Comparison of different methods for determining pyrimethamine susceptibility in P. falciparum malaria

Resistance against pyrimethamine was determined for Plasmodium falciparum adapted to continuous culture and for 'wild' isolates. The IC50 values were found by incubating for 48 hours or 48 + 24 hours (the last 24 hours without drug) and counting either the total parasites or the ring forms only. It was found that in continuous, asynchronous cultures the IC50 values were not significantly influenced by the choice of method. The IC50 value found when counting ring forms only were slightly lower than those obtained by counting the total number of parasites, but the confidence limits did overlap. The same was the case for the 48 hours and 48 + 24 hours tests. The isolates collected directly from patients with acute malaria were incubated for 48 hours. The percentage of ring forms in the control wells varied between 21% and 63% after 48 hours, indicating that the plasmodial cycle could be prolonged under test conditions. The IC50 values were estimated in two ways, by counting either the total parasitaemia or the ring forms only. On theoretical grounds, estimating the IC50 value from the number of ring forms is the most satisfactory method, as this number corresponds to the number of undamaged schizonts. The IC50 values found by 3H-hypoxanthine incorporation were lower than those found by any other method.