title: A new high-throughput method for simultaneous detection of drug resistance associated mutations in Plasmodium vivax dhfr, dhps and mdr1 genes.
abstract: 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.
authors.affiliation: Vector Borne Diseases Unit, Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea.
authors.forename: Céline
authors.initials: C
authors.lastname: Barnadas
authors.forename: David
authors.initials: D
authors.lastname: Kent
authors.forename: Lincoln
authors.initials: L
authors.lastname: Timinao
authors.forename: Jonah
authors.initials: J
authors.lastname: Iga
authors.forename: Laurie R
authors.initials: LR
authors.lastname: Gray
authors.forename: Peter
authors.initials: P
authors.lastname: Siba
authors.forename: Ivo
authors.initials: I
authors.lastname: Mueller
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Thomas
authors.forename: Peter A
authors.initials: PA
authors.lastname: Zimmerman
mesh_terms: D002675:Child, Preschool; D016054:DNA, Protozoan / Q000235:genetics; D004094:Dihydropteroate Synthase / Q000235:genetics*; D004351:Drug Resistance; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007223:Infant; D016780:Malaria, Vivax / Q000469:parasitology; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D009154:Mutation; D010219:Papua New Guinea; D021261:Parasitic Sensitivity Tests / Q000379:methods; D010966:Plasmodium vivax / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
chemical_list: D016054:DNA, Protozoan; D004094:Dihydropteroate Synthase; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; D013762:Tetrahydrofolate Dehydrogenase; C541684:Mdr1 protein, Plasmodium vivax