Molecular epidemiology of malaria in Cameroon.: XX.: Experimental studies on various factors of in vitro drug sensitivity assays using fresh isolates of Plasmodium falciparum

The influence of several factors on parasite growth and 50% inhibitory concentration (IC50) for chloroquine was assessed. Most isolates stored at 4degreesC up to 72 hours grew when they were subsequently cultivated. However, parasite viability sharply decreased from 24 hours, and the mean chloroquine IC50 decreased significantly (P < 0.05). There was no evidence for selection of pre-culture populations due to storage alone. The time point when H-3-hypoxanthine was added (0 versus 18 hours) had no effect on the IC50 during the 42-hour incubation, but was associated with a lower IC50 when H-3-hypoxanthine was added after the initial 42-hour incubation during the 72-hour incubation. An increase in H-3-hypoxanthine incorporation and chloroquine IC50 was observed as the hematocrit was increased from 1.0% to 2.5%. For the same isolates, chloroquine IC50 values were generally similar when the initial parasitemia was between 0.1% and 0.5% but increased at higher ( > 0.75%) parasitemias. Based on these results, we recommend immediate cultivation after blood collection, a 42-hour incubation period with the addition of H-3-hypoxanthine at the beginning of incubation, a 1.5% hematocrit, and an initial parasitemia 0.1-0.5%. Further studies on serum substitutes, gas mixture, and comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol.