Recrudescence, Reinfection, or Relapse? A More Rigorous Framework to Assess Chloroquine Efficacy for Plasmodium vivax Malaria.

Background: Plasmodium vivax resistance to chloroquine (CQ) has been reported worldwide, although the World Health Organization clinical drug efficacy studies protocol does not permit classification of patient outcomes. Methods: We enrolled 40 patients with P. vivax malaria in northeastern Cambodia, where >17% treatment failures were previously reported. Patients were treated with CQ (30 mg/kg) and followed for 2 months, with frequent clinical examination and capillary blood sample collection for microscopy, molecular parasite detection and genotyping, and drug concentration measurements. Reinfections were prevented by relocating patients to a transmission-free area. Results: P. vivax parasites were eliminated in all patients by day 3. Genomic analyses revealed that all clones in polyclonal infections were cleared at the same rate, indicating their equal susceptibility to CQ. CQ blood concentrations were below the therapeutic level in all recurrent infections (24 of 40 patients), which were efficiently cleared by a second course of CQ treatment. Genotyping (128 SNPs barcode) and sequences of entire parasite genome (Whole-Genome Sequencing, Illumina) indicated that two thirds (6 of 8) of the recurrent parasites resulted from heterologous relapses whose 50% are from by sibling/recombinant clones. Conclusions: No evidence of CQ resistance was observed. Our data suggest that P. vivax antimalarial drug resistance is likely overestimated and that the current guidelines for clinical drug studies of P. vivax malaria need to be revised.

Therapeutic and Transmission-Blocking Efficacy of Dihydroartemisinin/Piperaquine and Chloroquine against Plasmodium vivax Malaria, Cambodia.

We assessed the efficacy of standard 3-day courses of chloroquine and dihydroartemisinin/piperaquine against Plasmodium vivax malaria. Compared with chloroquine, dihydroartemisinin/piperaquine was faster in clearing asexual P. vivax parasites and blocking human-to-mosquito transmission. This drug combination was also more effective in preventing potential recurrences for >2 months.

Reduced polymorphism in the Kelch propeller domain in Plasmodium vivax isolates from Cambodia.

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.

Genomic Analyses Reveal the Common Occurrence and Complexity of Plasmodium vivax Relapses in Cambodia.

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)IMPORTANCEP. vivax parasites can remain dormant in the liver and relapse weeks or months after the initial infection, greatly complicating malaria control and elimination efforts. The few investigations of this dormant stage have relied on travelers and military personnel returning from areas of malaria endemicity. However, it is not clear whether these individuals, exposed to a limited number of infections, accurately represent the patterns of relapses of individuals living in areas of endemicity, who are repeatedly infected by P. vivax parasites. Our study combined tightly controlled fieldwork with comprehensive genomic analyses, and our report provides a first opportunity to investigate the patterns, dynamics, and diversity of P. vivax relapses directly with individuals living in areas of endemicity.

Genetic diversity in two Plasmodium vivax protein ligands for reticulocyte invasion.

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.

Characterization of P. vivax blood stage transcriptomes from field isolates reveals similarities among infections and complex gene isoforms.

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.