title: Alternative methods for the Plasmodium falciparum artemisinin ring-stage survival assay with increased simplicity and parasite stage-specificity.
abstract: BACKGROUND
  Artemisinin-based combination therapy is recommended to treat Plasmodium falciparum worldwide, but observations of longer artemisinin (ART) parasite clearance times (PCTs) in Southeast Asia are widely interpreted as a sign of potential ART resistance. In search of an in vitro correlate of in vivo PCT after ART treatment, a ring-stage survival assay (RSA) of 0-3 h parasites was developed and linked to polymorphisms in the Kelch propeller protein (K13). However, RSA remains a laborious process, involving heparin, Percoll gradient, and sorbitol treatments to obtain rings in the 0-3 h window. Here two alternative RSA protocols are presented and compared to the standard Percoll-based method, one highly stage-specific and one streamlined for laboratory application.
  
  
  METHODS
  For all protocols, P. falciparum cultures were synchronized with 5 % sorbitol treatment twice over two intra-erythrocytic cycles. For a filtration-based RSA, late-stage schizonts were passed through a 1.2 μm filter to isolate merozoites, which were incubated with uninfected erythrocytes for 45 min. The erythrocytes were then washed to remove lysis products and further incubated until 3 h post-filtration. Parasites were pulsed with either 0.1 % dimethyl sulfoxide (DMSO) or 700 nM dihydroartemisinin in 0.1 % DMSO for 6 h, washed twice in drug-free media, and incubated for 66-90 h, when survival was assessed by microscopy. For a sorbitol-only RSA, synchronized young (0-3 h) rings were treated with 5 % sorbitol once more prior to the assay and adjusted to 1 % parasitaemia. The drug pulse, incubation, and survival assessment were as described above.
  
  
  RESULTS
  Ring-stage survival of P. falciparum parasites containing either the K13 C580 or C580Y polymorphism (associated with low and high RSA survival, respectively) were assessed by the described filtration and sorbitol-only methods and produced comparable results to the reported Percoll gradient RSA. Advantages of both new methods include: fewer reagents, decreased time investment, and fewer procedural steps, with enhanced stage-specificity conferred by the filtration method.
  
  
  CONCLUSIONS
  Assessing P. falciparum ART sensitivity in vitro via RSA can be streamlined and accurately evaluated in the laboratory by filtration or sorbitol synchronization methods, thus increasing the accessibility of the assay to research groups.
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Room 3E - 10, Rockville, MD, 20852, USA. whitney.adana@gmail.com.
authors.forename: Whitney A
authors.initials: WA
authors.lastname: Kite
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Room 3E - 10, Rockville, MD, 20852, USA. viviana.aimee@gmail.com.|Universidad Central del Caribe School of Medicine, Bayamon, PR, 00960, USA. viviana.aimee@gmail.com.
authors.forename: Viviana A
authors.initials: VA
authors.lastname: Melendez-Muniz
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Room 3E - 10, Rockville, MD, 20852, USA. roberto.moraesbarros@nih.gov.
authors.forename: Roberto R
authors.initials: RR
authors.lastname: Moraes Barros
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Room 3E - 10, Rockville, MD, 20852, USA. twellems@niaid.nih.gov.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wellems
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Room 3E - 10, Rockville, MD, 20852, USA. jsa@niaid.nih.gov.
authors.forename: Juliana M
authors.initials: JM
authors.lastname: Sá
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D037621:Artemisinins / Q000494:pharmacology*; D004912:Erythrocytes / Q000469:parasitology; D006801:Humans; D008018:Life Cycle Stages / Q000502:physiology; D010274:Parasitology / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000502:physiology*
chemical_list: D000962:Antimalarials; D037621:Artemisinins