Optimal concentration of p-aminobenzoic acid and folic acid in the in vitro assay of antifolates against Plasmodium falciparum.

In vitro tests for Plasmodium falciparum sensitivity to pyrimethamine, sulfadoxine, and both drugs in combination were performed in four kinds of culture medium, each differing in p-amino benzoic acid (PABA) and folic acid concentrations. Results of the tests using pyrimethamine-sensitive and pyrimethamine-resistant isolates indicated that drug activity was reduced proportionally to the concentrations of these two growth factors in the medium. The optimal concentrations of PABA and folic acid for parasite growth and drug susceptibility, as evaluated by microscopic examination and by the extent of incorporation of radioactive 14C-pyrimethamine and 14C-sulfadoxine, were 10 ng/ml and 2 ng/ml, respectively. Depletion of PABA and folic acid from the medium had no effect on drug-resistant parasites but multiplication of drug-sensitive isolates was markedly reduced. Medium containing 0.5 ng/ml PABA and 10 ng/ml folic acid was the best for parasite growth regardless of the degree of drug sensitivity. Results obtained by using this medium agreed most closely with results from in vivo observations.

The influence of magnesium ion and ascorbic acid on the erythrocytic schizogony of Plasmodium vivax.

Asexual erythrocytic stages of Plasmodium vivax have been cultivated for one schizogonic cycle to investigate parasite requirements for metal ions and vitamins. Waymouth and RPMI 1640 (GIBCO) media were used in varying proportions resulting in varying concentrations of organic salts, vitamins, and growth factors. A 1:3 mixture gave the highest percentage (62.6%) of parasite development from the amoeboid forms to mature segmenters after 44 h of cultivation in a candle jar atmosphere at 38.5 degrees C. Nevertheless, the total parasite count was significantly higher (P less than 0.50) in the mixture which had a Waymouth: RPMI ratio of 1:2. Differentiation of schizonts to merozoites as well as parasite counts could be further enhanced by the addition of magnesium chloride to a final concentration of 1.8 mM magnesium ions. The minimal requirement for ascorbic acid which was studied in Science Mahidol (SCMI 612) medium appeared to vary among isolates. For example, all parasite population of four isolates tested declined proportionally with the decrease in concentration of ascorbic acid, the critical point being 3 micrograms/ml medium. However, two isolates used in this study could no longer differentiate to segmenters when the ascorbic acid concentration of the medium was less than 6 micrograms/ml.

Mefloquine-pyrimethamine-sulfadoxine combination delays emergence of mefloquine resistant Plasmodium falciparum in continuous culture.

The efficacy of mefloquine against Plasmodium falciparum in continuous culture was studied. The development of mefloquine resistance in P. falciparum was significantly inhibited by a combination of mefloquine, pyrimethamine and sulfadoxine. By contrast, more resistant variants were selected in continuous culture without drug pressure or with pyrimethamine-sulfadoxine pressure. The most mefloquine resistant variants were selected by step-wise increases in mefloquine pressure.