Development of a colorimetric assay to assess drug susceptibilities in trypanosoma congolense

Abstract

When propagated in vitro bloodstream forms of all Trypanosoma brucei subspecies give suspension cultures while T Congolense gives predominantly adherent ones. As a result, in vitro assays used in assessing drug sensitivity in T brucei species are unsuitable for T congolense unless a laborious step to detach the trypanosomes is included. A colorimetric cytotoxicity assay originally developed for cancer cells was investigated for its suitability in assessing drug susceptibilities in culture adapted T congolense populations. Bloodstream form trypomastigotes were propagated in a liquid medium at 34°C and 4.5% CO2 atmosphere. The trypanosome culture adapted assay required the use of trypomastigotes harvested from cultures in logarithmic growth phase. T congolense bloodstream forms were seeded at a density of lx l O' to lx l O" organismslml in 200~1 aliquotes per well of a 96 well plate. They were then incubated with various concentrations of drug for 48 hours without medium change. The endpoint of the assay was determined by staining fixed trypanosomeprotein with sultorhodamine B. The optical density measured at 540 nm was found to increase in direct proportion to the number oftrypanosomes in the range of 5.0xl04 to 8.0x105 organisms 200 sample, The modified assay was tested using seven antitrypanosomal drugs on two stocks and five clones of T. congolense. The trypanocides diminazene aceturate, isometamidium chloride, homidium chloride, quinapyramine sulphate. suramin, melarsoprol and difluoromethyl ornithine (DFMO) were used. The colorimetric assay demonstrated the antitrypanosornal activity of the drugs in vitro and was found to be effective in showing differences in sensitivities of different T. congolense populations to the drugs. This in vitro chemosensitivity assay is suitable for the screening of new compounds and for the identification of drug resistant trypanosome strains. It is easy to perform and enables one to process large numbers of samples