A comparative study of a flow-cytometry-based assessment of in vitro Plasmodium falciparum drug sensitivity

Karl, Stephan; Wong, Rina P. M.; Pierre, Timothy G. St.; Davis, Timothy M. E.

doi:10.1186/1475-2875-8-294

Cited by 33 publications

(29 citation statements)

References 31 publications

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“…A previous study done in Australia pointed to the difference of sensitivity of the Flow cytometry assay, the Sybr Green plate reader assay, the Plasmodium falciparum lactate dehydrogenase assay and light microscopy used in in vitro antiplasmodial activity assessments. 24 …”

Section: Discussionmentioning

confidence: 99%

<i>In vitro</i> antiplasmodial and cytotoxic properties of some medicinal plants from western Burkina Faso

et al. 2013

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BackgroundResistance of malaria parasites to existing drugs complicates treatment, but an antimalarial vaccine that could protect against this disease is not yet available. It is therefore necessary to find new effective and affordable medicines. Medicinal plants could be a potential source of antimalarial agents. Some medicinal plants from Burkina Faso were evaluated for their antiplasmodial and cytotoxic properties in vitro.MethodsCrude dichloromethane, methanol, water-methanol, aqueous and alkaloids extracts were prepared for 12 parts of 10 plants. Chloroquine-resistant malaria strain K1 was used for the in vitro sensibility assay. The Plasmodium lactacte dehydrogenase technique was used to determine the 50% inhibitory concentration of parasites activity (IC50). The cytotoxic effects were determined with HepG2 cells, using the tetrazolium-based colorimetric technique, and the selectivity index (SI) was calculated.ResultsSixty crude extracts were prepared. Seven extracts from Terminalia avicenoides showed IC50 < 5 µg/mL. The IC50 of dichloromethane, methanol, aqueous and alkaloids extracts ranged between 1.6 µg/mL and 4.5 µg/mL. Three crude extracts from Combretum collinum and three from Ficus capraefolia had an IC50 ranging between 0.2 µg/mL and 2.5 µg/mL. Crude extracts from these three plants had no cytotoxic effect, with SI > 1. The other plants have mostly moderate or no antimalarial effects. Some extracts from Cordia myxa, Ficus capraefolia and Opilia celtidifolia showed cytotoxicity, with an SI ranging between 0.4 and 0.9.ConclusionOur study showed a good antiplasmodial in vitro activity of Terminalia avicenoides, Combretum collinum and Ficus capraefolia. These three plants may contain antiplasmodial molecules that could be isolated by bio-guided phytochemical studies.

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Section: Discussionmentioning

confidence: 99%

<i>In vitro</i> antiplasmodial and cytotoxic properties of some medicinal plants from western Burkina Faso

et al. 2013

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“…In practice, detection of ring stages with non-GFP-fluorescent lines may require the application of an alternative staining method such as the combination of thiazole orange and hydroethidine (44) or Hoechst 33342 and (45). Previous studies have reported an encouraging correlation between flow cytometry and microscopy counts of drug-treated cultures in vitro (45,46) as well as application of flow cytometry to assess drug inhibition of late-stage parasite development (47). In this study, the correlation between numbers of free merozoites and parasite growth inhibition (newly invaded rings) has been described in detail.…”

Section: Discussionmentioning

confidence: 99%

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Wilson

Langer

Goodman

et al. 2013

Antimicrob Agents Chemother

129

135

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e Most current antimalarials for treatment of clinical Plasmodium falciparum malaria fall into two broad drug families and target the food vacuole of the trophozoite stage. No antimalarials have been shown to target the brief extracellular merozoite form of blood-stage malaria. We studied a panel of 12 drugs, 10 of which have been used extensively clinically, for their invasion, schizont rupture, and growth-inhibitory activity using high-throughput flow cytometry and new approaches for the study of merozoite invasion and early intraerythrocytic development. Not surprisingly, given reported mechanisms of action, none of the drugs inhibited merozoite invasion in vitro. Pretreatment of erythrocytes with drugs suggested that halofantrine, lumefantrine, piperaquine, amodiaquine, and mefloquine diffuse into and remain within the erythrocyte and inhibit downstream growth of parasites. Studying the inhibitory activity of the drugs on intraerythrocytic development, schizont rupture, and reinvasion enabled several different inhibitory phenotypes to be defined. All drugs inhibited parasite replication when added at ring stages, but only artesunate, artemisinin, cycloheximide, and trichostatin A appeared to have substantial activity against ring stages, whereas the other drugs acted later during intraerythrocytic development. When drugs were added to late schizonts, only artemisinin, cycloheximide, and trichostatin A were able to inhibit rupture and subsequent replication. Flow cytometry proved valuable for in vitro assays of antimalarial activity, with the free merozoite population acting as a clear marker for parasite growth inhibition. These studies have important implications for further understanding the mechanisms of action of antimalarials, studying and evaluating drug resistance, and developing new antimalarials.

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“…Artesunate has been reported to have an IC 50 of 108 nM for gametocytes (stage IV-V) when assayed by Giemsa smear [8], but the effect of DHA against gametocytes has not been published previously. Although DHA is more efficacious than the other drugs tested, the effective dose against asexual parasites is much lower than that required to kill gametocytes and this is reflected in the ability of gametocytes to remain infectious to mosquitoes for a week after artemisinin treatment [10, 18–19]. Together these results demonstrate that alamarBlue can be used to efficiently screen compounds for gametocytocidal activity.…”

Section: Gametocytocidal Activity Of Common Anti-malarial Drugsmentioning

confidence: 99%

A malaria gametocytocidal assay using oxidoreduction indicator, alamarBlue

Tanaka

Williamson

2011

Molecular and Biochemical Parasitology

101

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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.

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A comparative study of a flow-cytometry-based assessment of in vitro Plasmodium falciparum drug sensitivity

Cited by 33 publications

References 31 publications

<i>In vitro</i> antiplasmodial and cytotoxic properties of some medicinal plants from western Burkina Faso

<i>In vitro</i> antiplasmodial and cytotoxic properties of some medicinal plants from western Burkina Faso

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

A malaria gametocytocidal assay using oxidoreduction indicator, alamarBlue

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