Artemisinin Partial Resistance in Ethiopian Plasmodium Falciparum: A Multisite Clinical, Molecular and In Vitro Study

Confirmation of Artemisinin Partial Resistance in Plasmodium Falciparum Using WHO Criteria: A Multisite Clinical, Molecular and Phenotypic Study Across Five Sentinel Sites in Ethiopia, 2024-2025

Artemisinin-based combination therapies (ACTs) are the main treatment for falciparum malaria in Africa. Artemisinin partial resistance (ART-R), characterized by delayed parasite clearance after treatment, has been confirmed in four sub-Saharan African countries. In Ethiopia, molecular surveys have detected the Pfkelch13 R622I mutation associated with ART-R at multiple sites, but no study has yet combined clinical, molecular, and in vitro evidence to confirm ART-R per WHO criteria. This multisite study conducted across five sentinel sites in Ethiopia (2024-2025) assessed day-3 parasite positivity after artemether-lumefantrine treatment, Pfkelch13 genotyping, and ring-stage survival assay on culture-adapted field isolates, to determine whether ART-R is confirmed in Ethiopian Plasmodium falciparum populations.

This study integrated three WHO-required lines of evidence to confirm artemisinin partial resistance (ART-R) in Ethiopia: (1) clinical evidence through day-3 parasite positivity assessment after artemether-lumefantrine treatment in therapeutic efficacy studies; (2) molecular evidence through Pfkelch13 propeller domain genotyping; and (3) phenotypic in vitro evidence through ring-stage survival assay (RSA0-3h) on culture-adapted field isolates. Five sentinel sites were selected across malaria-endemic regions of Ethiopia. Blood samples were collected at enrollment (day 0) and day 3. Isolates were cryopreserved and shipped to France (Strasbourg) for RSA.

Inclusion Criteria:

• Age ≥ 6 months
• Microscopically confirmed uncomplicated Plasmodium falciparum monoinfection
• Axillary temperature ≥ 37.5°C or history of fever in the past 24 hours
• Ability to take oral medication
• Written informed consent from patient or parent/guardian
• Residence in the study area with intention to remain during follow-up

Exclusion Criteria:

• Severe or complicated malaria (WHO criteria)
• Mixed Plasmodium infection
• Pregnancy or breastfeeding
• Known hypersensitivity to artemether-lumefantrine
• Antimalarial treatment in the 4 weeks prior to enrollment
• Severe malnutrition
• Concomitant febrile illness other than malaria requiring systemic treatment