Low prevalence of the molecular markers of Plasmodium falciparum resistance to chloroquine and sulphadoxine/pyrimethamine in asymptomatic children in Northern Benin

Background: In Benin, very few studies have been done on the genetics of Plasmodium falciparum and the resistance markers of anti-malarial drugs, while malaria treatment policy changed in 2004. Chloroquine (CQ) and sulphadoxine pyrimethamine (SP) have been removed and replaced by artemisinin-combination therapy (ACT). The objective of this study was to determine the genetic diversity of P. falciparum and the prevalence of P. falciparum molecular markers that are associated with resistance to CQ and SP in northern Benin seven years after the new policy was instituted. Methods: The study was conducted in northern Benin, a region characterized by a seasonal malaria transmission. Blood samples were collected in 2012 from children presenting with asymptomatic P. falciparum infections. Samples collected in filter paper were genotyped by primary and nested PCR in block 2 of msp-1 and block 3 of msp-2 to analyse the diversity of P. falciparum. The prevalence of critical point mutations in the genes of Pfcrt (codon 76), Pfmdr1 (codon 86), Pfdhfr (codons, 51, 59 and 108) and Pfdhps (codons 437, 540) was examined in parasite isolates by mutation-specific restriction enzyme digestion. Results: Genotyping of 195 isolates from asymptomatic children showed 34 msp-1 and 38 msp-2 genotypes. The multiplicity of infection was 4.51 +/- 0.35 for msp-1 and 4.84 +/- 0.30 for msp-2. Only the codon 51 of Pfdhfr and codon 437 of Pfdhps showed a high mutation rate: I51: 64.4% (57.3; 71.2); G437: 47.4% (40.2; 54.7), respectively. The prevalence of Pfdhfr triple mutant IRN (I51, R59 and N108) was 1.5% (0.3; 3.9), and Pfdhfr/Pfdhps quadruple mutant IRNG (PfdhfrI51, R59, N108, and PfdhpsG437): 0. 5% (0; 2.5). No mutation was found with codon 540 of Pfdhps. Analysis of mutation according to age (younger or older than ten years) showed similar frequencies in each category without significant difference between the two groups. Conclusions: This study showed a high diversity of P. falciparum in northern Benin with a very low prevalence of resistance markers to CQ and SP that dramatically contrasted with the pattern observed in southern Benin. No influence of age on genetic diversity of P. falciparum and on distribution of the mutations was observed