UMR 5235, Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS Université Montpellier II, Case 107, Place Eugene Bataillon, 34095 Montpellier, Cedex 05, France.
J Clin Microbiol. 2010 May;48(5):1651-60. doi: 10.1128/JCM.02250-09. Epub 2010 Mar 10.
In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.
体外抗疟活性测试在疟疾药物研究或监测野外分离株的药物耐药性方面发挥着关键作用。我们应用了两种同位素测试、两种酶联免疫吸附测定（ELISA）和 SYBR 绿色 I 荧光测定，以测试青蒿琥酯和氯喹、代谢抑制剂阿托伐醌和嘧啶并恶嗪、我们的快速作用胆碱类似物 T3/SAR97276 和多西环素，后者具有延迟死亡特征。基于次黄嘌呤和乙醇胺掺入的同位素测试是最可靠的测试，当它们在一个完整的 48 小时寄生虫周期后应用时。测量 DNA 含量的 SYBR 绿色测定通常需要 72 小时的孵育才能获得可靠的结果。当怀疑存在延迟死亡时，需要使用特定的方案，孵育时间增加到 96 小时。相比之下，使用的两种 ELISA 测试（pLDH 和 HRP2）似乎存在问题，导致与青蒿琥酯样特征不同的分子产生令人失望甚至错误的结果。这些测试的可靠性与药物的作用模式有关，获得有意义结果所需的条件很难预测。我们的结果表明了一些应用这些测试的最低条件，无论化合物的作用机制如何，都应该产生一个标准的 50%抑制浓度，并强调最常用的体外抗疟活性测试没有相同的潜力。其中一些可能无法检测到具有创新作用模式的新型化合物的抗疟潜力，这些化合物随后可能成为有前途的新抗疟药物。
