Research Article Volume 8 • Issue 2 470 High Prevalence of Pfdhps and Pfdhfr Resistance Markers to Sulfadoxine- pyrimethamine During Subpatent Parasitemia in Doneguebougou, Mali Hamma Maiga *,1,2 , Robert D. Morrison 1 , Issaka Sagara 3 , Sara Healy 1 , Abdoulaye Katile 3 , Amatigue Zeguime 3 , and Patrick E. Duffy 1 Abstract Blood smear-positive asymptomatic infections are an important malaria parasite reservoir because they harbor gametocytes. However, asymptomatic infections are often submicroscopic, can infect mosquitoes and can only be detected by molecular methods. Malaria prevention programs pursue mass treatment of asymptomatic individuals, which may contribute to development and spread of drug resistance. Here, we investigated the prevalence of molecular markers of drug resistance of Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps) during subpatent parasitemia compared to patent parasitemia episodes in Mali. From July to December 2019, blood smears and filter paper blood spots were collected from children with subpatent parasitemia (BS-) and patent parasitemia (BS+) in Doneguebougou. Point mutations at codons (50, 51, 59, 108, 140 and 164) of the Pfdhfr gene and codons (431, 436, 437, 540, 581 and 613) of the Pfdhps gene were evaluated by nested PCR amplification followed by direct sequencing. A total of 84 children under five years of age were evaluated (27 BS- and 57 BS+). When assessed as a binary endpoint (any versus no detection of resistance allele), we found a significance higher prevalence of Pfdhps 581G (51.9% vs. 12.3%, p=0.001) and Pfdhfr at codons 50R (75.0% vs. 22.2%, p=0.001), 140L (66.7% vs. 25.0%, p=0.014) and 164L (88.9% vs. 21.2%, p=0.0004) in BS- vs. BS+, respectively. In BS- the prevalence of Pfdhps at codons (431V, 437G and 540E) and Pfdhfr at codon (51I) were higher but not significant in BS- vs. BS+. No Pfdhps 540E was detected in BS+. For all markers except one, mutations were more prevalent in BS- samples in Mali. Asymptomatic subpatent parasitemia can be a reservoir of drug-resistant parasites and should be regularly surveyed (in addition to BS+ episodes) to accurately monitor the spread of molecular markers of resistance. Affiliation: 1 Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 29 Lincoln Drive, Bethesda, MD, 20892, USA 2 Institut National de Sante Publique, Bamako, BP: 1771, Mali 3 Malaria Research and Training Center, Department of Epidemiology of Parasitic Diseases, Faculty of Medicine and Dentistry, Faculty of Pharmacy, University of Sciences, Techniques and Technologies of Bamako, BP: 1805, Mali *Corresponding author: Hamma Maiga, Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 29 Lincoln Drive, Bethesda, MD, 20892, USA. Citation: Hamma Maiga, Robert D. Morrison, Issaka Sagara, Sara Healy, Abdoulaye Katile, Amatigue Zeguime, and Patrick E. Duffy. High Prevalence of Pfdhps and Pfdhfr Molecular Markers of Sulfadoxine-Pyrimethamine Resistance During Subpatent Parasitemia in Doneguebougou, Mali. Fortune Journal of Health Sciences, 8 (2025): 470-478. Received: April 15, 2025 Accepted: April 22, 2025 Published: June 02, 2025 Keywords: Malaria elimination, pfdhps, pfdhfr, subpatent parasitemia, Mali Introduction Asymptomatic malaria is not often defined rigorously, but generally refers to an individual harboring blood-stage malaria parasites without experiencing fever or other symptoms that would lead the individual to seek treatment [1]. In most malaria-endemic settings, asymptomatic infections outnumber symptomatic infections [2]. The lack of rigorous definitions for asymptomatic malaria has clouded research to support elimination strategies, which rely on the ability to find and treat the asymptomatic reservoir [3]. As resources