Comparison between Two Amplification Sets for Molecular Diagnosis of Toxoplasmosis by Real-Time PCR

Cassaing, Sophie; Bessières, M H; Berry, Antoine; Berrébi, Alain; Fabre, Richard; Magnaval, Jean-François

doi:10.1128/jcm.44.3.720-724.2006

Cited by 114 publications

(69 citation statements)

References 22 publications

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“…As previously described in other studies, this can be explained by the difference in the number of repetitions of this sequence (about 7-to 10-fold more repeated than B1) (11), which results in a gain of about 4 C T (12,14), as observed here (P Ͻ 0.001), and allows for the detection of 10-fold lower parasite loads (14). However, few studies evaluated the impact in terms of the sensitivity of diagnosis in routine clinical use.…”

Section: Discussionmentioning

confidence: 64%

A 10-Year Retrospective Comparison of Two Target Sequences, REP-529 and B1, for Toxoplasma gondii Detection by Quantitative PCR

et al. 2015

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bThis study aimed to evaluate the repeated sequence REP-529 compared to that of the B1 gene in the molecular diagnosis of toxoplasmosis by quantitative PCR (qPCR) in routine diagnosis. Over a 10-year period (2003 to 2013), all patients prospectively diagnosed with a positive REP-529 qPCR result for toxoplasmosis were included. All DNA samples (76 samples from 56 patients) were simultaneously tested using the two qPCR methods (REP-529 and B1). The mean cycle threshold (C T ) obtained with the B1 qPCR was significantly higher (؉4.71 cycles) than that obtained with REP-529 qPCR (P < 0.0001). Thirty-one out of 69 extracts (45.6%) positive with REP-529 qPCR were not amplified with the B1 qPCR (relative sensitivity of 54.4% compared to that with REP-529), yielding false-negative results with 15/28 placenta, 5 cord blood, 2 amniotic fluid, 4 cerebrospinal fluid, 1 aqueous humor, 2 lymph node puncture, and 1 abortion product sample. This defect in sensitivity would have left 20/56 patients undiagnosed, distributed as follows: 12/40 congenital toxoplasmosis, 4/5 cerebral toxoplasmosis, 2/8 patients with retinochoroiditis, and 2 patients with chronic lymphadenopathy. This poor performance of B1 qPCR might be related to low parasite loads, since the mean Toxoplasma quantification in extracts with B1 false-negative results was 0.4 parasite/reaction. These results clearly show the superiority of the REP-529 sequence in the diagnosis of toxoplasmosis by PCR and suggest that this target should be adopted as part of the standardization of the PCR assay.T oxoplasmosis is a worldwide parasitic infection due to the intracellular parasite Toxoplasma gondii. The infection is usually asymptomatic in immunocompetent patients and more rarely results in fever, lymphadenopathy, or retinochoroiditis. In contrast, immunocompromised patients can experience severe neurologic, ocular, pulmonary, or disseminated disease (1). Yet, toxoplasmosis is well known for its pathogenicity during pregnancy. Indeed, when primary infection occurs in pregnant women, it can lead to congenital toxoplasmosis, with a frequency of transmission and a severity of fetal infection depending on the stage of pregnancy at which infection occurs (2). The diagnosis of toxoplasmosis is routinely based on serology. In some countries, such as France, seronegative pregnant women are monitored monthly by serology. In case of seroconversion, the detection of T. gondii DNA by PCR is a major diagnostic method for congenital toxoplasmosis and is performed on amniotic fluid (prenatal diagnosis) (3-5) and placenta or cord blood samples at birth (postnatal diagnosis) (6-8). In immunocompromised patients, DNA can also be found in cerebrospinal fluid (CSF), bronchoalveolar lavage (BAL) fluid, or other samples, as guided by clinical signs. The 35-fold repeated B1 gene (9) has commonly been used for this molecular diagnosis since 1989, with acceptable sensitivity (3, 5, 10), but another sequence (REP-529, GenBank accession no. AF146527) was described more recently as being repeated 200 ...

