Novel Phenotypic Assays for the Detection of Artemisinin-Resistant Plasmodium Falciparum Malaria in Cambodia: In-Vitro and Ex-Vivo Drug-Response Studies

Lancet Infect Dis. 2013 Dec;13(12):1043-9. doi: 10.1016/S1473-3099(13)70252-4. Epub 2013 Sep 11.

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.

Findings: 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).

Interpretation: 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.

Funding: Institut Pasteur du Cambodge and the Intramural Research Program, NIAID, NIH.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimalarials / pharmacology*
  • Artemisinins / pharmacology*
  • Cambodia / epidemiology
  • Drug Resistance
  • Genotype
  • Humans
  • Malaria, Falciparum / drug therapy*
  • Malaria, Falciparum / epidemiology
  • Malaria, Falciparum / parasitology*
  • Phenotype
  • Plasmodium falciparum / drug effects*
  • Plasmodium falciparum / isolation & purification
  • Prospective Studies
  • Survival Analysis
  • Survival Rate

Substances

  • Antimalarials
  • Artemisinins
  • dihydroartemisinin

Associated data

  • ClinicalTrials.gov/NCT00341003
  • ClinicalTrials.gov/NCT01736319