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Background   Plasmodium falciparum  drug resistance surveillance is key to successful disease control and eradication. Contemporary methods that only allow determination of prevalence of resistance are expensive, time consuming and require ethical considerations. A newer method involving Next Generation Sequencing (NGS) permits obtaining frequency of resistance while allowing to detect minority variants in mixed infections. Here, NGS was tested for  P. falciparum  resistance marker detection in mosquito samples as a feasible and suitable alternative for molecular resistance surveillance.  Anopheles funestus  were collected in southern Mozambique using CDC light traps and manual collections. DNA was extracted from either whole mosquito, head-thorax and abdomen separately or pools of five mosquitoes. These samples were screened for  P. falciparum  and if positive for  k13 ,  pfcrt ,  pfmdr1 ,  pfdhps  and  pfdhfr  mutations related to anti-malarial drug resistance with Sanger sequencing and NGS.    Results  Among the 846 samples screened for  P. falciparum , 122 were positive by 18S ssrDNA qPCR with an infection rate of 23.6%. No mutations were observed for  k13  and  pfcrt 72-76 and almost zero for  pfmdr 86, but quintuple  pfdhfr/pfdhps  mutations were near fixation and about half of the isolates contained the  pfmdr 184F polymorphism. Similar allele frequencies of resistance markers were estimated with NGS in comparison with the prevalence of markers obtained with the gold standard Sanger sequencing.    Conclusions  Pooled deep sequencing of  P. falciparum  isolates extracted from mosquitoes is a promising, efficient and cost-effective method to quantify allele frequencies at population level which allows to detect known and unknown markers of resistance in single and mixed infections in a timelier manner. Using mosquitoes as sentinel group and focusing on allele frequency opposed to prevalence, permits active surveillance across a more homogeneous geographical range.

Mosquitoes as a feasible sentinel group for anti-malarial resistance surveillance by Next Generation Sequencing of Plasmodium falciparum