63835

Chloroquine (CQ) uptake and efflux studies in clinical Plasmodium falciparum and P. vivax field isolates reveal fundamental differences between species

Richard N Price 1 ; Jutta Marfurt 2 ; Grennady Wirjanata 2 ; Pak Prayoga 3 ; Leo Leonardo 3 ; Dwin Apriyanti 4 ; Ruland Wandosa 3 ; Basbak Gobay 3 ; Enny Kenangalem 5 ; Jeanne R. Poespoprodjo 6 ; Rintis Noviyanti 4 ; Dennis E. Kyle 7 ; Qi Cheng 8
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Background. Although chloroquine (CQ) is still the first-line treatment of P. vivax malaria in most countries, reports of CQ resistant (CQR) P. vivax cases are increasing. Whereas molecular determinants of the CQR phenotype in P. falciparum, defined by decreased accumulation of CQ in the digestive vacuole of the parasite, are characterized, the mechanisms of CQ resistance in P. vivax are elusive. The aim of this study was to quantify CQ uptake and efflux parameters in both ring and trophozoite stages of P. falciparum and P. vivax field isolates, and to assess the impact of CQ resistance reversal agents (CQRRA) on CQ uptake and efflux in both species. Materials and Methods. Ex vivo drug susceptibility of CQ and CQ+CQRRAs verapamil, mibefradil, L703,606, or primaquine was determined by using a modified schizont maturation assay. For the quantification of CQ uptake and efflux parameters, a novel solid-phase and field-applicable assay using the fluorescent CQ probe NBD-CQ and flow cytometry was developed. Results and Conclusions. The assay was validated with P. falciparum laboratory strains and results were in-line with previous studies, highlighting greater and faster accumulation of CQ in CQS isolates compared to CQR isolates. The findings confirmed that CQ resistance in P. falciparum is associated with reduced CQ accumulation, but not increased CQ efflux. CQ uptake and efflux in P. vivax clinical isolates was demonstrated for the first time, highlighting a marked difference in CQ uptake parameters between P. falciparum and P. vivax which were modifiable by CQRRAs in P. falciparum, but not in P. vivax. Furthermore, CQ accumulation in P. vivax occurred at a faster rate, and CQ efflux at a slower rate, implying that P. vivax is able to tolerate intrinsically much higher concentrations of CQ than P. falciparum. Taken together, the study supports a growing body of evidence that the mechanisms of CQ resistance, and possibly also the mode of CQ action, are different between these two species.