Clinical, molecular, and in vitro evidence of artemisinin partial resistance in Ethiopian Plasmodium falciparum: a prospective, multisite, surveillance study

International audience Background: Artemisinin partial resistance (ART-R) has been confirmed in four sub-Saharan African countries since 2020, but evidence from Ethiopia is limited to molecular surveys without phenotypic confirmation. We aimed to determine whether ART-R met WHO criteria in Ethiopian Plasmodium falciparum populations during 2024-25, integrating day-3 parasite positivity after artemether-lumefantrine treatment, Pfkelch13 genotyping, and the ring-stage survival assay (RSA)0-3 h on culture-adapted field isolates.Methods: We conducted a prospective, multisite, surveillance study at five sentinel health facilities in Ethiopia (Rama, Werkamba, Mehoni, Bako, and Metehara). Patients aged 6 months or older with uncomplicated P falciparum malaria confirmed by microscopy received artemether-lumefantrine according to bodyweight (six doses during 3 days). Pfkelch13 was genotyped by Nanopore sequencing and Pfhrp2/Pfhrp3 deletions were assessed by quantitative PCR. The RSA0-3 h was performed on culture-adapted field isolates. Follow-up occurred on days 0 and 3. The main outcome was day-3 parasite positivity rate (defined as microscopically detectable asexual P falciparum parasitaemia on day 3 after initiation of artemether-lumefantrine). This study is registered with ClinicalTrials.gov, NCT07527182 (completed).Findings: Patients were enrolled from June 6 to Dec 8, 2024, during the 2024 P falciparum transmission season at all five sentinel sites and from July 1 to Nov 8, 2025, during the 2025 transmission season at Werkamba. 3207 febrile patients were assessed for malaria, of whom 2771 were excluded and 436 (14%) were P falciparum-positive on microscopy. 277 (64%) patients were enrolled, of whom 153 (55%) returned for the day-3 parasitological assessment and 124 (45%) were lost to follow-up. 172 (62%) of 277 patients were male and 105 (38%) were female. The median age was 20·0 years (IQR 10·0-30·0). 243 (88%) had P falciparum monoinfection by PCR and 34 (12%) had P falciparum and Plasmodium vivax co-infections undetected by microscopy at enrolment. The day-3 parasite positivity rate was 19·6% ([95% CI 14·1-26·6] in 30 of 153 patients with available data). 26 (9%) of 277 enrolled patients carried a validated Pfkelch13 mutation and were positive on day 3, exceeding the WHO 5% threshold for confirmation of ART-R. A strong age-dependent gradient was observed in a post-hoc analysis, with six (38%) of 16 patients younger than 5 years, 14 (35%) of 40 aged 5-15 years, and ten (10%) of 97 older than 15 years (p=0·00035). R622I was detected in 143 (53%) samples of 271 genotyped isolates. Carrying an R622I mutation was associated with day-3 positivity (26 [33%] of 79) in a univariate analysis (crude odds ratio [OR] 7·85 [95% CI 2·58-23·91]; p=0·0003). After adjustment, the association remained strong and independent (adjusted OR 9·96 [95% CI 3·39-36·35]; p<0·0001). Of 70 P falciparum field isolates on day 0 collected for culture adaptation, only six were successfully maintained. Five R622I isolates carried the Pfkelch13 R622I mutation and exceeded the 1% in vitro threshold for ART-R (mean survival rates of 1·41% [SD 0·27] for EW04, 2·69% [0·90] for EW14, 1·07% [0·52] for EW23, 3·76% [0·35] for EW38, and 1·29% [0·04] for EW56). Pooled with the wild-type strains, all five R622I isolates exceeded the 1% threshold compared with three wild-type isolates that did not exceed this threshold (p=0·018). Pfhrp3 was the most frequent deletion (39·8% [95% CI 32·6-47·4]; in 66 of 166 patients), followed by double Pfhrp2/Pfhrp3 deletion (23·5% [17·7-30·5]; in 39), wild-type (23·5% [17·7-30·5]; in 39), and Pfhrp2 deletion (13·3% [8·9-19·3]; in 22). R622I prevalence was similar across all four deletion categories (range 42-56%; p=0·47).Interpretation: Ethiopia is the fifth sub-Saharan African country now meeting WHO confirmation criteria for ART-R. High R622I prevalence and double deletion rates, consistent with distinct selective pressures, represent a dual threat to treatment and HRP2-based diagnosis of P falciparum malaria in this region.