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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

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.

Received July 14, 2004. Accepted for publication October 9, 2004.

Acknowledgments: We thank the staff of TPC Hospital, particularly Dr. H. Mwerinde for sample collection, and Jimmy Weng for excellent technical assistance in performing the PCR-ELISA and dot blotting. We also thank David Wallikers (University of Edinburgh, Edinburgh, United Kingdom) for providing P. falciparum isolates K1, 7G8, and DD2, and Trine Staalsoe and Kirsten Pihl (Centre for Medical Parasitology) for providing isolates 3D7 and FCR3.

Financial support: This study was supported by the Danish International Development Agency Research Council (RUF, grant no. 91106) and the Wellcome Trust (GR063516) and conducted as part of the Joint Malaria Programme, a collaboration between the National Institute for Medical Research, Kilimanjaro Christian Medical College, the London School of Hygiene and Tropical Medicine, and the Centre for Medical Parasitology, University of Copenhagen.

Authors’ addresses: Michael Alifrangis, Insaf F Khalil, Anita M. Rønn, and Ib C. Bygbjerg, Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark and Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark, E-mails: Alifrangis{at}cmp.dk, Insafk{at}immu.ku.dk, ARN{at}euro-alarm.dk, and I.Bygbjerg{at}pubhealth.ku.dk. Sonia Enosse, Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology and Institute of Public Health, University of Copenhagen, Copenhagen, Denmark and Instituto Nacional de Saúde, Ministério de Saúde, Maputo, Mozambique, E-mail: Senosse{at}hotmail.com. Richard Pearce and Cally Roper, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom, E-mails: Richard.Pearce{at}lshtm.ac.uk and Cally.Roper{at}lshtm.ac.uk. Chris Drakeley, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom and The Joint Malaria Programme, Moshi, Tanzania, E-mail: Chris.Drakely{at}lshtm.ac.uk. Watoky M. M. M. Nkya, Kilimanjaro Christian Medical Centre, Moshi, Tanzania and The Joint Malaria Programme, Moshi, Tanzania, E-mail: Nkya10{at}hotmail.com. Thor G. Theander, The Joint Malaria Programme, Moshi, Tanzania, E-mail: Theander{at}cmp.dk.

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