A second European collaborative study on polymerase chain reaction for<i>Toxoplasma gondii</i>, involving 15 teams

Pelloux, H; Guy, Edward; Angelici, Maria Cristina; Aspöck, Horst; Bessières, Marie-Hélène; Blatz, R; Pezzo, Mariassunta Del; Girault, Véronique; Gratzl, R.; Holberg-Petersen, Mona; Johnson, Julie; Krüger, Dominique; Lappalainen, Maija; Naessens, Anne; Olsson, Mats Joakim Robert

doi:10.1111/j.1574-6968.1998.tb13151.x

Cited by 82 publications

(6 citation statements)

References 17 publications

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“…In this study, T. gondii DNA was detected only in 2/5 IgM positive women who have had spontaneous abortions and 3/11 women showed no evidence of infection by PCR, though IgG antibodies were detected. This can be attributed to the presence of a long standing immunity to toxoplasmosis or cross-reactive antibodies [ 58 , 59 ] and confirm the sensitivity and specificity of PCR analysis for detecting recent infection in early pregnancy [ 60 ]. This is in agreement with previous reports that PCR is recommended over serologic techniques for diagnosis of toxoplasmosis [ 61 – 63 ].…”

Section: Discussionmentioning

confidence: 68%

Molecular diagnosis of Toxoplasma gondii infection in Libya

et al. 2016

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BackgroundToxoplasma gondii infections are prevalent in humans and animals throughout Libya. Current diagnosis is based on detection of Toxoplasma-specific IgM and IgG. In this study, we established and optimized a diagnostic PCR assay for molecular diagnosis of T. gondii in Libya.MethodsFrom January to December, 2010, 177 blood and serum samples were collected from suspected patients. This includes: 140 women who have had spontaneous abortions, 26 HIV-positive patients, nine patients with leukemia and lymphoma, and two infants with ocular infection. Samples were screened for anti-Toxoplasma IgG and IgM antibodies before DNA extraction. The surface antigen gene 2 (SAG2) was targeted in a semi-nested PCR to amplify a 999 bp and a 614 bp fragment in the first and the second run respectively.ResultsA total of 54/140 (38.5 %) women who have had spontaneous abortions, 23/26 (88 %) HIV patients, 6/9 (66.6 %) of the leukaemia and lymphoma patients, and one child with ocular infection were seropositive for anti-Toxoplasma IgG and/or IgM. Genomic DNA was extracted from 38 selected seropositive samples. The PCR was sensitive enough to detect DNA concentration of 12 ng/μL. PCR analysis was performed for 38 selected seropositive patients (16 women who have had spontaneous abortions, 15 positive HIV patients, six leukaemia patients and one child with ocular infection). Our designed primers were successfully amplified in 22/38 (57.9 %) samples; 5/12 (35.7 %) from serum and 17/26 (65.8 %) from whole blood samples. All PCR positive samples were IgG-positive except two samples which were IgM and IgG & IgM-positive serum samples respectively. The semi-nested PCR confirmed five more samples. These included two leukaemia and two HIV-positive whole blood samples and one serum sample from an aborted woman.ConclusionThe ability of PCR to diagnose active toxoplasmosis is needed in immunocompromised patients and congenital toxoplasmosis cases, especially when serological techniques fail. For the first time in Libya, we established and optimized semi-nested PCR of SAG2 gene. The developed PCR method was able to detect as little as 12 ng/μL of T. gondii DNA and was useful to diagnose the diseases in women who have had spontaneous abortions, HIV-positive patients, patients with leukemia and lymphoma, and infants with ocular infection.

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Section: Discussionmentioning

confidence: 68%

Molecular diagnosis of Toxoplasma gondii infection in Libya

et al. 2016

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“…It is strongly suggested that individual laboratories should carry out their own characterization if these parameters are modified, and even if no parameter is modified at all. To this aim, a series of “reference samples” should be maintained and provided by a “reference laboratory” to any laboratory intending to establish and maintain an accurate diagnostic test 41 . The organization of training sessions by these reference laboratories would also help to share experience and knowledge about the use of a given protocol, and would harmonize the practices and decision rules.…”

Section: Discussionmentioning

confidence: 99%

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum

Ioos¹,

Aloi

Piškur

et al. 2019

Sci Rep

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Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii , causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F . circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F . circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F . circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

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“…The development of quantitative real-time PCR (qrt-PCR) has therefore brought to this diagnosis not only robustness but also the possibility of quantifying parasitic loads (4), with the aim of establishing a correlation with the severity of the disease (5,6). 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.…”

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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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A second European collaborative study on polymerase chain reaction forToxoplasma gondii, involving 15 teams

Cited by 82 publications

References 17 publications

Molecular diagnosis of Toxoplasma gondii infection in Libya

Molecular diagnosis of Toxoplasma gondii infection in Libya

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum

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

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