title: A flow cytometry-based quantitative drug sensitivity assay for all Plasmodium falciparum gametocyte stages.
abstract: BACKGROUND
  Malaria elimination/eradication campaigns emphasize interruption of parasite transmission as a priority strategy. Screening for new drugs and vaccines against gametocytes is therefore urgently needed. However, current methods for sexual stage drug assays, usually performed by counting or via fluorescent markers are either laborious or restricted to a certain stage. Here we describe the use of a transgenic parasite line for assaying drug sensitivity in all gametocyte stages.
  
  
  METHODS
  A transgenic parasite line expressing green fluorescence protein (GFP) under the control of the gametocyte-specific gene α-tubulin II promoter was generated. This parasite line expresses GFP in all gametocyte stages. Using this transgenic line, we developed a flow cytometry-based assay to determine drug sensitivity of all gametocyte stages, and tested the gametocytocidal activities of four antimalarial drugs.
  
  
  RESULTS
  This assay proved to be suitable for determining drug sensitivity of all sexual stages and can be automated. A Z' factor of 0.79 ± 0.02 indicated that this assay could be further optimized for high-throughput screening. The daily sensitivity of gametocytes to three antimalarial drugs (chloroquine, dihydroartemisinin and pyronaridine) showed a drastic decrease from stage III on, whereas it remained relatively steady for primaquine.
  
  
  CONCLUSIONS
  A drug assay was developed to use a single transgenic parasite line for determining drug susceptibility of all gametocyte stages. This assay may be further automated into a high-throughput platform for screening compound libraries against P. falciparum gametocytes.
authors.affiliation: Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Zenglei
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America; Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong, P.R. China.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Liang
authors.affiliation: Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Salil
authors.initials: S
authors.lastname: Siriwat
authors.affiliation: Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Xiaolian
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong, P.R. China.
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Parker
authors.affiliation: Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Jun
authors.initials: J
authors.lastname: Miao
authors.affiliation: Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Liwang
authors.initials: L
authors.lastname: Cui
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D006801:Humans; D020128:Inhibitory Concentration 50; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development*
chemical_list: D000962:Antimalarials
references.citation: Arch Pharm Res. 2004 May;27(5):576-80
references.pmid: 15202566
references.citation: Malar J. 2010 Feb 11;9:49
references.pmid: 20149239
references.citation: Am J Trop Med Hyg. 1998 Oct;59(4):505-8
references.pmid: 9790418
references.citation: Mol Biochem Parasitol. 2007 Jul;154(1):119-23
references.pmid: 17521751
references.citation: Malar J. 2006 Jan 18;5:1
references.pmid: 16420686
references.citation: Mol Biochem Parasitol. 2005 Sep;143(1):67-79
references.pmid: 16005087
references.citation: Nature. 2010 May 20;465(7296):297-8
references.pmid: 20485420
references.citation: Spectrochim Acta A Mol Biomol Spectrosc. 2005 Jan 1;61(1-2):163-70
references.pmid: 15556435
references.citation: Proc Natl Acad Sci U S A. 2011 Nov 22;108(47):E1214-23
references.pmid: 22042867
references.citation: Southeast Asian J Trop Med Public Health. 1999 Dec;30(4):636-42
references.pmid: 10928353
references.citation: Ann Trop Med Parasitol. 1992 Jun;86(3):207-15
references.pmid: 1449270
references.citation: J Biomol Screen. 1999;4(2):67-73
references.pmid: 10838414
references.citation: Antimicrob Agents Chemother. 2009 Oct;53(10):4080-5
references.pmid: 19651911
references.citation: Mol Biochem Parasitol. 2006 Nov;150(1):118-21
references.pmid: 16901558
references.citation: Br J Clin Pharmacol. 1984 Apr;17(4):441-6
references.pmid: 6721990
references.citation: PLoS One. 2010 Dec 30;5(12):e14470
references.pmid: 21209927
references.citation: J Phys Chem B. 2009 Jun 18;113(24):8391-401
references.pmid: 19472980
references.citation: Eur J Med Chem. 2009 Mar;44(3):937-53
references.pmid: 18930565
references.citation: Mol Biochem Parasitol. 2009 Feb;163(2):123-6
references.pmid: 19027798
references.citation: J Struct Biol. 2012 Feb;177(2):224-32
references.pmid: 21945653
references.citation: Parasite. 2000 Mar;7(1):19-26
references.pmid: 10743643
references.citation: Mol Biochem Parasitol. 2010 Aug;172(2):57-65
references.pmid: 20381542
references.citation: Int J Parasitol. 1997 Sep;27(9):975-87
references.pmid: 9363480
references.citation: PLoS One. 2012;7(4):e35019
references.pmid: 22514702
references.citation: PLoS Med. 2011 Jan 25;8(1):e1000406
references.pmid: 21311579
references.citation: Trans R Soc Trop Med Hyg. 1977;71(6):526-9
references.pmid: 343314
references.citation: Mol Biochem Parasitol. 2011 Jun;177(2):160-3
references.pmid: 21316401
references.citation: Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11405-10
references.pmid: 21709259
references.citation: Clin Infect Dis. 2004 Nov 1;39(9):1336-45
references.pmid: 15494911
references.citation: Cochrane Database Syst Rev. 2012 Sep 12;(9):CD008152
references.pmid: 22972117
references.citation: Parasitology. 2009 Oct;136(12):1427-34
references.pmid: 19660156
references.citation: Malar J. 2013 Jun 07;12:190
references.pmid: 23758788
references.citation: J Infect Dis. 2009 Nov 15;200(10):1518-21
references.pmid: 19848586
references.citation: Lancet. 2010 Nov 6;376(9752):1566-78
references.pmid: 21035842
references.citation: BMC Biol. 2013 Jun 13;11:67
references.pmid: 23763941
references.citation: Mol Biochem Parasitol. 2011 Dec;180(2):127-31
references.pmid: 21939693
references.citation: Parasitol Int. 2000 Feb;48(4):275-80
references.pmid: 10725690
references.citation: J Biosci. 2007 Sep;32(6):1139-45
references.pmid: 17954974
references.citation: J Infect Dis. 2011 May 15;203(10):1445-53
references.pmid: 21502082
references.citation: Annu Rev Microbiol. 1998;52:561-90
references.pmid: 9891808
references.citation: Exp Parasitol. 2013 Nov;135(3):541-5
references.pmid: 24055216
references.citation: Nucleic Acids Res. 2001 Feb 1;29(3):850-3
references.pmid: 11160909
references.citation: Mol Biochem Parasitol. 2008 Aug;160(2):148-51
references.pmid: 18490066
references.citation: Southeast Asian J Trop Med Public Health. 2010 Nov;41(6):1306-11
references.pmid: 21329302
references.citation: Lancet. 2010 Dec 4;376(9756):1883-5
references.pmid: 21035840
references.citation: Malar J. 2008 Dec 11;7 Suppl 1:S8
references.pmid: 19091042
references.citation: Science. 1976 Aug 20;193(4254):673-5
references.pmid: 781840
references.citation: Mol Biochem Parasitol. 1992 Dec;56(2):239-50
references.pmid: 1484548