Molecular epidemiology of resistance to antimalarial drugs in the Greater Mekong subregion: an observational study

Abstract

Background: The Greater Mekong subregion is a recurrent source of antimalarial drug resistance in Plasmodium falciparum malaria. This study aimed to characterise the extent and spread of resistance across this entire region between 2007 and 2018.

Methods: P falciparum isolates from Myanmar, Thailand, Laos, and Cambodia were obtained from clinical trials and epidemiological studies done between Jan 1, 2007, and Dec 31, 2018, and were genotyped for molecular markers (pfkelch, pfcrt, pfplasmepsin2, and pfmdr1) of antimalarial drug resistance. Genetic relatedness was assessed using microsatellite and single nucleotide polymorphism typing of flanking sequences around target genes.

Findings: 10 632 isolates were genotyped. A single long pfkelch Cys580Tyr haplotype (from -50 kb to +31·5 kb) conferring artemisinin resistance (PfPailin) now dominates across the eastern Greater Mekong subregion. Piperaquine resistance associated with pfplasmepsin2 gene amplification and mutations in pfcrt downstream of the Lys76Thr chloroquine resistance locus has also developed. On the Thailand-Myanmar border a different pfkelch Cys580Tyr lineage rose to high frequencies before it was eliminated. Elsewhere in Myanmar the Cys580Tyr allele remains widespread at low allele frequencies. Meanwhile a single artemisinin-resistant pfkelch Phe446Ile haplotype has spread across Myanmar. Despite intense use of dihydroartemisinin-piperaquine in Kayin state, eastern Myanmar, both in treatment and mass drug administrations, no selection of piperaquine resistance markers was observed. pfmdr1 amplification, a marker of resistance to mefloquine, remains at low prevalence across the entire region.

Interpretation: Artemisinin resistance in P falciparum is now prevalent across the Greater Mekong subregion. In the eastern Greater Mekong subregion a multidrug resistant P falciparum lineage (PfPailin) dominates. In Myanmar a long pfkelch Phe446Ile haplotype has spread widely but, by contrast with the eastern Greater Mekong subregion, there is no indication of artemisinin combination therapy (ACT) partner drug resistance from genotyping known markers, and no evidence of spread of ACT resistant P falciparum from the east to the west. There is still a window of opportunity to prevent global spread of ACT resistance.

Funding: Thailand Science Research and Innovation, Initiative 5%, Expertise France, Wellcome Trust.

Figure 1

Frequency distributions of mutations found in the pfkelch gene in Plasmodium falciparum isolates obtained from four countries of the Greater Mekong subregion, 2007–18

Figure 2

Polyallelic marker data from all Cys580Tyr mutant Plasmodium falciparum isolates (A) Data from all mutant isolates from Myanmar (n=113) with matched wild-type isolates (n=95). (B) Data from all mutant isolates from western Cambodia (n=189) with matched wild-type isolates (n=87). Shown are data at position 580 on the pfkelch gene (0 on the x-axis) and in an interval from −56 kb to +225 kb surrounding the pfkelch gene. The colours correspond to the different alleles, whereby an independent colouring scheme was applied to each polyallelic marker separately. The colour scheme is based on all observed alleles for all samples from the Greater Mekong subregion (not only those shown in this figure), so that comparisons can be made across figures. When multiple alleles were observed at a single locus, the column is broken into subcolumns with the corresponding colours. White corresponds to missing data. For the pfkelch gene, green is wild-type and red is Cys580Tyr. The number of distinct alleles observed in all the data for each marker is given by the number in parentheses.

Figure 3

The spread of pfkelch haplotypes across the Greater Mekong subregion (A) Map of the Greater Mekong subregion showing the spread of pfkelch haplotypes. The single long pfkelch Cys580Tyr haplotype (from −50 kb to +31·5 kb; PfPailin; red arrows) emerged in western Cambodia in 2008 and spread across the eastern Greater Mekong subregion., Cys580Tyr bearing parasites of a different lineage have spread widely in Myanmar but have not dominated, except on the Thailand–Myanmar border. There the previously dominant lineage almost disappeared as falciparum malaria was eliminated by intensive targeted activities in Kayin state. A single pfkelch Phe446Ile haplotype (blue arrows), which probably originated in the north, has spread widely across Myanmar. Two haplotypes were evident, although one has predominated since 2017. (B) Polyallelic marker data (single nucleotide polymorphisms and microsatellites) at the amino acid 446 position on the pfkelch gene (0 on the x-axis), and in an interval of −56 kb to +225 kb surrounding the pfkelch gene. This plot shows the marker data for all isolates (one row per isolate) with the Phe446Ile mutation (n=202) found in Myanmar and 95 matched wild-type isolates. For pfkelch, green is wild-type and orange is Phe446Ile.

Figure 4

Frequency distributions of pfplasmepsin2 gene amplification in four countries of the Greater Mekong subregion, 2007–18 CNV=copy number variation.

Figure 5

Frequency distributions of pfmdr1 gene amplification in four countries of the Greater Mekong subregion, 2007–18 CNV=copy number variation.

Figure 6

Frequency distributions of pfplasmepsin2 gene amplification, pfcrt, and pfkelch gene mutations in Plasmodium falciparum isolates from submicroscopic parasitaemias in eastern Myanmar and western Cambodia, before (n=142) and after (n=38) mass drug administration with dihydroartemisinin and piperaquine M inside the circles refers to the month of study. Areas the isolates were collected from are shown on the map in pink. CNV=copy-number variation.