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

Uwimana, Aline; Legrand, Eric; Stokes, Barbara H.; Ndikumana, Jean-Louis Mangala; Warsame, Marian; Umulisa, Noella; Ngamije, Daniel; Munyaneza, Tharcisse; Mazarati, Jean-Baptiste; Munguti, Kaendi; Campagne, Pascal; Criscuolo, Alexis; Ariey, Frédéric; Murindahabi, Monique; Ringwald, Pascal; Fidock, David A.; Mbituyumuremyi, Aimable; Ménard, Didier

doi:10.1038/s41591-020-1005-2

Cited by 594 publications

(613 citation statements)

References 39 publications

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“…Drug resistance monitoring also led the country to adopt different anti-malarial drugs, including artemisinin-based combinations (93-97%), that remain efficacious following recent studies carried out in 2018 [73].…”

Section: Discussionmentioning

confidence: 99%

History of malaria control in Rwanda: implications for future elimination in Rwanda and other malaria-endemic countries

Karema

Wen

Sidibe

et al. 2020

Malar J

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Background Malaria was first reported in Rwanda in the early 1900s with significant heterogeneity and volatility in transmission over subsequent decades. Here, a comprehensive literature review of malaria transmission patterns and control strategies in Rwanda between 1900 and 2018 is presented to provide insight into successes and challenges in the country and to inform the future of malaria control in Rwanda. Methods A systematic literature search of peer-reviewed publications (Web of Knowledge, PubMed, Google Scholar, and the World Health Organization Library (WHOLIS) and grey literature on malaria control in Rwanda between 1900 and 2019 was conducted with the following search terms: “malaria”“, “Rwanda”, “epidemiology”, “control”, “treatment”, and/or “prevention.” Reports and other relevant documents were also obtained from the Rwanda National Malaria Control Programme (NMCP). To inform this literature review and evidence synthesis, epidemiologic and intervention data were collated from NMCP and partner reports, the national routine surveillance system, and population surveys. Results Two hundred sixty-eight peer-reviewed publications and 56 grey literature items were reviewed, and information was extracted. The history of malaria control in Rwanda is thematically described here according to five phases: 1900 to 1954 before the launch of the Global Malaria Eradication Programme (GMEP); (2) Implementation of the GMEP from 1955 to 1969; (3) Post- GMEP to 1994 Genocide; (4) the re-establishment of malaria control from 1995 to 2005, and (5) current malaria control efforts from 2006 to 2018. The review shows that Rwanda was an early adopter of tools and approaches in the early 2000s, putting the country ahead of the curve and health systems reforms created an enabling environment for an effective malaria control programme. The last two decades have seen unprecedented investments in malaria in Rwanda, resulting in significant declines in disease burden from 2000 to 2011. However, in recent years, these gains appear to have reversed with increasing cases since 2012 although the country is starting to make progress again. Conclusion The review shows the impact and fragility of gains against malaria, even in the context of sustained health system development. Also, as shown in Rwanda, country malaria control programmes should be dynamic and adaptive to respond and address changing settings.

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Section: Discussionmentioning

confidence: 99%

History of malaria control in Rwanda: implications for future elimination in Rwanda and other malaria-endemic countries

Karema

Wen

Sidibe

et al. 2020

Malar J

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“…Nevertheless, ACTs have been threatened by the emergence of Plasmodium falciparum (PF) resistance to ARTs in Southeast Asia (SEA), and resistance has the potential to spread to other malaria-endemic regions, as has been a historical trend with earlier firstline antimalarial drugs (2,5,6). Indeed, K13 variants that are able to mediate ART resistance in vitro have recently been identified in PF parasites in French Guiana and in Rwanda (7,8). Moreover, recent aggressive expansion of a parasite lineage carrying the genetic determinants of resistance to both ART derivatives and the ACT partner drug piperaquine has been reported across SEA, resulting in a dramatic loss of clinical efficacy (9,10).…”

Section: Introductionmentioning

confidence: 99%

Plasmodium bergheiK13 Mutations MediateIn VivoArtemisinin Resistance That is Reversed by Proteasome Inhibition

