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Background  Reports of severe cases and increasing levels of drug resistance highlight the importance of improved  Plasmodium vivax  case management. Whereas monitoring  P. vivax  resistance to anti-malarial drug by  in vivo  and  in vitro  tests remain challenging, molecular markers of resistance represent a valuable tool for high-scale analysis and surveillance studies. A new high-throughput assay for detecting the most relevant markers related to  P. vivax  drug resistance was developed and assessed on Papua New Guinea (PNG) patient isolates.    Methods   Pvdhfr, pvdhps  and  pvmdr1  fragments were amplified by multiplex nested PCR. Then, PCR products were processed through an LDR-FMA (ligase detection reaction - fluorescent microsphere assay). 23 SNPs, including  pvdhfr  57-58-61 and 173,  pvdhps  382-383, 553, 647 and  pvmdr1  976, were simultaneously screened in 366 PNG  P. vivax  samples.    Results  Genotyping was successful in 95.4% of the samples for at least one gene. The coexistence of multiple distinct haplotypes in the parasite population necessitated the introduction of a computer-assisted approach to data analysis. Whereas 73.1% of patients were infected with at least one wild-type genotype at codons 57, 58 and 61 of  pvdhfr , a triple mutant genotype was detected in 65.6% of the patients, often associated with the 117T mutation. Only one patient carried the 173L mutation. The mutant 647P  pvdhps  genotype allele was approaching genetic fixation (99.3%), whereas 35.1% of patients were infected with parasites carrying the  pvmdr1  976F mutant allele.    Conclusions  The LDR-FMA described here allows a discriminant genotyping of resistance alleles in the  pvdhfr ,  pvdhps , and  pvmdr1  genes and can be used in large-scale surveillance studies.

A new high-throughput method for simultaneous detection of drug resistance associated mutations in Plasmodium vivax dhfr, dhps and mdr1 genes