Invasion of human red blood cells (RBCs) by P. falciparum merozoites is a complex, multistage process. It has been known for some time that the condition of the targeted RBC may affect the efficiency of invasion and hence the severity of the disease. The work reported here arose from a casual observation which suggested that the volume of RBCs has a profound effect on parasite invasion.

To investigate this volume effect, invasion and growth parameters of P. falciparum parasites were compared in cultures sustained with normal-volume RBCs and with RBCs whose volume had been altered experimentally. The volume of target RBCs was modified isotonically, by transient activation of the RBC Ca²⁺-sensitive K⁺ channels (Gardos channels). Venous blood from healthy volunteers (blood group O) was drawn into heparinized syringes after informed, written consent. After washes, RBCs were suspended in isotonic Hepes-buffered salines containing 10 mM SCN^–, 50 µM Ca²⁺ and different K⁺ concentrations, from 2 to 137 mM, and incubated at 37 °C. A uniform Ca²⁺ load was induced by addition of a high concentration of a Ca²⁺ ionophore (A23187). This activated the Gardos channels allowing rapid K⁺ equilibration with cell shrinkage or swelling by net KCl loss or gain, respectively, depending on the set K⁺ gradients. Addition to the suspension of EGTA in excess of Ca²⁺ extracted the cell Ca²⁺. Subsequent washes with albumin removed the ionophore from the cells, thus restoring the normal low Ca²⁺ and K⁺ permeabilities. P. falciparum parasites of the A4 clone were cultured in human RBCs and synchronized by standard methods. Schizont-infected RBCs were enriched to about 90 % parasitaemia, mixed with uninfected RBCs of different volumes to final parasitaemias of about 6 %, and cultured in standard conditions. Invasion and growth were followed by microscopic inspection of Giemsa stained thin smears after 6 h, and by ³H-hypoxanthine incorporation after 24 h.

In one experiment, typical of five, ring-parasitaemias at 6 h in controls and in cells equilibrated at [K⁺]_o of 2, 45, 80 and 137 mM, were 10, 0, 4.8, 9.6 and 11.4 %, respectively. Invasion was similar in untreated controls and in K⁺-equilibrated normal-volume RBCs indicating that brief exposures to ionophore and high internal Ca²⁺ had no effect on invasion. ³H-hypoxanthine incorporation showed an identical trend in three experiments: maximal inhibition in profoundly dehydrated cells, and normal or elevated incorporation in swollen cells, with graded responses in between. Although the mechanism of this volume-effect is yet to be investigated, our results may help explain the selective advantage in malaria endemic regions of mutations which generate subpopulations of relatively dense RBCs in the circulation. The reduced infectivity of dehydrated RBCs would prevent the development of high parasitaemias and hence the incidence of severe malaria.

This work was supported by The Wellcome Trust