Genomic Surveillance of <i>Plasmodium falciparum</i> Drug Resistance Markers Between October 2021 and June 2023 in Kigali, Rwanda

Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment in Rwanda, but the emergence of drug resistance threatens their efficacy. This study conducted genomic surveillance of <i>Plasmodium falciparum</i> isolates collected in Kigali between October 2021 and June 2023 to assess resistance markers. Using Oxford Nanopore Technology and Sanger sequencing methods, we analyzed 250 clinical isolates focusing on mutations in the <i>pfcrt</i>, <i>pfmdr1</i>, <i>pfdhfr</i>, <i>pfdhps</i>, and <i>Pfkelch13</i> genes. Resistance-associated mutations were highly prevalent: <i>pfcrt</i> 76T (26%) and <i>pfmdr1</i> 184F (72.8%) were common, indicating continued lumefantrine pressure. All isolates carried mutations in <i>pfdhfr</i> and <i>pfdhps</i>, with the <b>IRN</b>I-SA<b>E</b>AA and <b>IRN</b>I-SA<b>EG</b>A haplotypes found in 45.6% and 24.8% of samples, respectively, suggesting sustained antifolate resistance. <i>Pfkelch13</i> mutations were present in 50.4% of isolates, including validated R561H (25.6%), A675V and candidate P441L mutations. Novel haplotypes, including K189T + R561H (24.8%), were identified for the first time in Rwanda. The BTB/POZ domain mutation H384R was observed in 6.4% of isolates, raising questions about its potential functional role. These findings highlight complex and evolving resistance patterns and emphasize the urgent need for continued molecular surveillance and functional validation to inform malaria control strategies in Rwanda.