Oxford Nanopore Targeted sequencing of Plasmodium falciparum isolates from Kigali, Rwanda to monitor drug resistance markers and CSP genetic diversity (2021–2023)
Artemisinin-based combination therapies (ACTs) and emerging malaria vaccines remain central to malaria control in Rwanda, yet the effectiveness of these interventions is increasingly challenged by parasite genetic adaptation. To generate a comprehensive genomic surveillance baseline, we analyzed Plasmodium falciparum isolates collected between October 2021 and June 2023 in Kigali, Rwanda. Using Oxford Nanopore Technology complemented with Sanger sequencing, we screened over 250 clinical isolates for drug resistance markers in pfcrt, pfmdr1, pfdhfr, pfdhps, and pfkelch13 genes. High frequencies of resistance-associated mutations were detected, including pfcrt 76T (26%), pfmdr1 184F (72.8%), and widespread antifolate resistance haplotypes such as IRNI-SAEAA and IRNI-SAEGA, indicating persistent drug pressure. Pfkelch13 mutations, including validated R561H (25.6%) and emerging A675V and P441L variants, were detected alongside novel combinations such as K189T + R561H, suggesting ongoing diversification under selection pressure.  In parallel, we assessed vaccine-relevant diversity by characterizing the circumsporozoite protein (Pfcsp) gene in 245 isolates from the same genomic dataset. A total of 48 distinct Pfcsp haplotypes were identified, reflecting high haplotype diversity (Hd = 0.8899) but moderate nucleotide diversity, consistent with immune-mediated balancing selection. While the N-terminal region, including the KLKQP motif, remained conserved, the central NANP/NVNP repeat region and C-terminal Th2R/Th3R epitopes displayed extensive polymorphism. Fewer than 1% of sequences matched the 3D7 vaccine strain, and several positions linked to vaccine escape exhibited high mutation frequencies.  Together, these findings reveal concurrent drug resistance evolution and antigenic diversification in P. falciparum populations circulating in urban Kigali. This dataset provides an integrated genomic resource to inform both therapeutic policy and vaccine deployment strategies in Rwanda and similar transmission settings.