Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes

Costa, Jean-Marc; Ernault, Pauline; Gautier, E; Bretagne, Stéphane

doi:10.1002/1097-0223(200102)21:2<85::aid-pd18>3.0.co;2-1

Cited by 90 publications

(82 citation statements)

References 26 publications

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“…2,10,[12][13][14]29 Currently, the diagnosis of toxoplasmosis in transplant patients is commonly based on nested PCR and, more recently, real-time PCR because these have been reported to have high sensitivity and specificity. 2,9,12,[15][16][17][18][19][21][22][23] Thus, in the present study, a group of liver recipients under immunosuppressive treatment were screened for 4 months with real-time PCR, nested PCR, the Sabin-Feldman dye test, EIA IgM, and ISAGA IgM in order to compare the diagnostic value of these assays. We believe that this is the first time such a systematic comparison of this range of methods has been presented.…”

Section: Discussionmentioning

confidence: 99%

“…[21][22][23][24] Briefly, the primers used for amplifying The PCR amplification reactions were performed with the following calculated control protocol: a 10-minute preincubation step at 95°C followed by 45 cycles of 10 seconds at 95°C, 5 seconds at 60°C, and 5 seconds at 72°C. As positive controls, T. gondii genomic DNA, serially 10-fold diluted and ranging from 5000 to 0.5 parasites per microliter (TIB Molbiol), and 1 negative control, prepared by the replacement of template DNA with distilled water, were used in each nested and real-time PCR run.…”

Section: Dna Extraction and Pcr Analysismentioning

confidence: 99%

“…15,20 More recently, quantitative real-time PCR has been employed to assess changes in the levels of T. gondii DNA in order to monitor the efficacy of treatment. 17,18,[21][22][23] Although toxoplasmosis has been reported to be uncommon in liver transplant recipients, there is only limited evidence addressing the rate of transplant-mediated transmission. 2,5,6,14 Thus, the present study was designed to assess the rate of primary or reactivated toxoplasmosis associated with liver transplantation.…”

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confidence: 99%

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Incidence and diagnosis of active toxoplasma infection among liver transplant recipients in Western Turkey

et al. 2008

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Toxoplasmosis is a serious and potentially life-threatening disease in liver transplant recipients while they are immunosuppressed. We report the clinical and laboratory findings related to active toxoplasma infection associated with 40 immunosuppressed liver transplant procedures that took place over a 12-month period at a major transplant unit in Izmir, Turkey. Twenty-seven (67.5%) of the 40 transplant recipients were found to be seropositive for toxoplasma infection and therefore at risk of reactivated infection. From the serological status of the donors, which was ascertained in 38 of 40 cases, we identified 3 (7.9%) of 38 transplants to be from a seropositive donor to a seronegative recipient. In 10 (26.3%) of 38 transplants, both the donor and recipient were seronegative, and this excluded toxoplasma as a risk. A comparison of real-time polymerase chain reaction (PCR) and nested PCR was undertaken in combination with a range of serological assays (the Sabin-Feldman dye test, enzyme immunoassay immunoglobulin M, and immunosorbent agglutination assay immunoglobulin M). Ethylene diamine tetraacetic acid blood samples from 3 of the 30 recipients at risk from toxoplasma were found positive by PCR, but only 1 of these was found positive in both assays. Among the 3 PCR-positive patients, immunoglobulin M and immunoglobulin G antibody levels increased in only 1 patient. Correlations between symptoms, laboratory findings, and clinical management (use of anti-toxoplasma therapy) are presented. Our findings suggest that toxoplasma presents a significant risk to our liver transplant population and that PCR is a helpful addition in identifying active infections and hence in informing clinical management decisions. Liver Transpl 14:1526Transpl 14: -1532Transpl 14: , 2008

