Assessment of Droplet Digital PCR for the Detection and Absolute Quantification of Toxoplasma gondii: A Comparative Retrospective Study.

Autor:	Nabet C; Sorbonne University, INSERM, Pierre-Louis Institute of Epidemiology and Public Health (IPLESP), Assistance Publique-Hôpitaux de Paris (AP-HP), Pitié-Salpêtrière Hospital, Parasitology and Mycology Department, Paris, France. Electronic address: cecile.nabet@aphp.fr., Brossas JY; Sorbonne University, Parasitology and Mycology Department, AP-HP, Pitié-Salpêtrière Hospital, Paris, France., Poignon C; Sorbonne University, Parasitology and Mycology Department, AP-HP, Pitié-Salpêtrière Hospital, Paris, France., Bouzidi A; Sorbonne University, INSERM, Research Unit on Cardiovascular and Metabolic Disease, Institut of Cardiometabolism and Nutrition (ICAN), Department of Endocrine Biochemistry and Oncology, AP-HP, Pitié-Salpêtrière Hospital, Paris, France., Paris L; Sorbonne University, Parasitology and Mycology Department, AP-HP, Pitié-Salpêtrière Hospital, Paris, France., Touafek F; Sorbonne University, Parasitology and Mycology Department, AP-HP, Pitié-Salpêtrière Hospital, Paris, France., Varlet-Marie E; University of Montpellier, Centre National de la Recherche Scientifique (CNRS), Institut de Rechercher pour le Développement (IRD), MiVEGEC, University Hospital of Montpellier, Molecular Biology Pole of the National Reference Centre (CNR) for Toxoplasmosis, Montpellier, France., Sterkers Y; University of Montpellier, Centre National de la Recherche Scientifique (CNRS), Institut de Rechercher pour le Développement (IRD), MiVEGEC, University Hospital of Montpellier, Molecular Biology Pole of the National Reference Centre (CNR) for Toxoplasmosis, Montpellier, France., Passebosc-Faure K; National Reference Centre (CNR) for Toxoplasmosis/Toxoplasma Biological Research Centre (BRC), Dupuytren University Hospital Centre, Limoges, France., Dardé ML; National Reference Centre (CNR) for Toxoplasmosis/Toxoplasma Biological Research Centre (BRC), Dupuytren University Hospital Centre, Limoges, France; Limoges University, INSERM, University Hospital Centre Limoges, Institut de Recherche pour le Développement (IRD), Tropical Neuroepidemiology Unit, Institute of Epidemiology and Tropical Neurology, Limoges, France., Piarroux R; Sorbonne University, INSERM, Pierre-Louis Institute of Epidemiology and Public Health (IPLESP), Assistance Publique-Hôpitaux de Paris (AP-HP), Pitié-Salpêtrière Hospital, Parasitology and Mycology Department, Paris, France., Denis JA; Sorbonne University, INSERM, Saint-Antoine Research Centre, Cancer Biology and Therapeutics, Department of Endocrine Biochemistry and Oncology, AP-HP, Pitié-Salpêtrière Hospital, Paris, France.
Jazyk:	angličtina
Zdroj:	The Journal of molecular diagnostics : JMD [J Mol Diagn] 2023 Jul; Vol. 25 (7), pp. 467-476. Date of Electronic Publication: 2023 Apr 15.
DOI:	10.1016/j.jmoldx.2023.03.006
Abstrakt:	Accurate tools for Toxoplasma gondii detection and quantification can be valuable for the early and effective management of toxoplasmosis. Droplet digital PCR (ddPCR) is a next-generation end-point PCR technique with high performance. The objective of the study was to evaluate the performance of ddPCR for the detection and absolute quantification of T. gondii. From January 2019 to October 2020, DNA samples collected at the Laboratory of Parasitology and Mycology of Pitié-Salpêtrière Hospital in Paris were retrospectively analyzed by ddPCR and real-time quantitative PCR (qPCR). To detect T. gondii with the best sensitivity possible, the REP-529 multicopy target was used. For absolute quantification of T. gondii, a specific single-copy target of α-tubulin was designed. T. gondii detection by ddPCR and qPCR was strongly correlated (R 2  = 0.93), with a total concordance of 96.7% (n = 145/150). Quantification of T. gondii using ddPCR was successful for 15 of 35 samples showing a parasite load ≥170 copies/mL of DNA eluate using the α-tubulin target. The qPCR REP-529 quantification based on a standard curve was approximate and dependent on the strain genotype, which led to an estimate of parasite copy number 14- to 160-fold superior to the ddPCR result. In total, ddPCR is an effective molecular method for T. gondii detection that shows equivalent performance to qPCR. For robust T. gondii quantification, ddPCR is clearly more accurate than semiquantitative qPCR methods. (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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