基于流式细胞术的体外疟原虫药物敏感性评估的比较研究。

A comparative study of a flow-cytometry-based assessment of in vitro Plasmodium falciparum drug sensitivity.

机构信息

School of Physics, M013, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia.

出版信息

Malar J. 2009 Dec 14;8:294. doi: 10.1186/1475-2875-8-294.

BACKGROUND

Recently developed Sybr Green-based in vitro Plasmodium falciparum drug sensitivity assays provide an attractive alternative to current manual and automated methods. The present study evaluated flow cytometry measurement of DNA staining with Sybr Green in comparison with the P. falciparum lactate dehydrogenase assay, the tritiated hypoxanthine incorporation assay, a previously described Sybr Green based plate reader assay and light microscopy.

METHODS

All assays were set up in standardized format in 96-well plates. The 50% inhibitory concentrations (IC50) of chloroquine, mefloquine and dihydroartemisinin against the laboratory adapted P. falciparum strains 3D7, E8B, W2mef and Dd2 were determined using each method.

RESULTS

The resolution achieved by flow cytometry allowed quantification of the increase in individual cell DNA content after an incubation period of only 24 h. Regression, and Bland and Altman analyses showed that the IC50 values determined using the flow cytometry assay after 24 h agreed well with those obtained using the hypoxanthine incorporation assay, the P. falciparum lactate dehydrogenase assay, the Sybr Green plate reader assay and light microscopy. However the values obtained with the flow cytometry assay after 48 h of incubation differed significantly from those obtained with the hypoxanthine incorporation assay, and the P. falciparum lactate dehydrogenase assay at low IC50 values, but agreed well with the Sybr Green plate reader assay and light microscopy.

CONCLUSIONS

Although flow cytometric equipment is expensive, the necessary reagents are inexpensive, the procedure is simple and rapid, and the cell volume required is minimal. This should allow field studies using fingerprick sample volumes.

背景

最近开发的基于 Sybr Green 的体外疟原虫药物敏感性检测方法为目前的手动和自动化方法提供了一种有吸引力的替代方法。本研究评估了用 Sybr Green 对 DNA 染色的流式细胞术测量，与疟原虫乳酸脱氢酶检测法、氚标记次黄嘌呤掺入检测法、之前描述的基于 Sybr Green 的平板读数仪检测法和相差显微镜进行比较。

方法

所有检测法均以标准化格式在 96 孔板中设置。使用每种方法确定氯喹、甲氟喹和双氢青蒿素对实验室适应的疟原虫株 3D7、E8B、W2mef 和 Dd2 的 50%抑制浓度（IC50）。

结果

流式细胞术的分辨率允许在仅孵育 24 小时后定量个体细胞 DNA 含量的增加。回归和 Bland 和 Altman 分析表明，24 小时后使用流式细胞术检测法确定的 IC50 值与氚标记次黄嘌呤掺入检测法、疟原虫乳酸脱氢酶检测法、Sybr Green 平板读数仪检测法和相差显微镜获得的 IC50 值吻合良好。然而，孵育 48 小时后使用流式细胞术检测法获得的值与氚标记次黄嘌呤掺入检测法以及疟原虫乳酸脱氢酶检测法在低 IC50 值时差异显著，但与 Sybr Green 平板读数仪检测法和相差显微镜吻合良好。