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Find video protocols related to scientific articles indexed in Pubmed.
Multicentric comparative assessment of the Bio-Evolution® Toxoplasma gondii detection kit with eight laboratory-developed PCR assays for the molecular diagnosis of congenital toxoplasmosis.
J. Clin. Microbiol.
PUBLISHED: 10-24-2014
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Detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and today is essentially based upon PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR have been recently developed and offer certain advantages; however they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed at comparing the Bio-Evolution® Toxoplasma gondii detection kit with laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found a concordance of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated at 86% (54/63); specificity was 100% for all. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target 'rep529', use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of Uracil-N-glycosylase, as well as small defects in the reliability of the production of different reagents.
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Characterization and Multicentric Validation of a Common Standard for Toxoplasma gondii Detection Using Nucleic Acid Amplification Assays.
J. Clin. Microbiol.
PUBLISHED: 09-03-2014
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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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Freezing and storage at -20 °C provides adequate preservation of Toxoplasma gondii DNA for retrospective molecular analysis.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 04-04-2014
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Nucleic acid-based testing has become crucial for toxoplasmosis diagnosis. For retrospective (forensic or scientific) studies, optimal methods must be employed for DNA long-term storage. We compared Toxoplasma gondii detection before and after DNA storage using real-time PCR. No significant differences were found depending on duration or storage conditions at -20 °C or -80 °C.
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New description of Toxoplasma gondii genotypes from French Polynesia.
Acta Trop.
PUBLISHED: 01-22-2014
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We report here the first isolation and genotyping of two human Toxoplasma gondii strains from French Polynesia. The parasites had new and atypical genotypes, and were responsible for asymptomatic congenital toxoplasmosis. Both genotypes were divergent from the common strains isolated in Europe, North America, South America, Africa and China.
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Plasmodium falciparum Rab5B is an N-terminally myristoylated Rab GTPase that is targeted to the parasite's plasma and food vacuole membranes.
PLoS ONE
PUBLISHED: 01-01-2014
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Plasmodium falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport. However, PfRab5B is unique in lacking a C-terminal geranyl-geranylation motif, while having N-terminal palmitoylation and myristoylation motifs. We show that the N-terminal glycine is required for PfRab5B myristoylation in vitro and when an N-terminal PfRab5B fragment possessing both acylation motifs is fused to GFP and expressed in transgenic P. falciparum parasites, the chimeric PfRab5B protein localizes to the plasma membrane. Upon substitution of the modified glycine by alanine the staining becomes diffuse and GFP is found in soluble subcellular fractions. Immuno-electron microscopy shows endogenous PfRab5B decorating the parasite's plasma and food vacuole membranes. Using reverse genetics rab5b couldn't be deleted from the haploid genome of asexual blood stage P. berghei parasites. The failure of PbRab5A or PbRab5C to complement for loss of PbRab5B function indicates non-overlapping roles for the three Plasmodium Rab5s, with PfRab5B involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane. We discuss similarities between Plasmodium Rab5B and Arabidopsis thaliana ARA6, a similarly unusual Rab5-like GTPase of plants.
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First identification of eggs of the Asian fish tapeworm Bothriocephalus acheilognathi (Cestoda: Bothriocephalidea) in human stool.
Parasitol. Int.
PUBLISHED: 02-10-2013
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We report the first case of egg isolation of the Asian fish tapeworm Bothriocephalus acheilognathi (Bothriocephalidea) from human stool. A male patient from Saint Laurent du Maroni (French Guiana) presenting abdominal pain was examined in France for the diagnosis of intestinal parasites. Diphyllobothrium-like eggs were observed in his stool. However, molecular phylogenetic analyses based on sequences of rDNA and COI genes showed that the eggs observed belong to a bothriocephalidean cestode B. acheilognathi. The adult life stages of B. acheilognathi cestodes are known as invasive parasites of a wide spectrum of fish; however, they have not been described to parasitize any mammals. This human infection seems to be accidental and represents a parasite passage through human intestine after the consumption of an infected fish host.
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Contribution of molecular diagnosis to congenital toxoplasmosis.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 01-16-2013
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We evaluated the performance of three real-time polymerase chain reaction (PCR) assays on 73 samples from mothers and children with congenital toxoplasmosis. PCR assays had significantly higher sensitivity in prenatal period than in birth period when targeting the 529-bp repeat element (81.3% versus 36.0%) or the B1 gene (64.6% versus 20.0%).
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Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites.
Biol. Cell
PUBLISHED: 07-20-2011
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The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized.
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Toxoplasma gondii infection in slaughter pigs in Serbia: seroprevalence and demonstration of parasites in blood.
Vet. Res.
