Autor/a:
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Smith Aguasca, Rebecca; Gupta, Himanshu; Uberegui, Estefania; Maquina, Mara; Saute, Francisco; Paaijmans, Krijn P.; Mayor Aparicio, Alfredo Gabriel; Huijben, Silvie
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Abstract:
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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 collec‑
tions. DNA was extracted from either whole mosquito, head-thorax and abdomen separately or pools of fve mosqui‑
toes. 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 pfcrt72-76 and almost zero for pfmdr86, but quintuple pfdhfr/
pfdhps mutations were near fxation and about half of the isolates contained the pfmdr184F 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, efcient
and cost-efective 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 homogene‑
ous geographical range. |