Characterization and Multicentric Validation of a Common Standard for Toxoplasma gondii Detection Using Nucleic Acid Amplification Assays

Varlet‐Marie, Emmanuelle; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaès, Laurence; Filisetti, Denis; Pelloux, Hervé; Touafek, Fériel; Yera, Hélène; Bastien, Patrick

doi:10.1128/jcm.01906-14

Cited by 26 publications

(22 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Due to this great heterogeneity, it is difficult to infer any recommendations. However, since 2011, the PCR method described by Reischl et al (laboratory-developed FRET) and the Bio-Evolution® kit (TaqMan®) are the most widely used methods (Figure 2.C); both have been shown to be highly sensitive methods in previous studies (Filisetti et al, 2015;Sterkers et al, 2010a;Varlet-Marie et al, 2014) and (Filisetti et al, 2015). The commercial Toxoplasma ELITe MGB kit (Elitech®) appeared recently as a robust alternative to Bio-Evolution® (Robert-Gangneux et al, 2017), but its use has not spread in French laboratories yet.…”

Section: Pcr Methodsmentioning

confidence: 99%

Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines

Roux

Varlet‐Marie

Bastien

et al. 2018

International Journal for Parasitology

Self Cite

View full text Add to dashboard Cite

The molecular diagnosis of toxoplasmosis lacks standardisation due to the use of numerous methods with variable performance. This diversity of methods also impairs robust performance comparisons between laboratories. The harmonisation of practices by diffusion of technical guidelines is a useful way to improve these performances. The knowledge of methods and practices used for this molecular diagnosis is an essential step to provide guidelines for Toxoplasma-PCR. In the present study, we aimed (i) to describe the methods and practices of Toxoplasma-PCR used by clinical microbiology laboratories in France and (ii) to propose technical guidelines to improve molecular diagnosis of toxoplasmosis. To do so, a yearly self-administered questionnaire-based survey was undertaken in proficient French laboratories from 2008 to 2015, and guidelines were proposed based on the results of those as well as previously published work. This period saw the progressive abandonment of conventional PCR methods, of Toxoplasma-PCR targeting the B1 gene and of the use of two concomitant molecular methods for this diagnosis. The diversity of practices persisted during the study, in spite of the increasing use of commercial kits such as PCR kits, DNA extraction controls and PCR inhibition controls. We also observed a tendency towards the automation of DNA extraction. The evolution of practices did not always go together with an improvement in those, as reported notably by the declining use of Uracil-DNA Glycosylase to avoid carry-over contamination. We here propose technical recommendations which correspond to items explored during the survey, with respect to DNA extraction, Toxoplasma-PCR and good PCR practices.

show abstract

Section: Pcr Methodsmentioning

confidence: 99%

Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines

Roux

Varlet‐Marie

Bastien

et al. 2018

International Journal for Parasitology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Briefly, the PCR target is rep529l; the sensitivity was described as detecting down to 2.5 Â 10 À2 parasite equivalents. In the present authors' hands, using spiked amniotic fluid, the limit of detection in the serial dilution assay was 0.1 Toxoplasma/ml (Varlet-Marie et al, 2014). b Architect Abbott, Germany; limit of positivity 3 IU/ml.…”

Section: Discussionmentioning

confidence: 53%

Molecular diagnosis of toxoplasmosis at the onset of symptomatic primary infection: A straightforward alternative to serological examinations

Lévêque

Chiffré

Galtier

et al. 2019

International Journal of Infectious Diseases

Self Cite

View full text Add to dashboard Cite

A B S T R A C TMyopericarditis is a rare but well-documented clinical presentation of primary Toxoplasma gondii infection in immunocompetent patients. Here, early detection of Toxoplasma DNA in the peripheral blood by PCR allowed the diagnosis of acute toxoplasmosis while serological tests were negative. Additional serological evaluations 2 weeks later confirmed the diagnosis and showed that cardiac manifestations occurred before seroconversion. This highlights the importance of a second serological control in the case of a suspected active infection. Overall, we show here that PCR testing for Toxoplasma is a sensitive and straightforward alternative to serological examinations.

