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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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Characteristic and clinical relevance of Candida mannan test in the diagnosis of probable invasive candidiasis.
Med. Mycol.
PUBLISHED: 06-15-2014
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The gold standard laboratory tests used to diagnose invasive Candida infection (ICI) are based on the in vitro culture of blood or samples from other sterile sites. However, these tests have limited sensitivity (Se) and are generally not diagnostic until late in the infectious process. The Serion Candida mannan kit was evaluated for the diagnosis of ICI at Grenoble University Hospital (France) between 2007 and 2011. The results were then compared with worldwide data published between 1997 and 2011. This retrospective study was based on follow-up from the investigation of 162 patients of whom 91 had proven ICI; 13 had Candida colonization index (CCI) scores ?0.42, positive mannan tests, with nonconcomitant infections; and 58 had no evidence of Candida infection. Candida albicans, C. glabrata, C. tropicalis, and C. parapsilosis were the etiologic agents in 104 patients. For patients with or without ICI, the 12-week mortality rates were 35/104 (33.7%) and 6/58 (10.3%), respectively. The mannan diagnostic specificity was 51% and Se was 77%. However, in the meta-analysis (n = 1,536), values were 86% and 62%, respectively. Positive mannan test results may appear early (median 6 days) in the development of candidemia and have moderate diagnostic value for ICI, with a negative predictive value of 83%. In patients at risk of ICI with negative candidemia, the combination of Candida mannan test data with a CCI score ?0.42 may improve the diagnosis of probable ICI.
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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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miR-146a and miR-155 delineate a MicroRNA fingerprint associated with Toxoplasma persistence in the host brain.
Cell Rep
PUBLISHED: 01-23-2014
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microRNAs were recently found to be regulators of the host response to infection by apicomplexan parasites. In this study, we identified two immunomodulatory microRNAs, miR-146a and miR-155, that were coinduced in the brains of mice challenged with Toxoplasma in a strain-specific manner. These microRNAs define a characteristic fingerprint for infection by type II strains, which are the most prevalent cause of human toxoplasmosis in Europe and North America. Using forward genetics, we showed that strain-specific differences in miR-146a modulation were in part mediated by the rhoptry kinase, ROP16. Remarkably, we found that miR-146a deficiency led to better control of parasite burden in the gut and most likely of early parasite dissemination in the brain tissue, resulting in the long-term survival of mice.
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Characteristics and outcomes of chronic pulmonary aspergillosis: a retrospective analysis of a tertiary hospital registry.
Clin Respir J
PUBLISHED: 01-04-2014
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Our objective was to investigate characteristics risk factors and outcomes of patients with chronic pulmonary aspergillosis (CPA).
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Usefulness of pan-fungal NASBA test for surveillance of environmental fungal contamination in a protected hematology unit.
Med. Mycol.
PUBLISHED: 01-03-2014
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A pan-fungal nucleic acid sequence based applification (NASBA) test was adapted and used for the first time to detect and quantify the level of filamentous fungi in environmental samples. Surface samples (n = 356) collected in a controlled air flow hematology ward were tested by mycological culture and the pan-fungal NASBA test. The overall percentage of agreement between culture and NASBA was 88%, the Kappa coefficient was equal to 0.61 (95%CI = [0.51; 0.72]). This pan-fungal NASBA test could be a promising tool to rapidly monitor the absence of molds in controlled environments.
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Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.
PLoS ONE
PUBLISHED: 01-01-2014
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In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS) complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.
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Human toxoplasmosis: which biological diagnostic tests are best suited to which clinical situations?
Expert Rev Anti Infect Ther
PUBLISHED: 09-24-2013
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The Toxoplasma gondii parasite is a worldwide threat most particularly in fetal life and immunosuppression. In most clinical situations (except in some ocular cases), correct detection or identification of toxoplasmosis requires biological analysis. This article considers the laboratory tools that have been developed in this field since the discovery of the pathogen, with emphasis on the most recent tests and how they can or should be used in different clinical situations. The authors also discuss the requirements and pitfalls that one should be aware of when biologically investigating this intriguing parasitosis.
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A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation.
