A novel live-dead staining methodology to study malaria parasite viability

Erica M Pasini,Henri J Vial,Clemens Hm Kocken,Denise Van Den Ierssel

doi:10.1186/1475-2875-12-190

Abstract

BackgroundMalaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world. Several methodologies have been used to assess parasite viability during the adaption of field strains to culture or the assessment of drug potential, but these are in general not able to provide an accurate real-time assessment of whether parasites are alive or dead.MethodsDifferent commercial dyes and kits were assessed for their potential to allow for the real-time detection of whether a blood stage malaria parasite is dead or alive.ResultsHere, a methodology is presented based on the potential-sensitive mitochondrial probe JC-1, which allows for the real-time visualization of live (red staining) and/or dead (absence of red staining) blood stage parasites in vitro and ex vivo. This method is applicable across malaria parasite species and strains and allows to visualize all parasite blood stages including gametocytes. Further, this methodology has been assessed also for use in drug sensitivity testing.ConclusionsThe JC-1 staining approach is a versatile methodology that can be used to assess parasite viability during the adaptation of field samples to culture and during drug treatment. It was found to hold promise in the assessment of drugs expected to lead to delayed death phenotypes and it currently being evaluated as a method for the assessment of parasite viability during the adaptation of patient-derived Plasmodium vivax to long-term in vitro culture.

Highlights

Malaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world
A methodology is presented based on the potential-sensitive mitochondrial probe JC-1, which allows for the real-time visualization of live and/ or dead parasites in vitro and ex vivo
Parasite staining with JC-1 and drug assays using LDH or JC-1 staining as read-out Drug assays were performed in culture flasks or 96-well plates by adding serial drug dilutions to synchronized P. falciparum or P. knowlesi ring-stage cultures as previously described [13,26,27]

Summary

Introduction

Malaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world. A disease caused by apicomplexan parasites of the genus Plasmodium, with Plasmodium falciparum causing 250–500 million clinical cases and up to 1.2 million deaths every year, is a major health- and socioeconomical problem in the tropical and sub-tropical areas of the world [1]. As the focus shifted towards malaria eradication, there is a need to adapt new field strains and additional parasite species. There are challenges derived from the rapidly spreading resistance to existing anti-malarial drugs, mainly in P. falciparum and from the need to tackle emerging zoonoses (e.g. Plasmodium knowlesi [10,11]). The search for potent anti-malarials with innovative mechanism of action remains a priority

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