To develop new drugs and vaccines for malaria elimination， it will be necessary to discover biological interventions， including small molecules that act against Plasmodium vivax exoerythrocytic forms. However， a robust in vitro culture system for P. vivax is still lacking. Thus， to study exoerythrocytic forms， researchers must have simultaneous access to fresh， temperature-controlled patient blood samples， as well as an anopheline mosquito colony. In addition， researchers must rely on native mosquito species to avoid introducing a potentially dangerous invasive species into a malaria-endemic region. Here， we report an in vitro culture system carried out on site in a malaria-endemic region for... More
To develop new drugs and vaccines for malaria elimination， it will be necessary to discover biological interventions， including small molecules that act against Plasmodium vivax exoerythrocytic forms. However， a robust in vitro culture system for P. vivax is still lacking. Thus， to study exoerythrocytic forms， researchers must have simultaneous access to fresh， temperature-controlled patient blood samples， as well as an anopheline mosquito colony. In addition， researchers must rely on native mosquito species to avoid introducing a potentially dangerous invasive species into a malaria-endemic region. Here， we report an in vitro culture system carried out on site in a malaria-endemic region for liver stage parasites of P. vivax sporozoites obtained from An. darlingi， the main malaria vector in the Americas. P. vivax sporozoites were obtained by dissection of salivary glands from infected An. darlingi mosquitoes and purified by Accudenz density gradient centrifugation. HC04 liver cells were exposed to P. vivax sporozoites and cultured up to 9 days. To overcome low P. vivax patient parasitemias， potentially lower mosquito vectorial capacity， and humid， nonsterile environmental conditions， a new antibiotic cocktail was included in tissue culture to prevent contamination. Culturing conditions supported exoerythrocytic (EEF) P. vivax liver stage growth up to 9 days and allowed for maturation into intrahepatocyte merosomes. Some of the identified small forms were resistant to atovaquone (1 μM) but sensitive to the phosphatidylinositol 4-kinase inhibitor， KDU691 (1 μM). This study reports a field-accessible EEF production process for drug discovery in a malaria-endemic site in which viable P. vivax sporozoites are used for drug studies using hepatocyte infection. Our data demonstrate that the development of meaningful， field-based resources for P. vivax liver stage drug screening and liver stage human malaria experimentation in the Amazon region is feasible.
