Progress and challenges in the use of fluorescence‐based flow cytometric assays for anti‐malarial drug susceptibility tests

Abstract

Drug-resistant Plasmodium is a frequent global threat in malaria eradication programmes, highlighting the need for new anti-malarial drugs and efficient detection of treatment failure. Plasmodium falciparum culture is essential in drug discovery and resistance surveillance. Microscopy of Giemsa-stained erythrocytes is common for determining anti-malarial effects on the intraerythrocytic development of cultured Plasmodium parasites. Giemsa-based microscopy use is conventional but laborious, and its accuracy depends largely on examiner skill. Given the availability of nucleic acid-binding fluorescent dyes and advances in flow cytometry, the use of various fluorochromes has been frequently attempted for the enumeration of parasitaemia and discrimination of P. falciparum growth in drug susceptibility assays. However, fluorochromes do not meet the requirements of being fast, simple, reliable and sensitive. Thus, this review revisits the utility of fluorochromes, notes previously reported hindrances, and highlights the challenges and opportunities for using fluorochromes in flow cytometer-based drug susceptibility tests. It aims to improve drug discovery and support a resistance surveillance system, an essential feature in combatting malaria.

Trio fluorophore-based phenotypic assay for the detection of artemisinin-induced growth-arrested Plasmodium falciparum in human erythrocytes

“…Advances in fluorescence-based cell analysis allow more specific and sensitive detection of organelles and biomolecules of a given cell type, including Plasmodium spp 25 . Given the lack of nuclei, the detection of intraerythrocytic parasites primarily relies on the use of any fluorescent probe that binds to the DNA of P. falciparum .…”

Antimalarial Activity of Aqueous Extracts of Nasturtium (Tropaeolum majus L.) and Benzyl Isothiocyanate

“…Flow cytometry was first used to evaluate the aqueous extracts and BITC for antimalarial activity against the asexual blood stage GFP-expressing P. falciparum (3D7-GFP), a chloroquine-sensitive strain. Using the 3D7-GFP strain obviates any staining procedure with a fluorescent dye since the parasites are auto-fluorescent, simplifying culture procedures [30,31]. The number of fluorescent events after drug exposure detected by flow cytometry, i.e., the percentage of surviving GFP parasites, allows the determination of the growth inhibition percentage, as described in Teixeira de Morais Gomes et al (2020).…”

Antimalarial Activity of Aqueous Extracts of Nasturtium (Tropaeolum majus L.) and Benzyl Isothiocyanate

“…Flow cytometry was first used to evaluate the aqueous extracts and BITC for antimalarial activity against the asexual blood stage GFP-expressing P. falciparum (3D7 − GFP), a chloroquine-sensitive strain. Using the 3D7 − GFP strain obviates any staining procedure with a fluorescent dye since the parasites are auto-fluorescent, simplifying culture procedures [33,34]. The number of fluorescent events after drug exposure detected by flow cytometry, i.e., the percentage of surviving GFP parasites, allows for the determination of the growth inhibition percentage, as described in Teixeira de Morais Gomes et al (2020).…”