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Malaria outbreaks are an important public health concern in endemic regions approaching elimination. Genetic surveillance of malaria parasites can elucidate the population dynamics of an outbreak, and help identify its causes. We investigated the epidemiology of a Plasmodium falciparum outbreak in Attapeu Province, Laos, during the 2020-2021 season. An analysis of 249 samples, collected by routine genetic surveillance during the outbreak, revealed a massive loss of genetic diversity in the parasite population, primarily caused by the rapid expansion of a multidrug resistant strain, named LAA1. This strain carried the kelch13 R539T mutation and expanded clonally, replacing the previously dominant kelch13 C580Y mutants (KEL1/PLA1) resistant to dihydroartemisinin-piperaquine. Identity by descent (IBD) patterns showed that LAA1 was a recombinant that inherited 60% of its genome from a strain first sampled in Cambodia over a decade ago. A less common outbreak strain (LAA2) carried the kelch13 C580Y allele, but was distinct from KEL1/PLA1, its genome essentially identical to that of a Cambodian parasite from 2009. A third, low-frequency strain (LAA7) was a recombinant of KEL1/PLA1 with a R539T mutant, the latter providing the kelch13 variant. These results strongly suggest that the outbreak was driven by a selective sweep, possibly associated with drug-resistant phenotypes of the outbreak strains. The observation that new variants of established multidrug resistant populations can overwhelm previously dominant strains so rapidly has implications for elimination of malaria. Genetic surveillance provides the tools for characterizing outbreaks, and for monitoring the evolution and spread of the populations involved.

The emergence and spread of artemisinin resistant Plasmodium falciparum, first in the Greater Mekong Subregion (GMS), and now in East Africa, is a major threat to global malaria eliminations ambitions. To investigate the artemisinin resistance mechanism, transcriptome analysis was conducted of 577 P. falciparum isolates collected in the GMS between 2016-2018. A specific artemisinin resistance-associated transcriptional profile was identified that involves a broad but discrete set of biological functions related to proteotoxic stress, host cytoplasm remodeling and REDOX metabolism. The artemisinin resistance-associated transcriptional profile evolved from initial transcriptional responses of susceptible parasites to artemisinin. The genetic basis for this adapted response is likely to be complex.One sentence summaryThe transcriptional profile that characterize artemisinin resistant infections with malaria parasites Plasmodium falciparum originates in the initial transcriptional response to the drug.

Imported cases present a considerable challenge to the elimination of malaria. Traditionally, patient travel history has been used to identify imported cases, but the long-latency liver stages confound this approach in Plasmodium vivax. Molecular tools to identify and map imported cases offer a more robust approach, that can be combined with drug resistance and other surveillance markers in high-throughput, population-based genotyping frameworks. Using a machine learning approach incorporating hierarchical FST (HFST) and decision tree (DT) analysis applied to 831 P. vivax genomes from 20 countries, we identified a 28-Single Nucleotide Polymorphism (SNP) barcode with high capacity to predict the country of origin. The Matthews correlation coefficient (MCC), which provides a measure of the quality of the classifications, ranging from −1 (total disagreement) to 1 (perfect prediction), exceeded 0.9 in 15 countries in cross-validation evaluations. When combined with an existing 37-SNP P. vivax barcode, the 65-SNP panel exhibits MCC scores exceeding 0.9 in 17 countries with up to 30% missing data. As a secondary objective, several genes were identified with moderate MCC scores (median MCC range from 0.54-0.68), amenable as markers for rapid testing using low-throughput genotyping approaches. A likelihood-based classifier framework was established, that supports analysis of missing data and polyclonal infections. To facilitate investigator-lead analyses, the likelihood framework is provided as a web-based, open-access platform (vivaxGEN-geo) to support the analysis and interpretation of data produced either at the 28-SNP core or full 65-SNP barcode. These tools can be used by malaria control programs to identify the main reservoirs of infection so that resources can be focused to where they are needed most.

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants’ genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites’ drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance., Author summary Artemisinin is the most widely used drug against Plasmodium falciparum malaria. In southeast Asia, parasites have evolved genetic changes making them resistant to artemisinin. The elimination of resistant strains is a global priority, since their global spread could result in massive loss of lives. In Papua New Guinea, we found three patients infected with parasites carrying the most widespread resistant variant in southeast Asia, and they were confirmed to be artemisinin resistant. We established that the mutations were not imported from southeast Asia, and found other drug resistance variants in their genetic background, including some shared with parasites in Indonesia. This indicates that artemisinin resistance has emerged in New Guinea separately from southeast Asia, not by a chance event, but by a gradual process of evolution which may still be ongoing undetected on the island. These resistant strains could undermine malaria local control efforts, and constitute a global threat.

