Frequent and complex relapses confound assessment of chloroquine resistance in Cambodian Plasmodium vivax

Abstract

Background: Plasmodium vivax resistance to chloroquine has been observed in several endemic countries. In Cambodia, up to 17% clinical treatment failure following 3-days standard chloroquine treatment was reported in vivax malaria patients in 2009. The loss of chloroquine efficacy was solely described in northeast area of Cambodia while chloroquine seemed to remain fully effective in other provinces. This led to the withdrawn of chloroquine and its replacement by dihydroartemisinin-piperaquine in 2012. To rigorously assess the extent of P. vivax chloroquine-resistance in Cambodia, we conducted a comprehensive therapeutic efficacy study with extensive genotyping of the parasites.Methods & Materials: The study was conducted in Rattanakiri, in northeastern Cambodia in 2014. 40 enrolled patients were treated with chloroquine (30 mg/kg) for three days and followed for two months. Reinfection was controlled for half of the patients by relocating them to a no-transmission area. The 2-months followup consisted in frequent clinical examination and capillary blood collection for microscopic, molecular parasite detection and drug concentration measure. The entire genomes of the initial and recurrent parasites were sequenced and complemented by genotyping of more than 100 SNPs for each PCR-positive blood samples collected during follow-up.Results: Recurrences occurred in 24/40 (60%) patients within the follow-up. No difference was observed between relocated and non-relocated patients. Recurrent parasites were always detected when chloroquine concentration in blood was below therapeutic level. Genotyping revealed that all P. vivax clones, within a given infection, responded similarly to CQ. In addition, whole genome sequencing unambiguously showed that most relapsing parasites were different from those in the initial infections.Conclusion: Recurrences within two months are frequent among Cambodian vivax malaria patients and originate from relapsing parasites from the liver. Pharmacological and genetic analyses revealed no evidence of CQ resistance and suggest that CQ is fully effective against P. vivax episodes in Rattanakiri Cambodia. Our results suggest that CQ resistance might be over-diagnosed and confounded with relapses from liver parasites. Our clinical and analytical framework has the potential to differentiate between relapse and resistance and should be implemented in vivax malaria endemic areas with suspected drug resistance.