title: Analysis of the dihydrofolate reductase-thymidylate synthase gene sequences in Plasmodium vivax field isolates that failed chloroquine treatment.
abstract: BACKGROUND
  To use pyrimethamine as an alternative anti-malarial drug for chloroquine-resistant malaria parasites, it was necessary to determine the enzyme's genetic variation in dihydrofolate reductase-thymidylate syntase (DHFR-TS) among Korean strains.
  
  
  METHODS
  Genetic variation of dhfr-ts genes of Plasmodium vivax clinical isolates from patients who did not respond to drug treatment (n = 11) in Korea were analysed. The genes were amplified using the polymerase chain reaction (PCR) with genomic DNA as a template.
  
  
  RESULTS
  Sequence analysis showed that the open reading frame (ORF) of 1,857 nucleotides encoded a deduced protein of 618 amino acids (aa). Alignment with the DHFR-TS genes of other malaria parasites showed that a 231-residue DHFR domain and a 286-residue TS domain were seperated by a 101-aa linker region. This ORF shows 98.7% homology with the P. vivax Sal I strain (XM001615032) in the DHFR domain, 100% in the linker region and 99% in the TS domain. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed that nine isolates belonged to the sensitive strain, whereas two isolates met the criteria for resistance. In these two isolates, the amino acid at position 117 is changed from serine to asparagine (S117N). Additionally, all Korean isolates showed a deletion mutant of THGGDN in short tandem repetitive sequences between 88 and 106 amino acid.
  
  
  CONCLUSIONS
  These results suggest that sequence variations in the DHFR-TS represent the prevalence of antifolate-resistant P. vivax in Korea. Two of 11 isolates have the Ser to Asn mutation in codon 117, which is the major determinant of pyrimethamine resistance in P. vivax. Therefore, the introduction of pyrimethamine for the treatment of chloroquine-resistant vivax malaria as alternative drug in Korea should be seriously considered.
authors.affiliation: Division of Malaria and Parasitic diseases, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul 122-701, Republic of Korea.
authors.forename: Won-Ja
authors.initials: WJ
authors.lastname: Lee
authors.forename: Hyung-Hwan
authors.initials: HH
authors.lastname: Kim
authors.forename: Yien-Kyoung
authors.initials: YK
authors.lastname: Choi
authors.forename: Kyung-Mi
authors.initials: KM
authors.lastname: Choi
authors.forename: Mi-A
authors.initials: MA
authors.lastname: Kim
authors.forename: Jung-Yeon
authors.initials: JY
authors.lastname: Kim
authors.forename: Jetsumon
authors.initials: J
authors.lastname: Sattabongkot
authors.forename: Youngjoo
authors.initials: Y
authors.lastname: Sohn
authors.forename: Hyuck
authors.initials: H
authors.lastname: Kim
authors.forename: Jong-Koo
authors.initials: JK
authors.lastname: Lee
authors.forename: Han-Sook
authors.initials: HS
authors.lastname: Park
authors.forename: Hyeong-Woo
authors.initials: HW
authors.lastname: Lee
mesh_terms: D000962:Antimalarials / Q000627:therapeutic use*; D002738:Chloroquine / Q000627:therapeutic use*; D004252:DNA Mutational Analysis; D016054:DNA, Protozoan / Q000235:genetics; D006801:Humans; D016780:Malaria, Vivax / Q000188:drug therapy* / Q000469:parasitology; D020125:Mutation, Missense; D010966:Plasmodium vivax / Q000187:drug effects* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics; D056910:Republic of Korea; D017422:Sequence Analysis, DNA; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*; D017211:Treatment Failure
chemical_list: D000962:Antimalarials; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D002738:Chloroquine; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase