A SIMPLE, HIGH-THROUGHPUT METHOD TO DETECT PLASMODIUM FALCIPARUM SINGLE NUCLEOTIDE POLYMORPHISMS IN THE DIHYDROFOLATE REDUCTASE, DIHYDROPTEROATE SYNTHASE, AND P. FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER GENES USING POLYMERASE CHAIN REACTION– AND ENZYME-LINKED IMMUNOSORBENT ASSAY–BASED TECHNOLOGY

MICHAEL ALIFRANGIS Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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SONIA ENOSSE Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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RICHARD PEARCE Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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CHRIS DRAKELEY Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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CALLY ROPER Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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INSAF F. KHALIL Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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WATOKY MMM NKYA Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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ANITA M. RØNN Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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THOR G. THEANDER Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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IB C. BYGBJERG Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; Kilimanjaro Christian Medical Centre, Moshi, Tanzania; The Joint Malaria Programme, Moshi, Tanzania

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Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine. However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)– and enzyme-linked immunosorbent assay (ELISA)–based technology. Biotinylated PCR products of dhfr, dhps, or Pfcrt were captured on streptavidin-coated microtiter plates and sequence-specific oligonucleotide probes (SSOPs) were hybridized with the PCR products. A stringent washing procedure enabled detection of remaining bound SSOPs and distinguished between the SNPs of dhfr, dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR–restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P. falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt SNPs on 88 samples can be performed in a single day and provides quick and reproducible results. The system can potentially be modified to detect SNPs in other genes.

Author Notes

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