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

1Malaria represents a major public health issue in the tropics, with an estimated 228 million cases and 405,000 deaths in 2018 (refs.,). Of increasing concern is P. falciparum resistance to artemisinin (ART) derivatives, used worldwide as the core components of ART-based combination therapies (ACTs). ART resistance (ART-R), characterized by delayed P. falciparum clearance following treatment with artemisinin monotherapy or an ACT,, is now widespread in the Greater Mekong subregion (GMS), which consists of Cambodia, Thailand, Vietnam, Myanmar and Laos,. Resistance to the partner drugs piperaquine and mefloquine is also now common in the GMS, causing high rates of ACT treatment failure,.

2The appearance of ART-R parasites in Africa would pose a major public health threat. Resistance to the former first-line antimalarial chloroquine first arose in the GMS in the 1960s before spreading to Africa. Resistance to pyrimethamine (used in association with sulfadoxine) followed shortly thereafter. The lost clinical efficacy of these compounds is suspected to have contributed to millions of additional malaria deaths in young African children in the 1980s. In addition to the risk of imported resistance, the likelihood of resistance emerging locally in Africa has increased in areas where control measures have reduced the disease transmission intensity. The resulting attenuation in naturally acquired human immunity can increase the frequency of symptomatic infections and the need for treatment, while decreasing parasite genetic diversity and reducing competition between sensitive and resistant parasites. To date, the efficacy of ACTs has remained high outside Southeast Asia (SEA). Early detection of resistance provides the best chance of minimizing its lethal impact.

3Mutations in the Pfkelch13 propeller domain (PF3D7_1343700) constitute the primary determinant of ART-R,,. These mutations are suspected to reduce Pfkelch13 function, which is required for parasite-mediated endocytosis of host hemoglobin in the newly invaded intra-erythrocytic ring stages,. Pfkelch13 C580Y is the most widespread allele in SEA, and has recently been detected in Guyana and Papua New Guinea. In Africa, slow-clearing infections after ACT treatment have been observed at frequencies of <1%. Previously we observed nonsynonymous Pfkelch13 mutations in <5% of African isolates, with >50% of the polymorphisms present in only a single P. falciparum infection. The most frequent Pfkelch13 mutation in Africa was A578S, which did not confer ART-R in vivo or in vitro. Nonsynonymous Pfkelch13 mutations associated with delayed parasite clearance or day 3 positivity (day 3+) in the GMS (F446I, Y493H, R539T, I543T, P553L, R561H, P574L, C580Y, A675V) have only been rarely reported, if at all, in African parasites,.

4Here we conducted an in-depth genetic analysis of P. falciparum samples collected from 2012 to 2015 at six Rwandan sites and performed gene-editing studies to evaluate the in vitro resistance phenotypes of parasites harboring the Pfkelch13 R561H or P574L mutations identified in these samples.

5From September 2013 to December 2015, clinical drug efficacy studies to assess the efficacy of artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) for the treatment of uncomplicated P. falciparum malaria were conducted in patients enrolled at the Masaka and Ruhuha health facilities in Rwanda (Fig. 1; http://www.isrctn.com/ISRCTN63145981). The overall 42-day PCR-corrected efficacies of AL (95.2% (196 of 206); 95% CI, 91.3% to 97.7%) and DP (97.5% (212 of 217); 95% CI, 94.4% to 99.1%) were similar between both sites (P = 0.17, log-rank test). The day 3 positivity rate (day 3+), defined as the proportion of patients who were still parasitemic on day 3 after initiation of treatment, was low for both treatments: 1 of 263 (0.4%) for AL and 0 of 264 for DP (Table 1).

6Pfkelch13 propeller domain genotyping was performed on 534 pretreatment samples collected at Masaka and Ruhuha. Of the 507 successfully genotyped samples, 35 (6.9%) harbored 1 of 14 different Pfkelch13 nonsynonymous mutations. These included M460I, C469Y, R513L, V555A, R561H, P574L, R575I, A578S, G592E, E605K, A626E, V637I, E651K and P667R. One sample contained two clones, each with a different mutation (G592E or V637I). The Pfkelch13 561H variant, previously associated with delayed parasite clearance following ART monotherapy or ACT treatment in the GMS, was the most predominant mutant. This R561H mutation was observed exclusively in Masaka, where it was present in 7 of 58 samples in 2013–2014 and 12 of 199 samples in 2015 (19 of 257, 7.4%). We also detected two additional Pfkelch13 mutations (P574L and C469Y) previously associated with delayed parasite clearance. We did not observe significant changes in the proportion of Pfkelch13 nonsynonymous mutations over time (P = 0.3, chi-squared test, 1.92, d.f., 2) at either study site (Table 1).

