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J. Clin. Microbiol. doi:10.1128/JCM.01683-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

The Center for Global Health and Diseases, Case Western Reserve University, Wolstein Research Building, Room 4-125, 2103 Cornell Road, Cleveland, OH 44106-7286; PNG Institute of Medical Research, PO Box 60, Goroka, EHP 441

^* To whom correspondence should be addressed. Email: paz{at}case.edu.

	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 anti-malarial drug resistance complicates efficient monitoring of parasite populations and limits the success of malaria control efforts in endemic regions. To improve surveillance of Plasmodium falciparum (Pf) drug resistance we developed a multiplex ligase detection reaction/fluorescent microsphere based assay (LDR-FMA) that identifies single nucleotide polymorphisms (SNPs) in the Pf dhfr (9 alleles), dhps (10 alleles), and pfcrt (3 alleles) genes associated with resistance to Fansidar and chloroquine. We evaluated 1121 blood samples from study participants in the malaria-endemic Wosera region of Papua New Guinea (PNG). Results showed that 468 samples were Pf-negative, and 453 samples were Pf-positive by a Plasmodium species and all three gene assays (concordance = 82.2%). In Pf infections where assays for each gene was positive, two samples carried resistance alleles for all three genes; 301 infections carried resistance alleles for dhfr and pfcrt; 131 infections carried resistance alleles for only one gene (dhfr [n=40], dhps [n=1], and pfcrt [n=90]); 21 infections carried only sensitive alleles at all three genes. Mixed strain infections characterized 100 samples. Overall 95.4% (432/453) of Pf infections carried at least one allele associated with resistance to Fansidar or chloroquine. As 82% (371/453) of Pf infections carried pfcrt mutations, chloroquine is largely ineffective against Pf in PNG. Surveillance of additional dhfr and dhps polymorphism is recommended to monitor continued effectiveness of Fansidar.