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      Detection of Toxoplasma gondii DNA by qPCR in the feces of a cat that recently ingested infected prey does not necessarily imply oocyst shedding Translated title: La détection d’ADN de Toxoplasma gondii dans les fèces d’un chat qui a récemment ingéré des tissus infectés ne signifie pas nécessairement émission d’oocystes

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          Abstract

          Detection of Toxoplasma gondii DNA in cat feces is considered indicative of the presence of T. gondii oocysts. This study aims to demonstrate that the high sensitivity of qPCR can lead to T. gondii DNA detection in cat feces in the absence of oocysts. A cat immune to toxoplasmosis was fed with a mouse experimentally infected with T. gondii. Detection of DNA of this parasite was performed by qPCR on feces passed: (i) on the day the cat ingested the infected prey; (ii) during the three previous days; and (iii) during the three following days. The kinetics of qPCR results are clearly not linked to oocyst shedding and this result demonstrates that qPCR can detect T. gondii DNA related to bradyzoites from an infected prey, in the absence of oocysts. Caution is thus recommended when interpreting T. gondii qPCR results for samples of cat feces.

          Translated abstract

          La détection d’ADN de Toxoplasma gondii dans les fèces de chats est considérée comme révélatrice de la présence d’oocystes. Cette étude a pour objectif de démontrer que la grande sensibilité de la qPCR peut conduire à la détection de l’ADN de T. gondii dans les fèces de chat en l’absence d’oocystes. Un chat immunisé contre la toxoplasmose a été nourri avec une souris expérimentalement infectée par T. gondii. La détection d’ADN de ce parasite a été réalisée par qPCR sur les fèces émises : (i) le jour où le chat a ingéré la proie infectée ; (ii) durant les trois jours précédents ; (iii) durant les trois jours suivants. La cinétique des résultats de qPCR n’est clairement pas celle d’une émission d’oocystes et ce résultat démontre que la qPCR peut détecter l’ADN de T. gondii lié aux bradyzoïtes d’une proie infectée, en l’absence d’oocystes. La prudence est donc recommandée pour l’interprétation des résultats de qPCR de T. gondii sur fèces de chats.

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          Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii.

          The development of simple, sensitive and rapid methods for the detection and identification of Toxoplasma gondii is important for the diagnosis and epidemiological studies of the zoonotic disease toxoplasmosis. In the past 2 decades, molecular methods based on a variety of genetic markers have been developed, each with its advantages and limitations. The application of these methods has generated invaluable information to enhance our understanding of the epidemiology, population genetics and phylogeny of T. gondii. However, since most studies focused solely on the detection but not genetic characterization of T. gondii, the information obtained was limited. In this review, we discuss some widely used molecular methods and propose an integrated approach for the detection and identification of T. gondii, in order to generate maximum information for epidemiological, population and phylogenetic studies of this key pathogen.
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            Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

            Background Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. Methods Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. Results Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. Conclusion We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.
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              Evaluation of a strategy for Toxoplasma gondii oocyst detection in water.

              Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.
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                Author and article information

                Journal
                Parasite
                Parasite
                parasite
                Parasite
                EDP Sciences
                1252-607X
                1776-1042
                2016
                22 July 2016
                : 23
                : ( publisher-idID: parasite/2016/01 )
                Affiliations
                [1 ] Université de Reims Champagne-Ardenne, SFR Cap-Santé, EA3800-PROTAL, UFR de Médecine 51 rue Cognacq-Jay 51095 Reims Cedex France
                [2 ] Université de Reims Champagne-Ardenne, CERFE 5 rue de la Héronnière 08240 Boult-aux-Bois France
                Author notes
                [a]

                Equal contribution of these authors to this paper.

                Article
                parasite160040 10.1051/parasite/2016029
                10.1051/parasite/2016029
                4958142
                27449050
                © M.-L. Poulle et al., published by EDP Sciences, 2016

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 0, Tables: 1, Equations: 0, References: 17, Pages: 4
                Categories
                Short Note

                qpcr, toxoplasma gondii, domestic cat, oocyst, coprodiagnosis

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