Sutherland D V, Taylor A M, Ross C A
Centre for Tropical Veterinary Medicine, University of Edinburgh, Roslin, Midlothian, Scotland.
Trop Med Parasitol. 1993 Sep;44(3):208-12.
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.
研究了两种体外检测系统在检测伊氏锥虫株对锥虫杀灭剂苏拉明敏感性方面的有效性。这些检测系统测量了：1）放射性标记核酸前体次黄嘌呤的掺入；以及2）丙酮酸的产生。将它们与直接计数法进行比较，直接计数法是使用Neubauer血细胞计数板估算活动锥虫的数量。对三株伊氏锥虫进行了测试，两株来自印度尼西亚的苏拉明敏感株TREU 1840和TREU 1981，以及一株来自苏丹的苏拉明耐药株TREU 2136。每种检测系统都能区分苏拉明敏感株和耐药株。然而，与未处理的对照培养物相比，根据培养上清液中丙酮酸浓度评估的抑制作用比直接计数法要小。在加入放射性标记之前与药物孵育的时间是次黄嘌呤掺入检测中最重要的变量。发现加入标记前先与药物预孵育16小时，然后再进行8小时检测是最敏感的。在这些条件下，IC50值（引起50%抑制的药物浓度）与直接计数法得到的值相似。在加入标记前将寄生虫与药物预孵育会导致IC50降低。这些结果表明，在药物开始抑制细胞生长之前的检测初始阶段，寄生虫的代谢导致了在抑制浓度下丙酮酸产生水平与相对生长之间的差异。