J. Exp. Med.
PUBLISHED: 09-16-2013
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Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite that resides inside a parasitophorous vacuole. During infection, Toxoplasma actively remodels the transcriptome of its hosting cells with profound and coupled impact on the host immune response. We report that Toxoplasma secretes GRA24, a novel dense granule protein which traffics from the vacuole to the host cell nucleus. Once released into the host cell, GRA24 has the unique ability to trigger prolonged autophosphorylation and nuclear translocation of the host cell p38? MAP kinase. This noncanonical kinetics of p38? activation correlates with the up-regulation of the transcription factors Egr-1 and c-Fos and the correlated synthesis of key proinflammatory cytokines, including interleukin-12 and the chemokine MCP-1, both known to control early parasite replication in vivo. Remarkably, the GRA24-p38? complex is defined by peculiar structural features and uncovers a new regulatory signaling path distinct from the MAPK signaling cascade and otherwise commonly activated by stress-related stimuli or various intracellular microbes.
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Comparison of the Vidas system and two recent fully automated assays for diagnosis and follow-up of toxoplasmosis in pregnant women and newborns.
Clin. Vaccine Immunol.
PUBLISHED: 06-05-2013
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Serological testing to detect toxoplasmosis is of major importance to avoid the possible effects of the disease in newborns. This study assessed anti-Toxoplasma IgG and IgM with the Vidas (bioMérieux), Architect (Abbott), and Liaison (DiaSorin) systems in 631 sera from pregnant women and newborns as well as anti-Toxoplasma IgG avidity with these three systems on 54 sera from pregnant women with positive IgG and IgM. The IgG and IgM results were in agreement in, respectively, 95.2% and 98.3% (Vidas versus Architect) and 96.9% and 95.3% (Vidas versus Liaison) of the samples. Specificities were excellent for all the assays, while Vidas sensitivities ranged (depending on the classification of gray zone results) from 93.8 to 98.4% for IgG (Architect, 84.4 to 93.8%; Liaison, 93.8%) and from 81.8 to 90.9% for IgM (Architect, 63.6%; Liaison, 81.8 to 90.9%). In seroconversion sequences, IgMs were generally detected simultaneously by the three assays, while Architect was the earliest assay to detect IgG. In noninfected children, maternally transmitted IgGs were detected for a longer time with Architect than with the other systems. IgMs were positive in only one infected child with the Vidas and Liaison systems. Significantly more sera were classified in the high-avidity category with Vidas than with Architect. This evaluation shows similar performances for Vidas and more recent systems. The Vidas system adequately detects toxoplasmosis in pregnant women and newborns. This system fits the needs of laboratories working on small routine series for first-line testing as well as expert laboratories, due to a high specificity and a powerful avidity test.
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Flexible synthesis and evaluation of diverse anti-Apicomplexa cyclic peptides.
J. Org. Chem.
PUBLISHED: 04-04-2013
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A modular approach to synthesize anti-Apicomplexa parasite inhibitors was developed that takes advantage of a pluripotent cyclic tetrapeptide scaffold capable of adjusting appendage and skeletal diversities in only a few steps (one to three steps). The diversification processes make use of selective radical coupling reactions and involve a new example of a reductive carbon-nitrogen cleavage reaction with SmI2. The resulting bioactive cyclic peptides have revealed new insights into structural factors that govern selectivity between Apicomplexa parasites such as Toxoplasma and Plasmodium and human cells.
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Borrelia crocidurae meningoencephalitis, West Africa.
Emerging Infect. Dis.
PUBLISHED: 01-26-2013
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Borrelia crocidurae-associated relapsing fever is endemic to West Africa and is considered benign. We report 4 patients with B. crocidurae-associated neurologic symptoms; 2 of their cases had been misdiagnosed. Frequency and severity of this disease could be underestimated; molecular methods and serodiagnostic tests for Lyme disease might be helpful in its detection.
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Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.
Cell Host Microbe
PUBLISHED: 01-17-2013
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After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.
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Susceptibility of 100 filamentous fungi: comparison of two diffusion methods, Neo-Sensitabs and E-test, for amphotericin B, caspofungin, itraconazole, voriconazole and posaconazole.
Med. Mycol.
