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

Abstract

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 RSA 0–3 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. Funding Fondation pour la Recherche Médicale, Institut Universitaire de France, Agence Nationale de la Recherche, Université de Strasbourg, and the National Natural Science Foundation of China. Translation For the Amharic translation of the abstract see Supplementary Material section.