Kasem Kulkeaw1, Nutpakal Ketprasit2, Anchalee Tungtrongchitr3, Duangdao Palasuwan4. 1. Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2, Wanglang Road, Bangkoknoi, Bangkok, 10700, Thailand. kasem.kuk@mahidol.edu. 2. Oxidation in Red Cell Disorders and Health Research Unit, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, 154 Rama 1 Road, Pathumwan, Bangkok, 10330, Thailand. 3. Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2, Wanglang Road, Bangkoknoi, Bangkok, 10700, Thailand. 4. Oxidation in Red Cell Disorders and Health Research Unit, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, 154 Rama 1 Road, Pathumwan, Bangkok, 10330, Thailand. nantadao@gmail.com.
Abstract
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 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. 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.
BACKGROUND: Gold standard microscopic examination of Plasmodiumfalciparum 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 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. 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.