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Purification and in Vitro Selection of Rosette-Positive (R+) and Rosette-Negative (R-) Phenotypes of Knob-Positive Plasmodium falciparum Parasites

We have developed methods for in vitro selection of Plasmodium falciparum parasites that bear knob protrusions (K+) and are either of the rosette-positive (K+ R+) or rosette-negative (K+ R-) phenotypes. Cryopreserved parasites from spleen-intact Aotus monkeys that were K+, C32 cell adherence-positive (C+), CD36 adherence-positive, and R- with Aotus erythrocytes were adapted to continuous growth in human erythrocytes, and selected initially for adherence to C32 melanoma cells. In the absence of independent selection for rosettes, K+ R - C+ parasites were produced that adhered to both C32 cells and CD36. Without selection for the C+ phenotype, K+ R- C- parasites eventually predominated in such cultures. The R+ parasites were selected using differences in sedimentation behavior of rosette-infected cells versus non-rosette-infected cells. Methods were devised for selection of the R+ or R- phenotypes and for the purification of R+ or R- infected cells of high parasitemia that were suitable for molecular studies. With the repeated selection for K+ R+ parasites, we were able to maintain the K+ R+ phenotype for several months in vitro. These methods will allow systematic study of the molecular basis of the K+ R+ and K+ R- phenotypes.