Plasmodium falciparum Genomic Surveillance Reveals a Diversity of Kelch 13 Mutations in Zambia.

Antimalarials are central to Zambia's strategies for malaria control and elimination. Antimalarial drug resistance poses a significant threat to the effectiveness of artemisinin-based combination therapies and preventive strategies such as sulfadoxine-pyrimethamine chemoprevention in pregnant women. In this genomic surveillance study, dried blood spots and epidemiological data were collected from confirmed Plasmodium falciparum cases at 61 health facilities across all 10 Zambian provinces from March to July 2023. A total of 2,486 samples were genotyped by using multiplexed amplicon sequencing to identify mutations in 12 genes associated with resistance to seven antimalarial drugs. Several mutations potentially associated with artemisinin partial resistance were identified, including the validated kelch 13 (k13) P574L marker (0.66% adjusted national prevalence) and the candidate k13 P441L marker (1.39%). The distribution of mutations was heterogeneous, with many health facilities reporting resistance markers in more than 5% of infections, and in some instances, up to 46% of infections. The multidrug resistance protein 1 N86 genotype, which is associated with decreased lumefantrine susceptibility, was found in all samples. Very high levels of sulfadoxine-pyrimethamine resistance markers were observed, including dihydropteroate synthetase K540E (93.26%). The variable prevalence of resistance markers underscores the need for routine molecular surveillance to detect emergent resistance and guide malaria control strategies. These results also call for studies designed to help us understand the clinical implications of these mutations and ensure the continued efficacy of antimalarial interventions in Zambia.