Short technical report
A malaria gametocytocidal assay using oxidoreduction indicator, alamarBlue

https://doi.org/10.1016/j.molbiopara.2011.02.005Get rights and content

Abstract

Efforts to move from malaria control to eradication will require new approaches to block malaria transmission, such as the development of anti-malarial drugs with gametocytocidal activity. Here fluorescent oxidoreduction indicator alamarBlue is used to develop a screen for gametocyte viability. The fluorescent signal increases linearly with gametocyte number (R2 = 0.99) and determination of the IC50 of epoxomicin demonstrated the assay was reproducible and sensitive (IC50 2.16 ± 0.57 nM, Z′-factor 0.81 ± 0.01). Six anti-malarials were also tested and at 10 μM only primaquine and dihydroartemisinin (DHA) had gametocytocidal activity. This new assay provides an important tool to efficiently screen compounds for gametocytocidal activity.

Graphical abstract

. An efficient and reliable gametocytocidal drug screening assay was developed using cell viability marker, alamarBlue.

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Research highlights

► Inhibition data from alamarBlue assays are similar to traditional optical assays. ►Assay time for 96 samples is decreased from 10 h to 1 min. ► Reproducibility is increased. ► Any parasite line, including field isolates, can be tested for drug sensitivity.

Section snippets

Assay optimization

AlamarBlue was first screened to ensure that it did not interfere with gametocyte development or the gametocytocidal activity of proteasome inhibitor, epoxomicin [14]. Gametocyte (3D7 strain) cultures were set up in complete media (RPMI 1640 supplemented with l-Glutamine, 25 mM HEPES, 50 μg ml−1 hypoxanthine (KD Biomedical), 10 μg ml−1 gentamicin, 0.25% NaHCO3, and 10% human serum) as described [15] and the parasites were treated with N-acetyl glucosamine (NAG) from day 9 to day 11 to eliminate

Final assay conditions

Once the basic conditions were established for the alamarBlue assay it was directly compared with the current standard assay, optical microscopy of Giemsa-stained smears. The epoxomicin dose response was used for comparison. For the optical assay, 10 μl of DMSO alone or in combination with the indicated concentration of epoxomicin were added to NAG-treated gametocyte cultures (1.45% gametocytemia (stages III–V)/3% hematocrit on day 13). Giemsa-stained smears were prepared at 72 and 96 h after

Gametocytocidal activity of common anti-malarial drugs

As a further test, the alamarBlue assay was used to screen the gametocytocidal activity of a panel of current anti-malarial drugs, pyrimethamine, chroloquine, quinine, mefloquine, dihydroartemisinine (DHA) and primaquine (Fig. 2). Consistent with previous reports pyrimethamine, chloroquine, quinine or mefloquine did not show gametocytocidal activity, while primaquine showed a reduction of 69.4 ± 17.2% activity at 10 μM. DHA, the active metabolite of artemisinin was the most effective against late

Acknowledgements

This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank Dr. S. Desai for use of the fluorescent plate reader and Dr. B. Morahan for critical reading of the manuscript.

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