Field-based evidence of fast and global increase of Plasmodium falciparum drug-resistance by DNA-microarrays and PCR/RFLP in Niger

BACKGROUND: Over the last years, significant progress has been made in the comprehension of the molecular mechanism of malaria resistance to drugs. Together with in vivo tests, the molecular monitoring is now part of the survey strategy of the Plasmodium sensitivity. Currently, DNA-microarray analysis allows the simultaneous study of many single nucleotide polymorphisms (SNP) of Plasmodium isolates. In December 2005, the International Federation of the Red Cross distributed two million three hundred thousand long-lasting insecticide nets to pregnant women and mothers of under five years children in the whole Niger. Then, Niger adopted artemisinin-based combination therapy as first-line treatment. METHODS: Thirty four SNPs of pfcrt, pfdhfr, pfdhps, pfmdr and pfATPase were analysed by DNA-microarray and PCR/RFLP in two villages – Zindarou and Banizoumbou – with different durations of malaria transmission. The main objective of the study was to measure the dynamics of Plasmodium falciparum resistant strains and associated factors. RESULTS: This study shows a global and clear increase of the drug-resistance associated molecular markers frequencies during a relatively short-time period of four years. Markers associated with resistance to chloroquine and sulphonamids were more frequently found in the short transmission zone than in the long transmission one. The pfcrt76T mutation is significantly more present at Banizoumbou than Zindarou (38.3% vs 25.2%, p = 0.013). This work allowed the screening of several field strains for five SNPs of PfATPase6 gene. The pfATPase6S769N, candidate mutation of resistance to artemisinin was not found. However the pfATPsaeA623E mutation was found in 4.7% of samples. CONCLUSION: A significant increase of several SNPs frequencies was highlighted over a four-year period. The polymorphism of five PfATPase6 gene SNPs was described. The global, large and fast increase of the molecular resistance is discussed in the context of current changes of health policy and malaria control in Niger.