Indigenous emergence and spread of kelch13 C469Y artemisinin-resistant Plasmodium falciparum in Uganda

Abstract

Artemisinin-based combination therapies (ACTs) were introduced as the standard of care for uncomplicated malaria in Africa almost two decades ago. Recent studies in East Africa have reported a gradual increase in kelch13 (k13) mutant parasites associated with reduced artesunate efficacy. As part of the Community Access to Rectal Artesunate for Malaria project, we collected blood samples from 697 children with signs of severe malaria in northern Uganda between 2018 and 2020, before and after the introduction of rectal artesunate (RAS) in 2019. K13 polymorphisms were assessed, and parasite editing and phenotyping were performed to assess the impact of mutations on parasite resistance. Whole-genome sequencing was performed, and haplotype networks were constructed to determine the geographic origin of k13 mutations. Of the 697 children, 540 were positive for Plasmodium falciparum malaria by PCR and were treated with either RAS or injectable artesunate monotherapy followed in most cases by ACT. The most common k13 mutation was C469Y (6.7%), which was detected more frequently in samples collected after RAS introduction. Genome editing confirmed reduced in vitro susceptibility to artemisinin in C469Y-harboring parasites compared to wild-type controls (P < 0.001). The haplotypic network showed that flanking regions of the C469Y mutation shared the same African genetic background, suggesting a single and indigenous origin of the mutation. Our data provide evidence of selection for the artemisinin-resistant C469Y mutation. The realistic threat of multiresistant parasites emerging in Africa should encourage careful monitoring of the efficacy of artemisinin derivatives and strict adherence to ACT treatment regimens.

Keywords: Uganda; artemisinin resistance; k13; malaria; rectal artesunate.

Fig 1

Proportion and evolution of k13 mutations observed in the different study sites. (A) Proportion of WT and mutant k13 parasites before and after RAS introduction (sampled either on day 0 or day 28) in the different study sites. K13 mutations found in less than three isolates were grouped together (“Others” group). The map was generated using QGIS. (B) Frequency of k13 mutations between 2018 and 2020. (C) Frequency of k13 mutations before and after RAS implementation (sampled either on day 0 or day 28). The depicted Kwania district area includes the area of Apac district, from which it was administratively separated in mid-2018.

Fig 2

Results of the ring-stage survival assay (RSA_0-3h). 3D7^WT (green), 3D7^SLI-WT (yellow), 3D7^SLI-C469Y (red), and 3D7^SLI-C580Y (blue) parasites were measured with the in vitro RSA_0-3h assay. Error bars represent the standard deviation of RSA values. ****P-value < 0.0001 (Student’s t-test). Values in parentheses indicate the number of replicates.

Fig 3

Minimum spanning network of 331 k13-associated haplotypes. The analysis used SNPs located within ±100 kb of the k13 gene to minimize the effect of recombination. Haplotypes are represented by filled circles, colored according to the geographic origin of the sample or the k13 mutation. The size of the circle indicates the number of samples represented. The gray lines connecting the circles indicate genetic relatedness: the darker the link, the greater the genetic distance between the isolates.