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The spread of artemisinin-resistant Plasmodium falciparum in the Greater Mekong subregion: a molecular epidemiology observational study

Summary Background Evidence suggests that the PfKelch13 mutations that confer artemisinin resistance in falciparum malaria have multiple independent origins across the Greater Mekong subregion, which has motivated a regional malaria elimination agenda. We aimed to use molecular genotyping to assess antimalarial drug resistance selection and spread in the Greater Mekong subregion. Methods In this observational study, we tested Plasmodium falciparum isolates from Myanmar, northeastern Thailand, southern Laos, and western Cambodia for PfKelch13 mutations and for Pfplasmepsin2 gene amplification (indicating piperaquine resistance). We collected blood spots from patients with microscopy or rapid test confirmed uncomplicated falciparum malaria. We used microsatellite genotyping to assess genetic relatedness. Findings As part of studies on the epidemiology of artemisinin-resistant malaria between Jan 1, 2008, and Dec 31, 2015, we collected 434 isolates. In 2014–15, a single long PfKelch13 C580Y haplotype (−50 to +31·5 kb) lineage, which emerged in western Cambodia in 2008, was detected in 65 of 88 isolates from northeastern Thailand, 86 of 111 isolates from southern Laos, and 14 of 14 isolates from western Cambodia, signifying a hard transnational selective sweep. Pfplasmepsin2 amplification occurred only within this lineage, and by 2015 these closely related parasites were found in ten of the 14 isolates from Cambodia and 15 of 15 isolates from northeastern Thailand. C580Y mutated parasites from Myanmar had a different genetic origin. Interpretation Our results suggest that the dominant artemisinin-resistant P falciparum C580Y lineage probably arose in western Cambodia and then spread to Thailand and Laos, outcompeting other parasites and acquiring piperaquine resistance. The emergence and spread of fit artemisinin-resistant P falciparum parasite lineages, which then acquire partner drug resistance across the Greater Mekong subregion, threatens regional malaria control and elimination goals. Elimination of falciparum malaria from this region should be accelerated while available antimalarial drugs still remain effective. Funding The Wellcome Trust and the Bill and Melinda Gates Foundation.


Introduction
Artemisinin combination therapies (ACTs) have become the cornerstone of the treatment of falciparum malaria throughout the malaria endemic world. 1 New antimalarial compounds will not become widely available for several more years. 2 The emergence and geographic spread of artemisinin resistant P falciparum in the Greater Mekong subregion (GMS) represents a serious threat to global malaria control and to aspirations to eliminate malaria. Artemisinin resistance results in reduced ring stage susceptibility and manifests as slow parasite clearance. 3,4 As a result, fewer malaria parasites are killed during the 3 days of ACT treatment, leaving more for the partner drug to remove. This action increases the risk of treatment failure and facilitates selection of partner drug resistance. 2 Resistance to both components of the ACT results in substantial reductions in cure rates. ACTs containing mefloquine and piperaquine are now failing across increasing areas of the GMS. [5][6][7][8] Retrospective genotyping of archived samples showed presence of the artemisinin resistance marker at the beginning of the millennium. The PfKelch13 mutations that mediate artemisinin resistance appear to have arisen independently on multiple occasions. 9,10 Some have taken this to suggest that a radical firewall approach to prevent the spread of these mutations might not work, and so prompted abandonment of containment and the launch of a regional malaria elimination effort in the GMS. 11,12 On the other hand, the spread of artemisinin resistance has been geographically contiguous, and there is no evidence yet for independent emergence of resistance in India or Africa. 4,13 Resistance in P falciparum to the antimalarials chloroquine and sulfadoxine-pyrimeth amine also arose on multiple occasions in the GMS and in the case of pyrimethamine, resistance eventually a single parasite lineage dominated (a so-called hard selective sweep) and it was this dominant resistant lineage that spread across India and Africa. 14,15 Using microsatellite genotyping around the PfKelch13 gene in P falciparum isolates collected over 7 years from four countries in the GMS, we test the hypothesis that history is being repeated-ie, that dominant lineages of artemisininresistant falciparum malaria parasites are now spreading through the GMS. Using molecular genotyping we also investigate the relation between artemisinin resistance (PfKelch13 mutation) 4,9,10,13 and piperaquine resistance (Pfplasmepsin2 amplification) 16,17 and we discuss the implications of the findings for the regional and global containment of multidrug-resistant falciparum malaria.

