Alterations in Plasmodium falciparum Genetic Structure Two Years after Increased Malaria Control Efforts in Western Kenya

Anne M. Vardo-Zalik 0 1 Guofa Zhou 0 1 Daibin Zhong 0 1 Yaw A. Afrane 0 1 Andrew K. Githeko 0 1 Guiyun Yan 0 1 0 State University , 1031 Edgecomb Avenue, York, PA 17403 1 Program in Public Health, College of Health Sciences, University of California Irvine, Irvine, California; Climate and Human Health Research Unit, Kenya Medical Research Institute , Kisumu , Kenya The impact of malaria intervention measures (insecticide-treated net use and artemisinin combination therapy) on malaria genetics was investigated at two sites in western Kenya: an endemic lowland and an epidemic highland. The genetic structure of the parasite population was assessed by using microsatellites, and the prevalence of drugresistant mutations was examined by using the polymerase chain reaction-restriction fragment length polymorphism method. Two years after intervention, genetic diversity remained high in both populations. A significant decrease in the prevalence of quintuple mutations conferring resistance to sulfadoxine-pyrimethamine was detected in both populations, but the mutation prevalence at codon 1246 of the Plasmodium falciparum multidrug resistance 1 gene had increased in the highland population. The decrease in sulfadoxine-pyrimethamine-resistant mutants is encouraging, but the increase in P. falciparum multidrug resistance 1 gene mutations is worrisome because these mutations are linked to resistance to other antimalarial drugs. In addition, the high level of genetic diversity observed after intervention suggests transmission is still high in each population. - The World Health Organization estimates that malaria causes 300500 million clinical cases annually, with more than one million deaths per year.1 Recently, major funding from the Presidents Malaria Initiative and the Global Fund to Fight against AIDS, Tuberculosis and Malaria has significantly enhanced malaria control efforts in sub-Saharan Africa where malaria morbidity and mortality are highest.2 The main malaria control tools include treating infected persons with antimalarial drugs and reducing human-mosquito contact rates through mosquito control.36 The large-scale distribution of insecticide-treated nets (ITNs) and a change in the first-line antimalarial drug from sulfadoxine-pyrimethamine (SP) or chloroquine (CQ) to artemisinin combination therapy (ACT) marked the beginning of a new era of malaria control campaign in sub-Saharan Africa.4,5 The goal of any malaria control program is to reduce overall transmission of the parasite and consequently malariainduced morbidity and mortality. In Kenya, the current strategy involves a two-tiered system. First, more than seven million ITNs were distributed free of charge in 2006 to children and pregnant women.1,510 The ITNs minimize contact rates of the mosquito vector, thereby reducing transmission and malaria incidence.1113 This reduced transmission may have an important effect on the parasites genetic variability, a factor that might influence its adaptive ability. With reduced transmission, the malaria parasite has fewer opportunities to sexually recombine, and therefore lose favorable drug-resistant combinations.14 This may result in lower population genetic diversity. Second, the use of ACT to treat uncomplicated malaria targets the transmission stages of malaria and the asexual stages.1 The prescription of a drug that affects both blood stages of the parasite could also lead to reduced transmission. The previous long-term usages of CQ and SP have resulted in a rapid spread of drug resistant malaria genotypes.4,15 In 2004, artemether-lumefantrine (Coartem; Roche, Basel, Switzerland) was introduced to replace SP and in 2006 was provided free of charge in health facilities with the support from the Global Fund to Fight AIDS, Tuberculosis and Malaria.2 The introduction of artemether-lumefantrine has relaxed the selection pressure on resistance to CQ and SP, but has posed additional selection pressure on the genes related to artemether-lumefantrine resistance. Drug resistance in malaria parasites is associated with genetic mutations in target genes and can be monitored using molecular methods. Chloroquine resistance is determined by the major point mutation at codon 76 of the P. falciparum CQ resistance transporter ( pfcrt) gene.16 This mutation highly correlates with increased clinical CQ tolerance and treatment failure.1619 In addition, point mutations in the P. falciparum multidrug resistance 1 ( pfmdr1) gene (e.g., N86Y, Y183F, S1034C, N1042D, and D1246Y) have been shown to modulate CQ resistance.20 Resistance to antifolates is associated with point mutations in the dihydrofolate reductase ( pfdhfr) and dihydropteroate synthetase ( pfdhps) genes.21,22 The quintuple mutations in pfdhfr and pfdhps (triple S108N/N51I/C59R mutations in dhfr and double A437G/K540E mutations in dhps) are associated with the clinical failure of SP treatment for P. falciparum malaria.21,22 Resistance to artemisinins has been reported in Southeast Asia, but it has not been detected in Africa.23,24 The objective of this study was to determine the effects of the new two-tiered control program on the genetic diversity and the prevalence of malaria drug-resistant mutations in western Kenya. Two sentinel sites, one in an endemic lowland (Kombewa) and one in an epidemic highland (Kakamega), were examined. Data on malaria prevalence during this period have been published.5 In brief, at the beginning of the study period in 2005, the prevalence of P. falciparum was 41% in Kakamega and this significantly decreased throughout the four-year study period; the prevalence in 2008 was 6.8%.5 The prevalence of P. falciparum in 2005 in Kombewa was 48%; the prevalence decreased to 31% in 2007, but increased in 2008, and the prevalence surpassing pre-intervention levels (49%).5 We hypothesized that malaria parasite genetic diversity would be significantly reduced in Kakamega because of decreases in overall transmission as measured by malaria prevalence, but this effect would not be as noticeable in Kombewa because of a lack of a sustained reduction in malaria prevalence. In addition, as SP has been phased out, we expected to see a reduction in the point mutation frequencies in the codons of pfdhfr and pfdhps genes coding for resistance to this drug in both study populations. Study areas. This study was conducted in two sites in western Kenya with differing levels of transmission. Kakamega, a highland site 1,5001,600 meters above sea level, is characterized by valleys and depressions surrounded by densely populated hills and is hyperendemic for malaria. Kombewa, a lowland site with a mean altitude of 1,200 meters above sea level, has a rolling terrain bisected by small streams and is holoendemic for malaria. Plasmodium falciparum is the primary malaria parasite species, with the predominant malaria mosquito vector species being Anopheles gambiae, An. arabiensis, and An. funestus.25,26 The estimated entomologic i (...truncated)