, permitting growth to be quantitated using a scintillation counter. While there are a number of other methods available t...