title: Nowhere to hide: interrogating different metabolic parameters of Plasmodium falciparum gametocytes in a transmission blocking drug discovery pipeline towards malaria elimination.
abstract: BACKGROUND
  The discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions.
  
  
  METHODS
  Gametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable.
  
  
  RESULTS
  A highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4%. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident.
  
  
  CONCLUSIONS
  With this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broad-diversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.
authors.affiliation: Malaria Parasite Molecular Laboratory, Centre for Sustainable Malaria Control, Department of Biochemistry, University of Pretoria, Private Bag x20, Hatfield, Pretoria, 0028, South Africa. Janette.reader@up.ac.za.
authors.forename: Janette
authors.initials: J
authors.lastname: Reader
authors.affiliation: Malaria Parasite Molecular Laboratory, Centre for Sustainable Malaria Control, Department of Biochemistry, University of Pretoria, Private Bag x20, Hatfield, Pretoria, 0028, South Africa. mariette.botha@up.ac.za.
authors.forename: Mariëtte
authors.initials: M
authors.lastname: Botha
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, PO Box 395, Pretoria, 0001, South Africa. ANTheron@csir.co.za.
authors.forename: Anjo
authors.initials: A
authors.lastname: Theron
authors.affiliation: Plasmodium Molecular Research Unit, Wits Research Institute for Malaria, Department of Molecular Medicine and Haematology, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand and National Health Laboratory Service, Johannesburg, 2193, South Africa. sonja.lauterbach@gmail.com.
authors.forename: Sonja B
authors.initials: SB
authors.lastname: Lauterbach
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, PO Box 395, Pretoria, 0001, South Africa. clairerossouw@gmail.com.
authors.forename: Claire
authors.initials: C
authors.lastname: Rossouw
authors.affiliation: Plasmodium Molecular Research Unit, Wits Research Institute for Malaria, Department of Molecular Medicine and Haematology, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand and National Health Laboratory Service, Johannesburg, 2193, South Africa. dewaldtengelbrecht@gmail.com.
authors.forename: Dewaldt
authors.initials: D
authors.lastname: Engelbrecht
authors.affiliation: Plasmodium Molecular Research Unit, Wits Research Institute for Malaria, Department of Molecular Medicine and Haematology, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand and National Health Laboratory Service, Johannesburg, 2193, South Africa. melfried88@gmail.com.
authors.forename: Melanie
authors.initials: M
authors.lastname: Wepener
authors.affiliation: Malaria Parasite Molecular Laboratory, Centre for Sustainable Malaria Control, Department of Biochemistry, University of Pretoria, Private Bag x20, Hatfield, Pretoria, 0028, South Africa. annel.smit@up.ac.za.
authors.forename: Annél
authors.initials: A
authors.lastname: Smit
authors.affiliation: Medicines for Malaria Venture, Geneva, Switzerland. leroyd@mmv.org.
authors.forename: Didier
authors.initials: D
authors.lastname: Leroy
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, PO Box 395, Pretoria, 0001, South Africa. DMancama@csir.co.za.
authors.forename: Dalu
authors.initials: D
authors.lastname: Mancama
authors.affiliation: Plasmodium Molecular Research Unit, Wits Research Institute for Malaria, Department of Molecular Medicine and Haematology, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand and National Health Laboratory Service, Johannesburg, 2193, South Africa. theresa.coetzer@nhls.ac.za.
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Coetzer
authors.affiliation: Malaria Parasite Molecular Laboratory, Centre for Sustainable Malaria Control, Department of Biochemistry, University of Pretoria, Private Bag x20, Hatfield, Pretoria, 0028, South Africa. lbirkholtz@up.ac.za.
authors.forename: Lyn-Marie
authors.initials: LM
authors.lastname: Birkholtz
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D060740:Disease Eradication; D055808:Drug Discovery; D008288:Malaria / Q000517:prevention & control*; D021261:Parasitic Sensitivity Tests / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects*
chemical_list: D000962:Antimalarials