Molecular surveillance of act drug resistance markers and genetic diversity in plasmodium falciparum in Gasabo district

Abstract

The emergence of Plasmodium falciparum resistance to artemisinin-based combination therapies (ACTs) threatens malaria control in sub-Saharan Africa, which accounts for 94% of global malaria cases and over 95% of malaria-related deaths(WHO, 2023). This study assessed ACT drug resistance markers and the genetic diversity of P. falciparum in Gasabo District, Kigali, Rwanda. A cross-sectional molecular epidemiological study was conducted from April to July 2025, during which 392 venous blood samples were collected. Of these, 290 samples were successfully amplified for target genes, and 54 samples were sequenced using Oxford Nanopore Technologies. The study targeted four genes: pfk13, pfmdr1, and pfcrt, which are involved in ACT metabolism, and msp2, used to assess parasite genetic diversity. Bioinformatics processing and statistical analysis using SPSS were performed to identify patterns of drug resistance and parasite diversity. Molecular analysis revealed that 70% (n = 38) of sequenced isolates harbored single nucleotide polymorphisms (SNPs) in pfk13, pfmdr1, or pfcrt. The WHO-validated resistance marker R561H in pfk13 was detected in 39% (n = 21) of isolates, the pfmdr1 Y184F mutation occurred in 54% (n = 29), and pfcrt K76T, historically associated with chloroquine resistance, was observed in 7% (n = 4). Structural modeling of the novel A569V mutation in pfk13 suggested a destabilizing effect on the protein. Co-occurrence of resistance markers, particularly between pfk13 and pfmdr1, was observed in 31% of isolates, indicating a possible reduction in the efficacy of Coartem, the first-line treatment used in Rwanda. MSP2 genotyping revealed limited allelic diversity, with predominant 3D7 strains. Only a subset of the amplified samples (54 of 250) was sequenced due to academic constraints, which limited the available time. These findings highlight ongoing transmission and emerging drug resistance, emphasizing the need to integrate molecular surveillance into national malaria control strategies to support evidence-based policy decisions and preserve ACT efficacy.