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

Kasem Kulkeaw,Duangdao Palasuwan,Nutpakal Ketprasit,Anchalee Tungtrongchitr

doi:10.1186/s12936-020-03156-1

Kasem Kulkeaw, Duangdao Palasuwan + Show 2 more

Open Access

https://doi.org/10.1186/s12936-020-03156-1

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Abstract

BackgroundGold 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.MethodsIn 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.ResultsFluorescence 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.ConclusionsTaken 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.

Highlights

Gold standard microscopic examination of Plasmodium falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; microscopy is laborious and its accuracy is dependent upon the skill of the examiner
Cell permeability of ViSafe Green (VSG) dye To ensure that VSG is cell-permeable and that it binds to nucleic acid, a non-synchronized culture of P. falciparum (Fig. 1a) was incubated with VSG dye without fixation and subjected to laser-scanning confocal microscope imaging in which the emitted fluorescent signal of VSG was displayed as green
To deny the possibility of autofluorescence, a sample of unstained P. falciparuminfected erythrocytes was used as a control

Summary

Introduction

Gold standard microscopic examination of Plasmodium falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; microscopy is laborious and its accuracy is dependent upon the skill of the examiner. Plasmodium falciparum remains a wide-spreading and highly virulent parasitic protozoan worldwide [1]. Microscopic examination is an effective method for assessing in vitro growth of malaria parasites in P. falciparum culture, as well as for drug sensitivity testing [3,4,5]. The counting of malaria-infected erythrocytes under a microscope is tedious and timeconsuming. This method requires a well-trained and experienced microscopist to enumerate and differentiate various stages of malaria parasites. Inter-rater variability among microscopists is, a drawback of the microscopic examination method

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