Gabrielle C. Ngwana-Joseph: Genomic analysis of global Plasmodium vivax populations reveals insights into the evolution of drug resistance

However, the molecular drivers of CQR remain unclear. Using a genome-wide approach, we perform a genomic analysis of 1534 P. vivax isolates across 29 endemic countries, detailing population structure, patterns of relatedness, selection, and resistance profiling, providing insights into potential drivers of CQR. Selective sweeps in a locus proximal to pvmdr1, a putative marker for CQR, along with transcriptional regulation genes, distinguish isolates from Indonesia from those in regions where chloroquine remains highly effective. 

In 106 isolates from Indonesian Papua, the epicentre of CQR, we observe an increasing prevalence of novel SNPs in the candidate resistance gene pvmrp1 since the introduction of dihydroartemisinin-piperaquine. Overall, we provide novel markers for resistance surveillance, supported by evidence of regions under recent directional selection and temporal analysis in this continually evolving parasite.

Introduction

Plasmodium vivax is the most geographically widespread human malaria parasite and the leading cause of malaria outside of sub-Saharan Africa. In 2022, there were 6.9 million cases across 49 endemic countries across Central and South America, East Africa, Asia, and Oceania1. Intensive efforts to combat the deadlier Plasmodium falciparum, particularly in areas that are co-endemic with P. vivax, has distributed resources away from P. vivax control programs, leading to its emergence as the dominant species, particularly in the Greater Mekong Subregion1. The absence of a long term continuous in vitro culture system2 has meant that our understanding of the parasite’s life cycle, transmission, and biology has been limited.

Chloroquine, in combination with primaquine, is the front-line treatment for the radical cure of P. vivax malaria in most endemic countries. First documented in the late 1980s, chloroquine resistance (CQR) in P. vivax, also known as chloroquine treatment failure, is characterised by the World Health Organization (WHO) as the persistence of parasitaemia on day 28 following treatment, despite a blood concentration of chloroquine-desethylchloroquine at or above 100 ng/mL. CQR has consequently led to the adoption of artemisinin-based combination therapies to replace chloroquine in several countries, including dihydroartemisinin-piperaquine in Indonesia, artesunate-mefloquine in Cambodia, and artemether-lumefantrine in Papua New Guinea (PNG)3,4. Indonesian Papua and PNG have been the epicentre of high-grade, or category 1 CQR, defined as >10% recurrences by day 28 of treatment5. Over the past two decades, increasing reports of CQR in P. vivax beyond these countries have been characterised by parasite persistence 28 days post-treatment, spurring research into the molecular determinants of CQR5,6,7,8,9.

Evidence regarding the molecular drivers of CQR in P. vivax is both weak and conflicting. The major candidate gene, pvmdr1, was initially posited as a mediator of CQR due to its high sequence homology with the orthologous pfmdr1, which is involved in CQR in P. falciparum10. However, subsequent studies have produced contrasting reports, showing no consistent correlation between pvmdr1 and CQR11. Profiling of polymorphisms within the pvmdr1 gene led to the association of the Y976F mutation with CQR due to increased chloroquine IC50 in samples from Indonesian Papua and Thailand12. This mutation has since become a recognised marker for CQR in studies across P. vivax endemic regions9,13,14. At least 50 pvmdr1 SNPs have been documented globally, yet none have emerged as a definitive CQR marker, questioning the extent and relevance of pvmdr1 in modulating CQR. Although profiling the prevalence of pvmdr1 polymorphisms has increased our understanding of its evolution across different drug pressure backgrounds15,16, most studies focus solely on the pvmdr1 gene itself, biasing our understanding of the acquisition of CQR, which is hypothesised to be a multifaceted process involving numerous loci.

Extensive use of antimalarial drugs over several decades has resulted in high-resolution characterisations of recent selection events associated with drug resistance in Plasmodium spp. using genome-wide sequence data. Genome-wide analyses have shown evidence for recent positive selection in P. falciparum endemic regions with artemisinin resistance17,18,19 and revealed selective sweeps around the pfcrt, pfmdr1, and pfaat1 genes, which are explicitly linked to CQR20,21. Similar selection metrics applied to P. vivax genomes have revealed that the orthologous loci associated with antifolate resistance in P. falciparum have been subject to selective sweeps15,16,22. More recent work has found evidence of selective sweeps around pvmrp1 in East Asian isolates, a gene associated with chloroquine and mefloquine resistance in the orthologous P. falciparum pfmrp1, necessitating further investigation16.

Understanding the population genetics and dynamics of P. vivax malaria, particularly in the context of drug resistance, is essential for successful control and elimination planning. With the expanding repertoire of whole genome sequence data for P. vivax, we can now investigate the temporal dynamics of populations pre- and post-chloroquine contraindication, to provide increased insight into the markers of CQR. Here, we leverage publicly available whole genome sequences to present a large-scale population genomics study of P. vivax. We provide an expanded insight into population structure, global ancestry, relatedness, and genomic diversity. Using a genome-wide approach, we perform intra- and inter-population analyses between isolates from regions with different degrees of reported CQR to make inferences about both previously described and novel loci that could be mediating CQR and the evolutionary forces that shape P. vivax populations.

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