Evaluation of propidium monoazide-based qPCR to detect viable oocysts of Toxoplasma gondii

Authors: ROUSSEAU, ANGELIQUE - Actalia Securite Des Aliment; VILLENA, ISABELLE - Universite De Reims Champagne-Ardenne; DUMETRE, AURELIEN - Aix-Marseille University; ESCOTTE-BINET, SANDIE - Universite De Reims Champagne-Ardenne; FAVENNEC, LOIC - University Of Rouen; Dubey, Jitender; AUBERT, DOMINIQUE - Universite De Reims Champagne-Ardenne; LA CARBONA, STEPHANIE - Actalia Securite Des Aliment

Submitted to: Parasitology Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/17/2018
Publication Date: 2/7/2019
Citation: Rousseau, A., Villena, I., Dumetre, A., Escotte-Binet, S., Favennec, L., Dubey, J.P., Aubert, D., La Carbona, S. 2019. Evaluation of propidium monoazide-based qPCR to detect viable oocysts of Toxoplasma gondii. Parasitology Research. 118:999-1010. https://doi.org/10.1007/s00436-019-06220-1
DOI: https://doi.org/10.1007/s00436-019-06220-1

Interpretive Summary: Toxoplasmosis, caused by the single celled parasite, Toxoplasma gondii, continues to be a public health problem worldwide. This parasite infects all warm-blooded hosts, including humans. It causes mental retardation and loss of vision in children, and abortion in livestock. The ingestion of food and water contaminated with resistant stage of the parasite, the oocyst, is a major mode of transmission of this parasite. Of all the hosts infected, only cats are known to excrete oocysts in feces. Cats can excrete millions of oocysts after eating an infected prey, such as a mouse or a bird. Oocysts can survive outdoors for months and they are highly infectious to humans. Detection of oocysts in environment is difficult and tedious because of the low numbers in water and soil. Currently available molecular techniques do not distinguish between live and dead oocysts. The present study reports partial success in discriminating between dead and live oocysts by PCR. These results will be of interest to biologists and parasitologists.

Technical 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°C, 37°C and 45°C), amplicon length, selected targeted gene and dyes (PMA, PMAxxTM) 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 room temperature 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.