Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda

Abstract

Artemisinin resistance (delayed P. falciparum clearance following artemisinin-based combination therapy), is widespread across Southeast Asia but to date has not been reported in Africa^1-4. Here we genotyped the P. falciparum K13 (Pfkelch13) propeller domain, mutations in which can mediate artemisinin resistance^5,6, in pretreatment samples collected from recent dihydroarteminisin-piperaquine and artemether-lumefantrine efficacy trials in Rwanda⁷. While cure rates were >95% in both treatment arms, the Pfkelch13 R561H mutation was identified in 19 of 257 (7.4%) patients at Masaka. Phylogenetic analysis revealed the expansion of an indigenous R561H lineage. Gene editing confirmed that this mutation can drive artemisinin resistance in vitro. This study provides evidence for the de novo emergence of Pfkelch13-mediated artemisinin resistance in Rwanda, potentially compromising the continued success of antimalarial chemotherapy in Africa.

Fig. 1. Genome‐wide phylogenetic tree of 25 P. falciparum Rwandan isolates, together with 315 isolates collected worldwide (Africa, Asia and South America).

Isolates were sourced from the MalariaGEN P. falciparum Community Project (https://www.malariagen.net/apps/pf/4.0). Locations of clinical drug efficacy study sites where Rwandan isolates were collected are indicated. Patients enrolled at Masaka and Ruhuha (black) were treated with AL or DP, whereas patients enrolled at Bugarama, Kibirizi, Nyarurema and Rukara (gray) were treated with AL. Pfkelch13 nonsynonymous mutations identified in these regions and relative proportions of mutant alleles are detailed in Table 1. Each leaf represents one sample and is colored according to the country of collection. Rwandan parasites carrying the Pfkelch13 R561H mutation or the Pfkelch13 WT allele are identified by filled or unfilled red stars at the tip, respectively. Rwandan Pfkelch13 R561H mutants are closely related to other African samples at a genomic level, demonstrating that they are the product of a local emergence event. Scale bar, 0.0001 nucleotide substitutions per character. Only branch confidence supports <95% are indicated.

Fig. 2. Survival rates of Dd2^R561H, Dd2^P574L, Dd2^C580Y and Dd2^WT lines in the ring-stage survival assay (RSA_0–3h).

Mean ± s.e.m. RSA_0–3h survival rates (percentage of viable parasites) were as follows: Dd2^R561H 4.3 ± 0.1% (n = 7 assays); Dd2^P574L 2.1 ± 0.3% (n = 8 assays); Dd2^C580Y 4.7 ± 0.4% (n = 9 assays); Dd2^WT 0.6 ± 0.1% (n = 13 assays). All assays were performed in duplicate. Mann–Whitney U-tests (two-sided) were used to test for statistically significant differences between Pfkelch13-edited clones and the Dd2^WT comparator line. Survival rates of Dd2^R561H, Dd2^P574L and Dd2^C580Y all differed significantly from Dd2^WT (**** P < 0.0001). The limit of detection of viable parasites was estimated at 0.1% parasitemia (lower limit of 50 parasitized red blood cells per total number of 50,000 counted for each line in each assay).

Extended Data Fig. 1. Comparison of mutant pseudo-haplotypes in a 200 kb window around the R561H mutation (100 kb on both sides of the mutation, on chromosome 13).

Each cell represents a single SNP. The blocks of cells (grouped in columns) correspond to SNPs falling into the same 20 kb interval within the 200 kb window. The R561H mutation in Pfkelch13 (PF3D7_1343700) is flagged in the dark red cell at the top. Light blue cells correspond to the reference allele (that is the 3D7 genome), dark blue cells correspond to the alternate allele and grey cells to missing values. Each row corresponds to one isolate, with isolates color-coded according to the country of origin (red for Rwanda, cyan for Thailand and green for Myanmar). Mutant pseudo-haplotypes include eight P. falciparum monoclonal Rwandan samples and 18 Southeast Asian samples (from Myanmar and Thailand, sourced from the Plasmodium falciparum Community Project; https://www.malariagen.net/apps/pf/4.0). The presence of a single shared haplotype surrounding the R561H mutation in Rwandan P. falciparum isolates is consistent with a single epidemiological origin of the genetic background on which the mutation arose. This genetic background demonstrates no genetic relatedness to R561H mutants previously detected in Myanmar and Thailand.

Extended Data Fig. 2. Principal Coordinate Analysis (PCoA) based on pairwise genetic distances in a 494 kb window around the Pfkelch13 gene.

Principal Coordinate Analysis including Pfkelch13 wild type and 561H isolates including those sourced from a public database (small dots, the MalariaGEN Plasmodium falciparum Community Project, https://www.malariagen.net/apps/pf/4.0) and originating from different continents (Asia, Africa or South America). Isolates originating from populations where the Pfkelch13 R561H mutation was found are emphasized (large dots). Empty large dots correspond to Pfkelch13 wild-type isolates and filled large dots correspond to Pfkelch13 561H mutants. While the mutants tend to cluster with individuals of similar origin, axis 1 clearly discriminates African (Rwanda) from Asian (Thailand and Myanmar) Pfkelch13 561H mutants.

Extended Data Fig. 3. Extent of the common core haplotype in the eight Rwandan Pfkelch13 561H isolates (monoclonal isolates).

a, Recombination breakpoints estimated based on the accumulation of discrepancies between the consensus core sequence of mutants and each haplotype on both sides of the Pfkelch13 R561H mutation. The analysis was performed on the eight isolates that appeared monoclonal. Genomic positions are indicated relative to the Pfkelch13 mutation (0 kb). b, Length of the corresponding core mutant haplotypes (obtained based on (A)). Dotted lines delineate a common core region of 494 kb within which all mutant haplotypes appear identical. Genomic positions are indicated relative to the Pfkelch13 mutation (relative position 0 kb). In the larger haplotypes, no clear recombination breakpoint was observed on chromosome 13, indicating a sequence identity along the whole chromosome. Each of the eight isolates are represented by a specific color, consistent between panel (a) and panel (b).