Molecular Markers for Tracking the Origin and Spread of Sulfadoxine and Pyrimethamine Resistant Mutations Venkatachalam Udhayakumar, Andrea M. McCollum, Sean Griffing, Zhiyong Zhou, John Barnwell, Ananias A. Escalante Abstract Molecular markers are valuable tools for monitoring the emergence of drug resistance. Single nucleotide polymorphisms (SNPs) in the antimalarial drug target genes (eg: Pfcrt for chloroquine, dhfr for pyrimethamine) have been correlated with clinical resistance. Microsatellite markers, which are random repeats of nucleotides spread across different parts of the chromosomes, can be used as tools to track the origin and spread of drug resistant mutations. We have used several microsatellite markers to track the drug resistant dhfr and dhps genotypes in parasite isolates from different endemic areas of Africa and South America. In this presentation, how these molecular tools helped us to determine the lineage relationship between different dhfr genotypes in Agrica and South America will be described. I ntroduction The emergence of drug resistant Plasmodium falciparum is a serious public health problem in many countries where malaria is endemic (Gregson and Plowe, 2005; Talisuna, Bloland and D’Alessandro, 2004). Resistance to chloroquine (CQ), the least expensive and widely available antimalarial drug, has spread throughout the world except in Central America and the Caribbean. As a result, sulfadoxine-pyrimethamine (SP) became the next widely used drug treatment for uncomplicated P. falciparum malaria. Unfortunately, resistance to SP has become established in many parts of the world, as well. Currently, artemisinin based combination therapy (ACT) is advocated as the best treatment option to circumvent the rapid emergence of resistance to malaria. Understanding the mechanisms involved in drug resistance is critical to developing methods for the prevention of emergent drug resistance, the development of molecular surveillance tools, and the formulation of appropriate policy to respond to drug resistance. It has become evident that there is a genetic basis for drug resistance to CQ, SP and other drugs (Gregson and Plowe, 2005; Talisuna, Bloland and D’Alessandro, 2004). Resistance to CQ is strongly associated with mutations in the pfcrt gene which encodes a transmembrane protein in the digestive vacuole (Sidhu, Verdier-Pinard and Fidock, 2002). SP acts as an inhibitor of the P. falciparum folic acid pathway, and point mutations in the genes encoding dihydrofolate reductase ( dhfr) and dihydropteroate synthetase ( dhps) have been implicated in pyrimethamine and sufadoxine resistance, respectively (Hayton and Su , 2004). Microsatellite loci, random repeats of DNA bases (example: ATATATATATATATATATATAT), are distributed across the genome and are considered to be neutral with respect to the fitness of an organism; i.e. the microsatellite loci are assumed not to be under selection pressure because of the microsatellite sequence itself. However, these loci can be influenced by natural selection when they are physi- cally close to a sequence under selection. When a beneficial mutation spreads through a population, the regions of the genome surrounding the mutation can become “linked” to the selected site due to their proximity and will subsequently increase in frequency