PUBLISHED: 10-10-2011
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We compared the E-test method to that of the Neo-Sensitabs tablet diffusion assay for evaluating the in vitro susceptibility of 100 clinical isolates of filamentous fungi (Aspergillus spp., Fusarium spp., Scedosporium spp., zygomycetes and other molds) to amphotericin B, itraconazole, voriconazole, caspofungin, and posaconazole. We determined the categorical agreement level between E-test minimum inhibitory concentrations (MIC) and tablet end-points, as opposed to the following disagreement parameters: very major error - resistant parameter (R) in E-test and susceptible (S) in tablet; major error - S by E-test and R by tablet; minor error - shifts between S and susceptible dose-dependent (S-DD) or S-DD and R. We also performed linear regression analyses and computed Pearsons correlation coefficients (R values) between the log transforms of MICs and the inhibition zone diameters of the five studied antifungal agents. For itraconazole we obtained 97% categorical agreement and R = -0.727. Categorical agreement for caspofungin and voriconazole was 96% and R =-0.821 and R = -0.789, respectively. For posaconazole the categorical agreement was 94% and R =-0.743. Amphotericin B exhibited a lower degree of agreement (76%, R = -0.672), especially in studies of Aspergillus spp. Our results suggest a potential value of the Neo-Sensitabs assay for in vitro susceptibility testing of molds to itraconazole, voriconazole, caspofungin and posaconazole, while amphotericin B exhibited an overall lower degree of agreement.
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Antifungal use influences Candida species distribution and susceptibility in the intensive care unit.
J. Antimicrob. Chemother.
PUBLISHED: 10-06-2011
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Antifungal prescription practices have changed over the last decade, and the impact of these changes is unclear. Our objective here was to evaluate the effect of antifungal drug use on the distribution and drug susceptibility of Candida spp. in a French intensive care unit (ICU).
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Effects of age, gender and time on receptor expression and anti-Aspergillus functions of human phagocytes.
Cell. Immunol.
PUBLISHED: 05-06-2011
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Phagocytes play a central role in immune defense. Their dysfunction predisposes to infections. This study determined the expression level of nine receptors involved in Aspergillus immune response as well as the values of phagocytosis and production of radical oxygen species after Aspergillus stimulation, in a healthy adult population. The expression values of the CD11b, CD11c, CD14, CD18, CD35, CD181, CD182, CD282 and CD284 receptors on peripheral human monocytes and granulocytes was established. A heterogenous expression of the CD282 on granulocytes was observed as CD181, CD182 and CD284 on monocytes. Similarly, we observed considerable variation in the expression of these receptors over time. Only CD282 on granulocytes varied with sex. No variation with age was observed. Adherence of Aspergillus conidia to phagocytes was dependent of individual, sex, age and time. A better characterization of these innate immunity parameters is necessary to develop in the future an immunologic surveillance strategy for transplant recipients.
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The placenta: a main role in congenital toxoplasmosis?
Trends Parasitol.
PUBLISHED: 05-03-2011
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Systemic infections, such as toxoplasmosis, acquired during pregnancy can lead to placental infection and have profound effects on the mother-to-child relationship and the success of pregnancy. Placental permeability to Toxoplasma gondii is a main parameter that determines parasite transmission to the foetus, and the use of antibiotics to decrease placental parasite load and prevent congenital toxoplasmosis has been suggested for decades. Although parasitological examination of the placenta at birth is commonly used to diagnose neonatal congenital toxoplasmosis, this approach can be controversial. Here we argue in favour of placental examination for both diagnostic and epidemiological purposes.
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Slippery or sticky? Functional diversity in the trapping strategy of Nepenthes carnivorous plants.
New Phytol.
PUBLISHED: 03-24-2011
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The pitcher-shaped leaves of Nepenthes carnivorous plants have been considered as pitfall traps that essentially rely on slippery surfaces to capture insects. But a recent study of Nepenthes rafflesiana has shown that the viscoelasticity of the digestive fluid inside the pitchers plays a key role. Here, we investigated whether Nepenthes species exhibit diverse trapping strategies. We measured the amount of slippery wax on the pitcher walls of 23 taxa and the viscoelasticity of their digestive liquid and compared their retention efficiency on ants and flies. The amount of wax was shown to vary greatly between species. Most mountain species exhibited viscoelastic digestive fluids while water-like fluids were predominant in lowland species. Both characteristics contributed to insect trapping but wax was more efficient at trapping ants while viscoelasticity was key in trapping insects and was even more efficient than wax on flies. Trap waxiness and fluid viscoelasticity were inversely related, suggesting the possibility of an investment trade-off for the plants. Therefore Nepenthes pitcher plants do not solely employ slippery devices to trap insects but often employ a viscoelastic strategy. The entomofauna specific to the plants habitat may exert selective pressures, favouring one trapping strategy at the expense of the other.
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Characteristics and clinical relevance of the quantitative touch-down major surface glycoprotein polymerase chain reaction in the diagnosis of Pneumocystis pneumonia.
