Artemisinin resistantkelch13R622I and RDT negativity approaching predominance in northern Ethiopia and emerging C580Y of African origin threaten falciparum malaria control

Abstract The rise of antimalarial drug-resistant Plasmodium falciparum poses a major threat to malaria treatment, control, and elimination efforts. Mutations in the kelch13 (k13) gene confer artemisinin partial resistance (ART-R), compromising the efficacy of combination therapies. In the Horn of Africa, the validated mutation k13 R622I has rapidly emerged in parallel with other mutations elsewhere in Eastern Africa. To monitor and inform control efforts, we conducted in-depth sampling where R622I was first detected, using molecular inversion probe (MIP) targeted sequencing of key resistance mutations and informative SNPs across the genome. Samples were collected for a year within two districts, Gondar Zuria and Tach Armachiho, representing different ecological zones in northwest Ethiopia. Among 903 people with P. falciparum malaria, we observed a markedly higher prevalence of R622I (44.3%) compared to earlier reports, with significantly higher prevalence in Gondar Zuria (52%) than in Tach Armachiho (40%; p<0.001). Alarmingly, the prevalence of histidine rich protein 2 (HRP2) based rapid diagnostic test (RDT) negativity was higher in R622I mutants compared to wild types (48.3% vs 30.7%; p<0.001). Furthermore, 98.2% of R622I samples carried the multidrug resistance protein 1 NFD haplotype associated with reduced susceptibility to lumefantrine. We also detected the k13 C580Y mutation in two patients (0.4%) from Gondar Zuria, the first report in the Horn of Africa. Identity-by-descent (IBD) analysis showed C580Y mutant parasites were likely clonal (IBD=1). Whole-genome sequencing confirmed their clonal nature and revealed flanking haplotypes distinct from those seen in Southeast Asia where the mutation was first observed, suggesting a local, de novo emergence. These findings highlight increasing prevalence and types of ART-R mutations and the concerning association now with RDT negativity that will further challenge malaria control and elimination efforts. Competing Interest Statement The authors have declared no competing interest. Funding Statement This project has received funding from the European Union through the Horizon Europe research and innovation programme under grant agreement number CSA 2020NOE-3102, as part of the East Africa Consortium for Clinical Research (EACCR), with genotyping support provided by the United States National Institutes of Health (grant R01AI177791). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical clearance with reference number of VP/RTT/05/814/2022 was obtained from the institutional review board (IRB) of University of Gondar. Permissions from the respected Health Offices of the study districts and village administration were also obtained. Informed consent was obtained from all adult participants or from guardians in the case of children, while assent was also obtained from children aged 12 to 17 years prior to sample collection. When the study was being carried out, all appropriate safety measures and ethical methods were taken into account for both the participants and the researchers. Only authorized staff members have access to the protected site where all screening and case record forms are stored. The computer-based data entry employed unique numerical identifiers. Genotyping work in Brown University was regarded as non-human subjects research. For reporting individual level genotypes for publishing, only de-identified samples and aggregated clinical data were utilized I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced are available online at https://www.ncbi.nlm.nih.gov/sra https://www.ncbi.nlm.nih.gov/bioproject/ https://github.com/gtollefson/Northern_Ethiopia_Pf_ARTR_2025_Project