Sample collection
As part of studies on the epidemiology of artemisininresistant malaria (reported previously 3,4,18,19

Assessment of mutations in PfKelch13
We sequenced nested PCR products of the full length of the PfKelch13 gene (2181 bp including one exon), 4 with an ABI Sequencer (Macrogen Inc, Seoul, Korea). Cross con tamination was monitored by adding negative control samples in every run. We aligned sequencing results against PfKelch13 of reference strain 3D7 (putative 9PF13_0238 NCBI Reference Sequence [3D7]: XM_001350122.1), using Bioedit software (Abbott, Carlsbad, CA, USA). Polymorphic patterns were assessed by two people blinded to the origin of the sample.

Pfplasmepsin2 gene amplification
We quantified Pfplasmepsin2 copy number using relative-quantitative RT-PCR based on SYBR-Green fluorescence on an Applied Biosystems ViiA 7Real-Time-PCR-System 16 (Life technologies, Carlsbad, CA, USA). We did amplification in triplicate on a total volume of 20 μL as singleplex PCR using SsoFast

Research in context
Evidence before this study We searched PubMed on Nov 9, 2016, using the terms "artemisinin resistance", and "genotype" and "Cambodia", "Thailand", "Myanmar" and "Laos" without any date or language restrictions. This search identified 47 articles, of which 23 contained information on the epidemiology of artemisinin-resistant genotypes. Phyo and colleagues described the evolution of artemisinin resistance on the Thailand-Myanmar border, and how the E252Q PfKelch mutation predominated initially but was overtaken in recent years by C580Y as artemisinin combination therapies (ACTs) began to fail. Takala-Harrison and colleagues conducted a genome-wide association study which showed multiple independent emergences of PfKelch13 mutations in Plasmodium falciparum populations in southeast Asia, but haplotype analysis also suggested spread of the Y493H and C580Y mutations from Cambodia across the border to Vietnam. Menard and colleagues assessed the genetic relatedness of PfKelch13 mutant parasites in the Greater Mekong Subregion (GMS) and also identified "numerous emergence events alongside spreading of a small group of mutations for artemisinin resistance".

Added value of this study
This longitudinal study conducted over 8 years in four countries has two important findings; that over time single lineages of artemisinin-resistant parasites dominate, outcompeting the other parasites, and then under continued ACT therapies drug pressure, spreads-in one case across three countries-and that artemisinin resistance selects for piperaquine resistance.

Implications of all the available evidence
This study shows that artemisinin resistance has continued to evolve such that with time, and continued selection pressure, what started as multiple independent emergences of PfKelch13 mutations was overtaken by a few dominant lineages that spread and acquired partner drug resistance. This same sequence of events occurred previously when antimalarial drug resistance emerged in the GMS and later spread to Africa. Malaria elimination efforts need to be accelerated in the GMS while antimalarial drugs remain effective, particularly in areas where these successful dominant P falciparum lineages are found.
Evagreen Supermix (BIO-RAD, Hercules, CA, USA). Every amplification run contained three replicates of 3D7 calibrators and triplicates without template as negative controls. β-tubulin served as an internal standard for the amount of sample DNA added to the reactions. We calculated copy numbers using the formula: copy number=2 −ΔΔCt with ΔΔC t denoting the difference between ΔC t of the unknown sample and ΔC t of the reference sample.

Microsatellite genotyping of the regions flanking PfKelch13
Microsatellite genotyping of the -56 kb to +72 kb regions flanking the PfKelch13 gene was done as described previously. 20,21 For isolates carrying the PfKelch13 C580Y allele, we designed seven additional microsatellite markers covering -247 kb to +269 kb and for the PfKelch13 R539T allele 17 additional microsatellites covering -579 kb to +631 kb (appendix). We did single PCR for 35 cycles of 1 minute at 95°C, 1 minute annealing at 55°C, and 1 minute extension at 72°C. The lengths of the PCR-generated products were measured by comparison with internal size standards (Genescan 500 LIZ) on an ABI 3100 Genetic analyser, using Genescan and Genotyper software (PE Applied Biosystems) to measure allele lengths and to quantify peak heights. Negative control samples were included to monitor cross-contamination. We analysed a subset of ten samples in triplicate to assess intraassay consistency. 79 samples with multiple genotypes were included in all analyses except for the assessments of genetic relatedness.

