title: Island-wide diversity in single nucleotide polymorphisms of the Plasmodium vivax dihydrofolate reductase and dihydropteroate synthetase genes in Sri Lanka.
abstract: BACKGROUND
  Single nucleotide polymorphisms (SNPs) in the Plasmodium vivax dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pvdhps) genes cause parasite resistance to the antifolate drug combination, sulphadoxine/pyrimethamine (SP). Monitoring these SNPs provide insights into the level of drug pressure caused by SP use and presumably other antifolate drugs. In Sri Lanka, chloroquine (CQ) with primaquine (PQ) and SP with PQ is used as first and second line treatment, respectively, against uncomplicated Plasmodium falciparum and/or P. vivax infections. CQ/PQ is still efficacious against P. vivax infections, thus SP is rarely used and it is assumed that the prevalence of SNPs related to P. vivax SP resistance is low. However, this has not been assessed in Sri Lanka as in most other parts of Asia. This study describes the prevalence and distribution of SNPs related to P. vivax SP resistance across Sri Lanka.
  
  
  METHODS
  P. vivax-positive samples were collected from subjects presenting at government health facilities across nine of the major malaria endemic districts on the island. The samples were analysed for SNPs/haplotypes at codon 57, 58, 61 and 117 of the Pvdhfr gene and 383, 553 and 585 of the Pvdhps gene by applying PCR followed by a hybridization step using sequence specific oligonucleotide probes (SSOPs) in an ELISA format.
  
  
  RESULTS
  In the study period, the government of Sri Lanka recorded 2,149 P. vivax cases from the nine districts out of which, 454 (21.1%) blood samples were obtained. Pvdhfr haplotypes could be constructed for 373 of these. The FSTS wild-haplotype was represented in 257 samples (68.9%), the double mutant LRTS haplotype was the most frequently observed mutant (24.4%) while the triple mutation (LRTN) was only identified once. Except for two samples of the single mutated Pvdhps GAV haplotype, the remaining samples were wildtype. Geographical differences were apparent, notably a significantly higher frequency of mutant Pvdhfr haplotypes was observed in the Northern districts.
  
  
  CONCLUSIONS
  Since SP is rarely used in Sri Lanka, the high frequency and diversity of Pvdhfr mutations was unexpected indicating the emergence of drug resistant parasites despite a low level of SP drug pressure.
authors.affiliation: Centre for Medical Parasitology, Institute for International Health, Immunology and Microbiology, Øster Farimagsgade 5, 1014 Copenhagen K, Denmark. Mette.Leth@cmp.dk <Mette.Leth@cmp.dk>
authors.forename: Mette L
authors.initials: ML
authors.lastname: Schousboe
authors.forename: Rupika S
authors.initials: RS
authors.lastname: Rajakaruna
authors.forename: Ali
authors.initials: A
authors.lastname: Salanti
authors.forename: Hapuarachchige C
authors.initials: HC
authors.lastname: Hapuarachchi
authors.forename: Gawrie N L
authors.initials: GN
authors.lastname: Galappaththy
authors.forename: Ib C
authors.initials: IC
authors.lastname: Bygbjerg
authors.forename: Priyanie H
authors.initials: PH
authors.lastname: Amerasinghe
authors.forename: Flemming
authors.initials: F
authors.lastname: Konradsen
authors.forename: Michael
authors.initials: M
authors.lastname: Alifrangis
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology / Q000627:therapeutic use; D004094:Dihydropteroate Synthase / Q000235:genetics*; D004338:Drug Combinations; D004351:Drug Resistance / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D006239:Haplotypes; D006801:Humans; D016780:Malaria, Vivax / Q000453:epidemiology / Q000469:parasitology; D010966:Plasmodium vivax / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011739:Pyrimethamine / Q000494:pharmacology / Q000627:therapeutic use; D013188:Sri Lanka / Q000453:epidemiology; D013413:Sulfadoxine / Q000494:pharmacology / Q000627:therapeutic use; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
chemical_list: D000962:Antimalarials; D004338:Drug Combinations; C001205:fanasil, pyrimethamine drug combination; D013413:Sulfadoxine; D013762:Tetrahydrofolate Dehydrogenase; D004094:Dihydropteroate Synthase; D011739:Pyrimethamine