A flow cytometry-based quantitative drug sensitivity assay for all Plasmodium falciparum gametocyte stages.

Zenglei Wang,Salil Siriwat,Xiaoying Liang,Min Liu,Xiaoguang Chen,Daniel M Parker,Liwang Cui,Jun Miao,Xiaolian Li

doi:10.1371/journal.pone.0093825

Zenglei Wang, Salil Siriwat + Show 7 more

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https://doi.org/10.1371/journal.pone.0093825

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Abstract

BackgroundMalaria 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.MethodsA 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.FindingsThis 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.ConclusionsA 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.

Highlights

Malaria remains a major public health menace throughout the tropics and subtropics and is responsible for nearly one million deaths annually
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
We report a robust flow cytometry (FCM)-based method for quantitative measurement of responses of P. falciparum gametocytes to antimalarial drugs based on the combination of a transgenic green fluorescence protein (GFP)-expressing line and synchronous gametocyte culture technique

Summary

Introduction

Malaria remains a major public health menace throughout the tropics and subtropics and is responsible for nearly one million deaths annually. Control measures that target gametocytes need to be considered for the malaria elimination campaign. The World Health Organization (WHO) recommends a single dose of PMQ to artemisinin combination therapy (ACT) for interrupting malaria transmission [7]. This drug has serious drawbacks, which compromise its widespread use during the malaria elimination phase [8,9]. Screening for new drugs and vaccines that are active against both developing and mature gametocytes is urgently needed. Malaria elimination/eradication campaigns emphasize interruption of parasite transmission as a priority strategy. Screening for new drugs and vaccines against gametocytes is urgently needed. We describe the use of a transgenic parasite line for assaying drug sensitivity in all gametocyte stages

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