A simple and predictive phenotypic High Content Imaging assay for Plasmodium falciparum mature gametocytes to identify malaria transmission blocking compounds.

Leonardo Lucantoni,Maarten Eldering,Giulia Siciliano,Michele Signore,Francesco Silvestrini,Koen J. Dechering,Pietro Alano,Vicky M. Avery

doi:10.1038/srep16414

Abstract

Plasmodium falciparum gametocytes, specifically the mature stages, are the only malaria parasite stage in humans transmissible to the mosquito vector. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria and tools allowing the screening of large compound libraries with high predictive power are needed to identify new candidates. As gametocytes are not a replicative stage it is difficult to apply the same drug screening methods used for asexual stages. Here we propose an assay, based on high content imaging, combining “classic” gametocyte viability readout based on gametocyte counts with a functional viability readout, based on gametocyte activation and the discrimination of the typical gamete spherical morphology. This simple and rapid assay has been miniaturized to a 384-well format using acridine orange staining of wild type P. falciparum 3D7A sexual forms, and was validated by screening reference antimalarial drugs and the MMV Malaria Box. The assay demonstrated excellent robustness and ability to identify quality hits with high likelihood of confirmation of transmission reducing activity in subsequent mosquito membrane feeding assays.

Highlights

Malaria is a disease resulting from infection by the intracellular protozoan parasite Plasmodium
The major drawbacks in the current assays include the need to use gametocyte cultures highly purified from asexual parasites[18] or from uninfected red blood cells, or both[11], long incubation and assay times imposed by the slow decay of the enzymatic or transgenic reporter activity in unhealthy or dead mature gametocytes[18] or by the slow accumulation of sufficient signal intensity[15]
With the only assumption that this process requires the mature gametocyte to be alive, as it needs to sense the change in environment and to respond by modifying its cell shape, we hypothesized that effects of compounds on mature gametocyte viability could be phenotypically assessed from their ability to “round up” and that failure to undergo this developmental step would be predictive of the compounds’ transmission-blocking activity measured in experimental infections of mosquitoes

Summary

Introduction

Malaria is a disease resulting from infection by the intracellular protozoan parasite Plasmodium. The last four years have seen the emergence of a considerable number of different approaches to determine the impact of compounds on gametocyte development[8,9,10,11,12,13,14,15,16,17,18,19,20] Some of these assays are focused on stage IV–V gametocytes, with the aim to identify compounds active against the mature sexual stages, the only ones able to survive and further develop in the mosquito blood meal. The insensitive stage V gametocytes remain apparently quiescent but infectious in the peripheral bloodstream for at least three weeks[3,22] These cells are directly responsible for malaria parasite transmission as they are programmed to sense the environmental changes in the transition from human circulation to the mosquito midgut and to readily transform into male and female gametes. The existing assays able to quantify female gamete formation, all based on Pfs[25] antibody, require high number of parasites, which limits throughput[14], and/or additional 16h–24h incubation of activated gametocytes/gametes to achieve adequate signal intensities of fluorescent labelled Pfs[25] antibodies[13,14,20]

Methods

Results

Conclusion