show abstract

“…The laboratory is accredited according to ISO 15189:2012 for the Toxoplasma and Pneumocystis PCR assays. The Toxoplasma -qPCR assay is being used in routine since July 2009, >17000 clinical samples have been routinely tested, and PCR efficiency is regularly controlled and found at ≥97.5% [18]. For the Pneumocystis- qPCR assay, the corresponding features are March 2013, >2500, and 90 ±9%.…”

Section: Resultsmentioning

confidence: 99%

“…Amplification controls were made of reaction wells containing pathogen DNA on top of the patient DNA extract. For the Toxoplasma qPCR assay, this pathogen-specificamplification positive control was prepared from a 10 5 T. gondii tachyzoites/mL freeze-dried standard as described in Varlet-Marie et al [18]; 2 µL of T. gondii DNA extract were added to obtain a final concentration of 1.5 T. gondii genome/tube for which the expected Cp value is 35.2 ±1.5. The control was performed in duplicate.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Polymerase Chain Reaction: Pathogen-Specific Controls are better than Human Gene Amplification

Roux

Ravel

Varlet‐Marie

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

21PCR inhibition is frequent in medical microbiology routine practice and may lead to 22 false-negative results; however there is no consensus on how to detect it. Pathogen-specific 23 and human gene amplifications are widely used to detect PCR inhibition. We aimed at 24 comparing the value of PCR inhibitor detection using these two methods. We analysed Cp 25 shifts (Cp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific 26 sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples 27 including various matrixes were included. Pathogen-specific amplification and albumin qPCR 28 identified 62/3152 samples (2.0 %), and 409/3152 (13.0%) samples, respectively, as inhibited. 29Only 16 samples were detected using both methods. In addition, the use of the Youden's index 30 failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished 31 among the different sample matrixes. qPCR targeting the albumin gene therefore appears not 32 adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data 33 may be extrapolated to other heterologous targets and should discourage their use to assess 34 the presence of PCR inhibition in microbiological PCR assays. 4 60 due to increased amounts of amplicons and airborne contamination of reaction wells. 61Amplification controls may be prepared from genomic DNA (as in our Toxoplasma-PCR 62 assay) or from the target DNA sequence cloned in a plasmid (as in our Pneumocystis-PCR 63 assay). One development of the plasmid strategy is to clone a chimeric sequence to be 64 amplified by the same primers than the PCR target but detected by specific probe(s) [23][24][25][26]. 65 In the second type of method, albumin, beta-globin or human RNase P genes are targeted. 66This method is found attractive since (i) they can be implemented in all qPCR assays 67 involving human samples; and (ii) they constitute a complete process control for DNA 68 extraction and amplification. Yet, human gene qPCR cycle of positivity (Cp) depends also on 69 the initial human DNA content or cellularity in the clinical sample, which is highly variable. 70 Indeed, DNA content or cellularity depends on the size/volume of the sample, the matrix, i.e. 71 the nature of the sample, such as blood or cerebrospinal fluid, and on the pathophysiological 72 state of the patients. The control of DNA extraction is another critical step of molecular 73 diagnosis, but was not explored in our study. In addition, it is reported in the literature that 74 PCR methods differing by their primers and/or amplified sequence have variable 75 susceptibility to inhibitors [8-10]. These studies tend to invalidate the assumption that absence 76 of inhibition in a qPCR targeting any human gene has a good predictive value for assessing 77 inhibition in a qPCR targeting a pathogen. Consequently, it is critical to assess the 78 performances of human gene-based PCR inhibition screening methods in clinical samples in 79 clinical microbiology routine ...

show abstract

Characterization and Multicentric Validation of a Common Standard for Toxoplasma gondii Detection Using Nucleic Acid Amplification Assays

Cited by 26 publications

References 30 publications

Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines

Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines

Molecular diagnosis of toxoplasmosis at the onset of symptomatic primary infection: A straightforward alternative to serological examinations

Inhibition of Polymerase Chain Reaction: Pathogen-Specific Controls are better than Human Gene Amplification

Contact Info

Product

Resources

About