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

Rebecca Smith-Aguasca, Himanshu Gupta, Estefania Uberegui, Mara Maquina, Francisco Saute, Krijn P. Paaijmans, Alfredo Mayor, Silvie Huijben

Research output: Contribution to journalArticle

Abstract

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 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. 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.

Original languageEnglish (US)
Article number351
JournalMalaria journal
Volume18
Issue number1
DOIs
StatePublished - Oct 17 2019
Externally publishedYes

Fingerprint

Antimalarials
Plasmodium falciparum
Culicidae
Gene Frequency
Coinfection
Drug Resistance
Mutation
Mozambique
Disease Eradication
High-Throughput Nucleotide Sequencing
Anopheles
Centers for Disease Control and Prevention (U.S.)
Abdomen
Thorax
Head
cyhalothrin
Light
Costs and Cost Analysis
DNA
Infection

Keywords

  • Anopheles funestus
  • Drug resistance
  • k13
  • Malaria
  • Mozambique
  • pfcrt
  • pfdhfr
  • pfdhps
  • pfmdr1
  • Sequencing

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

Cite this

Mosquitoes as a feasible sentinel group for anti-malarial resistance surveillance by Next Generation Sequencing of Plasmodium falciparum. / Smith-Aguasca, Rebecca; Gupta, Himanshu; Uberegui, Estefania; Maquina, Mara; Saute, Francisco; Paaijmans, Krijn P.; Mayor, Alfredo; Huijben, Silvie.

In: Malaria journal, Vol. 18, No. 1, 351, 17.10.2019.

Research output: Contribution to journalArticle

Smith-Aguasca, Rebecca ; Gupta, Himanshu ; Uberegui, Estefania ; Maquina, Mara ; Saute, Francisco ; Paaijmans, Krijn P. ; Mayor, Alfredo ; Huijben, Silvie. / Mosquitoes as a feasible sentinel group for anti-malarial resistance surveillance by Next Generation Sequencing of Plasmodium falciparum. In: Malaria journal. 2019 ; Vol. 18, No. 1.
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abstract = "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 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. 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.",
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AU - Smith-Aguasca, Rebecca

AU - Gupta, Himanshu

AU - Uberegui, Estefania

AU - Maquina, Mara

AU - Saute, Francisco

AU - Paaijmans, Krijn P.

AU - Mayor, Alfredo

AU - Huijben, Silvie

PY - 2019/10/17

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N2 - 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 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. 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.

AB - 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 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. 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.

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KW - pfmdr1

KW - Sequencing

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