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system also makes it possible to identify the reemergence of drug-susceptible parasites once a drug has been withdrawn.

FIG. 1.

Flow diagram of the analytical procedure, starting from blood samples collected in the field. DNA is prepared from blood samples and is amplified by nested PCR; subsequently all amplicons are combined, and nucleotides are eliminated by SAP. Primer extension is performed in two aliquots of 20 μl from each sample, and the mixtures are combined for hybridization on the microarray. After being washed, the array is air dried, scanned in a microarray scanner, and subsequently analyzed using GenePix Pro and dedicated analysis software.

FIG. 2.

Sensitivity curves for SNP analysis of parasite samples diluted in uninfected blood. All data represent the percentages of fluorescence of the undiluted sample containing the genomic equivalent of 100,000 parasites per reaction. The upper panel represents values obtained for SNPs within the dhfr and dhps loci. The lower panel represents the values for mdr and crt loci.

FIG. 3.

Detection of SNPs in mixed parasite infections. The upper panel depicts arbitrary fluorescence values obtained when strain K1 was mixed with various dilutions of the 3D7 strain. K1 and 3D7 differ at codons 59 and 108 in the dhfr gene. The lower panel shows arbitrary fluorescence values obtained when strain 3D7 was mixed with various dilutions of the K1 strain.