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Background  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. 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.    Methods  For 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.    Results  SNP 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.    Conclusion  The 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.

malaria journal

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