Cloning of a fresh isolate of Plasmodium falciparum and drug sensitivity of the clones.

A freshly isolated strain of Plasmodium falciparum was cloned by limited dilution using a co-culture of infected erythrocytes on monolayers of functionally active rodent hepatocytes. 15 clones were isolated, and the anti-malarial activity of chloroquine, quinine, mefloquine and halofantrine against the clones, the original isolate, and a culture-adapted isolate was determined using a 48 h radioisotope microdilution method. The multiplication rates of all clones and the culture-adapted isolate were estimated by counting the number of parasitized cells on Giemsa-stained thin smears. Variations found in drug sensitivity and multiplication rate of different clones provided strong evidence of heterogeneity of a single strain parasite population. No morphological variation was detected by light microscopy. The use of a hepatocyte feeder layer improved the adaptation of cloned parasites to continuous culture conditions and thus enabled us to clone directly a fresh isolate, without losing clones during the culture-adaptation process.