Arias Maria H, Deharo Eric, Valentin Alexis, Garavito Giovanny
Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias, Departamento de Farmacia (DFUNC), Grupo de Investigación FaMeTra (Farmacología de la Medicina Tradicional y Popular), Carrera 30 45-03, Bogotá D.C., 111311, Colombia.
Institut de Recherche pour le Développement, Représentation IRD Ban Naxay, Saysettha District, P.O. Box 5992, Vientiane, Lao PDR.
Parasitol Res. 2017 Jul;116(7):1955-1962. doi: 10.1007/s00436-017-5477-z. Epub 2017 May 16.
The in vivo efficacy of potential antimalarials is usually evaluated by direct microscopic determination of the parasitaemia of Plasmodium-infected mice on Giemsa-stained blood smears. This process is time-consuming, requires experienced technicians and is not automatable. Therefore, we optimized a SYBR Green I (SYBRG I) fluorescence-based assay to fluorometers commonly available in many research laboratories. This technique was originally developed to assess parasitaemia in humans by cytometry. We defined optimal conditions with Plasmodium berghei-infected mice, standard lysis buffer (Tris, EDTA, saponin and Triton), whole blood cells and 2 h staining incubation with SYBRG I 2X. The fluorescence background generated by uninfected whole blood cells was low (around 4.6%), and the linearity high (r = 0.96), with parasitaemia ranging from 1.4 to 60%. The Bland-Altman plot showed a strong correlation between SYBRG I and Giemsa gold standard method; Z'-factor was >0.5. These findings suggest that our fluorescence-based assay is suitable for in vivo antimalarial drug assessment in a malaria murine model. It can help to overcome the human bias found with microscopic techniques.
潜在抗疟药物的体内疗效通常通过直接显微镜检查来评估，即在吉姆萨染色的血涂片上测定感染疟原虫小鼠的疟原虫血症。这个过程耗时，需要经验丰富的技术人员，且无法自动化。因此，我们针对许多研究实验室常用的荧光计优化了一种基于SYBR Green I（SYBRG I）荧光的检测方法。该技术最初是为通过细胞计数法评估人类疟原虫血症而开发的。我们用感染伯氏疟原虫的小鼠、标准裂解缓冲液（Tris、EDTA、皂苷和 Triton）、全血细胞以及与2X SYBRG I进行2小时染色孵育确定了最佳条件。未感染全血细胞产生的荧光背景较低（约4.6%），线性度较高（r = 0.96），疟原虫血症范围为1.4%至60%。Bland-Altman图显示SYBRG I与吉姆萨金标准方法之间有很强的相关性；Z'因子>0.5。这些发现表明，我们基于荧光的检测方法适用于疟疾小鼠模型中的体内抗疟药物评估。它有助于克服显微镜技术中存在的人为偏差。
