Clinical evidence of Kelch13-dependent and -independent malaria treatment failure with Artemether-Lumefantrine

Malaria remains a major public health threat, particularly in sub-Saharan Africa. In Rwanda, Artemether-Lumefantrine (AL; Coartem) has been the primary treatment for uncomplicated malaria, though its efficacy is increasingly in question. Using clinical data and Plasmodium biobanks from King Faisal Hospital Kigali, we analyzed 23 selected samples from patient with recurrent malaria. Genotyping confirmed recrudescence rather than reinfection. Following the initial AL treatment, 56.5% (microscopy) and 83.3% (qPCR) remained positive, with symptoms persisting. After a second treatment, positivity declined but was still notable. Sequencing analysis revealed widespread MDR1 Y184F mutations and cumulative Pfkelch13 mutations, including H384R in the BTB/POZ domain, associated with treatment failure. Four failing strains lacked kelch13 mutations, indicating alternative resistance mechanisms. These findings support the need for expanded genetic surveillance to preserve ACT efficacy. Overall design: Amplicon sequencing: gDNA from Plasmodium infected blood was extracted and specific plasmodium genes amplified and sequenced using MINION technology on R.10 flow cells.