SummaryBackgroundA multidrug resistant co-lineage of Plasmodium falciparum malaria, named KEL1/PLA1, spread across Cambodia c.2008-2013, causing high treatment failure rates to the frontline combination therapy dihydroartemisinin-piperaquine. Here, we report on the evolution and spread of KEL1/PLA1 in subsequent years.MethodsWe analysed whole genome sequencing data from 1,673 P. falciparum clinical samples collected in 2008-2018 from northeast Thailand, Laos, Cambodia and Vietnam. By investigating genome-wide relatedness between parasites, we inferred patterns of shared ancestry in the KEL1/PLA1 population.FindingsKEL1/PLA1 spread rapidly from 2015 into all of the surveyed countries and now exceeds 80% of the P. falciparum population in several regions. These parasites maintained a high level of genetic relatedness reflecting their common origin. However, several genetic subgroups have recently emerged within this co-lineage with diverse geographical distributions. Some of these emerging KEL1/PLA1 subgroups carry recent mutations in the chloroquine resistance transporter (crt) gene, which arise on a specific genetic background comprising multiple genomic regions.InterpretationAfter 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 elimination efforts.FundingWellcome Trust, Bill & Melinda Gates Foundation, UK Medical Research Council, UK Department for International Development.Research in contextEvidence before this studyThis study updates our previous work describing the emergence and spread of a multidrug resistant P. falciparum co-lineage (KEL1/PLA1) within Cambodia up to 2013. Since then, a regional genetic surveillance project, GenRe-Mekong, has reported that markers of dihydroartemisinin-piperaquine (DHA-PPQ) resistance have increased in frequency in neighbouring countries. A PubMed search (terms: “artemisinin”, “piperaquine”, “resistance”, “southeast asia”) for articles listed since our previous study (from 30/10/2017 to 05/01/2019) yielded 28 results, including reports of a recent sharp decline in DHA-PPQ clinical efficacy in Vietnam; the spread of genetic markers of DHA-PPQ resistance into neighbouring countries by Imwong and colleagues; and multiple reports associating mutations in the crt gene with piperaquine resistance, including newly emerging crt variants in Southeast Asia.Added value of this studyWe analysed P. falciparum whole genomes collected up to early 2018 from Eastern Southeast Asia (Cambodia and surrounding regions), describing the fine-scale epidemiology of multiple KEL1/PLA1 genetic subgroups that have spread out from Cambodia since 2015 and taken over indigenous parasite populations in northeastern Thailand, southern and central Vietnam and parts of southern Laos. Several newly emerging crt mutations accompanied the spread and expansion of KEL1/PLA1 subgroups, suggesting an active proliferation of biologically fit, multidrug resistant parasites.Implications of all the available evidenceThe problem of P. falciparum multidrug resistance has dramatically worsened in Eastern Southeast Asia since previous reports. KEL1/PLA1 has diversified and spread widely across Eastern Southeast Asia since 2015, becoming the predominant parasite group in several regions. This may have been fuelled by continued parasite exposure to DHA-PPQ, resulting in sustained selection after KEL1/PLA1 became established. Continued drug pressure enabled the acquisition of further mutations, resulting in higher levels of resistance. These data demonstrate the value of pathogen genetic surveillance and the urgent need to eliminate these dangerous parasites.

Malaria control programs face difficult resource allocation decisions. Of particular concern for countries aiming for malaria elimination, the regular movement of individuals to and from endemic areas undermines local interventions by reintroducing infections and sustaining local transmission. Quantifying this movement of malaria parasites around a country has become a priority for national control programs, but remains methodologically challenging, particularly in areas with highly mobile populations. Here, we combined multiple data sources to measure the geographical spread of malaria parasites, including epidemiological surveillance data, travel surveys, parasite genetic data, and anonymized mobile phone data. We collected parasite genetic barcodes and travel surveys from 2,090 patients residing in 176 unions in southeast Bangladesh. We developed a genetic mixing index to quantify the likelihood of samples being local or imported. We then inferred the direction and intensity of parasite flow between locations using an epidemiological model, and estimated the proportion of imported cases assuming mobility patterns parameterized using the travel survey and mobile phone calling data. Our results show that each data source provided related but different information about the patterns of geographic spread of parasites. We identify a consistent north/south separation of the Chittagong Hill Tracts region in Bangladesh, and found that in addition to imported infections from forested regions, frequent mixing also occurs in low transmission but highly populated areas in the southwest. Thus, unlike risk maps generated from incidence alone, our maps provide evidence that elimination programs must address ongoing movement of parasites around the lower transmission areas in the southwest.

BackgroundAntimalarial failure is rapidly spreading across parts of Southeast Asia where dihydroartemisinin-piperaquine (DHA-PPQ) is used as first line treatment. The first published reports came from western Cambodia in 2013. Here we analyse genetic changes in the Plasmodium falciparum population of western Cambodia in the six years prior to that.MethodsWe analysed genome sequence data on 1492 P. falciparum samples from Southeast Asia, including 464 collected in western Cambodia between 2007 and 2013. Different epidemiological origins of resistance were identified by haplotypic analysis of the kelch13 artemisinin resistance locus and the plasmepsin 2-3 piperaquine resistance locus.FindingsWe identified over 30 independent origins of artemisinin resistance, of which the KEL1 lineage accounted for 91% of DHA-PPQ-resistant parasites. In 2008, KEL1 combined with PLA1, the major lineage associated with piperaquine resistance. By 2012, the KEL1/PLA1 co-lineage had reached over 60% frequency in western Cambodia and had spread to northern Cambodia.InterpretationThe KEL1/PLA1 co-lineage emerged in the same year that DHA-PPQ became the first line antimalarial drug in western Cambodia and spread aggressively thereafter, displacing other artemisinin-resistant parasite lineages. These findings have significant implications for management of the global health risk associated with the current outbreak.FundingWellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, UK Department for International Development, and Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health.