title: K13 mutations and pfmdr1 copy number variation in Plasmodium falciparum malaria in Myanmar.
abstract: BACKGROUND
  Artemisinin-based combination therapy has been first-line treatment for falciparum malaria in Myanmar since 2005. The wide extent of artemisinin resistance in the Greater Mekong sub-region and the presence of mefloquine resistance at the Myanmar-Thailand border raise concerns over resistance patterns in Myanmar. The availability of molecular markers for resistance to both drugs enables assessment even in remote malaria-endemic areas.
  
  
  METHODS
  A total of 250 dried blood spot samples collected from patients with Plasmodium falciparum malarial infection in five malaria-endemic areas across Myanmar were analysed for kelch 13 sequence (k13) and pfmdr1 copy number variation. K13 mutations in the region corresponding to amino acids 210-726 (including the propeller region of the protein) were detected by nested PCR amplification and sequencing, and pfmdr1 copy number variation by real-time PCR. In two sites, a sub-set of patients were prospectively followed up for assessment of day-3 parasite clearance rates after a standard course of artemether-lumefantrine.
  
  
  RESULTS
  K13 mutations and pfmdr1 amplification were successfully analysed in 206 and 218 samples, respectively. Sixty-nine isolates (33.5 %) had mutations within the k13 propeller region with 53 of these (76.8 %) having mutations already known to be associated with artemisinin resistance. F446I (32 isolates) and P574L (15 isolates) were the most common examples. K13 mutation was less common in sites in western border regions (29 of 155 isolates) compared to samples from the east and north (40 of 51 isolates; p < 0.0001). The overall proportion of parasites with multiple pfmdr1 copies (greater than 1.5) was 5.5 %. Seven samples showed both k13 mutation and multiple copies of pfmdr1. Only one of 36 patients followed up after artemether-lumefantrine treatment still had parasites at day 3; molecular analysis indicated wild-type k13 and single copy pfmdr1.
  
  
  CONCLUSIONS
  The proportion of P. falciparum isolates with mutations in the propeller region of k13 indicates that artemisinin resistance extends across much of Myanmar. There is a low prevalence of parasites with multiple pfmdr1 copies across the country. The efficacy of artemisinin-based combination therapy containing mefloquine and lumefantrine is, therefore, expected to be high, although regular monitoring of efficacy will be important.
authors.affiliation: Department of Medicine, Institute of Medicine 1, Yangon, Myanmar. jujuchan05@gmail.com.
authors.forename: Aye A
authors.initials: AA
authors.lastname: Win
authors.affiliation: Department of Molecular Tropical Medicine and Genetics, Mahidol University, Bangkok, Thailand. noi@tropmedres.ac.|Mahidol Oxford Tropical Medicine Research Unit, Bangkok, Thailand. noi@tropmedres.ac.
authors.forename: Mallika
authors.initials: M
authors.lastname: Imwong
authors.affiliation: Department of Medical Research (Lower Myanmar), Yangon, Myanmar. Kyaw606@gmail.com.
authors.forename: Myat P
authors.initials: MP
authors.lastname: Kyaw
authors.affiliation: Mahidol Oxford Tropical Medicine Research Unit, Bangkok, Thailand. Charlie@tropmedres.ac.|Nuffield Department of Clinical Medicine, Centre for Tropical Medicine & Global Health, University of Oxford, Oxford, UK. Charlie@tropmedres.ac.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Woodrow
authors.affiliation: Mahidol Oxford Tropical Medicine Research Unit, Bangkok, Thailand. nok@tropmedres.ac.|Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. nok@tropmedres.ac.
authors.forename: Kesinee
authors.initials: K
authors.lastname: Chotivanich
authors.affiliation: Mahidol Oxford Tropical Medicine Research Unit, Bangkok, Thailand. Borimas@tropmedres.ac.|Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. Borimas@tropmedres.ac.
authors.forename: Borimas
authors.initials: B
authors.lastname: Hanboonkunupakarn
authors.affiliation: Mahidol Oxford Tropical Medicine Research Unit, Bangkok, Thailand. yon@tropmedres.ac.|Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. yon@tropmedres.ac.
authors.forename: Sasithon
authors.initials: S
authors.lastname: Pukrittayakamee
mesh_terms: D000962:Antimalarials / Q000627:therapeutic use; D000077611:Artemether, Lumefantrine Drug Combination; D037621:Artemisinins / Q000627:therapeutic use; D056915:DNA Copy Number Variations / Q000235:genetics; D016054:DNA, Protozoan / Q000097:blood / Q000235:genetics; D004338:Drug Combinations; D004983:Ethanolamines / Q000627:therapeutic use; D005449:Fluorenes / Q000627:therapeutic use; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000453:epidemiology* / Q000469:parasitology*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D002052:Myanmar / Q000453:epidemiology; D010963:Plasmodium falciparum / Q000235:genetics*; D011446:Prospective Studies
chemical_list: D000962:Antimalarials; D000077611:Artemether, Lumefantrine Drug Combination; D037621:Artemisinins; D016054:DNA, Protozoan; D004338:Drug Combinations; D004983:Ethanolamines; D005449:Fluorenes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C513498:Mdr1 protein, Plasmodium falciparum; C031327:artemisinin