Enhanced ex vivo Plasmodium vivax intraerythrocytic maturation from cryopreserved isolates for highly sensitive antimalarial susceptibility assays

Background: The clinical antimalarial options against Plasmodium vivax are limited, and emergence of P. vivax populations resistant to these drugs is globally widespread. While many variations of in vitro drug assays have been developed for P. vivax, none to date have been able to utilize small-volume, cryopreserved human isolates for testing more than a handful of drugs. Factors that make such an assay challenging include extremely low parasitemias from patients and poor parasite survival through in vitro intraerythrocytic maturation at all stages, which lead to suboptimal sensitivities of the current P. vivax drug assays. Thus, there is a serious demand for robust, sensitive and high-throughput, in vitro P. vivax drug assays. An improved assay would enable more rapid identification of novel antimalarials via larger-scale compound screening, and would enable the study of emerging and spreading drug resistant parasite populations in various geographic locations simultaneously. Materials and Methods: To address low parasitemias from patient isolates, various density-based reticulocyte enrichment methods were compared for their ability maximize enrichment and yield of parasites from small-volume, cryopreserved P. vivax isolates from human donors. To enhance P. vivax survival throughout intraerythrocytic growth, we designed and executed a P. vivax maturation screen of twenty different, commercially acquired media, with an emphasis on media developed for cultivation of erythropoietic cells. Finally, isotopic labeling was adapted to maximize sensitivity of drug assays. Results and Conclusions: We adapted reticulocyte enrichment protocols to establish a reproducible Percoll gradient enrichment method, increasing working parasitemias from small-volume, cryopreserved isolates by an average of 50-fold. The media screen identified a medium formulation that roughly doubles parasite survival through intraerythrocytic maturation compared to current media standards. We used this to optimize additional culture parameters including oxygen tension, hematocrit and serum supplementation. Finally, coupling these advances with hypoxanthine metabolic labeling permits highly-sensitive, robust, and scalable measurement of parasite survival and maturation. Applying this to multiple cryopreserved isolates enables assays to be done in batch on inherently synchronous parasites, minimizing experimental variation. This improved assay facilitates the assessment of P. vivax susceptibly to novel and existing antimalarials, permitting more rapid drug discovery and easier resistance monitoring.