A simple monochromatic flow cytometric assay for assessment of intraerythrocytic development of Plasmodium falciparum

Background 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. Methods 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. Results Fluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSG high population, and growing trophozoites and ring-shaped forms were observed in the VSG intermediate and VSG low 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. Conclusions 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.