title: Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia.
abstract: BACKGROUND
  Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission.
  
  
  METHODS
  The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket.
  
  
  RESULTS
  Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates.
  
  
  CONCLUSIONS
  Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.
authors.affiliation: Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia.|Infectious Diseases Society Sabah-Menzies School of Health Research Clinical Research Unit, Kota Kinabalu, Sabah, Malaysia.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Grigg
authors.affiliation: Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia.|Infectious Diseases Society Sabah-Menzies School of Health Research Clinical Research Unit, Kota Kinabalu, Sabah, Malaysia.
authors.forename: Bridget E
authors.initials: BE
authors.lastname: Barber
authors.affiliation: Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia.
authors.forename: Jutta
authors.initials: J
authors.lastname: Marfurt
authors.affiliation: Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.|Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand.
authors.forename: Mallika
authors.initials: M
authors.lastname: Imwong
authors.affiliation: Infectious Diseases Society Sabah-Menzies School of Health Research Clinical Research Unit, Kota Kinabalu, Sabah, Malaysia.|Clinical Research Centre, Queen Elizabeth Hospital, Sabah, Malaysia.|Jesselton Medical Centre, Kota Kinabalu, Sabah, Malaysia.
authors.forename: Timothy
authors.initials: T
authors.lastname: William
authors.affiliation: Infectious Diseases Society Sabah-Menzies School of Health Research Clinical Research Unit, Kota Kinabalu, Sabah, Malaysia.
authors.forename: Elspeth
authors.initials: E
authors.lastname: Bird
authors.affiliation: Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Piera
authors.affiliation: Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia.
authors.forename: Ammar
authors.initials: A
authors.lastname: Aziz
authors.affiliation: Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
authors.forename: Usa
authors.initials: U
authors.lastname: Boonyuen
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Drakeley
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Cox
authors.affiliation: Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: White
authors.affiliation: Australian Army Malaria Institute, Brisbane, Australia.|Clinical Tropical Medicine, Queensland Institute of Medical Research, Brisbane, Australia.
authors.forename: Qin
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia.|Infectious Diseases Society Sabah-Menzies School of Health Research Clinical Research Unit, Kota Kinabalu, Sabah, Malaysia.|Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore.
authors.forename: Tsin W
authors.initials: TW
authors.lastname: Yeo
authors.affiliation: Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia.
authors.forename: Sarah
authors.initials: S
authors.lastname: Auburn
authors.affiliation: Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia.|Infectious Diseases Society Sabah-Menzies School of Health Research Clinical Research Unit, Kota Kinabalu, Sabah, Malaysia.|Royal Darwin Hospital, Darwin, Northern Territory, Australia.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Anstey
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000962:Antimalarials / Q000494:pharmacology / Q000627:therapeutic use; D002648:Child; D002675:Child, Preschool; D004351:Drug Resistance; D005260:Female; D005493:Folic Acid Antagonists / Q000494:pharmacology / Q000627:therapeutic use*; D006801:Humans; D007223:Infant; D008288:Malaria / Q000188:drug therapy* / Q000453:epidemiology / Q000469:parasitology* / Q000635:transmission; D008296:Malaysia / Q000453:epidemiology; D008297:Male; D008875:Middle Aged; D062105:Molecular Docking Simulation; D016790:Plasmodium knowlesi / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D011739:Pyrimethamine / Q000494:pharmacology / Q000627:therapeutic use; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D055815:Young Adult
chemical_list: D000962:Antimalarials; D005493:Folic Acid Antagonists; D013762:Tetrahydrofolate Dehydrogenase; D011739:Pyrimethamine