Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

Abstract

Key words: Plasmodium falciparum -antimalarial compounds -flow cytometry -hypoxanthine uptake -chloroquine -quinolone Drug resistance of Plasmodium falciparum, the most deadly human malaria parasite, is a major factor in the widespread persistence of malaria (Ouellette & Kunding 1997, Macreadi et al. 2000. Current efforts focus on research into novel compounds and on measures to prevent or delay resistance once new drugs are introduced. However, malaria therapy has generally not taken into consideration the stage-specificity of action of different drugs. This is an important consideration, since inappropriate timing of administration of antimalarial drugs might limit drug efficacy and favor the selection of drug-resistant parasites.Few studies have focused on the in vitro stage-specific efficacy of antimalarial compounds (Chimanuka et al. 2001). Most in vitro studies monitoring resistance and susceptibility to antimalarial compounds have been performed by microscopy and by uptake of a radiolabelled nucleic acid precursor 3 [H]-hypoxanthine (Desjardins et al. 1979). They are poorly suited to discriminate stagespecific activity. More recently, flow cytometry analysis (FCA) using different fluorescent dyes has been reported (van Vianen et al. 1990, Pattanapanyasat et al. 1996.We used propidium iodide (PI) whose incorporation as fluorescent molecule has been shown to be proportional to the DNA parasite content. In the present study, results of in vitro effect of antimalarial compounds on P. falciparum parasitized erythrocytes obtained from FCA were compared with other assays. FCA results were comparable to optical microscopy observation and to [ 3 H]-hypoxanthine uptake assay. Moreover, FCA offers advantages of high throughput, ready automation, simplified data processing, and easy determination of the stage-specific effectiveness of potential antimalarial compounds.

<i>In Vitro</i > Culture of <i>Plasmodium falciparum</i>: Obtention of Synchronous Asexual Erythrocytic Stages

“…In agreement with other authors [11], [13] and [16], isolation of all erythrocytic asexual stages-early rings, late rings, early trophozoites, late trophozoites, early schizonts and late schizonts-is now available, and their specific time of production is defined as follows: early rings at 0 -7 hours, late rings at 15 -22 hours, early trophozoites at 20 -27 hours, late trophozoites at 25 -32 hours, early schizonts at 30 -37 hours, and late schizonts at 40 -45 hours (Figure 6(b)). The synchronous culture develops healthily throughout the 48 hours of the asexual cycle, reaching a final parasitemia of 7.64% at the end of the cycle (100% new rings), maintaining synchrony throughout the cycle (Figure 6(a)).…”

“…In most studies, the establishment of highly synchronized cultures of Plasmodium falciparum has become essential. These being isolation of early rings, late rings, early trophozoites, late trophozoites, early schizonts and late schizonts [13] and [16], for example, to study the changes in the structure of parasitized red blood cells for immunological, biochemical and physiological studies [17], [6], or to study the specific stage more susceptible to new antimalarials [13]. In fact, this optimized method has been used as a procedure to obtain the different stages of the asexual erythrocytic cycle, and to test novel compounds candidate to become antimalarial drugs.…”

Antimalarial Drug Resistance: A Threat to Malaria Elimination

“…The procedures for the parasite culture are those defined by Trager and Jensen (1976). The readout of the ex vivo or in vitro tests is parasite growth (at 48 h or later), evaluated by various methods including microscopy (Rieckmann et al 1978), radioisotopic activity (isotopic test) (Desjardins et al 1979), colorimetry (ELISA based on HRP2 and pLDH detection) (Makler and Hinrichs 1993;Brasseur et al 2001;Noedl et al 2002), fluorescence (Pico Green or Sybr Green dyes) (Smilkstein et al 2004;Bacon et al 2007;Rason et al 2008), or flow cytometry (Pattanapanyasat et al 1997;Contreras et al 2004). In vitro susceptibility parameters of P. falciparum isolates are expressed as the 50% inhibitory concentration (IC 50 ) or the 90% inhibitory concentration (IC 90 ) defined as the minimal concentration of antimalarial drug that inhibits parasite growth by 50% or 90% compared with the development in drug-free control wells.…”

Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay

“…Others methods can be used: the WHO schizont maturation tests by optical microscopy (Mark III) with pre-dosed plates [7], which was based on the method of Rieckmann et al [8] and of Wernsdorfer [9], a flow cytometric analysis of propidium iodide incorporation into parasite, which permits a stage-specific evaluation of antimalarial compounds [10], and colorimetric assays with the measurement of Histidine Rich Protein II (HRP2) by an enzyme-linked immunosorbent assay (ELISA) [11,12] and the DELI-microtest (Double-site Enzyme-linked Lactate dehydrogenase Immunosorbent assay) [13,14]. …”