Additional assessments of genetic diversity
To assess genetic relatedness in parasites with the long PfKelch13 haplotypes and between Pfplasmepsin2 amplified and non-amplified parasites, we genotyped parasite isolates for variable blocks within the msp1, msp2, and glurp genes, 22 and for nine microsatellites across the genome 23 using semi-nested PCR. Genetic similarity between isolates was analysed by unweighted

Role of the funding source
The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.
Microsatellite defined PfKelch13 R539T haplotypes from 39 parasite isolates from Cambodia, Laos, and Thailand were compared with the 87 PfKelch13 wild-type isolates. Mean H e in wild-type isolates was 0·691 (SE 0·190) compared with 0·077 (0·005) in R539T parasites (p<0·0001), signifying much reduced micro satellite diversity -50 to +242 kb around the R539T allele. The conserved region around the R539T allele was asymmetric, and was larger than in the C580Y haplotype. The R539T haplotype was the same in isolates from Ubon Ratchathani and Champasak, and was only a single locus different from those in Pailin, suggesting a common origin (appendix). Nine microsatellite markers flanking the PfKelch13 F446I allele were genotyped in six samples from Homalin, Sagaing and 20 samples from Kyainseikgyi, Kayin, Myanmar. A unique identical PfKelch13 haplotype was observed in Kayin with a mean H e 0 (SD 0), which was not observed elsewhere. By comparison, the mean H e assessed from 45 local PfKelch13 wild-type isolates was 0·599 (SE 0·010), signifying high diversity.
A median-joining network tree was generated using microsatellite defined haplotypes around PfKelch13 from 346 P falciparum isolates obtained from the four countries (appendix). There was strong genetic clustering of the C580Y allele in parasites from Cambodia, Thailand, and Laos, far from the dispersed haplotypes observed in northern and southern Myanmar and from local parasites containing wild type or the F446I allele. Clustering was also observed among isolates containing the R539T allele in samples from Cambodia, Thailand, and Laos. Using microsatellite typing across the genome, relatedness between the 49 isolates with concurrent Pfplasmepsin2 amplification was compared with the 40 Pfplasmepsin2 single copy isolates but the same PfKelch13 C580Y long haplotype. Overall mean H e of the Pfplasmepsin2 amplified isolates was significantly lower than that of single copy isolates (p=0 . 021) and cluster analysis by UPGMA showed that isolates containing Pfplasmepsin2 amplification diverged genetically from single copy isolates (appendix). Isolates with Pfplasmepsin2 amplification obtained from Pailin in 2008 and 2011 clustered separately from Pfplasmepsin2 amplified isolates obtained from Pailin and Phusing in 2015.

