Adapting a conventional pcr assay for<i>Toxoplasma gondii</i>detection to real-time quantitative pcr including a competitive internal control

Brenier-Pinchart, Marie-Pierre; Morand-Bui, V; Fricker‐Hidalgo, Hélène; Equy, V.; Marlu, Raphaël; Pelloux, H

doi:10.1051/parasite/2007142149

Cited by 20 publications

(14 citation statements)

References 21 publications

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“…Indeed, all of them are authorized by the French national authority Agence de la Biomédecine to perform prenatal diagnostics for congenital toxoplasmosis. They are also all members of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (http://cnrtoxoplasmose .chu-reims.fr), and each of them has developed a high-performing laboratory-developed PCR assay (1,6,(9)(10)(11)(16)(17)(18)(19)(20)(21) that targets the repetitive DNA element rep529, shown to be the most efficient target to date for this diagnosis (10,15,17,(22)(23)(24) (for details, see Table 1; see also Table S1 in the supplemental material). This same working group earlier recommended that laboratories work toward a sensitivity threshold of 0.75 to 2.5 tachyzoites/ml of AF (10) and 100% specificity.…”

Section: Resultsmentioning

confidence: 99%

Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

et al. 2015

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The detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-Evolution T. gondii detection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents.T oxoplasmosis is a worldwide infectious disease that is usually asymptomatic and not severe in humans, except in certain circumstances. Thus, when primarily acquired during pregnancy, Toxoplasma gondii infection in the mother can lead to fetal infection, i.e., congenital toxoplasmosis. The diagnosis of congenital toxoplasmosis may prove a difficult task, as it requires a combination of clinical criteria and results from a battery of serologic and molecular tests in the prenatal, neonatal, and postnatal periods (1). In France, the prenatal diagnosis of congenital toxoplasmosis was based on Toxoplasma isolation in fetal blood and amniotic fluid (AF) by mouse inoculation and the detection of specific antibodies in fetal blood until the 1990s, when these methods were superseded by PCR using amniotic fluid (2-4). In France, amniocentesis is performed Ն4 weeks after Toxoplasma infection of the mother but not before the 18th week of amenorrhea (see http: //cnrtoxoplasmose.chu-reims.fr); it is followed by PCR-based molecular diagnosis. A positive Toxoplasma PCR result affirms congenital toxoplasmosis; a combination treatment using pyrimethamine and sulfadiazine-sulfadoxine is then used in order to limit the presence of sequelae in the fetus, thus increasing the frequency of asymptomatic infection at birth. When a Toxoplasma PCR result is negative, congenital toxoplas...

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Section: Resultsmentioning

confidence: 99%

Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

et al. 2015

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“…Thus, for Toxoplasma gondii, another protozoan parasite, qrtPCR assays using exactly the same (B1 gene) target and different primers have been reported with analytical sensitivities varying by a 200-fold factor, i.e., from 0.05 to 10 genome equivalents per reaction tube (6,7,11,13,14,19,25,33,36). Still, such variations are due not only to the choice of the primers but also, at least as importantly, to the optimization of the method: indeed, identical primers may yield widely different performances in different laboratories (P. Bastien and the Molecular Biology Group of the French National Reference Center for Toxoplasmosis, unpublished data).…”

mentioning

confidence: 99%

Quantitative Real-Time PCR Is Not More Sensitive than “Conventional” PCR

2008

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“…This confirms that the use of two (or three) reaction tubes per patient in routine practice is an important measure to avoid falsely negative results in Toxoplasma PCR (8). The diagnosis of congenital toxoplasmosis was based on the results of a combination of both parasitological and serological tests and was established in each center that sent amniotic fluid (4,6). IC, immune charge; WB, Western blot.…”

Section: Resultsmentioning

confidence: 85%

“…Information was collected and analyzed in order (i) to estimate the gestational age at which maternal infection occurred and (ii) to establish the diagnosis of CT. The diagnosis of CT was established in each center that sent amniotic fluid, based on the results of a series of parasitological (including PCR and mouse inoculation using amniotic fluid, placenta, and cord blood) and serological (at birth and during a long-term serological and clinical follow-up of the infants during the first year of life) tests (6). According to the classification system and case definitions of congenital toxoplasmosis developed by the European Research Network on Congenital Toxoplasmosis (15), all cases were defined as definite or unlikely congenital toxoplasmosis, not infected, or lost to follow-up.…”

Section: Methodsmentioning

confidence: 99%

Comparative Assessment of a Commercial Kit and Two Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

et al. 2012

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Toxoplasmosis is a worldwide infection that may cause severe disease and is regarded as a serious health problem in France. Detection of the parasite by molecular methods is crucial for diagnosing the disease. The extreme diversity of methods and performances of Toxoplasma PCR assays makes the use of commercial PCR kits an attractive alternative, as they offer a chance for standardization. We compared the performances of three molecular methods for the detection of Toxoplasma gondii DNA in amniotic fluid: a commercial method using nested PCR and two laboratory-developed methods, one using conventional PCR and the other one real-time PCR. This evaluation was based upon a T. gondii DNA serial dilution assay, three amniotic fluid samples spiked with T. gondii at different concentrations, and a clinical cohort of 33 amniotic fluid samples. The T. gondii DNA serial dilution assay showed a much lower sensitivity for the commercial kit than for the laboratory-developed methods. Moreover, out of 12 proven congenital toxoplasmosis cases, 91.7% were detected by the laboratory-developed assays, whereas only 50% were detected by the commercial kit. A lack of sensitivity of the method, partly due to the presence of PCR inhibitors, was the main drawback of the commercial method. This study emphasizes that commercial PCR diagnostic kits do not systematically perform better than carefully optimized laboratory-developed methods. There is a need for thorough evaluation of such kits by proficient groups, as well as for performance standards that commercial kits can be tested against to improve confidence in those selected by health care providers.

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Adapting a conventional pcr assay forToxoplasma gondiidetection to real-time quantitative pcr including a competitive internal control

Cited by 20 publications

References 21 publications

Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

Quantitative Real-Time PCR Is Not More Sensitive than “Conventional” PCR

Comparative Assessment of a Commercial Kit and Two Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

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