Isolates of P. vivax were collected from symptomatic patients enrolled in the study after giving written informed consent at the Thai malaria clinic (SMRU) in Tak province, Mae Sod Malaria Clinic (Thailand Ministry of Public Health) in Tak province, and through recruitment via the village malaria workers network and the local health centers in the Mundolkiri Province in Cambodia. A sample of P. vivax-infected blood was drawn by venipuncture into heparin tubes. Thick and thin Giemsa-stained smears were prepared after removal of white blood cells (WBCs). A diagnostic PCR was used to confirm microscopic identification that P. vivax was the only Plasmodium species present in the samples used for the study77 (link).
The mosquito infections for P. vivax were performed using a colony of An. dirus (Bangkok strain), An. cracens78 (link) or An. dirus B in Cambodia. Briefly, 150 μL of RBC pellet from infected patient blood samples was suspended in pooled normal AB human serum to 50% hematocrit. Next, 300 μL of the suspension was fed for 30 min to 5–7 days old female mosquitoes via an artificial membrane attached to a water-jacketed glass feeder maintained at 37 °C. Engorged mosquitoes were kept on 10% sucrose solution and maintained at 26 °C and 80% humidity until dissected. Salivary glands of mosquitoes 14–16 days after infection were dissected and collected as described above. Studies including freshly dissected P. vivax sporozoites were performed at Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand, Shoklo Malaria Research Unit, Mae Sot, Thailand, and Institut Pasteur du Cambodge, Phnom Penh, Cambodia. For some LS studies, mosquitoes carrying P. vivax oocysts were shipped, still pre-infectious, from AFRIMS to the University of South Florida following permit approval by the Thai Ministry of Health, US Center for Disease Control, US Department of Agriculture, and Florida Department of Agriculture.
The mosquito infections for P. vivax were performed using a colony of An. dirus (Bangkok strain), An. cracens78 (link) or An. dirus B in Cambodia. Briefly, 150 μL of RBC pellet from infected patient blood samples was suspended in pooled normal AB human serum to 50% hematocrit. Next, 300 μL of the suspension was fed for 30 min to 5–7 days old female mosquitoes via an artificial membrane attached to a water-jacketed glass feeder maintained at 37 °C. Engorged mosquitoes were kept on 10% sucrose solution and maintained at 26 °C and 80% humidity until dissected. Salivary glands of mosquitoes 14–16 days after infection were dissected and collected as described above. Studies including freshly dissected P. vivax sporozoites were performed at Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand, Shoklo Malaria Research Unit, Mae Sot, Thailand, and Institut Pasteur du Cambodge, Phnom Penh, Cambodia. For some LS studies, mosquitoes carrying P. vivax oocysts were shipped, still pre-infectious, from AFRIMS to the University of South Florida following permit approval by the Thai Ministry of Health, US Center for Disease Control, US Department of Agriculture, and Florida Department of Agriculture.
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