Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014-2017

Sidsel Nag*, Johan Ursing, Amabelia Rodrigues, Marina Crespo, Camilla Krogsgaard, Ole Lund, Frank Møller Aarestrup, Michael Alifrangis, PouL-Erik Kofoed

*Corresponding author for this work

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Abstract

Large-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging. Between 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13. The study found a high frequency of the pfmdr1 codon 86N at 88-97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11-28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation. This study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.
Original languageEnglish
Article number252
JournalMalaria Journal
Volume18
Issue number1
Number of pages13
ISSN1475-2875
DOIs
Publication statusPublished - 2019

Keywords

  • Amplicon sequencing
  • Guinea-Bissau
  • Molecular markers of antimalarial resistance
  • Next-generation sequencing
  • Plasmodium falciparum
  • Rapid diagnostic tests
  • pfcrt
  • pfdhfr
  • pfdhps
  • pfk13
  • pfmdr1

Cite this

@article{ee77d27de483419b95d4bbb843cbe045,
title = "Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014-2017",
abstract = "Large-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging. Between 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13. The study found a high frequency of the pfmdr1 codon 86N at 88-97{\%}, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51{\%} between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66{\%}, interquartile range 11-28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87{\%} for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation. This study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.",
keywords = "Amplicon sequencing, Guinea-Bissau, Molecular markers of antimalarial resistance, Next-generation sequencing, Plasmodium falciparum, Rapid diagnostic tests, pfcrt, pfdhfr, pfdhps, pfk13, pfmdr1",
author = "Sidsel Nag and Johan Ursing and Amabelia Rodrigues and Marina Crespo and Camilla Krogsgaard and Ole Lund and Aarestrup, {Frank M{\o}ller} and Michael Alifrangis and PouL-Erik Kofoed",
year = "2019",
doi = "10.1186/s12936-019-2894-8",
language = "English",
volume = "18",
journal = "Malaria Journal",
issn = "1475-2875",
publisher = "BioMed Central",
number = "1",

}

Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014-2017. / Nag, Sidsel; Ursing, Johan; Rodrigues, Amabelia; Crespo, Marina; Krogsgaard, Camilla; Lund, Ole; Aarestrup, Frank Møller; Alifrangis, Michael; Kofoed, PouL-Erik.

In: Malaria Journal, Vol. 18, No. 1, 252, 2019.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014-2017

AU - Nag, Sidsel

AU - Ursing, Johan

AU - Rodrigues, Amabelia

AU - Crespo, Marina

AU - Krogsgaard, Camilla

AU - Lund, Ole

AU - Aarestrup, Frank Møller

AU - Alifrangis, Michael

AU - Kofoed, PouL-Erik

PY - 2019

Y1 - 2019

N2 - Large-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging. Between 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13. The study found a high frequency of the pfmdr1 codon 86N at 88-97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11-28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation. This study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.

AB - Large-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging. Between 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13. The study found a high frequency of the pfmdr1 codon 86N at 88-97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11-28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation. This study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.

KW - Amplicon sequencing

KW - Guinea-Bissau

KW - Molecular markers of antimalarial resistance

KW - Next-generation sequencing

KW - Plasmodium falciparum

KW - Rapid diagnostic tests

KW - pfcrt

KW - pfdhfr

KW - pfdhps

KW - pfk13

KW - pfmdr1

U2 - 10.1186/s12936-019-2894-8

DO - 10.1186/s12936-019-2894-8

M3 - Journal article

VL - 18

JO - Malaria Journal

JF - Malaria Journal

SN - 1475-2875

IS - 1

M1 - 252

ER -