7Pfkelch13 genotyping was also carried out on 420 additional blood samples collected before AL treatment from patients enrolled in a study following the same clinical protocol that was conducted in 2012–2015 across four sites in Rwanda (ISRCTN63145981; Fig. 1). Among these, ten (2.4%) carried a Pfkelch13 nonsynonymous mutation (C469F, V487I, V555A, R561H, A578S, A578V or P667R). The Pfkelch13 R561H mutation was found in a sample from Rukara from a patient who presented a negative Giemsa-stained blood film on day 3 after treatment (day 3−) but had recrudescent parasitemia on day 21. A blood sample collected on this day was also found to carry R561H parasites (Supplementary Table 1).

8All patients with uncomplicated P. falciparum infections that had Pfkelch13 mutant parasites were day 3−, with the exception of one patient who was day 3+ (80 parasites per µl) and had P. falciparum parasites harboring the Pfkelch13 574L variant (Table 2). Three patients (one in the AL arm and two in the DP arm) presented signs of severe malaria on day 1 and were treated with intravenous artesunate according to national treatment guidelines. These patients were all day 3+ and of these, two had Pfkelch13 mutant infections (either 561H or 626E).

9By excluding the intravenous artesunate-treated patients presenting with severe malaria from our final analysis, we did not find any association between Pfkelch13 nonsynonymous mutations and delayed parasite clearance as assessed by day 3+ (P = 0.06, Fisher’s exact test) or by clinical outcome at day 42 (cured versus recrudescent) (P = 1, Fisher’s exact test; Table 2). Furthermore, we did not observe any correlation between mutation status and clinical outcome in the samples from the second study conducted at the four additional sites in Rwanda (P = 1 for day 3+ and P = 0.3 for day 42 clinical outcome, Fisher’s exact test). This analysis included the patient mentioned above (treated with AL) who had a recrudescent parasitemia on day 21 with a Pfkelch13 561H infection (Table 2).

10To test the impact of the Pfkelch13 R561H and P574L mutations on ART-R in vitro, we used CRISPR-Cas9 to introduce these mutations into Dd2 parasites and subjected the recombinant mutant and wild-type (WT) control lines to phenotyping in the ring-stage survival assay (RSA0–3h). The Pfkelch13 R561H mutation was found to confer in vitro ART-R (increased RSA survival), with Dd2R561H parasites exhibiting a mean survival rate of 4.3% versus 0.6% for the Dd2WT line expressing WT Pfkelch13 (P < 0.0001, Mann–Whitney U-test). The survival of the 561H line was comparable to that of Dd2C580Y line (mean survival of 4.7%), which harbors the Pfkelch13 C580Y mutation (Fig. 2). These results demonstrate that the Pfkelch13 R561H mutation can yield ring-stage ART-R at a level that is comparable to the C580Y mutation that has swept across SEA,.

11Dd2P574L parasites displayed a mean RSA survival rate of 2.1%, which represented a modest but significant increase relative to the Dd2WT line (P < 0.0001, Mann–Whitney U-test; Fig. 2). These results provide evidence that the Pfkelch13 P574L mutation is able to confer a lesser degree of ART-R.

12To study the origin of the Pfkelch13 561H mutants found in Rwanda, we compared whole-genome sequences of 340 samples, comprising 25 Rwandan P. falciparum sequences generated for this study, and an earlier collection of 104 sequences from central, western and southern African locations, 164 from Bangladesh and SEA and 45 from South America, in addition to 2 reference genomes (3D7 from Africa and 7G8 from South America; Supplementary Table 2). Of the 25 Rwandan sequences, 16 were Pfkelch13 561H mutants and 9 were Pfkelch13 WT. The isolates from SEA (Myanmar and Thailand) included 17 561H mutants. All other parasite sequences either had distinct nonsynonymous Pfkelch13 mutations or were WT for Pfkelch13.

13A maximum-likelihood phylogenetic tree inferred from the 14 P. falciparum chromosomes showed clear separations between the African, Asian and South American parasites (Fig. 1). Additionally, the Rwandan Pfkelch13 561H mutants clustered unambiguously with the Rwandan Pfkelch13 WT parasites.