Simwela

Stokes

Aghabi

et al. 2020

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The recent emergence of Plasmodium falciparum (PF) parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the PF K13 protein, which enhance survival of early ring stage parasites treated with the artemisinin active metabolite dihydroartemisinin in vitro and associate with delayed parasite clearance in vivo. However, association of K13 mutations with in vivo artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous PF K13 mutations into the K13 gene of an artemisinin-sensitive, P. berghei (PB) rodent model of malaria. Introduction of the orthologous PF K13 F446I, M476I, Y493H and R539T mutations into PB K13 produced gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-hour in vitro assay and increased survival in an adapted in vitro ring-stage survival assay. Mutant PB K13 parasites also displayed delayed clearance in vivo upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into PB while the equivalent of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a Plasmodium-selective proteasome inhibitor strongly synergized dihydroartemisinin action in these PB K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome ART resistance. Taken together, our work provides clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins in vitro, and most importantly under in vivo conditions.IMPORTANCERecent successes in malaria control have been seriously threatened by the emergence of Plasmodium falciparum parasite resistance to the frontline artemisinin drugs in Southeast Asia. P. falciparum artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon treatment with the drug. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating P. falciparum artemisinin resistance in vitro under laboratory conditions. Nonetheless, the causal role of these mutations under in vivo conditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite, P. berghei. Phenotyping of these P. berghei K13 mutant parasites provides evidence of their role in mediating artemisinin resistance in vivo, which supports in vitro artemisinin resistance observations. However, we were unable to introduce some of the P. falciparum K13 mutations (C580Y, I543T) into the corresponding amino acid residues, while other introduced mutations (M476I, R539T equivalents) carried a pronounced fitness cost. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins in vitro and in vivo using the well-characterized P. berghei model. We also show that inhibition of the P. berghei proteasome offsets parasite resistance to artemisinins in these mutant lines.

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“…In 2014-2016, a new mutation (G533S) which showed signi cantly higher ring survival rates than the WT appeared and reached 44.1% along China-Myanmar border [61]. The Pfkelch13 R561H mutation which was con rmed of driving artemisinin was identi ed in 19 of 257 (7.4%) patients at Masaka in Rwandan and phylogenetic analysis revealed they were indigenous lineage [62]. Basing on these facts, it seemed that except of spreading, new mutations could appear among falciparum isolates at the places where they received accumulative pressures from different human populations, anti-malaria medicines or even vectors.…”

Section: Discussionmentioning

confidence: 99%

Genetic Features of P. falciparum Parasites Collected in 2012-2016 and Anti-Malaria Resistance Along China-Myanmar Border

Li¹,

Liu

Tang

et al. 2020

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Backgrounds The therapeutic efficacy studies (TES) of T(DHA-PIP) for uncomplicated P. falciparum patients were implemented during 2012-2016 along China (Yunnan province)-Myanmar border, which verified the high efficacy of DHA-PIP. The study focusing on the genetic features of falciparum parasites and basing on in vivo parasite clearance time (PCT) were carried out to explore if they had produced potential resistance to DHA and PIP at molecular level. Methods The genetic features were investigated based on K13 propeller genotypes, Copy numbers of genes pfPM2 and pfmdr1 and 9 microsatellite loci (Short Tandem Repeats, STR) flanking the K13 gene on chromosome 13. The PCT 50s basing on different K13 genotypes, sites, periods and copy numbers were compared.Results In NW (North-West Yunnan province bordering with Myanmar) area, F446I prevalence was 58.96% (79/134). No significant different PCT 50s presented among 3 K13 groups (Chi-Square=2.35, P=0.31, df=2) classified by K13 genotypes within NW area, but all of them were significantly shorter than that in SW (South-West Yunnan province bordering with Myanmar) area (P=0.036, t=-2.11, df=174) where isolates only showed wild K13 genotype. For the copy numbers of pfmdr1 gene, 14.63% (18/123) and 62.5% (10/16) parasite isolates showed double copies (≥ 1.6) in NW and SW areas separately, but for those of pfM2 gene, no parasite isolates did. Between isolates with single and double copies of pfmdr1 gene, no different PCT 50 presented. According to the mean He values, the four K13 groups were arranged as ML group (Menglian County in SW), F446I group, Others (Non-F446I K13 mutation) group and W (wild K13 genotype) group from low to high. The mean Fst values between ML and W groups were significantly higher than other 2 K13 group-W pairs (Paired-T Test: t=-2.659，df=8，P=0.029; t=-4.966, df=8，P=0.001). Conclusions According to our study, P. falciparum isolates in NW area and SW area are very different in genetic features. Artemisinin partial resistance, inferred from genetic marker, F446I, had independently appeared and spread in NW area during 2012-2016. With global application of ACTs, this pattern of artemisinin resistance producing was worth more attention. Fortunately, PIP-resistant feature basing on genetic analysis showed negative results in both areas. So, DHA-PIP was still recommended in antimalarial treatment along China-Myanmar border based on molecular data, which was agreed with the conclusion drawn from in vivo data obtained with same falciparum isolates. Trial registration: ISRCTN, ISRCTN 11775446. Registered 13 April 2020 - Retrospectively registered, http://www.isrctn.com/ISRCTN11775446.

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

Cited by 594 publications

References 39 publications

History of malaria control in Rwanda: implications for future elimination in Rwanda and other malaria-endemic countries

History of malaria control in Rwanda: implications for future elimination in Rwanda and other malaria-endemic countries

Plasmodium bergheiK13 Mutations MediateIn VivoArtemisinin Resistance That is Reversed by Proteasome Inhibition

Genetic Features of P. falciparum Parasites Collected in 2012-2016 and Anti-Malaria Resistance Along China-Myanmar Border

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