High prevalence and emerging positive association of kelch13 R622I and HRP2-based RDT negativity in Plasmodium falciparum in northern Ethiopia.

The rise of antimalarial drug-resistant Plasmodium falciparum threatens malaria elimination efforts. Mutations in the gene kelch13 (k13) confer artemisinin partial resistance (ART-R), compromising the efficacy of frontline artemisinin-based combination therapies (ACTs). The validated mutation k13 R622I has emerged and expanded rapidly in the Horn of Africa. We conducted a year-long genomic surveillance study in Gondar Zuria and Tach Armachiho, two ecologically distinct districts in northwestern Ethiopia where R622I was first identified. A total of 903 P. falciparum infections were sequenced using molecular inversion probe (MIP) panels targeting major drug resistance mutations and genome-wide informative SNPs. The R622I mutation was found in 44.3% of samples, more frequent in Gondar Zuria than Tach Armachiho (52% vs. 35%; p < 0.001), and persisted year-round in nearly all sites, indicating stable transmission with minimal seasonal variation. Histidine-rich protein 2 (HRP2) based rapid diagnostic test (RDT) negativity was also prevalent (39.3%), with significant district-level variation (48.7% vs. 27.6%; p < 0.001). Concerningly, R622I and HRP2-RDT co-occured in 22% of samples, higher in Gondar Zuria than in Tach Armachiho (28.9% vs. 12.9%; p < 0.001). Overall, HRP2-RDT negativity was significantly more common among R622I mutant parasites than wild-type (48.3% vs. 30.7%; p < 0.05). The k13 C580Y mutation was also detected at very low frequency (0.4%) in Gondar Zuria, representing the first report of this mutation in the Horn of Africa. Long-read whole-genome sequencing showed k13 flanking haplotypes of C580Y isolates were distinct from Southeast Asian lineages, suggesting a local, de novo emergence of African origin. These findings highlight the increasing prevalence and types of ART-R mutations, persistence of k13 R622I and its increasing association with HRP2-RDT negativity, representing a double threat to malaria control and elimination efforts.