Exploring genetic variability of Plasmodium falciparum: insights from PFCSP and PFK13 genes and their implications for malaria control in Rwanda

Abstract

Purpose and Scope: the purpose of this study was to characterize the genetic variability of plasmodium falciparum in Rwanda, focusing on two critical genes: the circumsporozoite protein gene (PfCSP), a target for malaria vaccine development, and kelch13 gene (Pfk13), which serves as a marker for artemisinin resistance. The research aims to assess the implications of genetic variability in these genes for malaria control strategies. Methods: A quantitative, cross-sectional molecular design was employed, analyzing 27 P. falciparum isolates. The study was conducted on samples from Huye and Kirehe districts, as malaria transmission areas. Blood samples were treated with QIAamp DNA Mini Kit to extract DNA, and PCR amplification of PfCSP and PfK13 genes. Sequencing was performed using Oxford Nanopore Technology, and bioinformatics tools (e.g., Minimap, Samtools, and Python packages) were used for variant calling, population genetic analysis and polygenetic tree construction. Statistical tests (Tajima’s D, Fu and Li’s D), and Principal component analysis (PCA) were used to assess population structure, selection pressures and genetic diversity. Principal Findings: the study identified significant single nucleotide polymorphisms (SNPs) in PfCSP, such as A98G, D199N and A361E observed at high frequencies (up to 69%). In Pfk13, the R561H mutation, a validated maker of artemisinin resistance was detected in 11 samples. Tajima’s D analysis indicated balancing selection in PfCSP (D=0.77), suggesting immune driven evolutionary pressure. Weak Linkage disequilibrium (LD) among SNPs suggested high genetic variability, while PCA revealed distinct clustering patterns, explaining 53% of genetic variation. Phylogenetic tree analysis showed genetic divergence between samples from Huye and Kirehe, likely influenced by regional transmission dynamics. Significance of Findings: The results demonstrate the genetic complexity of P. falciparum in Rwanda, which has consequences for the development of a vaccine against malaria and the tracking of resistance. The presence of R561H underscores the need for surveillance to mitigate artemisinin resistance. Balancing selection in PfCSP suggests ongoing immune evasion, which might affect how well the RTS, S/AS01, and R21 vaccinations work. This study provides the first district-level genetic analysis in Rwanda, offering important data to inform national malaria control programs and contributing to regional and global efforts in malaria elimination.