title: Detection of single-nucleotide polymorphisms in Plasmodium falciparum by PCR primer extension and lateral flow immunoassay.
abstract: The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries.
authors.affiliation: Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen University and Research Centre, Wageningen, The Netherlands.
authors.forename: A P H A
authors.initials: AP
authors.lastname: Moers
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: R L
authors.initials: RL
authors.lastname: Hallett
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Burrow
authors.affiliation: Koninklijk Instituut voor de Tropen/Royal Tropical Institute, Biomedical Research, Amsterdam, The Netherlands.
authors.forename: H D F H
authors.initials: HD
authors.lastname: Schallig
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom PHE Malaria Reference Laboratory, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: C J
authors.initials: CJ
authors.lastname: Sutherland
authors.affiliation: Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen University and Research Centre, Wageningen, The Netherlands aart.vanamerongen@wur.nl.
authors.forename: A
authors.identifier: http://orcid.org/0000-0002-2502-5744
authors.initials: A
authors.lastname: van Amerongen
mesh_terms: D000962:Antimalarials; D002738:Chloroquine / Q000627:therapeutic use; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D007118:Immunoassay / Q000379:methods*; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000469:parasitology; D026901:Membrane Transport Proteins / Q000235:genetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000235:genetics
chemical_list: D000962:Antimalarials; D002738:Chloroquine; D017931:DNA Primers; D016054:DNA, Protozoan; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; C513498:Mdr1 protein, Plasmodium falciparum; C416369:PfCRT protein, Plasmodium falciparum