Abstract
BACKGROUND: Understanding the origin and spread of mutations associated with drug resistance, especially in the context of combination therapy, will help guide strategies to halt and prevent the emergence of resistance. Unfortunately, studies have assessed these complex processes when resistance is already highly prevalent. Even further, information on the evolutionary dynamics leading to multidrug-resistant parasites is scattered and limited to areas with low or seasonal malaria transmission. This study describes the dynamics of strong selection for mutations conferring resistance against sulphadoxine-pyrimethamine (SP), a combination therapy, in western Kenya between 1992 and 1999, just before SP became first-line therapy (1999). Importantly, the study is based on longitudinal data, which allows for a comprehensive analysis that contrasts with previous cross-sectional studies carried out in other endemic regions. METHODS: This study used 236 blood samples collected between 1992 and 1999 in the Asembo Bay area of Kenya. Pyrosequencing was used to determine the alleles of dihydrofolate reductase (dhfr) and dihydropterote synthase (dhps) genes. Microsatellite alleles spanning 138 kb around dhfr and dhps, as well as, neutral markers spanning approximately 100 kb on chromosomes 2 and 3 were characterized. RESULTS: By 1992, the South-Asian dhfr triple mutant was already spreading, albeit in low frequency, in this holoendemic Kenyan population, prior to the use of SP as a first-line therapy. Additionally, dhfr triple mutant alleles that originated independently from the predominant Southeast Asian lineage were present in the sample set. Likewise, dhps double mutants were already present as early as 1992. There is evidence for soft selective sweeps of two dhfr mutant alleles and the possible emergence of a selective sweep of double mutant dhps alleles between 1992 and 1997. The longitudinal structure of the dataset allowed estimation of selection pressures on various dhfr and dhps mutants relative to each other based on a theoretical model tailored to P. falciparum. The data indicate that drug selection acted differently on the resistant alleles of dhfr and dhps, as evidenced by fitness differences. Thus a combination drug therapy such as SP, by itself, does not appear to select for "multidrug"-resistant parasites in areas with high recombination rate. CONCLUSIONS: The complexity of these observations emphasizes the importance of population-based studies to evaluate the effects of strong drug selection on Plasmodium falciparum populations.