Gold standard microscopic examination of Plasmodium falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; however, microscopy is laborious and its accuracy is dependent upon the skill of the examiner. In this study, ViSafe Green (VSG), which is a nucleic acid-binding fluorescent dye, was used for assessing in vitro development of P. falciparum using flow cytometry. Fluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSGhigh population, and growing trophozoites and ring-shaped forms were observed in the VSGintermediate and VSGlow populations. The efficacy of VSG-based assay was found to be comparable to the microscopic examination method, and it demonstrated an ability to detect as low as 0.001% of the parasitaemia estimated by Giemsa staining. Moreover, when applying VSG for anti-malarial drug test, it was able to observe the growth inhibitory effect of dihydroartemisinin, the front-line drug for malaria therapy. Taken together, the results of this study suggest the VSG-based flow cytometric assay to be a simple and reliable assay for assessing P. falciparum malaria development in vitro.

中文翻译：

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一种简单的单色流式细胞术，用于评估恶性疟原虫的红细胞内发育。

恶性疟原虫红细胞内阶段的金标准显微检查仍然是分期和枚举培养物中寄生红细胞的重要过程。但是，显微镜检查很费力，其准确性取决于检查者的技能。在这项研究中，ViSafe Green（VSG）是一种核酸结合荧光染料，用于通过流式细胞术评估恶性疟原虫的体外发育。发现VSG的荧光强度取决于寄生虫的发育阶段。具体来说，在VSGhigh种群中观察到了含有多核的裂殖体，在VSGintermediate和VSGlow种群中观察到了滋养体和环状形式的生长。发现基于VSG的检测方法的功效与显微镜检查方法相当，并且证明了通过Giemsa染色检测到的寄生虫血症的检出率低至0.001％。此外，将VSG应用于抗疟药测试时，能够观察到抗疟疾一线药物二氢青蒿素的生长抑制作用。两者合计，这项研究的结果表明基于VSG的流式细胞术是评估体外恶性疟原虫疟疾发展的一种简单可靠的方法。