A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection

Received 19 December 2012; returned 15 February 2013; revised 29 March 2013; accepted 2 April 2013Objectives:Plasmodiumgametocytes,responsibleformalariaparasitetransmissionfromhumanstomosquitoes,representacrucialtargetfornewantimalarialdrugstoachievemalariaelimination/eradication.Wedevelopedanovelcolorimetricscreeningmethodforanti-gametocytecompoundsbasedontheparasitelactatedehydrogen-ase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drugsusceptibility.Methods: Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexualparasitesweredepletedwithN-acetylglucosamine.Gametocytesweretreatedwithdihydroartemisinin,epoxomi-cin,methyleneblue,primaquine,puromycinorchloroquinein96-wellplatesandthepLDHactivitywasevaluatedusing a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay(SMFA).Results:AlinearcorrelationwasfoundbetweengametocytaemiadeterminedbyGiemsastainingandpLDHactiv-ity.Aconcentration-dependentreductioninpLDHactivitywasobservedafter72 hofdrugtreatment,whereasanadditional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability.SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reducedoocyst development in the mosquitovector.Conclusions: The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable forscreening novel transmission-blocking compounds, which does not require parasite transgenic lines.Keywords: malaria, gametocytes, drug screening