In vitro drug sensitivity test for Trypanosoma brucei subgroup bloodstream trypomastigotes

An in vitro test has been developed to determine drug sensitivities of bloodstream trypomastigotes of Trypanosoma (T.) brucei isolates. The incorporation of [14C]leucine, [3H]thymidine and [3H]hypoxanthine into bloodstream forms in vitro was compared and different sensitivity test procedures with trypanocidal drugs were evaluated. Bloodstream forms were added to a microtiter plate with serial dilutions of trypanocidal drugs containing a cell-free culture medium supplemented with a mammalian serum, which allows continuous cultivation of the bloodstream stages. After a preincubation period of 16 h, [3H]hypoxanthine was added, and after another 8 h, the cells were harvested with a cell harvester. The glass fiber filters on which the cells were collected were counted in a liquid scintillation counter and the percent inhibition determined as percentage of the control counts. This method gave accurate, reproducible results for T. (T.) brucei isolates tested with the trypanocidal drugs suramin, berenil, samorin, lomidine and Mel W.