Development of an in vitro microtest for determining the susceptibility of Plasmodium falciparum to sulfadoxine-pyrimethamine: laboratory investigations and field studies in Port-au-Prince, Haiti

Abstract

An in vitro microtest for assessing the susceptibility of Plasmodium falciparum to sulfadoxine-pyrimethamine (S-P) was developed following WHO guidelines. Paraaminobenzoic acid and folic acid were depleted in the culture medium used, the test wells were predosed with sulfadoxine and pyrimethamine at a constant ratio of 80:1, and the parasites were incubated for 48 hours. Optimum parasite multiplication was obtained with a 2% erythrocyte suspension in medium supplemented with 12% serum. During in vitro studies with laboratory-adapted isolates, response patterns were obtained which distinguished 3 isolates with documented in vivo sensitivity to S-P from 2 isolates with documented in vivo resistance to S-P. In addition, among the three S-P-sensitive isolates, one isolate that was pyrimethamine-resistant in vitro had a higher S-P inhibitory endpoint than 2 isolates that were pyrimethamine-sensitive in vitro. The S-P microtest was further evaluated in combined in vivo and in vitro studies in Port-au-Prince, Haiti. Twenty-six patients infected with P. falciparum were treated with standard doses of S-P, resulting in prompt clearance of parasitaemia, with no recurrence in the 24 patients who completed a 28-day follow-up period. Parallel in vitro tests with pyrimethamine alone showed 3 pyrimethamine-resistant isolates out of 22 successful tests on the patients' blood samples. In 23 successful S-P tests, the known in vivo S-P-sensitive parasites were inhibited at S-P concentrations that were generally lower for in vitro pyrimethamine-sensitive isolates than for in vitro pyrimethamine-resistant ones.