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An in vivo drug sensitivity study was conducted in Magoda village in northeastern Tanzania to evaluate the usefulness of polymerase chain reaction (PCR)-based genotyping of Plasmodium falciparum parasites to distinguish between re-infection and treatment failure. The study tested P. falciparum susceptibility to a combination of sulfadoxine/pyrimethamine (Fansidar; F. Hoffmann La Roche, Basel, Switzerland). Blood samples were collected before treatment and on days 7, 14, or 28 post-treatment in 51 asymptomatic children, of which 26 could not clear parasitemia within seven days post-treatment. Among the remaining 25 children who had no detectable parasites on day 7, only five remained parasite negative up to day 28. Primary and recrudescent P. falciparum parasites were analyzed by PCR using family specific primers for merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein (GLURP). All samples contained multiple P. falciparum infections. For all children with recrudescent P. falciparum, common alleles were detected in both the primary and recrudescent samples. However, in no child were the exact same alleles detected in both samples, indicating that probably at least some of the recrudescing parasites originated from new infections. The study demonstrates the general usefulness of PCR genotyping technique in distinguishing re-infections from true recrudescences following therapeutic drug treatment.