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

Abstract

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.

Evaluation of Serological and Molecular Tests Used for the Identification of Toxoplasma gondii Infection in Patients Treated in an Ophthalmology Clinic of a Public Health Service in São Paulo State, Brazil

“…Since there is no standardization to detect T. gondii by PCR, different protocols have been used (Roux et al, 2018;Greigert et al, 2019). Selection of primer, applied technology and a more suitable sample are some reasons for this challenge (Saadatnia and Golkar, 2012).…”

Serum IgG Anti-Toxoplasma gondii Antibody Concentrations Do Not Correlate Nested PCR Results in Blood Donors

“…Molecular methods, such as conventional PCR, Nested PCR, and real-time PCR have been used either in isolation or in association, to detect T. gondii parasitemia in acute and chronically infected individuals since they show high sensitivity (Brenier-Pinchart et al, 2015;Dard et al, 2016;Camilo et al, 2017;Roux et al, 2018;Botein et al, 2019;Greigert et al, 2019). In this study, we used the Nested PCR to target the B1 gene which is one of the most used tests in the literature for detecting T. gondii parasitemia (Okay et al, 2009;Mattos et al, 2011;Teixeira et al, 2013;Roux et al, 2018). Moreover, it has been demonstrated that the B1 gene might be targeted for molecular detection of T. gondii parasitemia in Brazilian samples, especially when the investigation is limited to one gene (Okay et al, 2009;Teixeira et al, 2013).…”

“…The diagnosis of infection by T. gondii is essentially serological but there are number of published papers demonstrating that the high sensitivity of molecular methods can offer more accurate results on the investigation of infection by this parasite (Mattos et al, 2011;Brenier-Pinchart et al, 2015;Robert-Gangneux et al, 2015;Camilo et al, 2017;Murata et al, 2017;Roux et al, 2018;Greigert et al, 2019;Lévêque et al, 2019;Pleyer et al, 2019). The combination of serology and molecular methods has been used to improve the diagnosis of infection by T. gondii.…”

Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens

“…Finally, this discrepancy could also be explained by the tandem between extraction method and qPCR assay. Indeed, as previously described for other pathogens, it is essential to check the correct match between the DNA extraction and the PCR method [ 20 ]. This can be observed in our study, as we found for manual extraction a sensitivity of 79% for the qPCR published by Stensvold et al and 71% for the method developed by Poirier et al However, the Poirier et al method detected significantly more positive samples (53%) with automated extracts compared to the qPCR published by Stensvold et al (34%).…”