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Detection of single nucleotide polymorphisms in Plasmodium falciparum by PCR primer extension and lateral flow immunoassay.

Moers AP, Hallett RL, Burrow R, Schallig HD, Sutherland CJ, van Amerongen A

Resistance of Plasmodium falciparum to some antimalarial drugs is linked to single nucleotide polymorphisms (SNPs). Currently there are no methods for identification of resistant parasites that are sufficiently simple, cheap and fast to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184 and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1), and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, qPCR or direct amplicon sequence analysis, in an initial open label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificity values of 98-100% were found for all 4 PCR-PEXT genotyping assays. Sensitivity values ranged from 75% to 100%, when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterised by the reference methods, but failed to generate a result in the PCR-PEXT-NALFIA, particularly samples with microscopy negative sub-patent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although requiring a standard thermal cycler, the procedure is cheap and rapid, and thus a potentially valuable tool for the point-of-care detection in developing countries.


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November, 2014


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