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.)

Our understanding of the structure and regulation of Plasmodium vivax genes is limited by our inability to grow the parasites in long-term in vitro cultures. Most P. vivax studies must therefore rely on patient samples, which typically display a low proportion of parasites and asynchronous parasites. Here, we present stranded RNA-seq data generated directly from a small volume of blood from three Cambodian vivax malaria patients collected before treatment. Our analyses show surprising similarities of the parasite gene expression patterns across infections, despite extensive variations in parasite stage proportion. These similarities contrast with the unique gene expression patterns observed in sporozoites isolated from salivary glands of infected Colombian mosquitoes. Our analyses also indicate that more than 10% of P. vivax genes encode multiple, often undescribed, protein-coding sequences, potentially increasing the diversity of proteins synthesized by blood stage parasites. These data also greatly improve the annotations of P. vivax gene untranslated regions, providing an important resource for future studies of specific genes.

The interaction between Plasmodium vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) has been described as critical for the invasion of human reticulocytes, although increasing reports of P. vivax infections in Duffy-negative individuals questions its unique role. To investigate the genetic diversity of the two main protein ligands for reticulocyte invasion, PvDBP and P. vivax Erythrocyte Binding Protein (PvEBP), we analyzed 458 isolates collected in Cambodia and Madagascar from individuals genotyped as Duffy-positive. First, we observed a high proportion of isolates with multiple copies PvEBP from Madagascar (56%) where Duffy negative and positive individuals coexist compared to Cambodia (19%) where Duffy-negative population is virtually absent. Whether the gene amplification observed is responsible for alternate invasion pathways remains to be tested. Second, we found that the PvEBP gene was less diverse than PvDBP gene (12 vs. 33 alleles) but provided evidence for an excess of nonsynonymous mutations with the complete absence of synonymous mutations. This finding reveals that PvEBP is under strong diversifying selection, and confirms the importance of this protein ligand in the invasion process of the human reticulocytes and as a target of acquired immunity. These observations highlight how genomic changes in parasite ligands improve the fitness of P. vivax isolates in the face of immune pressure and receptor polymorphisms.

Polymorphism in the ortholog gene of the Plasmodium falciparum K13 gene was investigated in Plasmodium vivax isolates collected in Cambodia. All of them were Sal-1 wild-type alleles except two (2/284, 0.7%), and P. vivax K12 polymorphism was reduced compared to that of the P. falciparum K13 gene. Both mutant allele isolates had the same nonsynonymous mutation at codon 552 (V552I) and were from Ratanak Kiri province. These preliminary data should encourage additional studies for associating artemisinin or chloroquine resistance and K12 polymorphism. In areas in which malaria is endemic and Plasmodium falciparum and Plasmodium vivax are present, such as in Southeast Asia and Pacific Oceania, both species share the same vectors and human hosts, either successively or concomitantly (mixed infections) (1, 2). These two species, in this context, often undergo similar mutation-driven evolution and natural selection. This includes nucleotide substitution, gene duplication/deletion, chromosomal change, and genome duplication (3). In terms of drug resistance, regardless of the fundamental biological differences between the two Plasmodium species, it is well known that antimalarial drug pressure induces a strong selection of resistant parasites for both of these parasite populations. For instance, sequencing of the dhfr gene in P. vivax isolates collected in areas where sulfadoxine-pyrimethamine was used to treat falciparum malaria and the alignment of these alleles with the P. falciparum dhfr gene have clearly demonstrated that mutations in codons 57, 58, 61, 117, and 173 were involved in pyrimethamine resistance and corresponded to the codons 51, 59, 108, and 164 found in P. falciparum pyrimethamine-resistant strains (4). More recently, we observed high frequencies of P. falciparum and P. vivax isolates with increased mdr-1 copy numbers in areas where mefloquine has been extensively used as the first-line treatment in falciparum-uncomplicated malaria, while in areas where mefloquine has never been used, P. falciparum and P. vivax isolates with increased mdr-1 copy numbers are rare (5). These studies clearly show that antimalarial drugs used to treat falciparum malaria have a significant impact on sympatric Plasmodium species, such as P. vivax.Since 2001, artemisinin combination therapies (ACTs) have been recommended as first-line treatment in the national treatment guidelines of most countries in which malaria is endemic. In 2008, the emergence of artemisinin-resistant P. falciparum parasites was observed in Southeast Asia (6-15). Recent molecular and biological studies showed that artemisinin resistance was associated with P. falciparum early ring stages and mutations in the PF3D7_1343700 kelch propeller domain (K13-propeller) (8,14,15). To date, although the role of the P. falciparum K13 protein remains unknown, two pieces of evidence suggest that it is involved in the cellular response to oxidative stress (8): (i) its homology to the KEAP1 human protein, which is involved in cell adaptation to oxidative stress (16), a...

No evidence of chloroquine resistance were observed. Our data suggest that P. vivax antimalarial drug resistance is likely overestimated and that the current guidelines for clinical drug studies in vivax malaria need to be revised.