Abstract
BackgroundPlasmodium 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.ResultsAmong 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 fixation 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.ConclusionsPooled 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.
Highlights
Plasmodium falciparum drug resistance surveillance is key to successful disease control and eradication
122 samples were quantitative polymerase chain reaction (qPCR)-positive, two of which were excluded from the infection rate calculations due to an error in the extraction procedure as precaution, but were included in Sanger and Next Generation Sequencing (NGS) analyses
Here, using a Generation Sequencing platform, mutant allele frequencies were obtained of P. falciparum parasites isolated from mosquitoes from southern Mozambique
Summary
Plasmodium falciparum drug resistance surveillance is key to successful disease control and eradication. DNA was extracted from either whole mosquito, head-thorax and abdomen separately or pools of five mosqui‐ toes These samples were screened for P. falciparum and if positive for k13, pfcrt, pfmdr, pfdhps and pfdhfr mutations related to anti-malarial drug resistance with Sanger sequencing and NGS. Due to lack of an alternative anti-malarial drug with the same level of efficacy and tolerability at present and in order to achieve successful disease control and eradication, it is fundamental to understand the prevalence and geographical distribution of drug resistance This requires (1) having up-to-date information on efficacy of these therapies in different areas, (2) establishing an early intervention system and (3) understanding more about the principles of spread of resistance in different areas [2, 3, 14]
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