Plasmodium vivax parasites have a unique dormant stage that can cause relapses weeks or months after the initial infection. These dormant parasites are among the main challenges of vivax malaria control as they constitute a reservoir that is difficult to eliminate. Since field studies are confounded by reinfections and possible recrudescence of drug-resistant parasites, most analyses of P. vivax relapses have focused on travelers returning from regions of malaria endemicity. However, it is not clear whether these individuals accurately recapitulate the relapse patterns of repeatedly infected individuals residing in areas of endemicity. Here, we present analyses of vivax malaria patients enrolled in a tightly controlled field study in Cambodia. After antimalarial drug treatment was administered, we relocated 20 individuals to a nontransmission area and followed them for 60 days, with blood collection performed every second day. Our analyses reveal that 60% of the patients relapsed during the monitoring period. Using whole-genome sequencing and high-throughput genotyping, we showed that relapses in Cambodia are often polyclonal and that the relapsing parasites harbor various degrees of relatedness to the parasites present in the initial infection. Our analyses also showed that clone populations differed dynamically, with new clones emerging during the course of the relapsing infections. Overall, our study data show that it is possible to investigate the patterns, dynamics, and diversity of P. vivax relapses of individuals living in a region of malaria endemicity and reveal that P. vivax relapses are much more pervasive and complex than previously considered. (This study has been registered at ClinicalTrials.gov under registration no. NCT02118090.)

BackgroundPlasmodium vivax is the most widely distributed human malaria parasite with 2.9 billion people living in endemic areas. Despite intensive malaria control efforts, the proportion of cases attributed to P. vivax is increasing in many countries. Genetic analyses of the parasite population and its dynamics could provide an assessment of the efficacy of control efforts, but, unfortunately, these studies are limited in P. vivax by the lack of informative markers and high-throughput genotyping methods.Methodology/Principal FindingsWe developed a sequencing-based assay to simultaneously genotype more than 100 SNPs and applied this approach to ~500 P. vivax-infected individuals recruited across nine locations in Cambodia between 2004 and 2013. Our analyses showed that the vast majority of infections are polyclonal (92%) and that P. vivax displays high genetic diversity in Cambodia without apparent geographic stratification. Interestingly, our analyses also revealed that the proportion of monoclonal infections significantly increased between 2004 and 2013, possibly suggesting that malaria control strategies in Cambodia may be successfully affecting the parasite population.Conclusions/SignificanceOur findings demonstrate that this high-throughput genotyping assay is efficient in characterizing P. vivax diversity and can provide valuable insights to assess the efficacy of malaria elimination programs or to monitor the spread of specific parasites.

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.