A malaria gametocytocidal assay using oxidoreduction indicator, alamarBlue

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 (R(2)=0.99) and determination of the IC(50) of epoxomicin demonstrated the assay was reproducible and sensitive (IC(50) 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.

Fig. 1

AlamarBlue assay development. (A) Giemsa-stained smear of DMSO control cultures with (+) and without (−) alamarBlue. (B) Gametocytemia of cultures in the presence of epoxomicin (0.3 – 30 nM) or DMSO as the carrier control (0 nM) with (■) and without (□) alamarBlue. NAG-treated gametocyte cultures were incubated with DMSO or epoxomicin for 72 h prior to the addition of alamarBlue. Giemsa-stained smears were prepared 24 h after alamarBlue addition. (C) Gametocyte-dependent alamarBlue fluorescence. NAG-Percoll-treated ametocyte cultures, 0 – 1 × 10⁷ total cells (45.6 % gametocytemia, stage III-V) (Gcyt/RBC) (♦) or 0 – 1 × 10⁷ RBCs (RBC only) (◊) were incubated in 96-well plates with alamarBlue. Fluorescence was measured 24 h after alamarBlue addition and the average signal and standard deviation from duplicate wells in a representative experiment are shown. The data from 0 and 2 to 10 × 10⁶ total cells is shown in the upper graph and the data from 0 to 2 × 10⁶ total cells is shown in the lower graph. (D) Timing of alamarBlue addition. NAG-Percoll-treated gametocyte cultures (4 × 10⁵ gametocytes, stage III-V/ and 6 × 10⁵ RBCs) or 6 × 10⁵ RBCs were incubated in 96-well plates with DMSO or 100 nM epoxomicin. Medium alone without cells was included as a negative control. AlamarBlue was added at 24, 48 or 72 h after epoxomicin addition, and fluorescence was measured 24 h later. Medium alone, open bar; RBC with DMSO, cross-hatched bar; RBC with epoxomicin, dotted bar; gametocyte culture with DMSO, grey bar; gametocyte culture with epoxomicin, black bar (E) Inhibition curves of epoxomicin by optical microscopy. NAG-treated gametocyte cultures (stage III-V) were incubated in a 24-well plate with DMSO or epoxomicin. Giemsa-stained smears were prepared at 72 and 96 h after epoxomicin addition. (F) Epoxomicin inhibition curves using the alamarBlue assay. NAG-Percoll-treated gametocyte cultures (stage III-V) were incubated in a 96-well plate with DMSO or epoxomicin. AlamarBlue was added at 72 h after epoxomicin addition and fluorescence was measured 24 h later. Statistical analysis was done with GraphPad Prism 5 software (GraphPad Software, Inc.).

Fig. 2

Anti-malarial gametocytocidal activity. AlamarBlue was added to NAG-Percoll-treated gametocyte cultures (stage III-V) 72 h after compound addition and fluorescence was measured 24 h later, Pyr, pyrimethamine; Qn, quinine; MQ, mefloquine; CQ, chloroquine; DHA, dihydroartemisinine; PriQ, primaquine. 10 µM, black bar; 1 µM, grey bar; 0.1 µM, open bar.