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

Abstract

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.

Molecular Diagnosis of Toxoplasmosis in Immunocompromised Patients: a 3-Year Multicenter Retrospective Study

“…Internal controls and negative and positive controls were included in each PCR run. All assays had been previously validated by multicenter evaluations (21,22) and are routinely evaluated through regular national external quality assessments (18).…”

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

“…Yet the molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and still suffers from lack of standardization (7,8). This in turn leads to variations in the performances (essentially in sensitivity) of the PCR assays (9,10) and hence in the quality of patient management. In France, where congenital toxoplasmosis is considered a public health problem and benefits from a national prevention program (11,12), the Molecular Biology "pole" of the French National Reference Center for Toxoplasmosis (NRC-T; http://cnrtoxoplasmose.chu-reims.fr) was created with the aim of standardizing this molecular diagnosis.…”

Quality Control for the Molecular Diagnosis of Toxoplasmosis

“…DNA can be used as such in its own extraction buffer. However, our experience demonstrated that the DNA extraction process and the PCR assay both contribute to the performance of the whole diagnostic method (Sterkers et al, 2010); thus, this form of EQA does not allow testing the DNA extraction method, and this can even lead to wrong assessments of the method used in the participating laboratories (Molecular biology working group of the CNRT, unpublished data). Therefore, the use of whole tachyzoites is preferred.…”

“…On top of these national 'core' EQAs, more limited and specific ('scientific') EQAs aiming at refining comparative studies between PCR methods were implemented at a smaller scale. A multicentric prospective study, involving eight laboratories proficient in the molecular prenatal diagnosis of toxoplasmosis, was a first step towards the harmonization of this diagnosis among university hospitals in France (Sterkers et al, 2010). It aimed at comparing the analytical performances of different PCR protocols used for Toxoplasma detection, and was reproduced over two consecutive years.…”

“…Each centre extracted the same concentrated T. gondii suspension and tested serial dilutions of the extracted DNA using its own assays. Since all reactions are not positive when the concentration of pathogens gets to the sensitivity limit of the method (Chabbert et al, 2004;Sterkers et al, 2010), original 'performance scores' were defined, taking into account the proportion of positive reactions over the number of reactions performed, which appeared as a good indicator of sensitivity. Differences in analytical sensitivities were observed between assays, particularly at low parasite concentrations (≤ 2 T. gondii genomes per reaction tube, i.e.…”