14We also explored haplotype diversity across a 200-kb region surrounding the R561H mutation. This analysis used sequences from eight Rwandan Pfkelch13 mutant infections that seemed to be predominantly monoclonal (allelic depth of the WT allele <0.05), as well as 17 sequences from Pfkelch13 561H mutants from SEA (Myanmar and Thailand). The presence of a single shared haplotype surrounding the 561H variant in the Rwandan samples was consistent with a single epidemiological origin for this mutation. These results confirmed that the Rwandan 561H mutants share no genetic relatedness to the 561H mutants previously detected in Myanmar and Thailand (Extended Data Fig. 1).

15Next, we performed a principal coordinate analysis (PCoA) based on a pairwise genetic distance matrix (computed from a 200-kb window around the Pfkelch13 gene). This analysis confirmed that the African samples (including both the Rwandan Pfkelch13 561H mutants and WT parasites) clustered together and were distinct from Asian samples (Extended Data Fig. 2). In the eight Pfkelch13 561H mutants from Rwanda we identified an extended 494-kb region, encompassing the mutation that was identical across isolates (Extended Data Fig. 3). Although an ancient common ancestry cannot strictly be ruled out, our data provide compelling evidence that Rwandan Pfkelch13 561H is the product of a recent de novo local emergence.

16To investigate further the genetic background of the Rwandan Pfkelch13 561H mutants, we searched for molecular signatures associated with resistance to other antimalarials, including the ACT partner drugs piperaquine and lumefantrine. We also screened for mutations that have been identified in founder populations common to SEA ART-R parasites (those that constitute a ‘genetic background’ for ART-R).

17First, we investigated 233 isolates from Masaka and Ruhuha for amplification of the plasmepsin2 (pfpm2; PF3D7_1408000) and multidrug resistance-1 (pfmdr1; PF3D7_0523000) genes, considered markers of reduced susceptibility to piperaquine and lumefantrine/mefloquine, respectively–. Of these, we found 4 isolates (1.7%) with two copies of pfpm2 and 12 isolates (5.1%) with two copies of pfmdr1. All isolates carrying two copies of pfpm2 or pfmdr1 were WT for Pfkelch13 (Supplementary Table 3). We also tested 14 of the 20 Pfkelch13 561H mutants for mutations in the chloroquine resistance transporter gene (pfcrt; PF3D7_0709000), whose variants can confer resistance to chloroquine or piperaquine,. All 561H mutants carried WT pfcrt (Supplementary Table 4).

18Second, we tested whether the proportions of single-nucleotide polymorphisms (SNPs) associated with the emergence of ART-R in the SEA genetic background varied between Rwandan Pfkelch13 561H mutants and WT parasites. For this analysis, we used 14 Rwandan Pfkelch13 561H mutants and 10 randomly selected WT parasites and tested for mutations in the six markers defining the SEA ART-R background. No significant differences were observed between the two groups of isolates. We detected four isolates with the D193Y mutation in the ferredoxin gene (pffd; PF3D7_1318100), two (15.4%) in Pfkelch13 561H mutant samples and two (22.2%) in WT samples (P = 0.69, Fisher’s exact test). No mutations were detected in the P. falciparum apicoplast ribosomal protein S10 precursor (pfarps10, PF3D7_1460900), multidrug resistance protein 2 (pfmdr2, PF3D7_1447900), pfpib7 (PF3D7_0720700), pfpph (PF3D7_1012700) or exonuclease (PF3D7_1362500) genes in either the Pfkelch13 561H or WT isolates (Supplementary Table 4).

19This study clearly shows early warning signs of ART-R in Rwanda. We provide evidence for the clonal expansion of an indigenous Pfkelch13 561H lineage in two localities 100 km apart in Rwanda (prevalence 7.4% in Masaka and 0.7% in Rukara). This expansion was not linked to delayed parasite clearance in vivo or clinical treatment failure following AL or DP treatments, likely due to the high efficacy of the partner drugs lumefantrine and piperaquine. Genetic analyses indicate that Rwandan Pfkelch13 561H mutants are the product of recent de novo local emergence. These findings contrast with previous scenarios from the 1980s in which the emergence of chloroquine- and pyrimethamine-resistant parasites in Africa resulted from the westward spread of these parasites from SEA, and confirm that local emergence of ART-R is possible in Africa.

20We used gene editing and the RSA0–3h, a clinically validated in vitro phenotypic analysis,, to demonstrate that the Pfkelch13 R561H mutation is sufficient to confer ART-R in vitro. These experiments employed Dd2, which has been the most widely used P. falciparum strain for Pfkelch13 gene editing,. Our results revealed that in Dd2 parasites, the R561H mutation confers survival at levels comparable to the C580Y mutation that predominates in SEA (with mean survival rates of 4.3% and 4.7%, respectively),,. Previous studies have shown that Pfkelch13 mutations that afford resistance do so across all strains, with the parasite genetic background modulating resistance levels and with mutations conferring less resistance in Dd2 compared to contemporary SEA strains. While we did not test the impact of this mutation in Rwandan parasites due to a lack of availability of culture-adapted strains, we are confident that the resistance phenotype observed herein would be maintained across strains.

