title: Rapid microarray-based method for monitoring of all currently known single-nucleotide polymorphisms associated with parasite resistance to antimalaria drugs.
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monitoring system also makes it possible to identify the reemergence of drug-susceptible parasites once a drug has been withdrawn.
authors.affiliation: Swiss Tropical Institute, Socinstrasse 57, CH 4002, Basel, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Crameri
authors.forename: Jutta
authors.initials: J
authors.lastname: Marfurt
authors.forename: Kefas
authors.initials: K
authors.lastname: Mugittu
authors.forename: Nicolas
authors.initials: N
authors.lastname: Maire
authors.forename: Attila
authors.initials: A
authors.lastname: Regös
authors.forename: Jean Yves
authors.initials: JY
authors.lastname: Coppee
authors.forename: Odile
authors.initials: O
authors.lastname: Sismeiro
authors.forename: Richard
authors.initials: R
authors.lastname: Burki
authors.forename: Eric
authors.initials: E
authors.lastname: Huber
authors.forename: Daniel
authors.initials: D
authors.lastname: Laubscher
authors.forename: Odile
authors.initials: O
authors.lastname: Puijalon
authors.forename: Blaise
authors.initials: B
authors.lastname: Genton
authors.forename: Ingrid
authors.initials: I
authors.lastname: Felger
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Beck
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D003365:Costs and Cost Analysis; D005838:Genotype; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity