Evaluation of propidium monoazide\u2013based qPCR to detect viable oocysts of Toxoplasma gondii

Angélique Rousseau,Stéphanie La Carbona,Isabelle Villena,Jitender P Dubey,Sandie Escotte-Binet,Dominique Aubert,Aurélien Dumètre,Loïc Favennec

doi:10.1007/s00436-019-06220-1

Abstract

Information on the viability of Toxoplasma gondii oocysts is crucial to establish the public health significance of this environmental transmission stage that can contaminate water and foods. Interest for molecular-based methods to assess viability is growing and the aim of our study was to assess, for the first time, a propidium monoazide (PMA)–qPCR approach to determine the viability of T. gondii oocysts. Untreated and heat-killed (99 °C, 5 min) oocysts were incubated with PMA, a photoreactive DNA binding dye, and analyzed by confocal microscopy and flow cytometry to characterize oocysts’ dye permeability. Different PMA concentrations (50 to 150 μM), incubation temperatures (22, 37, and 45 °C), amplicon length, selected targeted gene, and dyes (PMA, PMAxx™) were evaluated to define optimal conditions to discriminate specifically viable oocysts by PMA–qPCR. In theory, PMA binding to DNA would inhibit PCR amplification in dead but not in viable oocysts. Incubation at 22 °C with 100 μM PMA coupled to qPCR targeting a 123-bp sequence of the 529-bp repeat element allowed the distinction between viable and heated oocysts. However, the reduction of viability following heating of oocysts at high temperature was slight and, contrarily to reverse transcriptase-qPCR, the qPCR signal was not totally suppressed in heated suspensions. Therefore, PMA–qPCR is able to assess the impact of heating on T. gondii oocysts’ viability but underestimates the efficacy of this treatment. The relevance of this technique to evaluate the efficacy of other inactivation processes and assess exposure of humans to this pathogen requires further investigations.

Highlights

Toxoplasma gondii is a protozoan parasite that can infect humans either following ingesting meat containing cysts of the parasite or water and foods contaminated by oocysts
Untreated and heat-killed oocyst suspensions contained 22.9% and 91.1% propidium monoazide (PMA)+ oocysts, respectively (Fig. 1b, panels 3–4). These data demonstrated that PMA was able to penetrate within oocysts and sporozoites, and that heat-killed oocysts were more permeable to PMA than untreated oocysts in the tested conditions
At 22 °C, none of the tested PMA concentrations allow the abolition of the PCR signal in heat-killed oocysts, while heat-killed oocysts were not detected by reverse transcriptase-qPCR (RT-qPCR) (Table 1)

Summary

Introduction

Toxoplasma gondii is a protozoan parasite that can infect humans either following ingesting meat containing cysts of the parasite or water and foods contaminated by oocysts. Irrespective of the transmission route, T. gondii infections are usually asymptomatic in immunocompetent individuals, resulting in the formation of latent cysts in tissues and organs throughout the body (Robert-Gangneux and Dardé 2012). The parasite can sometimes lead to severe ocular, cerebral, or multivisceral complications, especially in congenitally infected infants and in immunocompromised individuals. T. gondii oocysts were responsible for 2% of parasitic protozoan waterborne outbreaks reported between January 2004 and December 2010 (Baldursson and Karanis 2011). Shellfishes are at risk due to their production in potentially contaminated waters (Tei et al 2016; Ghozzi et al 2017)

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