title: Temporal changes in Plasmodium falciparum anti-malarial drug sensitivity in vitro and resistance-associated genetic mutations in isolates from Papua New Guinea.
abstract: BACKGROUND
  In northern Papua New Guinea (PNG), most Plasmodium falciparum isolates proved resistant to chloroquine (CQ) in vitro between 2005 and 2007, and there was near-fixation of pfcrt K76T, pfdhfr C59R/S108N and pfmdr1 N86Y. To determine whether the subsequent introduction of artemisinin combination therapy (ACT) and reduced CQ-sulphadoxine-pyrimethamine pressure had attenuated parasite drug susceptibility and resistance-associated mutations, these parameters were re-assessed between 2011 and 2013.
  
  
  METHODS
  A validated fluorescence-based assay was used to assess growth inhibition of 52 P. falciparum isolates from children in a clinical trial in Madang Province. Responses to CQ, lumefantrine, piperaquine, naphthoquine, pyronaridine, artesunate, dihydroartemisinin, artemether were assessed. Molecular resistance markers were detected using a multiplex PCR ligase detection reaction fluorescent microsphere assay.
  
  
  RESULTS
  CQ resistance (in vitro concentration required for 50% parasite growth inhibition (IC₅₀) >100 nM) was present in 19% of isolates. All piperaquine and naphthoquine IC₅₀s were <100 nM and those for lumefantrine, pyronaridine and the artemisinin derivatives were in low nM ranges. Factor analysis of IC₅₀s showed three groupings (lumefantrine; CQ, piperaquine, naphthoquine; pyronaridine, dihydroartemisinin, artemether, artesunate). Most isolates (96%) were monoclonal pfcrt K76T (SVMNT) mutants and most (86%) contained pfmdr1 N86Y (YYSND). No wild-type pfdhfr was found but most isolates contained wild-type (SAKAA) pfdhps. Compared with 2005-2007, the geometric mean (95% CI) CQ IC₅₀ was lower (87 (71-107) vs 167 (141-197) nM) and there had been no change in the prevalence of pfcrt K76T or pfmdr1 mutations. There were fewer isolates of the pfdhps (SAKAA) wild-type (60 vs 100%) and pfdhfr mutations persisted.
  
  
  CONCLUSIONS
  Reflecting less drug pressure, in vitro CQ sensitivity appears to be improving in Madang Province despite continued near-fixation of pfcrt K76T and pfmdr1 mutations. Temporal changes in IC₅₀s for other anti-malarial drugs were inconsistent but susceptibility was preserved. Retention or increases in pfdhfr and pfdhps mutations reflect continued use of sulphadoxine-pyrimethamine in the study area including through paediatric intermittent preventive treatment. The susceptibility of local isolates to lumefantrine may be unrelated to those of other ACT partner drugs.
  
  
  BACKGROUND
  Australian New Zealand Clinical Trials Registry ACTRN12610000913077 .
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. tamarah.koleala@gmail.com.
authors.forename: Tamarah
authors.initials: T
authors.lastname: Koleala
authors.affiliation: School of Medicine and Pharmacology, University of Western Australia, Fremantle Hospital, PO Box 480, Fremantle, 6959, WA, Australia. stephanunkarl@googlemail.com.|Infection and Immunity Division, Walter and Eliza Hall Institute, Parkville, VIC, Australia. stephanunkarl@googlemail.com.|Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia. stephanunkarl@googlemail.com.
authors.forename: Stephan
authors.initials: S
authors.lastname: Karl
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. drmlaman@yahoo.com.|School of Medicine and Pharmacology, University of Western Australia, Fremantle Hospital, PO Box 480, Fremantle, 6959, WA, Australia. drmlaman@yahoo.com.
authors.forename: Moses
authors.initials: M
authors.lastname: Laman
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. brioni.moore@uwa.edu.au.|School of Medicine and Pharmacology, University of Western Australia, Fremantle Hospital, PO Box 480, Fremantle, 6959, WA, Australia. brioni.moore@uwa.edu.au.
authors.forename: Brioni R
authors.initials: BR
authors.lastname: Moore
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. jmbenjamin1606@gmail.com.
authors.forename: John
authors.initials: J
authors.lastname: Benjamin
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. barnadas@wehi.edu.au.|Infection and Immunity Division, Walter and Eliza Hall Institute, Parkville, VIC, Australia. barnadas@wehi.edu.au.|Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia. barnadas@wehi.edu.au.
authors.forename: Celine
authors.initials: C
authors.lastname: Barnadas
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. robinson@wehi.edu.au.|Infection and Immunity Division, Walter and Eliza Hall Institute, Parkville, VIC, Australia. robinson@wehi.edu.au.|Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia. robinson@wehi.edu.au.
authors.forename: Leanne J
authors.initials: LJ
authors.lastname: Robinson
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. kattenberg@wehi.edu.au.|Infection and Immunity Division, Walter and Eliza Hall Institute, Parkville, VIC, Australia. kattenberg@wehi.edu.au.
authors.forename: Johanna H
authors.initials: JH
authors.lastname: Kattenberg
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. sarah.javati@pngimr.org.pg.
authors.forename: Sarah
authors.initials: S
authors.lastname: Javati
authors.affiliation: School of Medicine and Pharmacology, University of Western Australia, Fremantle Hospital, PO Box 480, Fremantle, 6959, WA, Australia. rina.wong@uwa.edu.au.
authors.forename: Rina P M
authors.initials: RP
authors.lastname: Wong
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. anna.rosanas@gmail.com.|Institute of Tropical Medicine, Antwerp, Belgium. anna.rosanas@gmail.com.
authors.forename: Anna
authors.initials: A
authors.lastname: Rosanas-Urgell
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. ibetuela@gmail.com.
authors.forename: Inoni
authors.initials: I
authors.lastname: Betuela
authors.affiliation: Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea. Peter.Siba@pngimr.org.pg.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Siba
authors.affiliation: Infection and Immunity Division, Walter and Eliza Hall Institute, Parkville, VIC, Australia. ivomueller@fastmail.fm.|Center de Recerca en Salut Internacional de Barcelona (CRESIB), Barcelona, Spain. ivomueller@fastmail.fm.
authors.forename: Ivo
authors.initials: I
authors.lastname: Mueller
authors.affiliation: School of Medicine and Pharmacology, University of Western Australia, Fremantle Hospital, PO Box 480, Fremantle, 6959, WA, Australia. tim.davis@uwa.edu.au.
authors.forename: Timothy M E
authors.initials: TM
authors.lastname: Davis
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D002675:Child, Preschool; D004351:Drug Resistance / Q000235:genetics*; D006801:Humans; D007223:Infant; D016778:Malaria, Falciparum / Q000453:epidemiology / Q000469:parasitology*; D009154:Mutation / Q000235:genetics; D010219:Papua New Guinea / Q000453:epidemiology; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics*
chemical_list: D000962:Antimalarials