A Multiplex Ligase Detection Reaction-Fluorescent Microsphere Assay for Simultaneous Detection of Single Nucleotide Polymorphisms Associated with Plasmodium falciparum Drug Resistance

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Abstract
Incomplete malaria control efforts have resulted in a worldwide increase in resistance to drugs used to treat the disease. A complex array of mutations underlying antimalarial drug resistance complicates efficient monitoring of parasite populations and limits the success of malaria control efforts in regions of endemicity. To improve the surveillance of Plasmodium falciparum drug resistance, we developed a multiplex ligase detection reaction-fluorescent-microsphere-based assay (LDR-FMA) that identifies single nucleotide polymorphisms (SNPs) in the P. falciparum dhfr (9 alleles), dhps (10 alleles), and pfcrt (3 alleles) genes associated with resistance to Fansidar and chloroquine. We evaluated 1,121 blood samples from study participants in the Wosera region of Papua New Guinea, where malaria is endemic. Results showed that 468 samples were P. falciparum negative and 453 samples were P. falciparum positive by a Plasmodium species assay and all three gene assays (concordance, 82.2%). For P. falciparum infections where the assay for each gene was positive, 2 samples carried resistance alleles for all three genes, 299 carried resistance alleles for dhfr and pfcrt , 131 carried resistance alleles for only one gene ( dhfr [ n = 40], dhps [ n = 1], or pfcrt [ n = 90]), and 21 carried only sensitive alleles at all three genes. Mixed-strain infections characterized 100 samples. Overall, 95.4% (432/453) of P. falciparum -infected samples carried at least one allele associated with resistance to Fansidar or chloroquine. In view of the fact that 86.3% (391/453) of P. falciparum -infected samples carried pfcrt mutations, chloroquine is largely ineffective against P. falciparum in Papua New Guinea. Surveillance of additional dhfr and dhps polymorphisms in order to monitor the continued effectiveness of Fansidar is recommended.

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