PUBLISHED: 02-01-2011
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A seroepizootiological study of Toxoplasma gondii infection involving a total of 488 slaughter pigs (468 market-weight pigs and 20 sows) in the Belgrade area, also included examination of the presence of T. gondii in the blood. Blood sampled at the slaughter line was examined for specific antibodies by modified direct agglutination, and blood clots of those seropositive at titres of 1:50-1:12800 were bioassayed in mice. The overall seroprevalence was 9.2%, significantly higher (p = 0.0063) in sows (30.0%) than in market-weight pigs (8.3%). Amongst the 22 bioassays performed, a total of 16 (72.7%) were positive, by observation of T. gondii cysts (12), seropositivity (7, including 3 in which cysts were not detected), and/or detection of T. gondii DNA by real-time PCR (12, including one otherwise negative). The positive bioassays originated from the blood of 12 market-weight pigs and 4 sows. Despite a general increase in the rate of demonstration of T. gondii with the increase in the specific antibody level, the association was not significant (p = 0.101). The risk of infection was 41-fold increased in sows vs market-weight pigs, and 15-fold in pigs from smallholders finishing type farms vs those from large farrow-to-finish farms. The presence of viable T. gondii in a proportion of the samples indicates that some of the pigs had an active parasitaemia at the time of slaughter, which, along with the seroprevalence established, points to a potential source of human infection in Serbia. This is the first report on parasitaemia in naturally infected swine.
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Occupational asthma induced by Chrysonilia sitophila in a worker exposed to coffee grounds.
Clin. Vaccine Immunol.
PUBLISHED: 08-04-2010
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A new case of occupational asthma caused by Chrysonilia sitophila (asexual state of Neurospora sitophila) was diagnosed by molecular identification of the mold and confirmed by skin prick test, peak expiratory flow rate measurements, and experimental immunoglobulin E analysis.
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Multicentric comparative analytical performance study for molecular detection of low amounts of Toxoplasma gondii from simulated specimens.
J. Clin. Microbiol.
PUBLISHED: 07-07-2010
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Although screening for maternal toxoplasmic seroconversion during pregnancy is based on immunodiagnostic assays, the diagnosis of clinically relevant toxoplasmosis greatly relies upon molecular methods. A problem is that this molecular diagnosis is subject to variation of performances, mainly due to a large diversity of PCR methods and primers and the lack of standardization. The present multicentric prospective study, involving eight laboratories proficient in the molecular prenatal diagnosis of toxoplasmosis, was a first step toward the harmonization of this diagnosis among university hospitals in France. Its aim was to compare the analytical performances of different PCR protocols used for Toxoplasma detection. Each center extracted the same concentrated Toxoplasma gondii suspension and tested serial dilutions of the DNA using its own assays. Differences in analytical sensitivities were observed between assays, particularly at low parasite concentrations (
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Congenital toxoplasmosis after a preconceptional or periconceptional maternal infection.
Pediatr. Infect. Dis. J.
PUBLISHED: 06-30-2009
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We report a case of asymptomatic congenital toxoplasmosis following a periconceptional infection of the mother. Fetal infection is very uncommon in such a situation, and mostly associated with fetal damage. We recommend that newborns undergo postnatal screening, even after maternal periconceptional infection, and receive specific therapy to reduce long-term sequelae.
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Genotype of 88 Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings.
J. Infect. Dis.
PUBLISHED: 03-07-2009
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We report the genotyping analysis of Toxoplasma gondii isolates in samples collected from 88 immunocompromised patients, along with clinical and epidemiological data. Most of these samples were collected in France during the current decade by the Toxoplasma Biological Resource Center. Lack of specific anti-Toxoplasma treatment, pulmonary toxoplasmosis, and involvement of multiple organs were the 3 main risk factors associated with death for this patient group. Genotyping results with 6 microsatellite markers showed that type II isolates were predominant among patients who acquired toxoplasmic infection in Europe. Non-type II isolates included 13 different genotypes and were mainly collected from patients who acquired toxoplasmosis outside Europe. Type III was the second most common genotype recovered from patients, whereas type I was rare in our population. Three nonarchetypal genotypes were repeatedly recovered from different patients who acquired the infection in sub-Saharan Africa (genotypes Africa 1 and Africa 2) and in the French West Indies (genotype Caribbean 1). The distribution of genotypes (type II vs. non-type II) was not significantly different when patients were stratified by underlying cause of immunosuppression, site of infection, or outcome. We conclude that in immunocompromised patients, host factors are much more involved than parasite factors in patients resistance or susceptibility to toxoplasmosis.
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Congenital toxoplasmosis and reinfection during pregnancy: case report, strain characterization, experimental model of reinfection, and review.
J. Infect. Dis.
PUBLISHED: 03-07-2009
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We present a case of disseminated congenital toxoplasmosis in a newborn born to a mother who had been immunized against toxoplasmosis before conception. The mother was reinfected, likely by ingestion of imported raw horse meat during pregnancy. This clinical presentation is exceptional in France and raised the possibility of infection by a highly virulent Toxoplasma strain. The strain responsible was isolated from the peripheral blood of the newborn, and when genotyped with microsatellite markers, it exhibited an atypical genotype, one which is very uncommon in Europe but had been described in South America. We tested the hypothesis of a reinfection with a different genotype by using an experimental mouse model, which confirmed that acquired immunity against European Toxoplasma strains may not protect against reinfection by atypical strains acquired during travel outside Europe or by eating imported meat.
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A one-step multiplex PCR for acanthamoeba keratitis diagnosis and quality samples control.
Invest. Ophthalmol. Vis. Sci.
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As the number of cases of Acanthamoeba spp. keratitis (AK) is constantly growing, new diagnostic tools are needed to confirm and guide ophthalmologists in this clinically problematic diagnosis. Molecular diagnosis is particularly well adapted, although only a few real-time PCR techniques have been described recently. The aim of this study was to develop a new PCR technique for the diagnosis of AK by combining the detection of Acanthamoeba DNA with human DNA, thus allowing an accurate interpretation of the PCR result.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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