Optimization of a ligase detection reaction-fluorescent microsphere assay for characterization of resistance-mediating polymorphisms in African samples of Plasmodium falciparum

Abstract

Genetic polymorphisms in the malaria parasite Plasmodium falciparum mediate alterations in sensitivity to important antimalarial drugs. Surveillance for these polymorphisms is helpful in assessing the prevalence of drug resistance and designing strategies for malaria control. Multiple methods are available for the assessment of P. falciparum genetic polymorphisms, but they suffer from low throughput, technical limitations, and high cost. We have optimized and tested a multiplex ligase detection reaction-fluorescent microsphere (LDR-FM) assay for the identification of important P. falciparum genetic polymorphisms. For 84 clinical samples from Kampala, Uganda, a region where both transmission intensity and infection complexity are high, DNA was extracted from dried blood spots, genes of interest were amplified, amplicons were subjected to multiplex ligase detection reactions to add bead-specific oligonucleotides and biotin, fragments were hybridized to magnetic beads, and polymorphism prevalences were assessed fluorometrically in a multiplex format. A total of 19 alleles from the pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps genes were analyzed by LDR-FM and restriction fragment length polymorphism (RFLP) analyses. Considering samples with results from the two assays, concordance between the assays was good, with 78 to 100% of results identical at individual alleles, most nonconcordant results differing only between a mixed and pure genotype call, and full disagreement at individual alleles in only 0 to 3% of results. We estimate that the LDR-FM assay offers much higher throughput and lower cost than RFLP. Our results suggest that the LDR-FM system offers an accurate high-throughput means of classifying genetic polymorphisms in field samples of P. falciparum.

Fig 1

LDR-FM readings for 19 SNPs in P. falciparum reference strains. Values shown are uncorrected mean fluorescence readings for 3 to 5 assays, each run in triplicate. For pfcrt, the haplotype represents amino acids 72 to 76 in the gene product. Readings representing the known sequences at each allele are in bold type.

Fig 2

Mixing experiments. DNA from the indicated reference strains was mixed at the ratios shown, and the prevalences of the pfmdr1 86 and 184 alleles were assessed by LDR-FM. Mean fluorescence intensities, each based on triplicate readings, are shown. Error bars represent standard deviations from triplicate readings.

Fig 3

Representative LDR-FM results for 10 Ugandan samples. Results shown are for the same 10 clinical specimens for all studied alleles. Values shown are uncorrected mean fluorescence readings. For pfcrt, the haplotype represents amino acids 72 to 76 in the gene product. Readings representing the sequence call at each allele after correction by subtraction of background are in bold type.

Fig 4

Agreement between RFLP and LDR-FM assays. Results are shown for samples with readings from both assays at the indicated alleles. For the 84 samples studied, the RFLP assay was unsuccessful for 0 to 14 (mean, 6.2) samples, and the LDR-FM assay was unsuccessful for 0 to 11 (mean, 3.1) samples for the indicated alleles. Kappa statistics were 0.9 to 1 for all comparisons except for pfmdr1 1246 (0.72) and pfdhps 540 (0.55), with most discrepancies explained by differences in the percentages of mixed alleles. RFLP assays were not done for the other alleles studied by LDR-FM.