Individuals who visited the Service de Lutte Antipaludique (SLAP) clinic with signs and symptoms suggestive of uncomplicated P. falciparum malaria were screened by microscopic examination on Giemsa-stained blood smears. Malaria infected patients were treated with artemether-lumefantrine (AL, Coartem), according to the treatment guideline of the Senegal National Malaria Control Program (NMCP). For each patient, 5 mL vacutainer tubes of venous blood were collected for ex vivo RSA.
Parasite culture and ring-stage survival assay. Parasitemia was estimated by microscopy examination on Giemsa-stained blood smears. Venous blood samples were then processed by eliminating plasma, leukocytes and anticoagulant from red blood cells (RBCs), washed twice in RPMI 1640 medium (Gibco, Life technologies). Parasitemia were adjusted to 1% if greater by adding uninfected RBCs as previously described [10 (link)]. Then, 900 μL RBCs were loaded into wells, exposed to either 100 μL of 700 nM DHA or0.1% of dimethyl sulfoxide (DMSO) and cultivated at 37°C in incubator for six hours (conditions: 94% N2, 5% CO2, 1% O2) [30 (link)]. Finally, RBCs were washed and cultivated for 66 hours. The proportions of viable parasites were estimated independently by two expert malaria microscopists on Giemsa-stained thin smears. The number of viable parasites that developed into ring/trophozoite stages were determined, pyknotic forms were excluded. The average of the two counts was calculated. If any discrepancy was noted (either difference of parasite density of > 50%), slides were checked by a third independent reader, and parasite densities were calculated by averaging the two most close counts. Survival rates were calculated as the ratio of parasites in exposed and non-exposed cultures. Results were deemed as interpretable if the parasitemia in the sample exposed to DMSO was higher than the initial parasitemia at 0h [31 ].
Parasite culture and ring-stage survival assay. Parasitemia was estimated by microscopy examination on Giemsa-stained blood smears. Venous blood samples were then processed by eliminating plasma, leukocytes and anticoagulant from red blood cells (RBCs), washed twice in RPMI 1640 medium (Gibco, Life technologies). Parasitemia were adjusted to 1% if greater by adding uninfected RBCs as previously described [10 (link)]. Then, 900 μL RBCs were loaded into wells, exposed to either 100 μL of 700 nM DHA or0.1% of dimethyl sulfoxide (DMSO) and cultivated at 37°C in incubator for six hours (conditions: 94% N2, 5% CO2, 1% O2) [30 (link)]. Finally, RBCs were washed and cultivated for 66 hours. The proportions of viable parasites were estimated independently by two expert malaria microscopists on Giemsa-stained thin smears. The number of viable parasites that developed into ring/trophozoite stages were determined, pyknotic forms were excluded. The average of the two counts was calculated. If any discrepancy was noted (either difference of parasite density of > 50%), slides were checked by a third independent reader, and parasite densities were calculated by averaging the two most close counts. Survival rates were calculated as the ratio of parasites in exposed and non-exposed cultures. Results were deemed as interpretable if the parasitemia in the sample exposed to DMSO was higher than the initial parasitemia at 0h [31 ].
Free full text: Click here
