Plasmodium vivax trophozoites insensitive to chloroquine

Abstract

Background: Plasmodium vivax is a major cause of malaria and is still primarily treated with chloroquine. Chloroquine inhibits the polymerization of haem to inert haemozoin. Free haem monomers are thought to catalyze oxidative damage to the Plasmodium spp. trophozoite, the stage when haemoglobin catabolism is maximal. However preliminary in vitro observations on P. vivax clinical isolates suggest that only ring stages (early trophozoites) are sensitive to chloroquine. In this study, the stage specific action of chloroquine was investigated in synchronous cryopreserved isolates of P. vivax.

Methods: The in vitro chloroquine sensitivity of paired ring and trophozoite stages from 11 cryopreserved P. vivax clinical isolates from Thailand and two Plasmodium falciparum clones (chloroquine resistant K1 and chloroquine sensitive FC27) was measured using a modified WHO microtest method and fluorometric SYBR Green I Assay. The time each stage was exposed to chloroquine treatment was controlled by washing the chloroquine off at 20 hours after the beginning of treatment.

Results: Plasmodium vivax isolates added to the assay at ring stage had significantly lower median IC50s to chloroquine than the same isolates added at trophozoite stage (median IC50 12 nM vs 415 nM p < 0.01). Although only 36% (4/11) of the SYBR Green I assays for P. vivax were successful, both microscopy and SYBR Green I assays indicated that only P. vivax trophozoites were able to develop to schizonts at chloroquine concentrations above 100 nM.

Conclusion: Data from this study confirms the diminished sensitivity of P. vivax trophozoites to chloroquine, the stage thought to be the target of this drug. These results raise important questions about the pharmacodynamic action of chloroquine, and highlight a fundamental difference in the activity of chloroquine between P. vivax and P. falciparum.

Figure 1

The inhibition of Plasmodium vivax schizont development at increasing concentrations of chloroquine dependent on the initial stage (Ring or Trophozoite) exposed to 20 hours of chloroquine over a 40 hour culture period. The photomicrographs of thick films, show representative examples of drug effect on Plasmodium vivax at low medium and high concentrations of chloroquine. The black (microscopic) and green lines (SYBR Green I) represent the median inhibition of schizont development relative to a drug free control. Medians lines are only derived from assays with paired SYBR Green I and microscopic results.(N = 4).

Figure 2

The inhibition of Plasmodium falciparum (Chloroquine resistant K1 clone) schizont development at increasing concentrations of chloroquine dependent on the initial stage (Ring or Trophozoite) exposed to 20 hours of chloroquine over a 40 hour culture period. The photomicrographs of thick films, show representative examples of drug effect on Plasmodium falciparum at low medium and high concentrations of chloroquine. The black (microscopic) and green lines (SYBR Green I) represent the median inhibition of schizont development relative to a drug free control. Medians lines are only derived from assays with paired SYBR Green I and microscopic results.(N = 6).

Figure 3

The sensitivity of Plasmodium vivax isolates and Plasmodium falciparum clones (K1 and FC27) to chloroquine (CQIC⁵⁰nM) when; rings and trophozoites are exposed to chloroquine for 40 hours (R&T); exposed to chloroquine for 20 hours at the ring stage (R) and trophozoite stage (T). Red dotted lines connect related samples. Solid black lines represent the median CQIC⁵⁰nM for each treatment.