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

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.