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system also makes it possible to identify the reemergence of drug-susceptible parasites once a drug has been withdrawn.

Optimization of a Ligase Detection Reaction-Fluorescent Microsphere Assay for Characterization of Resistance-Mediating Polymorphisms in African Samples of Plasmodium falciparum

“…Thus, a number of groups have attempted to develop higher-throughput methods for the analysis of P. falciparum genotypes. A microarray-based method proved robust, sensitive, and accurate, but this methodology requires access to microarray slides and a fluorescence scanner, which may not be available in many laboratories (21). Melting curve analysis (23) and quantitative PCR (25,26) offer other high-throughput systems for detection of P. falciparum SNPs for laboratories with appropriate infrastructure, and these systems are particularly suited to identify small minority populations in mixed samples.…”

“…Other relatively lowthroughput methodologies include direct DNA sequencing, mutation-specific PCR (17), dot blot probe hybridization (18), molecular beacons (19), and single-nucleotide primer extension (20). Systems examined to provide improved throughput have included polymorphism-specific microarrays (21), melting curve analysis (22,23), quantitative PCR (24)(25)(26), and a ligase detection reaction-fluorescent microsphere (LDR-FM) assay (27,28). Each of these assays has shown success, although each has challenges, including the cost of equipment and reagents and the uncertainty of success with field samples, which are typically stored on filter paper at room temperature and commonly consist of polyclonal infections.…”

Decreased In Vitro Susceptibility of Plasmodium falciparum Isolates to Artesunate, Mefloquine, Chloroquine, and Quinine in Cambodia from 2001 to 2007

“…Nine SNPs of four genes-pfdhfr codons N51I, C59R, S108(N/T), and I164L, pfcrt codon K76T, pfmdr1 codons N86Y, Y184F, and N1042D, and pfatpase6 codon S769N-were analyzed using a microarray-based assay as described previously (4). Cutoff values were determined by an algorithm.…”

Polymorphism of Pfatpase6 in Cte dIvoire: Detection of a four new point mutations

“…They described the analysis of four SNPs in isolates from Pará in nucleotide positions: 110, 1916, 2306 and 2694. The same previous study was checked for five PfATPase6 gene SNPs (538, 574, 623, 683 and 769) by DNA-microarrays (Crameri et al, 2007). By DNAmicroarrays, the PfATPaseA623E mutation was found in 4.7% of the Niger samples, but sequencing did not confirm this.…”