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
Citation
Master of Science in Clinical StudiesPublisher
University of Nairobi Faculty of Agriculture, University of Nairobi