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

Abstract

Objective: The main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC50) for chloroquine.

Methods: The study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC50 for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake.

Results: From 5% O2 to 21% O2 conditions, no parasiticide effect of O2 concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoïtes and schizonts at 21% O2 with an increase in the length of schizogony. The chloroquine IC50 at 10% O2 were significantly higher than those at 21% O2, means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC50 > 100 nM) at 10% O2, 17 were sensitive to chloroquine (IC50 < 100 nM) at 21% O2.

Conclusion: Based on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance.

Figure 1

Evolution of asexual parasitaemia of synchronous cultures of Plasmodium falciparum under different oxygen tensions. Each point represents the mean ± standard deviation of three experiments. A: 3D7 clone. B: W2 clone.

Figure 2

Percentages evolution of asexual stages of synchronous culture of 3D7 Plasmodium falciparum clone under different oxygen tensions during 86 hours. Each percentage represents the mean ± standard deviation of three experiments. R: Ring. MT: Mature Trophozoïte: S: Schizont. RO: Rosace. Blue histograms correspond to 5% O₂; Purple histograms correspond to 10% O₂. Yellow histograms correspond to 21% O₂.

Figure 3

Cell cycle evolution of asexual blood stages of Plasmodium falciparum. A: 3D7 clone. B: W2 clone. 1: 5% O₂. 2: 21% O₂.

Figure 4

Evolution of asexual parasitaemia of synchronous cultures of Plasmodium falciparum exposed to 21% O₂ during ten hours at ring, trophozoïte or schizont stage. Each point represents the mean ± standard deviation of three experiments.

Figure 5

Tritiated hypoxanthine uptake of synchronous Plasmodium falciparum cultures under 10% O₂ and 21% O₂. Each point represents the mean ± standard deviation of three experiments. A: 3D7 clone. B: W2 clone.

Figure 6

Chemosusceptibility of Plasmodium falciparum isolates to chloroquine under 10% O₂ and 21% O₂. A: chloroquine IC₅₀ of 120 isolates. B: red plots corresponds to isolates with a switch from susceptible to chloroquine at 10% O₂ to resistant at 21% O₂; blue plots corresponds to isolates with a switch from resistant at 10% O₂ to susceptible at 21% O₂.