Functional assessment of prevalent <i>kelch13</i> mutations reveals high-level artemisinin resistance potential in Bangladeshi <i>Plasmodium falciparum</i>

Artemisinin partial resistance (ART-R), driven by mutations in the <i>Plasmodium falciparum kelch13</i> (<i>k13</i>) gene, has contributed to malaria treatment failures in the Greater Mekong Subregion (GMS). The Chittagong Hill Tracts (CHTs) of Bangladesh account for nearly 90% of national malaria cases. Although K13-mediated ART-R has not been reported in the CHTs, its emergence remains a concern due to the region's proximity to K13-mutation-endemic areas in Myanmar and India, and the presence of moderate K13-independent ART-R. A meta-analysis of global K13 mutation frequency identified C580Y, F446I, and R561H as the most prevalent substitutions, particularly in the GMS. To evaluate their potential impact, we introduced these mutations into two recent CHT isolates via CRISPR-Cas9: an artemisinin-sensitive strain and a moderately <i>in vitro</i> resistant, K13-independent strain (CHT-R). Edited lines were phenotyped using ring-stage survival assays (RSAs), post-treatment recovery, and competitive fitness assays. C580Y and R561H conferred marked increases in ART-R in both strains, while F446I had only a modest effect in CHT-R. All three mutations incurred minimal asexual fitness costs, except for F446I in the CHT-R background. R561H, the dominant allele at the Thai-Myanmar border and now expanding in Rwanda, conferred extreme resistance in CHT-R (RSA: 30.09% ± 1.9%), alongside an asexual growth advantage (fitness: 5.1% per generation) and the highest post-treatment recovery. This work provides the first functional evidence from South Asian parasites, including the first Asian analysis of K13 mutations in a resistant background, demonstrating that CHT parasites are genetically primed to sustain high-level ART-R, warranting enhanced surveillance during Bangladesh's malaria elimination phase.IMPORTANCEArtemisinin resistance threatens global malaria control and elimination efforts. While the Greater Mekong Subregion is already entrenched with kelch13 (K13)-mediated resistance, Bangladesh, though not yet reporting K13 mutations, has confirmed K13-independent artemisinin resistance in its endemic Chittagong Hill Tracts, which border resistance hotspots in Myanmar. This study is the first to functionally test globally dominant K13 mutations in Bangladeshi <i>Plasmodium falciparum</i> using genome editing. We show that mutations such as R561H and C580Y significantly increase resistance and asexual fitness, particularly in strains already carrying K13-independent resistance. These findings suggest that local parasites are genetically primed to acquire and sustain high-level resistance in asexual stages if these mutations emerge. Our work provides a timely warning and a framework for assessing drug resistance risk in other low-transmission regions with ongoing artemisinin use, critical for national elimination programs and global efforts to prevent the spread of resistance beyond Southeast Asia.