Abstract:
Malaria remains one of the most significant public health challenges globally, particularly in sub-Saharan Africa, where Plasmodium falciparum accounts for the majority of malariarelated morbidity and mortality. In Rwanda, malaria remains endemic. Despite the widespread implementation and success of artemisinin-based combination therapies (ACTs), the emergence of artemisinin resistance primarily associated with mutations in the P. falciparum Kelch 13 (K13) gene poses a serious threat to current treatment efficacy. K13 mutations have been increasingly reported in Rwanda, warranting continuous molecular surveillance. This study aimed to investigate the prevalence of P. falciparum K13 mutations and their association with parasitemia levels in Gisagara District. A cross-sectional study was conducted using 215 Rapid diagnostic test (RDT)-positive samples which were collected under the Artemisinin Resistance Monitoring in East Africa (ARMEA) project from November to December 2024. Malaria-positive participants were initially identified using the BiolineTM Malaria Ag P. f/Pan, Abbott Diagnostics Korea Inc RDTs and subsequently referred to health centers for venous blood collection. Samples were transported to the University Teaching Hospital of Butare (CHUB) for further laboratory analysis. Thick and thin smears were used for parasitemia identification and quantification, while polymerases chain reaction assay (PCR) and capillary-based Sanger sequencing were employed to detect K13 mutations. Of the 215 samples, 195 were confirmed positive by microscopy. Among these 190 were identified as P. falciparum. The majority of P. falciparum cases were trophozoites, while 17 samples presented with gametocytes. Additionally, five samples were identified as P. malariae, with 3 containing schizonts. The 195 microscopy-positive samples were classified into 3 categories: 31 exhibited low parasitemia,73 fell into the moderate range and 91 shower high parasitemia levels PCR amplification was successful in 203 samples, and sequencing revealed K13 mutations in 2.8% of cases, with R561H being the most frequently observed mutation. Notably, mutants samples exhibited moderate to high parasitemia, with no cases in the low parasitemia category. These findings highlight the significance of continued molecular surveillance for early detection of K13 mutations to combat outbreaks due to artemisinin resistance
