title: Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies.
abstract: BACKGROUND
  Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin monotherapy or artemisinin-based combination therapy. Absence of in-vitro and ex-vivo correlates of artemisinin resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate the stage-dependent susceptibility of parasites to dihydroartemisinin in the in-vitro RSA, and to assess whether an ex-vivo RSA can identify artemisinin-resistant P falciparum infections.
  
  
  METHODS
  We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study done in Pursat, Cambodia, in 2010 (registered with ClinicalTrials.gov, number NCT00341003) and used novel in-vitro survival assays to explore the stage-dependent susceptibility of slow-clearing and fast-clearing parasites to dihydroartemisinin. In 2012, we implemented the RSA in prospective parasite clearance studies in Pursat, Preah Vihear, and Ratanakiri, Cambodia (NCT01736319), to measure the ex-vivo responses of parasites from patients with malaria. Continuous variables were compared with the Mann-Whitney U test. Correlations were analysed with the Spearman correlation test.
  
  
  RESULTS
  In-vitro survival rates of culture-adapted parasites from 13 slow-clearing and 13 fast-clearing infections differed significantly when assays were done on 0-3 h ring-stage parasites (10·88% vs 0·23%; p=0·007). Ex-vivo survival rates significantly correlated with in-vivo parasite clearance half-lives (n=30, r=0·74, 95% CI 0·50-0·87; p<0·0001).
  
  
  CONCLUSIONS
  The in-vitro RSA of 0-3 h ring-stage parasites provides a platform for the molecular characterisation of artemisinin resistance. The ex-vivo RSA can be easily implemented where surveillance for artemisinin resistance is needed.
  
  
  BACKGROUND
  Institut Pasteur du Cambodge and the Intramural Research Program, NIAID, NIH.
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodia.
authors.forename: Benoit
authors.initials: B
authors.lastname: Witkowski
authors.forename: Chanaki
authors.initials: C
authors.lastname: Amaratunga
authors.forename: Nimol
authors.initials: N
authors.lastname: Khim
authors.forename: Sokunthea
authors.initials: S
authors.lastname: Sreng
authors.forename: Pheaktra
authors.initials: P
authors.lastname: Chim
authors.forename: Saorin
authors.initials: S
authors.lastname: Kim
authors.forename: Pharath
authors.initials: P
authors.lastname: Lim
authors.forename: Sivanna
authors.initials: S
authors.lastname: Mao
authors.forename: Chantha
authors.initials: C
authors.lastname: Sopha
authors.forename: Baramey
authors.initials: B
authors.lastname: Sam
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Anderson
authors.forename: Socheat
authors.initials: S
authors.lastname: Duong
authors.forename: Char Meng
authors.initials: CM
authors.lastname: Chuor
authors.forename: Walter R J
authors.initials: WR
authors.lastname: Taylor
authors.forename: Seila
authors.initials: S
authors.lastname: Suon
authors.forename: Odile
authors.initials: O
authors.lastname: Mercereau-Puijalon
authors.forename: Rick M
authors.initials: RM
authors.lastname: Fairhurst
authors.forename: Didier
authors.initials: D
authors.lastname: Menard
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D037621:Artemisinins / Q000494:pharmacology*; D002160:Cambodia / Q000453:epidemiology; D004351:Drug Resistance; D005838:Genotype; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000453:epidemiology / Q000469:parasitology*; D010641:Phenotype; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000302:isolation & purification; D011446:Prospective Studies; D016019:Survival Analysis; D015996:Survival Rate
chemical_list: D000962:Antimalarials; D037621:Artemisinins; C039060:artenimol
references.citation: Antimicrob Agents Chemother. 2010 May;54(5):1872-7
references.pmid: 20160056
references.citation: Proc Natl Acad Sci U S A. 2011 Jan 4;108(1):397-402
references.pmid: 21173254
references.citation: Science. 2010 May 14;328(5980):844-6
references.pmid: 20466917
references.citation: Malar J. 2011 Sep 22;10:278
references.pmid: 21939506
references.citation: Lancet Infect Dis. 2013 Feb;13(2):113-4
references.pmid: 23347631
references.citation: Proc Natl Acad Sci U S A. 2013 Jan 2;110(1):240-5
references.pmid: 23248304
references.citation: Antimicrob Agents Chemother. 2012 Jan;56(1):428-31
references.pmid: 21986828
references.citation: Mol Microbiol. 2012 Oct;86(1):111-28
references.pmid: 22812578
references.citation: Antimicrob Agents Chemother. 2013 Feb;57(2):914-23
references.pmid: 23208708
references.citation: N Engl J Med. 2011 Sep 22;365(12):1073-5
references.pmid: 21992120
references.citation: Nat Genet. 2013 Jun;45(6):648-55
references.pmid: 23624527
references.citation: J Infect Dis. 2013 Jun 1;207(11):1655-63
references.pmid: 23448727
references.citation: Antimicrob Agents Chemother. 1979 Dec;16(6):710-8
references.pmid: 394674
references.citation: Malar J. 2012 Oct 26;11:355
references.pmid: 23101492
references.citation: Science. 2012 Apr 6;336(6077):79-82
references.pmid: 22491853
references.citation: Lancet Infect Dis. 2012 Nov;12(11):851-8
references.pmid: 22940027
references.citation: Trans R Soc Trop Med Hyg. 1993 Jul-Aug;87(4):436-43
references.pmid: 8249075
references.citation: BMC Genomics. 2011 Aug 03;12:391
references.pmid: 21810278
references.citation: N Engl J Med. 2008 Dec 11;359(24):2619-20
references.pmid: 19064625
references.citation: Lancet. 2012 May 26;379(9830):1960-6
references.pmid: 22484134
references.citation: PLoS One. 2013;8(3):e57689
references.pmid: 23520478
references.citation: Comput Methods Programs Biomed. 2011 Oct;104(1):10-8
references.pmid: 20828858
references.citation: N Engl J Med. 2009 Jul 30;361(5):455-67
references.pmid: 19641202
references.citation: Malar J. 2011 Nov 10;10:339
references.pmid: 22074219
references.citation: Lancet Infect Dis. 2002 Apr;2(4):209-18
references.pmid: 11937421
references.citation: Malar J. 2008 Oct 29;7:223
references.pmid: 18959790
references.citation: Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5157-62
references.pmid: 23431146
references.citation: Antimicrob Agents Chemother. 2006 Oct;50(10):3343-9
references.pmid: 17005815