Rapid Microarray-based Method for Monitoring of All Currently Known Single-nucleotide Polymorphisms Associated with Parasite Resistance to Antimalaria Drugs

Overview

Journal J Clin Microbiol

Specialty Microbiology

Date 2007 Sep 7

PMID 17804664

Citations 27

Authors

Andreas Crameri

Jutta Marfurt

Kefas Mugittu

Nicolas Maire

Attila Regos

Jean Yves Coppee

Odile Sismeiro

Richard Burki

Eric Huber

Daniel Laubscher

Odile Puijalon

Blaise Genton

Ingrid Felger

Hans-Peter Beck

Affiliations

Soon will be listed here.

Abstract

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monitoring system also makes it possible to identify the reemergence of drug-susceptible parasites once a drug has been withdrawn.

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