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 Plasmodium falciparum 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 pfcrt, pfmdr1, pfdhfr, pfdhps, and Pfkelch13 genes. Resistance-associated mutations were highly prevalent: pfcrt 76T (26%) and pfmdr1 184F (72.8%) were common, indicating continued lumefantrine pressure. All isolates carried mutations in pfdhfr and pfdhps, with the IRNI-SAEAA and IRNI-SAEGA haplotypes found in 45.6% and 24.8% of samples, respectively, suggesting sustained antifolate resistance. Pfkelch13 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.

Malaria remains a significant public health challenge, particularly in endemic regions. The extensive genetic diversity of Plasmodium falciparum (Pf) complicates outbreak prediction and transmission control. One of its most polymorphic markers, merozoite surface protein 2 (MSP2), presents a potential target for molecular surveillance. This cross-sectional study, conducted at King Faisal Hospital Rwanda (KFHR) from October 2021 to June 2023, assessed MSP2’s utility in malaria prediction. PfMSP2 was sequenced, and selected amplicons were cloned, expressed in bacteria, and purified. These antigens were tested against sera from malaria patients and geographically diverse healthy individuals, with complementary surveys contextualizing serological findings. Of the 75 processed monoallelic clinical isolates, 3D7 strains predominated over FC27. Three MSP2-derived biomarkers were produced, eliciting significantly low IgG responses in malaria patients and Belgian controls, but a complex pattern emerged in healthy individuals, with significant differences between Rwandan and Cameroonian samples. IgG3 was the predominant subclass in individuals with high IgG responses. Notably, Rwandan individuals with weak humoral responses to the tested antigens but also other with high responses experienced malaria episodes in the subsequent year. These findings highlight MSP2 polymorphism as a valuable tool for malaria surveillance and outbreak prediction. Integrating genotyping and serology could enable precise, community-specific malaria risk assessments, strengthening control strategies.

Background: Malaria cases in Rwanda resurged markedly after more than a decade of decline under artemisinin-based combination therapy (ACT) deployment. Whether this resurgence reflected the emergence of a novel Plasmodium falciparum lineage or adaptive evolution within preexisting parasite populations has direct implications for resistance surveillance and treatment policy across East Africa.Methods: We conducted a longitudinal targeted genomic surveillance study of 1,176 P. falciparum isolates collected in Rwanda between October 2021 and December 2025, from urban (Kigali) and high-transmission rural settings. Targeted amplicon sequencing of kelch13, crt, mdr1, dhfr, and dhps resistance loci, together with msp2 and csp polymorphic genomic regions was integrated with principal-coordinate analysis, permutational multivariate analysis of variance, phylogenetic reconstruction, and temporal selection scans. Selected kelch13 variants were further characterized through ex vivo dihydroartemisinic susceptibility assays and in vitro serial-passage competition experiments.Findings: Parasite populations were genetically continuous across the study period, with no evidence of clonal lineage replacement. Mutations in dhfr and dhps remained near fixation. The combined R561H-K189T kelch13 genotype was associated with significant increase during the post-resurgence period (2024-2025; P=4·91×10⁻⁵), particularly in Kigali, and was enriched across multiple phylogenetic backgrounds, consistent with soft selective processes rather than clonal expansion. Of particular concern, a small cluster of isolates carried validated mutations across all analyzed resistance loci, more likely an alarming emerging threat against malaria control in case of amplification and spread of such multidrug-resistant genomic backgrounds. Temporal selection scans identified possible directional allele-frequency associated with enrichment at R561H, K189T, and A675V. Preliminary ex vivo assays were associated with higher dihydroartemisinic survival among K13-mutant isolates, and in vitro competition experiments suggest progressive expansion of K189T- and K189N-containing parasites over successive culture passages.Interpretation: Malaria resurgence in Rwanda occurred through adaptive enrichment of artemisinin-associated genomic backgrounds within a genetically continuous, recombining parasite population, rather than through emergence of a single dominant resistant lineage. These findings suggest that artemisinin adaptation in sub-Saharan Africa follows an incremental, population-wide evolutionary trajectory distinct from the lineage-restricted selective sweeps documented in Southeast Asia, with direct implications for the design of resistance surveillance and treatment-efficacy monitoring programmes across the region. These conclusions are derived from targeted surveillance of major antimalarial resistance loci and polymorphic markers rather than whole-genome analyses.

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.