Use of the polymerase chain reaction to detect Toxoplasma gondii in human blood samples.

Ho-Yen, D. O.; Joss, A. W. L.; Balfour, A H; Smyth, E.T.M.; Baird, Douglas; Chatterton, J. M. W.

doi:10.1136/jcp.45.10.910

Cited by 55 publications

(52 citation statements)

References 12 publications

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“…Ho-Yen (28) suggested that the samples should be collected prior to antibiotic treatment, but not later that 2 to 3 days after the fi rst examination. Antibiotic treatment usually leads to a rapid elimination of the parasite and the DNA degradation, thus producing false negative PCR results (2).…”

Section: Discussionmentioning

confidence: 99%

Molecular diagnosis of Toxoplasma gondii in pregnant women

Turčeková

Spišák²,

Dubinský³

et al. 2012

BLL

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Abstract:Background: Novel approaches in the diagnostics of T. gondii have enabled a detection of the parasite in the amniotic fl uid or blood of pregnant women. Objective: The high titres of IgM and IgG antibodies against T. gondii are not always indicative of the presence of this parasite in pregnant women, therefore the molecular assays can be used to diagnose and genetically characterise T. gondii in amniotic fl uids and blood samples. Methods: The study analysed 15 samples of amniotic fl uid and 1 sample of the blood from pregnant women suspected for toxoplasmosis. The serological ELISA test was used for the immunological study and molecular analyses, PCR at SAG2 locus followed by RFLP analysis were used for Toxoplasma gondii genotyping. Conclusion: Using PCR assay with TGR1E gene we have confi rmed the presence of T. gondii in the blood of a pregnant woman. The parasite was typed as genotype I, belonging to virulent strains (Tab. 1, Fig. 2

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Section: Discussionmentioning

confidence: 99%

Molecular diagnosis of Toxoplasma gondii in pregnant women

Turčeková

Spišák²,

Dubinský³

et al. 2012

BLL

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“…More recently, immunoblotting (19,28) and gene amplification techniques (5,20) have been implemented.…”

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confidence: 99%

Comparison of Enzyme-Linked Immunosorbent Assay, Immunoblotting, and PCR for Diagnosis of Toxoplasmic Chorioretinitis

et al. 2003

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Ocular toxoplasmosis is the major cause of posterior uvetis in European populations. The clinical diagnosis of toxoplasmic chorioretinitis is based upon ophthalmoscopic findings, which are often but not always typical. Laboratory testing is therefore important to confirm the etiology of the disease. In the present 2-year prospective study, the relative diagnostic sensitivities of the three analytical techniques (enzyme-linked immunosorbent assay [ELISA], immunoblotting, and PCR) were compared by using a group of patients (n ‫؍‬ 19) with suspected ocular toxoplasmosis. The relative specificities of the three techniques were assessed by including two control groups of patients: one with nontoxoplasmic and noninflammatory ocular disease (n ‫؍‬ 48) and the other with nontoxoplasmic and inflammatory ocular disease (n ‫؍‬ 20). All 19 of the clinically suspect patients had serological evidence of exposure to Toxoplasma gondii: 17 had been previously infected, and 2 had current infection. The analysis of paired aqueous humor and serum samples by ELISA and immunoblotting revealed the local production of specific antibodies of the immunoglobulin G type in 63% (12 of 19) and 53% (10 of 19) of patients, respectively. PCR analysis of aqueous humor samples confirmed the presence of T. gondii DNA in 28% (5 of 18) of cases. When combined, ELISA, immunoblotting, and PCR findings confirmed the toxoplasmic origin of retinal lesions in 83% (15 of 18) of patients. The relative specificities of the three techniques were 89% for ELISA and immunoblotting and 100% for PCR.

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“…A nested PCR was performed as described previously [8,9] but with a different source of Taq polymerase, reaction buffer (1 0 X concentrated) and MgC12 (all from Advanced Biotechnologies Ltd, Leatherhead, Surrey). Each PCR reaction contained 0.8 u of enzyme, 1 X buffer, 1.5 mM MgC12, 0.2 mM each dATP, dCTP, dGTP and dTTP (Advanced Biotechnologies) and 20 pmols of each primer.…”

Section: Pcrmentioning

confidence: 99%

“…Samples (20 p1) were tested in a final reaction volume of 50 p l as described previously [8,9]. The PCR positive controls were suspensions of 10 and 1 RH tachyzoite/20 p l of water Compared to the method described previously [8,9], the number of cycles in the first stage PCR was increased from 30 to 40 cycles of 94°C for 1 min, 53°C for 1 min, 72°C for 1 min, followed by a 5-min extension at 72°C in a Techne PHCl or PHC3 programmable Dri-block (Scotlab, Aberdeen). For the nested PCR, the number of cycles was reduced from 17 to 14 cycles of 94°C for 1 min, 53°C for 30 s, 72°C for 30 s, followed by a 5-min extension, performed on a 1 in 80 dilution in SDW of the primary product.…”

Section: Pcrmentioning

confidence: 99%

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The effects of sample storage on polymerase chain reaction-based detection of Toxoplasma gondii in amniotic fluids

Joss

Ho-Yen

1997

Journal of Medical Microbiology

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The effects of sample storage, target preparation and annealing temperature on a nested polymerase chain reaction (PCR) test for Toxoplasma gondii DNA were investigated with experimentally seeded amniotic fluids to simulate congenital infection. Aliquots of 17 amniotic fluid samples remaining after investigation for conditions other than toxoplasmosis and seeded to contain small numbers of T. gondii tachyzoites, were tested after storage for up to 2 weeks. There was no deterioration in test sensitivity on samples stored for 1-2 weeks at room temperature; thus, samples sent by post to reference laboratories are acceptable for examination. There was no significant difference in the results from two target preparation methods or two different annealing temperatures. One-to-ten parasites were detectable in 13 of 17 test samples. The significant feature in the remaining four samples was the use of washed stored parasites to seed the amniotic fluids rather than any feature of the amniotic fluids used or the test. False positive results were found in up to 15% of unseeded amniotic fluid samples, which contrasts with only 1.9% for the same PCR test in other routine or negative control samples tested as part of the laboratory's reference diagnostic work. The difference illustrates the increased risk of cross-contamination in PCR when the majority of specimens tested are positive. The results suggest that PCR techniques are likely to be sensitive and effective tools for the routine diagnosis of congenital toxoplasma infection.

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Use of the polymerase chain reaction to detect Toxoplasma gondii in human blood samples.

Abstract: Aims: To assess the value of detecting Toxoplasma gondii in human blood samples using the polymerase chain reaction (PCR). (7 Clin Pathol 1992;45:910-913)

Cited by 55 publications

References 12 publications

Molecular diagnosis of Toxoplasma gondii in pregnant women

Molecular diagnosis of Toxoplasma gondii in pregnant women

Comparison of Enzyme-Linked Immunosorbent Assay, Immunoblotting, and PCR for Diagnosis of Toxoplasmic Chorioretinitis

The effects of sample storage on polymerase chain reaction-based detection of Toxoplasma gondii in amniotic fluids

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