21At a genomic level, Rwandan Pfkelch13 561H mutants were phylogenetically closely related to other African samples and clustered unambiguously with Rwandan Pfkelch13 WT parasites. Haplotype analysis revealed that Rwandan Pfkelch13 561H mutants shared an identical haplotype surrounding the R561H mutation that differed from the haplotypes of SEA 561H mutants, strongly suggesting a single de novo epidemiological origin and recent spread of the mutation. No genetic relatedness was observed between Rwandan Pfkelch13 561H parasites and Pfkelch13 561H mutants previously detected in Myanmar and Thailand by PCoA.

22The current rise and expansion of the in vitro ART-R Pfkelch13 R561H mutation in Rwanda is particularly notable in light of the observed absence of clinical outcomes typically associated with ART-R. We suspect that the absence of delayed parasite clearance in Rwandan patients harboring Pfkelch13 561H mutant parasites is due to high levels of naturally acquired immunity to P. falciparum in the study participants. Indeed, it has been shown that P. falciparum antibody titers are strongly associated with faster parasite clearance rates in patients living in high-transmission areas like Rwanda and that antibodies against P. falciparum blood stages enhance antimalarial efficacy. In our study, the ages of patients enrolled at both sites ranged from 1 to 14 years, with an estimated median age of 8 years (interquartile range (IQR): 5–11 years). Given that immunity is acquired gradually with age, a clinical drug efficacy trial limited to younger populations (≤5 years of age) might reveal a significant association between the presence of Pfkelch13 561H mutants in pretreatment isolates and delayed parasite clearance. We hypothesize that early signs of clinical ART-R can lie undetected in populations with high levels of immunity, calling into question the relevance of the current clinical metrics used to detect ART-R in Africa. This hypothesis is supported by data from population-based mathematical modeling that showed that ART-R parasites might be able to circulate up to 10 years longer without detection in high-transmission areas than in low-transmission areas.

23To date, the Pfkelch13 R561H mutation has been reported multiple times in SEA (Cambodia until 2006, Myanmar and Thailand), once in India and a few times in Africa (Democratic Republic of the Congo, Rwanda and Tanzania), but has only been associated with slow-clearing infections in SEA. Thus, the degree to which Pfkelch13 561H mutant parasites are able to withstand exposure to ART in vivo and how Pfkelch13 561H is successfully transmitted between patients in the absence of clinical recrudescence (Table 2) requires further elucidation. It is possible that the resistance advantage afforded by the Pfkelch13 561H mutation is slight and undetectable based on day 3+ and recrudescence metrics, and thus would be evident only with ART monotherapy trials. Regarding transmission, we can offer several hypotheses. First, Pfkelch13 561H mutants could be less susceptible to ART due to an ability to enter into a dormant state and later produce transmissible gametocytes. Second, Pfkelch13 561H parasites may have a higher capacity to be transmitted due to an unknown genetic feature or Pfkelch13 561H gametocytes may be less susceptible to the gametocytocidal activity of artemisinin. However, it is most likely that the transmission of Pfkelch13 561H mutants in Rwanda is maintained by asymptomatic individuals or mildly symptomatic untreated patients with circulating Pfkelch13 561H mutants that have been selected by low levels of circulating drugs.

24We did not detect the combination of background mutations earlier suspected to be linked to the ART-R phenotype in SEA in the Pfkelch13 561H Rwandan isolates. This suggests that the emergence of mutant Pfkelch13 that drives in vitro resistance is not dependent on the presence of secondary mutations within the parasite genome. So far, no gene-editing and in vitro phenotyping experiments have been performed to test the importance of these secondary mutations for resistance. Data from this study suggest that mutations in fd, mdr2, arps10 and others represent the genetic architecture of regional ART-R in P. falciparum SEA parasite populations rather than secondary determinants of resistance.

25The findings of this study have substantial implications for public health in confirming the de novo emergence and clonal expansion of an ART-R Pfkelch13 R561H lineage in Rwanda and in validating this mutation as a mediator of ART-R in vitro. In the absence of effective strategies to contain the spread of resistance across Rwanda and to neighboring countries, we may soon witness a rise of resistance to ACT partner drugs, which will in turn lead to high treatment failure rates, as has occurred in SEA. Recent studies have predicted that ACT treatment failures in Africa could be responsible for an additional 78 million cases and 116,000 deaths over a 5-year period.

