Genetic heterogeneity of Plasmodium falciparum across areas of varying transmission intensity in Ethiopia based on merozoite surface protein (msp) genes msp1 and msp2

Background Plasmodium falciparum malaria continues to be a significant threat in Ethiopia. Understanding the genetic diversity of this parasite is crucial for informing public health strategies, including vaccine development, diagnostic accuracy, treatment efficacy, and targeted control interventions. Methods Samples for this study were collected from three malaria-endemic sites in Ethiopia with varying transmission intensities - Metema (northwest), Wondogenet (south), and Metehara (central-east). A consecutive convenient sampling technique was employed to recruit outpatients initially enrolled in an uncomplicated malaria therapeutic efficacy study in 2015 from these sites, along with additional samples collected from malaria suspects attending the Metehara health centre in 2019. Overall, 661 finger-prick blood samples were collected for malaria microscopy and rapid diagnostic tests (RDTs), while dried blood spot (DBS) samples were obtained on Whatman 903® filter paper for molecular analysis. This study specifically utilized selected 150 quantitative real-time polymerase chain reaction (qPCR)-confirmed P. falciparum monoinfection-positive samples from the previously reported total of 661 samples to investigate previously unreported merozoite surface protein ( msp1 and msp2 ) genes-based genetic heterogeneity. Genotyping of P. falciparum msp1 and msp2 genes was conducted using nested PCR, and statistical analysis was performed using SPSS software to assess mean multiplicity of infection (MOI), polyclonal infections and expected heterozygosity index (H _e ). Results Out of the total 150 samples analyzed, successful genotyping rates of 45.3% was revealed for msp1 and 43.3% for msp2 across the sites. Notable variations in allele frequencies were observed, with the K1 allele predominating for msp1 and the FC27 allele for msp2 . Metema exhibited the highest complexity of infections, with mean MOI values of 1.53 for msp1 and 2.14 for msp2 , alongside the greatest genetic diversity. The analysis indicated that polyclonal infections were prevalent, particularly in Metema and Wondogenet, with notable differences in allele frequencies over time between samples collected in 2015 and 2019 in Metehara, reflecting shifts in population dynamics. Conclusion This study highlights the genetic diversity and regional variations of P. falciparum in Ethiopia, emphasizing the importance of continuous monitoring to facilitate malaria control strategies. The findings emphasize the need for tailored intervention approaches and ongoing investigation to address the evolving landscape of malaria in the country.