Evolution and expansion of multidrug-resistant malaria in southeast Asia: a genomic epidemiology study

Abstract

Background: A multidrug-resistant co-lineage of Plasmodium falciparum malaria, named KEL1/PLA1, spread across Cambodia in 2008-13, causing high rates of treatment failure with the frontline combination therapy dihydroartemisinin-piperaquine. Here, we report on the evolution and spread of KEL1/PLA1 in subsequent years.

Methods: For this genomic epidemiology study, we analysed whole genome sequencing data from P falciparum clinical samples collected from patients with malaria between 2007 and 2018 from Cambodia, Laos, northeastern Thailand, and Vietnam, through the MalariaGEN P falciparum Community Project. Previously unpublished samples were provided by two large-scale multisite projects: the Tracking Artemisinin Resistance Collaboration II (TRAC2) and the Genetic Reconnaissance in the Greater Mekong Subregion (GenRe-Mekong) project. By investigating genome-wide relatedness between parasites, we inferred patterns of shared ancestry in the KEL1/PLA1 population.

Findings: We analysed 1673 whole genome sequences that passed quality filters, and determined KEL1/PLA1 status in 1615. Before 2009, KEL1/PLA1 was only found in western Cambodia; by 2016-17 its prevalence had risen to higher than 50% in all of the surveyed countries except for Laos. In northeastern Thailand and Vietnam, KEL1/PLA1 exceeded 80% of the most recent P falciparum parasites. KEL1/PLA1 parasites maintained high genetic relatedness and low diversity, reflecting a recent common origin. Several subgroups of highly related parasites have recently emerged within this co-lineage, with diverse geographical distributions. The three largest of these subgroups (n=84, n=79, and n=47) mostly emerged since 2016 and were all present in Cambodia, Laos, and Vietnam. These expanding subgroups carried new mutations in the crt gene, which arose on a specific genetic background comprising multiple genomic regions. Four newly emerging crt mutations were rare in the early period and became more prevalent by 2016-17 (Thr93Ser, rising to 19·8%; His97Tyr to 11·2%; Phe145Ile to 5·5%; and Ile218Phe to 11·1%).

Interpretation: After emerging and circulating for several years within Cambodia, the P falciparum KEL1/PLA1 co-lineage diversified into multiple subgroups and acquired new genetic features, including novel crt mutations. These subgroups have rapidly spread into neighbouring countries, suggesting enhanced fitness. These findings highlight the urgent need for elimination of this increasingly drug-resistant parasite co-lineage, and the importance of genetic surveillance in accelerating malaria elimination efforts.

Funding: Wellcome Trust, Bill & Melinda Gates Foundation, UK Medical Research Council, and UK Department for International Development.

Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.

Figures

Figure 1

Rise in KEL1/PLA1 prevalence over time in eastern southeast Asia (A) Proportions of different combinations of KEL1 and PLA1 alleles, across three time periods (2007–11, 2012–15, and 2016–18) in the eastern southeast Asia regions surveyed in this study. (B) Change in the frequency of KEL1/PLA1 parasites during the same time periods in different geographical regions within eastern southeast Asia.

Figure 2

Genetic similarity among KEL1/PLA1 parasites across geographical regions (A) Boxplot comparing the distribution of pairwise genetic distance in non-KEL1/PLA1 parasites (ie, carrying neither KEL1 nor PLA1 haplotypes, n=777) with the distribution in KEL1/PLA1 parasites (n=551). (B) Boxplot comparing the distribution of pairwise distance between KEL1/PLA1 and non-KEL1/PLA1 parasites in the same geographical region (blue); and between KEL1/PLA1 parasites in the region and KEL1/PLA1 parasites outside the region (red). The number of samples analysed (in the following order: KEL1/PLA1 in the region, KEL1/PLA1 outside the region, and non-KEL1/PLA1 in the region) was 22, 529, and 14 in northeastern Thailand; 32, 519, and 193 in Laos; and 162, 389, and 207 in Vietnam. In both plots, pairwise genetic distance is expressed in an arbitrary unit, which is a function of the number of genetic differences observed among variant single-nucleotide polymorphisms (SNPs) in this dataset between pairs of samples, after correcting for linkage disequilibrium and heterozygous genotypes. Thick lines represent median values, boxes show the IQR, and whiskers represent extremes of the distribution, discounting outliers.

Figure 3

KEL1/PLA1 family tree The dendrogram shows a hierarchical clustering tree of pairwise genetic distances for all 551 KEL1/PLA1 samples across eastern southeast Asia; longer branches indicate more distant relationships. The six largest subgroups of highly related parasites are shown in red and blue, and labelled below the tree. The alternating colours highlight the different subgroups. These subgroups, numbered in order of decreasing size (subgroup 1, n=84; subgroup 2, n=79; subgroup 3, n=47; subgroup 4, n=36; subgroup 5, n=24; and subgroup 6, n=19), were identified by grouping samples with pairwise genetic distances in the lowest quartile (delimited by a dotted line). Pairwise genetic distance is expressed in an arbitrary unit, which is a function of the number of genetic differences observed among variant single-nucleotide polymorphisms (SNPs) in this dataset between pairs of samples, after correcting for linkage disequilibrium and heterozygous genotypes.

Figure 4

Distinct epidemiological and genetic properties of KEL1/PLA1 subgroups Sample proportions by sampling time period (A) and location (B) in the six largest groups of high-similarity KEL1/PLA1 parasites. Subgroups 1–3 emerged recently and are internationally distributed, whereas subgroups 4 and 6 are older and confined to western Cambodia. Proportion of crt haplotypes in the same groups (C): newly emerging crt mutations are highly prevalent in the newer subgroups 1–3, but absent from the older geographically restricted subgroups 4 and 6, and also in subgroup 5, which has recently expanded in northeastern Cambodia. Numbers of samples are as follows: n=84 for subgroup 1, n=79 for subgroup 2, n=47 for subgroup 3, n=36 for subgroup 4, n=24 for subgroup 5, and n=19 for subgroup 6. Together, these samples comprise more than 50% of the 551 analysed KEL1/PLA1 samples.