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

Abstract

bGenetic 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.

Impact of Antimalarial Treatment and Chemoprevention on the Drug Sensitivity of Malaria Parasites Isolated from Ugandan Children

“…S2 in the supplemental material). Specifically, the pfdhfr 51I, 59R, and 108N, and pfdhps 437G and 540E mutations were all common, but other mutations associated with higher levels of drug resistance (pfdhfr 164L, and pfdhps 581G and 613T/S), which have been uncommon in earlier surveys (39,45,47), were also rare in these samples. Factors that likely contribute to the continued high prevalence of resistance-mediating antifolate mutations include use of antifolates to treat malaria (although this is no longer national policy), as intermittent preventive therapy against malaria, to treat bacterial infections, and as prophylaxis against opportunistic infections in those with HIV infection.…”

“…The formal assessments described here were performed with a newer technique, utilizing a ligase detection reaction-fluorescent microsphere assay. Assays were performed as previously described (39), with the modifications of nested-PCR for all reactions, and with a new primer to detect pfdhfr 164 polymorphisms (see Tables S1 and S2 in the supplemental material).…”

Intermittent Preventive Treatment with Dihydroartemisinin-Piperaquine in Ugandan Schoolchildren Selects for Plasmodium falciparum Transporter Polymorphisms That Modify Drug Sensitivity

“…Gene fragments spanning all loci of interest were amplified in nested reactions (26), and failed reactions were repeated. To detect polymorphisms, multiplex ligase detection reactionfluorescent microsphere assays were performed as previously described (26,41).…”

Lack of Artemisinin Resistance in Plasmodium falciparum in Uganda Based on Parasitological and Molecular Assays

“…K13-propeller-encoding domains (codons 440 to 726) were dideoxy sequenced, as previously de-scribed (4). To detect polymorphisms in pfcrt and pfmdr1, multiplex ligase detection reaction-fluorescent microsphere assays were performed, as previously described (19,20).…”