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Journal of Clinical Microbiology, November 2007, p. 3685-3691, Vol. 45, No. 11
0095-1137/07/$08.00+0     doi:10.1128/JCM.01178-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid Microarray-Based Method for Monitoring of All Currently Known Single-Nucleotide Polymorphisms Associated with Parasite Resistance to Antimalaria Drugs ^,

Andreas Crameri,^1,, Jutta Marfurt,^1, Kefas Mugittu,² Nicolas Maire,¹ Attila Regös,^1,¶ Jean Yves Coppee,³ Odile Sismeiro,³ Richard Burki,^1,|| Eric Huber,^1, Daniel Laubscher,^1, Odile Puijalon,⁴ Blaise Genton,^1, Ingrid Felger,¹ and Hans-Peter Beck^1*

Swiss Tropical Institute, Socinstrasse 57 Basel, Switzerland,¹ Ifakara Health Research and Development Centre, P.O. Box 53, Ifakara, Tanzania,² Plate-forme puces a ADN, Genopole/Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France,³ Unité d'Immunologie Moléculaire des Parasites, CNRS URA 2581, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France⁴

Received 12 June 2007/ Returned for modification 8 August 2007/ Accepted 23 August 2007

Parasite drug resistance is partly conferred by single-nucleotide polymorphisms (SNPs), and monitoring them has been proposed as an alternative to monitoring drug resistance. Therefore, techniques are required to facilitate analyses of multiple SNPs on an epidemiological scale. We report a rapid and affordable microarray technique for application in epidemiological studies of malaria drug resistance. We have designed a multiwell microarray that is used in conjunction with PCR-amplified target genes implicated in the drug resistance of malaria with subsequent one-tube minisequencing using two fluorochromes. The drug-resistance-associated genes pfdhfr, pfdhps, pfcrt, pfmdr1, and pfATPase were amplified and analyzed for cultured Plasmodium falciparum strains and from field samples. We obtained a specificity of 94%, and comparison of field sample results to those of restriction fragment length polymorphism (RFLP) typing resulted in an overall consistency of >90%, except for pfdhfr51, for which most discrepancies were due to false determinations by RFLP of mixed infections. The system is sufficiently sensitive to assay parasites in clinical malaria cases and in most asymptomatic cases, and it allows high throughput with minimal hands-on time. The cost for the assay has been calculated as 0.27 euros/SNP (US$0.33), which is below the cost incurred with other systems. Due to the simplicity of the approach, newly identified SNPs can be incorporated rapidly. Such a monitoring system also makes it possible to identify the reemergence of drug-susceptible parasites once a drug has been withdrawn.

Published ahead of print on 5 September 2007.

Supplemental material for this article may be found at http://jcm.asm.org/.

These authors contributed equally to the study.

Present address: Amunix, 500 Ellis Street, Mountain View, CA 94043.

^¶ Present address: Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria 3010, Australia.

^|| Present address: Centre for Fish and Wildlife Health, University of Bern, Langgassstrasse 122, CH-3001 Bern, Switzerland.

Present address: F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland.

Present address: Ifakara Health Research & Development Centre, P.O. Box 78373, Dar es Salaam, Tanzania.