A comparison of in vitro assay systems for the measurement of drug sensitivity of Trypanosoma evansi.

Two in vitro assay systems were investigated for their effectiveness in detecting the sensitivity of Trypanosoma evansi stocks to the trypanocide suramin. These assay systems measured 1) incorporation of radiolabelled nucleic acid precursor, hypoxanthine; and 2) pyruvate production. They were compared with the direct counting method in which numbers of motile trypanosomes were estimated using a Neubauer haemocytometer chamber. Three stocks of T. evansi were tested-2 suramin sensitive stocks from Indonesia, TREU 1840 and TREU 1981, and a suramin resistant stock from the Sudan, TREU 2136. Each assay system distinguished between the suramin sensitive and resistant stocks. However, inhibition compared to untreated control cultures was less when assessed from pyruvate concentration in culture supernatants than by direct counting. The length of incubation with drug before addition of radio-label was the most important variable in the hypoxanthine incorporation assay. A pre-incubation time of 16 hours with the drug before adding the label for the further 8 hours of the assay was found to be the most sensitive. Under these conditions, the IC50 values (drug concentrations causing 50% inhibition) were similar to those obtained from direct counts. Pre-incubation of parasites with drug before adding the label resulted in a decrease of the IC50. These results suggest that the discrepancy between the levels of pyruvate production and relative growth at inhibitory concentrations of the drug are due to metabolism by the parasites during the initial stages of the assay, before the drug has began to inhibit cell growth.