Three years of multi-laboratory external quality control for the molecular detection of Toxoplasma gondii in amniotic fluid in France

Bastien, Patrick; Jumas-Bilak, E.; Varlet‐Marie, Emmanuelle; Marty, Pierre

doi:10.1111/j.1469-0691.2006.01642.x

Cited by 35 publications

(40 citation statements)

References 21 publications

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“…Here, we used calibrated samples of human AF because (i) it perfectly mimics diagnostic samples for congenital toxoplasmosis, (ii) it constitutes an easily obtainable and abundant source of matrix, and (iii) the development of standard material from this type of matrix is more advanced than the development of that from blood samples (9,28). A minor drawback is that AF drawn from hydramnios is often naturally diluted and hence paucicellular and may not precisely reproduce the composition of AF samples tested in routine.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2014

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The molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and suffers from lack of standardization, hence the large diversity of performances between laboratories. Moreover, quantifications of parasitic loads differ among centers, a fact which prevents the possible prediction of the severity of this disease as a function of parasitic loads. The objectives of this multicentric study performed in eight proficient laboratories of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (NRC-T) were (i) to assess the suitability of a lyophilized preparation of Toxoplasma gondii as a common standard for use in this PCR-based molecular diagnosis and (ii) to make this standard available to the community. High-quality written procedures were used for the production and qualification of this standard. Three independent batches of this standard, containing concentrations ranging from 10 4 to 0.01 T. gondii genome equivalents per PCR, were first assessed: the linear dynamic range was >6 log, the intra-assay coefficients of variation (CV) from a sample containing 10 T. gondii organisms per PCR were 0.3% to 0.42%, and the interassay CV over a 2-week period was 0.76% to 1.47%. A further assessment in eight diagnostic centers showed that the standard is stable, robust, and reliable. These lyophilized standards can easily be produced at a larger scale when needed and can be made widely available at the national level. To our knowledge, this is the first quality control assessment of a common standard which is usable both for self-evaluation in laboratories and for accurate quantification of parasitic loads in T. gondii prenatal infections.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2014

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“…This situation has well-known drawbacks, particularly a lack of standardization and variations in efficiency (12,17,21,25). In addition to this diversity, external quality assessments or interlaboratory comparative studies for the molecular detection of Toxoplasma gondii are scarce: five of these have been carried out in the last 10 years, all in Europe and all demonstrating wide divergences in the performances of PCR methods (2,12,17,21,25). In France, a network has been set up for the improvement and standardization of this molecular diagnosis within the framework of the recently created National Reference Centre for Toxoplasmosis (http://www.chu-reims.fr /professionnels/cnr-toxoplasmose-1/).…”

mentioning

confidence: 99%

Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens

et al. 2010

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Although screening for maternal toxoplasmic seroconversion during pregnancy is based on immunodiagnostic assays, the diagnosis of clinically relevant toxoplasmosis greatly relies upon molecular methods. A problem is that this molecular diagnosis is subject to variation of performances, mainly due to a large diversity of PCR methods and primers and the lack of standardization. The present multicentric prospective study, involving eight laboratories proficient in the molecular prenatal diagnosis of toxoplasmosis, was a first step toward the harmonization of this diagnosis among university hospitals in France. Its aim was to compare the analytical performances of different PCR protocols used for Toxoplasma detection. Each center extracted the same concentrated Toxoplasma gondii suspension and tested serial dilutions of the DNA using its own assays. Differences in analytical sensitivities were observed between assays, particularly at low parasite concentrations (<2 T. gondii genomes per reaction tube), with "performance scores" differing by a 20-fold factor among laboratories. Our data stress the fact that differences do exist in the performances of molecular assays in spite of expertise in the matter; we propose that laboratories work toward a detection threshold defined for a best sensitivity of this diagnosis. Moreover, on the one hand, intralaboratory comparisons confirmed previous studies showing that rep529 is a more adequate DNA target for this diagnosis than the widely used B1 gene. But, on the other hand, interlaboratory comparisons showed differences that appear independent of the target, primers, or technology and that hence rely essentially on proficiency and care in the optimization of PCR conditions.

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“…Considering the 6-10 T/mL concentration which corresponds to the parasitic load in half of the amniotic fluids according to Costa et al (Costa et al, 2001), false-negative results represented 7.1% of 126 samples. It is interesting to note that over the whole period of our study, except in 2007, the number of false negatives decreased over the years from 6.7% in 2002(Bastien et al, 2007 (Table 1). The falsenegative results did not appear to be related to a particular PCR method (e.g., real-time, DNA target or primer pair), but can be attributed to technical proficiency, PCR optimization and laboratory practices.…”

Section: Qualitative Resultsmentioning

confidence: 72%

“…Briefly, on a yearly basis, a panel of positive and negative amniotic fluid samples prepared in Montpellier was sent blinded to participating centers for PCR testing, allowing each center to assess and follow its own performances in the molecular detection of CT (Bastien et al, 2007). This EQA was then continued by the working group for molecular diagnosis of the CNRT.…”

Section: Eight Years Of National Eqasmentioning

confidence: 99%

“…Yet, we believe further testing is required to assess the extraction efficiency of low concentrations of T. gondii in plasma. All EQAs for Toxoplasma-PCR published to date used panels consisting of samples of human amniotic fluid (Bastien et al, 2007, Kaiser et al, 2007. Although this avoids tackling with the problem of PCR inhibition, it allows good extraction efficiency and perfect mimicry of a diagnostic sample.…”

Section: Different Protocols For Eqa Of Toxoplasma-pcrmentioning

confidence: 99%

See 1 more Smart Citation

Quality Control for the Molecular Diagnosis of Toxoplasmosis

Varlet‐Marie¹,

Sterkers²,

Bastien³

2011

Applications and Experiences of Quality Control

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Three years of multi-laboratory external quality control for the molecular detection of Toxoplasma gondii in amniotic fluid in France

Cited by 35 publications

References 21 publications

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

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

Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens

Quality Control for the Molecular Diagnosis of Toxoplasmosis

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