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Section: Discussionmentioning

confidence: 99%

Section: Dna Extraction and Pcr Analysismentioning

confidence: 99%

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Incidence and diagnosis of active toxoplasma infection among liver transplant recipients in Western Turkey

et al. 2008

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“…Second, this gave a strong impulse towards freeze drying as the most adapted preparation method for EQAs (see below). The demonstration of low parasite loads in a large proportion of infected AF samples (<10 tachyzoites per mL) (Costa et al, 2001;Romand et al, 2004) makes it necessary to test sensitivity with low Toxoplasma concentrations. Moreover, this choice allows a finer and more stringent assessment of the performances of the methods used by each laboratory year by year, as diagnostic methods for pathogens are particularly fallible with low concentrations of pathogens in the biological sample (Chernesky et al, 2002;Kaiser et al, 2007;Lachaud et al, 2001;Lachaud et al, 2002;Pelloux et al, 1998).…”

Section: Specificities Of Eqas For Toxoplasma Molecular Detectionmentioning

confidence: 99%

“…The choice of the parasite concentrations depends upon the objectives of the EQA and the proficiency of the participants. In France, we opted for low concentrations of parasites (i) because it has been established that a large proportion of infected AFs contain Toxoplasma loads < 10 tachyzoites per ml (Costa et al, 2001;Romand et al, 2004), (ii) because diagnostic methods for pathogens are particularly fallible with low concentrations of pathogens in the biological sample (Pelloux et al, 1998), (Chabbert et al, 2004;Chernesky et al, 2002;Kaiser et al, 2007;Lachaud et al, 2001;Lachaud et al, 2002) and (iii) because most participating laboratories were proficient in this diagnosis. The highest concentrations that were ever sent were 100 and 50 tachyzoites per mL.…”

Section: Different Protocols For Eqa Of Toxoplasma-pcrmentioning

confidence: 99%

Quality Control for the Molecular Diagnosis of Toxoplasmosis

Varlet‐Marie¹,

Sterkers²,

Bastien³

2011

Applications and Experiences of Quality Control

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Real-Time Quantitative Polymerase Chain Reaction Diagnosis of Infectious Posterior Uveitis

Dworkin

Gibler²,

Gelder³

2002

Arch Ophthalmol

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To validate a real-time polymerase chain reaction (PCR) assay allowing rapid and sensitive detection and quantitation of 4 common infectious posterior uveitis pathogens. Methods: A real-time PCR assay using previously validated primer sets for cytomegalovirus, herpes simplex virus, varicella-zoster virus, and Toxoplasma gondii was developed. A standard curve for quantitation of pathogen load was generated for each pathogen using SYBR Green I fluorescence detection. Ocular samples from patients with posterior uveitis and from negative control samples were assayed and compared with standards to identify pathogens and quantify infectious load. Results: Sensitivity for detection of purified pathogen DNA by PCR was not reduced by application of the real-time method. Standard curves for the quantitation of pathogen loads showed sensitivity to fewer than 10 organisms for all pathogens. The technique was applied to 2 clinical problems. First, sensitivities of existing monoplex and multiplex PCR were compared by real-time PCR. No significant difference in sensitivity was observed between multiplex and monoplex techniques. Second, pathogen loads of vitreous specimens from patients previously diagnosed as having infectious posterior uveitis were calculated. Pathogen loads were found to be generally higher for patients with disease caused by varicella-zoster virus than those caused by cytomegalovirus or herpes simplex virus. Conclusions: Real-time PCR may be applied to infectious agents responsible for posterior uveitis. This technique will likely prove useful for the diagnosis of posterior uveitis as well as the linkage of pathogen to disease. Clinical Relevance: Real-time PCR provides a rapid technique for quantitatively evaluating ocular samples for the presence of infectious pathogens.

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Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes

Cited by 90 publications

References 26 publications

Incidence and diagnosis of active toxoplasma infection among liver transplant recipients in Western Turkey

Incidence and diagnosis of active toxoplasma infection among liver transplant recipients in Western Turkey

Quality Control for the Molecular Diagnosis of Toxoplasmosis

Real-Time Quantitative Polymerase Chain Reaction Diagnosis of Infectious Posterior Uveitis

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