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Section: Discussionmentioning

confidence: 64%

A 10-Year Retrospective Comparison of Two Target Sequences, REP-529 and B1, for Toxoplasma gondii Detection by Quantitative PCR

et al. 2015

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“…DNA sequence was chosen [27] and the formalin-fixation was restricted to 24 h. However, the fixation procedure might have accounted for a lower rate of positive PCR results due to DNA fragmentation, leading to an underestimation of the real infection frequency.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Involvement of Toxoplasma gondii in reproductive disorders in Swiss pig farms

Basso

Handke

Sydler

et al. 2015

Parasitology International

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To determine the role of Toxoplasma gondii in reproductive failure, 108 of 113 sows that had aborted or delivered stillborn or weak piglets from 58 Swiss farms were serologically tested for specific antibodies against T. gondii tachyzoite antigens by ELISA. Additionally, formalin-fixed and paraffin-embedded tissues from 123 foetuses or stillborn piglets derived from 25 seropositive and 27 seronegative sows were analyzed by real-time PCR for T. gondii DNA. Tissues from animals showing a positive reaction in real-time PCR were subsequently tested by immunohistochemistry for the presence of T. gondii antigens. Antibodies against T. gondii were detected in 24.1% (26 out of 108) of sows with reproductive failure, and 37.3% (22 of 58) of the 58 tested farms had seropositive sows. No significant differences in the prevalences were observed in relation to the housing system (exclusive indoor housing, indoor housing with outdoor yard and exclusive outdoor housing) neither at the individual nor at the farm levels. By real time-PCR, T. gondii DNA was detected in three placentas from one seropositive sow (abortion at 71 gestation days [gd]), and in brain tissues from one foetus (abortion at 76 gd), one stillborn (116 gd) and one mummy (112 gd) delivered by three further seropositive sows, but in no sample A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPTderived from seronegative dams. By immunohistochemical staining, the presence of T. gondii could be confirmed only in placenta samples. In one of the cases, a co-infection with porcine parvovirus (PPV) was detected. These results suggest vertical transmission of T. gondii and/or placental infection in at least 3.5% (4 of 113) of sows with reproductive disorders. Therefore, T. gondii should be more frequently included in the routine differential diagnosis of reproductive failure in sows. In addition, a proper disposal of placentas and abortion material beyond the reach of cats could help to interrupt the further dissemination of this parasite at the farm level.

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“…The sensitivity was higher with a real-time PCR assay targeting the T. gondii repeat element of 529 base pairs [68] than with real-time PCR targeting the B1 gene (40% and 36% respectively). Real-time PCR has been shown to be more sensitive on a variety of samples when the 529-bp repeat element rather than the B1 gene was used as a target [71]. In contrast to the IB and GWC results, the results of PCR are not influenced by the interval between symptom onset and paracentesis.…”

Section: Diagnosis Is Based On Clinical Signs and Some Selected Biolomentioning

confidence: 72%

“…We showed [68] that a combination of all three methods had a 85% sensitivity and a 93% specificity for the diagnosis of atypical or extensive toxoplasmic retinochoroiditis. The sensitivity of GWC alone for atypical uveitis (based mainly on aqueous humor samples) ranges from 39% to 93% [60,63,65,67,[69][70][71]. Discrepancies could be explained by differences in (i) the interval between symptom onset and paracentesis, (ii) the characteristics of the uveitis (typical or atypical), (iii) underlying immunological status, and (iv), the chosen GWC positivity threshold, which ranges from 2 to 8 in the literature.…”

Section: Diagnosis Is Based On Clinical Signs and Some Selected Biolomentioning

confidence: 99%

Risk Factors, Pathogenesis and Diagnosis of Ocular Toxoplasmosis

Dupouy‐Camet¹,

Talabani²,

Delair³

et al. 2012

Toxoplasmosis - Recent Advances

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Comparison between Two Amplification Sets for Molecular Diagnosis of Toxoplasmosis by Real-Time PCR

Cited by 114 publications

References 22 publications

A 10-Year Retrospective Comparison of Two Target Sequences, REP-529 and B1, for Toxoplasma gondii Detection by Quantitative PCR

A 10-Year Retrospective Comparison of Two Target Sequences, REP-529 and B1, for Toxoplasma gondii Detection by Quantitative PCR

Involvement of Toxoplasma gondii in reproductive disorders in Swiss pig farms

Risk Factors, Pathogenesis and Diagnosis of Ocular Toxoplasmosis

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