Discussion
Artemisinin resistance in P falciparum is evolving in the GMS. This study suggests that the previously described soft selective sweeps, 9,10 which resulted from the multiple independent emergences of mutant PfKelch13 alleles, have now been superseded by hard selective sweeps by presumably fitter parasites. A single long haplotype PfKelch13 C580Y mutant parasite lineage has now spread across three countries. The dominant haplotype prevalent in 2014 and 2015 in both northeastern Thailand (Ubon and Srisaket provinces) and southern Laos (Champasak province) is the same as that observed from 2008 onwards in western Cambodia, indicating a common origin. Given the time course of artemisinin resistance in the region, 4 and the low prevalence of falciparum malaria in northeastern Thailand 8 years ago, this suggests resistance spread from western Cambodia to Laos and Thailand, probably along with the cross-border movement of people (figure 2). Pfplasmepsin2 amplification, which is one of the genes associated with piperaquine resistance, 16,17 has emerged on the back of the PfKelch13 C580Y mutation, and this multidrugresistant P falciparum lineage is spreading widely, causing high ACT failure rates. From 2008 to 2015 in Pailin, western Cambodia, where artemisinin resistance was first described, parasites containing the C580Y mutation have now replaced parasites containing other PfKelch13 mutations, including the temporarily successful R539T haplotype. Malaria incidence has fallen, so this represents a relative rather than an absolute increase.
Earlier genetic epidemiological analysis from study sites in the GMS east of Bangkok showed clustering of long haplotypes around the PfKelch13 C580Y allele, consistent with a common origin in different sub populations across Cambodia and some samples from Vietnam and eastern Thailand. 10 Our study uses micro satellite genotyping and shows further selection and reduction in variability over time, with a common origin of PfKelch13 C580Y parasite isolates sharing a similar long haplotype in all isolates surveyed from Cambodia and adjacent countries, by contrast with the high diversity in the flanking regions of parasites carrying wild-type PfKelch13. A similar trend, in which C580Y mutant parasites overtook other PfKelch13 propeller region mutations, has been observed on the Thailand-Myanmar border. 7 The C580Y mutation does not confer a higher level of artemisinin resistance than many other PfKelch13 mutations (including R539T), but this lineage appears to be fitter or more transmissible. PfKelch13 mutations are found throughout the malaria endemic world, but artemisinin resistance has emerged and spread only in the GMS. Genome-wide association studies have shown that in addition to the PfKelch13 mutations, a combination of backbone mutations are closely linked to the artemisinin-resistant phenotype and are present in association with different PfKelch13 variants. 24 These other genetic changes presumably confer an additional fitness advantage, facilitating selection and spread.
The emergence and spread of piperaquine resistance in Cambodia is a sinister development. More than half of the parasite isolates obtained from Pailin in Cambodia and Phusing in nearby Thailand in 2015 also carried a marker for piperaquine resistance; gene amplification of the Pfplasmepsin2 genes on chromosome 14. 16,17 Until 2016, dihydroartemisinin-piperaquine was the first-line ACT in Cambodia, but its efficacy has decreased markedly since 2008. 5,6,8,25 High treatment failure rates facilitate the spread of resistance. P falciparum isolates from northeastern Thailand also carried the piperaquine resistance marker, yet first-line treatment there has been artesunate-mefloquine, which suggests recent importation rather than local selection. The Pfplasmepsin2 amplified parasites all carried the same PfKelch13 C580Y long haplotype, but were less diverse than the Pfplasmepsin2 single copy isolates with this haplotype, which suggests that Pfplasmepsin2 amplification occurred after the selection of the successful PfKelch13 C580Y lineage.
A limitation of the study is that geographic spread to other areas of the GMS was not investigated. Assessment of parasite diversity was also compromised by the low prevalence now of wild-type PfKelch13 in some study sites. However, this does not challenge the main conclusion of the study: parasites originating from artemisinin resistance soft selective sweeps 26 have been replaced by a single dominant genotype (a hard sweep) that is spreading widely. This change occurred previously with antifolate resistance in P falciparum. 15 Pfdhfr mutant genotypes (including the triple 108, 51, and 59 genotype) emerged de novo on multiple occasions but over time a single triple genotype parasite lineage dominated in the GMS and this spread across continents. 15 It is also possible that the dominant Pfcrt K76T genotype, conferring chloroquine resistance, was preceded by multiple soft sweeps.
The findings in this study have important implications for public health and containment strategies. Artemisinin resistance in this region was clearly a prelude to partner drug resistance. The combination of artemisinin and piperaquine resistance is now spreading quickly throughout Cambodia. Fitter multidrug-resistant parasites are spreading throughout western Cambodia, southern Laos, and northeastern Thailand. This evidence reinforces the urgency for P falciparum elimination in areas of artemisinin and partner drug resistance since containment will become increasingly difficult as drug efficacy declines. Further spread of these successful multidrug resistant parasites through India to sub-Saharan Africa would be a public health disaster. Border crossings between countries in the region are frequent, and mobile populations who carry parasites asymptomatically are a likely source of transnational spread. 27,28 In conclusion, this study shows that over a large area of the GMS multiple independently originating PfKelch13 mutant P falciparum parasites have been replaced by a single dominant long haplotype PfKelch13 C580Y mutant lineage. This mutant had its likely origin in western Cambodia, and then spread to northeastern Thailand and southern Laos. These dominant artemisinin-resistant parasites are developing resistance to the ACT partner drugs, and are causing high rates of malaria treatment failure. What appeared initially to be an unusual pattern of multiple events has transformed into a more familiar scenario in which successful closely genetically related malaria parasites, which are highly drug resistant, spread and threaten malaria control and elimination throughout the region and beyond.