title: Plasmodium falciparum dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations.
abstract: BACKGROUND
  The declining efficacy of dihydroartemisinin-piperaquine against Plasmodium falciparum in Cambodia, along with increasing numbers of recrudescent cases, suggests resistance to both artemisinin and piperaquine. Available in vitro piperaquine susceptibility assays do not correlate with treatment outcome. A novel assay using a pharmacologically relevant piperaquine dose/time exposure was designed and its relevance explored in retrospective and prospective studies.
  
  
  METHODS
  The piperaquine survival assay (PSA) exposed parasites to 200 nM piperaquine for 48 hours and monitored survival 24 hours later. The retrospective study tested 32 culture-adapted, C580Y-K13 mutant parasites collected at enrolment from patients treated with a 3-day course of dihydroartemisinin-piperaquine and having presented or not with a recrudescence at day 42 (registered ACTRN12615000793516). The prospective study assessed ex vivo PSA survival rate alongside K13 polymorphism of isolates collected from patients enrolled in an open-label study with dihydroartemisinin-piperaquine for uncomplicated P. falciparum malaria in Cambodia (registered ACTRN12615000696594).
  
  
  RESULTS
  All parasites from recrudescent cases had in vitro or ex vivo PSA survival rates ≥10%, a relevant cut-off value for piperaquine-resistance. Ex vivo PSA survival rates were higher for recrudescent than non-recrudescent cases (39.2% vs. 0.17%, P <1 × 10(-7)). Artemisinin-resistant K13 mutants with ex vivo PSA survival rates ≥10% were associated with 32-fold higher risk of recrudescence (95% CI, 4.5-224; P = 0.0005).
  
  
  CONCLUSIONS
  PSA adequately captures the piperaquine resistance/recrudescence phenotype, a mainstay to identify molecular marker(s) and evaluate efficacy of alternative drugs. Combined ex vivo PSA and K13 genotyping provides a convenient monitor for both artemisinin and piperaquine resistance where dihydroartemisinin-piperaquine is used.
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. vduru@pasteur-kh.org.
authors.forename: Valentine
authors.initials: V
authors.lastname: Duru
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. knimol@pasteur-kh.org.
authors.forename: Nimol
authors.initials: N
authors.lastname: Khim
authors.affiliation: National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia. rithealeang@gmail.com.
authors.forename: Rithea
authors.initials: R
authors.lastname: Leang
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. ksaorin@pasteur-kh.org.
authors.forename: Saorin
authors.initials: S
authors.lastname: Kim
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. adomergue@pasteur-kh.org.
authors.forename: Anais
authors.initials: A
authors.lastname: Domergue
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. klnimol@pasteur-kh.org.
authors.forename: Nimol
authors.initials: N
authors.lastname: Kloeung
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. ksopheakvatey@pasteur-kh.org.
authors.forename: Sopheakvatey
authors.initials: S
authors.lastname: Ke
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. chsophy@pasteur-kh.org.
authors.forename: Sophy
authors.initials: S
authors.lastname: Chy
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. rotha@pasteur-kh.org.
authors.forename: Rotha
authors.initials: R
authors.lastname: Eam
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. kchanra@pasteur-kh.org.
authors.forename: Chanra
authors.initials: C
authors.lastname: Khean
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. lkaknika@pasteur-kh.org.
authors.forename: Kaknika
authors.initials: K
authors.lastname: Loch
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. kmalen@pasteur-kh.org.
authors.forename: Malen
authors.initials: M
authors.lastname: Ken
authors.affiliation: National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia. soleycnm@gmail.com.
authors.forename: Dysoley
authors.initials: D
authors.lastname: Lek
authors.affiliation: Department of Parasites and Insect Vectors, Institut Pasteur, Paris, France. johann.beghain@wanadoo.fr.
authors.forename: Johann
authors.initials: J
authors.lastname: Beghain
authors.affiliation: Department of Parasites and Insect Vectors, Institut Pasteur, Paris, France. frederic.ariey@pasteur.fr.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Ariey
authors.affiliation: WorldWide Antimalarial Resistance Network, Oxford, UK. philippe.guerin@wwarn.org.|Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, Oxford University, Oxford, UK. philippe.guerin@wwarn.org.
authors.forename: Philippe J
authors.initials: PJ
authors.lastname: Guerin
authors.affiliation: National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia. kolhuy@gmail.com.
authors.forename: Rekol
authors.initials: R
authors.lastname: Huy
authors.affiliation: Department of Parasites and Insect Vectors, Institut Pasteur, Paris, France. omp@pasteur.fr.
authors.forename: Odile
authors.initials: O
authors.lastname: Mercereau-Puijalon
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. bwitkowski@pasteur-kh.org.
authors.forename: Benoit
authors.initials: B
authors.lastname: Witkowski
authors.affiliation: Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. dmenard@pasteur-kh.org.
authors.forename: Didier
authors.initials: D
authors.lastname: Menard
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D000962:Antimalarials / Q000627:therapeutic use; D037621:Artemisinins / Q000494:pharmacology*; D002160:Cambodia; D005260:Female; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000401:mortality; D008297:Male; D010271:Parasites; D010963:Plasmodium falciparum / Q000235:genetics*; D011446:Prospective Studies; D011804:Quinolines / Q000494:pharmacology*; D012189:Retrospective Studies; D015996:Survival Rate; D017211:Treatment Failure; D016896:Treatment Outcome; D055815:Young Adult; D010879:Piperazines
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D011804:Quinolines; C039060:artenimol; C034759:piperaquine; D010879:Piperazines