Validation of the ligase detection reaction fluorescent microsphere assay for the detection of Plasmodium falciparum resistance mediating polymorphisms in Uganda

Sheila Nankoberanyi,Stephen Tukwasibwe,Samuel L Nsobya,Jordan Tappero,Moses R Kamya,Patrick Tumwebaze,Norbert P Leclair,George W Mbogo,Philip J Rosenthal,Melissa D Conrad

doi:10.1186/1475-2875-13-95

Abstract

BackgroundMalaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. Several techniques have been studied to assess resistance markers. The most widely used methodology is restriction fragment length polymorphism (RFLP) analysis. The ligase detection reaction fluorescent microsphere (LDR-FM) assay was recently shown to provide high throughput assessment of P. falciparum SNPs associated with drug resistance. The aim of this study was to validate the reliability and accuracy of the LDR-FM assay in a field setting.MethodsFor 223 samples from a clinical trial in Tororo, Uganda in which P. falciparum was identified by blood smear, DNA was extracted from dried blood spots, genes of interest were amplified by PCR, amplicons were analysed by both RFLP and LDR-FM assays, and results were compared.ResultsSNP prevalence (wild type/mixed/mutant) with RFLP analysis was 8/5/87% for pfcrt K76T, 34/37/29% for pfmdr1 N86Y, 64/17/19% for pfmdr1 Y184F, and 42/21/37% for pfmdr1 D1246Y. These prevalences with the LDR-FM assay were 7/5/88%, 31/24/45%, 62/20/18%, and 48/19/33% for the four SNPs, respectively. Combining mixed and mutant outcomes for analysis, agreement between the assays was 97% (K = 0.77) for pfcrt K76T, 79% (K = 0.55) for pfmdr1 N86Y, 83% (K = 0.65) for pfmdr1 Y184F, and 91% (K = 0.82) for pfmdr1 D1246Y, with most disagreements due to discrepant readings of mixed genotypes.ConclusionThe LDR-FM assay provides a high throughput, relatively inexpensive and accurate assay for the surveillance of P. falciparum SNPs associated with drug resistance in resource-limited countries.

Highlights

Malaria remains a major public health problem, and its control has been hampered by drug resistance
Analysis of plasmodium falciparum reference strains The goal of this study was to validate the ligase detection reaction fluorescent microsphere (LDR-FM) assay, using samples from a recent clinical trial, with assays performed at a molecular laboratory in Kampala, Uganda
Assays were unsuccessful for six restriction fragment length polymorphism (RFLP) and zero LDR-FM assays for pfcrt, seven RFLP and seven LDR-FM assays at pfmdr1 86, seven RFLP and seven LDR-FM assays at pfmdr1 184, and three RFLP and one LDR-FM assays at pfmdr1 1246

Summary

Introduction

Malaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. The K76T mutation in the pfcrt gene, which encodes a putative drug transporter, is the principal mediator of resistance [2] Polymorphisms in another gene, pfmdr, which encodes a protein homologous to transporters that mediate drug resistance in other organisms, modulate levels of sensitivity to multiple drugs [3]. Given the limited arsenal of effective ACT and early signs of artemisinin resistance in Southeast Asia [9,10], there is a need for efficient surveillance of Ugandan parasites for genetic polymorphisms that may mediate resistance to the most important anti-malarial drugs

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