26Molecular surveillance of Pfkelch13-related ART-R currently implemented by the National Malaria Control Programme in Rwanda needs to be sustained and strengthened so that mutations can be identified before clinical phenotypes become apparent. Our findings argue for the need for more rapid collection of data, analysis and dissemination of information using new high-throughput field-based surveillance tools operable at a national level. Likewise, we have to reappraise the performances of the current clinical phenotypic metrics (delayed parasite clearance and day 3+) to detect the warning signs of ART-R in African populations with high immunity early on.

27The clinical drug efficacy trial (ISRCTN63145981, http://www.isrctn.com/ISRCTN63145981) was conducted by the Rwanda National Malaria Program between 2013 and 2015 at two health facilities in Rwanda (Masaka and Ruhuha, in the Kicukiro and Bugesera districts, respectively) to assess the efficacy of AL or DP for the treatment of uncomplicated P. falciparum malaria in children 1–14 years of age, presenting with suspected uncomplicated P. falciparum malaria. Patients at both sites were randomly assigned to receive a full course of AL (Co-artem, 20 mg artemether and 120 mg lumefantrine per tablet) or DP (Duo-cotecxin, 40 mg dihydroartemisinin and 320 mg piperaquine per tablet) according to the manufacturer’s dosing schedule.

28The primary outcome of the study was the PCR-adjusted clinical response to the designated treatment on day 42 (ref. ). The secondary outcome was the day 3+, defined as the proportion of patients who were still parasitemic on day 3 after initiation of treatment as assessed by thick blood smear (Supplementary Methods 1).

29Genomic investigations were carried out on blood samples collected before ACT treatment (AL or DP) from patients enrolled at Masaka and Ruhuha. We also analyzed blood samples collected before AL treatment from patients enrolled in clinical drug efficacy studies conducted at four additional sites (Bugarama, Kibirizi, Nyarurema and Rukara) across Rwanda between 2012 and 2015.

30Parasite DNA was extracted from dried blood samples (Fig. 1) using the QIAamp DNA Blood Mini Kit (Qiagen). Mutations in the propeller domain of Pfkelch13 (PF3D7_1343700, codons 440–680, 720 bp) were identified by capillary sequencing of PCR products. Parasite whole-genome sequences were obtained by Illumina paired-end sequencing after capture-based enrichment of parasite DNA (Supplementary Methods 2).

31For each sequenced sample, read alignments against the chromosome sequences of P. falciparum 3D7 v45 were processed to infer consensus sequences. These consensus sequences were pooled and concatenated, leading to 17,313,072 aligned nucleotide characters that were used to infer a maximum-likelihood phylogenetic tree (Supplementary Methods 3).

32The Genome Analysis Toolkit Haplotype Caller was used to identify SNPs in each isolate. We assessed the genetic identity of Pfkelch13 561H mutants from Rwanda and Asia by comparing alleles at loci within a 200-kb window around the mutation and recording the number of discrepancies between each sample and the mutant consensus sequence. PCoA was performed by computing pairwise Euclidean genetic distances between samples in an extended 494-kb window (Supplementary Methods 4).

33Dd2R561H and Dd2P574L gene-edited parasite lines, as well as Dd2WT and the Dd2C580Y lines used as controls, were generated by CRISPR-Cas9-mediated editing of the Pfkelch13 locus using the pDC2-cam-coSpCas9-U6-hdhfr vector. In vitro ART susceptibilities of these lines were assessed using RSA0–3h (Supplementary Methods 5–7).

34Sample size calculations and clinical data management methods have been previously described. PCR-adjusted clinical efficacy rates at day 42 were calculated using Kaplan–Meier survival analysis. Survival curves were compared using the Mantel–Haenszel log-rank test (one-sided). Patients with new infections during the 42-day follow-up period and patients with undetermined or noninterpretable PCR genotyping data were excluded from the final analysis. Data were reported in Microsoft Excel (Office 2016) and analyzed with MedCalc v.12 (MedCalc Software) and Prism 8 (GraphPad Software). Mann–Whitney U-tests (two-sided) were used for nonparametric comparisons. For frequency data (expressed with percentages and 95% CIs), we used chi-squared or Fisher’s exact tests (one-sided). Relative risks were estimated using the Mantel–Haenszel test. All P values <0.05 were deemed significant.

35Further information on research design is available in the Nature Research Reporting Summary linked to this article.

36Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41591-020-1005-2.