Enhanced ex vivo Plasmodium vivax intraerythrocytic enrichment and maturation for rapid and sensitive parasite growth assays [PublishAheadOfPrint]

Plasmodium vivax chloroquine resistance has been documented in nearly every region endemic for this malaria-causing parasite. Unfortunately, P. vivax resistance surveillance and drug discovery is challenging due to low parasitemias of patient isolates, and poor parasite survival through ex vivo maturation, that reduce the sensitivity and scalability of current P. vivax antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for P. vivax parasites. We next performed a media screen for formulations that enhance ex vivo survival. Finally, we optimized an isotopic metabolic labelling assay for measuring P. vivax maturation and sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume ex vivo isolates by an average of >40-fold. Using Iscove's Modified Dulbecco's Medium for P. vivax ex vivo culture approximately doubled parasite survival through maturation. Coupling these with ³H-hypoxanthine metabolic labeling permitted sensitive and robust measurement of parasite maturation, which was used to measure the sensitivities of Brazilian P. vivax isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the P. vivax isolate sensitivities to antimalarials for resistance surveillance and drug discovery.

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