Cytometric measurement of in vitro inhibition of Plasmodium falciparum field isolates by drugs: a new approach for re-invasion inhibition study

Marie Louise Varela,Jean Christophe Barale,Ronan Jambou,Romy Razakandrainibe,Delphine Aldebert

doi:10.1186/1475-2875-13-110

Abstract

BackgroundA flow cytometric method is proposed to study in vitro drug sensitivity of Plasmodium falciparum. Standard [3H]-hypoxanthine incorporation assay gives only information on inhibition of maturation by drugs. This method is usable on field isolates and provides data on both inhibition of maturation and re-invasion.MethodsThe method is based on the staining of parasites with hydroethidine (HE) and thiazole orange (TO) which allow differential identification of early, trophozoite and late stage of the parasite by flow cytometry. Late stages of the parasites are obtained by incubation in culture for 24 hours. Reinvasion is followed by culturing parasitized red blood cells for 24 h more.ResultsCompared to the standard [3H]-hypoxanthine incorporation assay, it gave similar results as expressed by 50% inhibitory concentrations for chloroquine of laboratory strains and “field” isolates. The effect of quinine on the schizont-ring transition was also explored using this method. First data on the inhibition of re-invasion induced by quinine are presented for both P. falciparum-cultured strains and field isolates.DiscussionThis method is simple to use event for field isolate study. It is suitable to analyse effect of drugs on steps of the parasite life cycle different for the maturation one. Using this method quinine was found to have a inhibitory effect on re-invasion of red cells by Plasmodium.

Highlights

A flow cytometric method is proposed to study in vitro drug sensitivity of Plasmodium falciparum
Standardization of a flow cytometry method for the detection of parasites To estimate the parasitaemia in whole blood, count was done on the three gates of the HE/thiazole orange (TO) histogram and compared with the count done on the Giemsa-stained smear
26 field isolates were analysed but due to lake of maturation of the parasites, four isolates were excluded from analysis

Summary

Introduction

A flow cytometric method is proposed to study in vitro drug sensitivity of Plasmodium falciparum. Standard [3H]-hypoxanthine incorporation assay gives only information on inhibition of maturation by drugs. This method is usable on field isolates and provides data on both inhibition of maturation and re-invasion. To fight the burden associated with growing resistance to available anti-malarial drugs, the development of new drugs is mandatory, as well as surveying sensitivity to available drugs in field isolates [1]. Following theoretical mechanisms of action of drugs on P. falciparum, most in vitro drug sensitivity tests measure the effects on parasite growth [2]. Such tests allow the discovery of anti-malarial molecules, such as quinolines and artemisinin.

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