Med. Mycol.
PUBLISHED: 03-18-2011
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The evaluation of quantitative polymerase chain reaction (PCR) characteristics can increase the accuracy of the laboratory diagnosis of Pneumocystis pneumonia (PCP). Between July 2008 and September 2009, 66 non-sequential prospective bronchoalveolar lavage (BAL) samples, obtained from five HIV-infected and 49 non HIV-infected patients were investigated, using a quantitative-touch-down-PCR to determine the number of copies of major surface glycoprotein (MSG) genes of Pneumocystis jirovecii (q-TD-MSG-PCR). PCP was confirmed by microscopic observation of Pneumocystis, radio-clinical and therapeutic data in 18/54 patients. For PCP, the cut-off was 54.3 MSG copies per ml of BAL fluid. The PCR was positive in these same 18 cases and it was the only positive assay in two cases and the earliest diagnosis test in one case of PCP relapse. The likelihood positive ratio, sensitivity and specificity of the q-TD-MSG-PCR were 44, 100% and 97.7%, respectively. The Predictive Negative Value was 100% and the Predictive Positive Value of 95.5%, the intra- and inter-assay variability values were 2.7% (at more than 30 MSG copies) and 11.7% (at 10,000 MSG copies), respectively. Quantitative PCR can help diagnose PCP even in cases of low Pneumocystis load and might decrease morbidity in association with very early specific treatments.
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Comparative proteomic profiles of Aspergillus fumigatus and Aspergillus lentulus strains by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS).
BMC Microbiol.
PUBLISHED: 02-15-2011
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Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was applied to analyze the protein profiles in both somatic and metabolic extracts of Aspergillus species. The study was carried out on some Aspergillus species within the Fumigati section (Aspergillus fumigatus wild-types and natural abnormally pigmented mutants, and Aspergillus lentulus). The aim was to validate whether mass spectrometry protein profiles can be used as specific signatures to discriminate different Aspergillus species or even mutants within the same species.
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Activity of the histone deacetylase inhibitor FR235222 on Toxoplasma gondii: inhibition of stage conversion of the parasite cyst form and study of new derivative compounds.
Antimicrob. Agents Chemother.
PUBLISHED: 08-16-2010
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Bradyzoite-to-tachyzoite conversion plays a role in the pathogenesis of recrudescence of ocular toxoplasmosis and disease in immunocompromised persons. The currently available medicines are ineffective on cysts and fail to prevent reactivation of latent toxoplasmosis. A previous study showed that the histone deacetylase inhibitor FR235222 has a dramatic effect on tachyzoite growth and induces tachyzoite-to-bradyzoite conversion in vitro. The present study shows that FR235222 can target in vitro-converted cysts and bradyzoites. Moreover, the compound is active on ex vivo T. gondii cysts. Free bradyzoites isolated after lysis of the cell wall did not proliferate in vitro when the cyst was treated with FR235222. The results imply that this compound is able to cross the T. gondii cystic cell wall. Fluorescent labeling shows that the compound impairs the capacity of the bradyzoites to convert without damaging the cyst wall integrity. In vivo inoculation of formerly treated cysts fails to infect mice when these cysts were treated with FR235222. We used our structural knowledge of FR235222 and its target, T. gondii HDAC3, to synthesize new FR235222 derivative compounds. We identified two new molecules that are highly active against tachyzoites. They harbor a better selectivity index that is more suitable for a future in vivo approach. These results identify FR235222 and its derivatives as new lead compounds in the range of therapeutics available for acute and chronic toxoplasmosis.
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Use of dense granule antigen GRA6 in an immunoglobulin G avidity test to exclude acute Toxoplasma gondii infection during pregnancy.
Clin. Vaccine Immunol.
PUBLISHED: 07-14-2010
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The usefulness of a specific immunoglobulin G (IgG) avidity enzyme-linked immunosorbent assay (ELISA) based on recombinant GRA6 antigen for distinguishing between acute and chronic Toxoplasma infection was investigated. Two sets of serum samples obtained from pregnant women with acute, chronic, or no Toxoplasma infection collected in France and Iran were used. Among the French subjects, 19 of 20 (95%) women who experienced seroconversion during the past 4 months before sampling displayed low-avidity IgG antibodies against GRA6, while all 17 (100%) women with chronic infection had high-avidity antibodies. When the Euroimmun IgG avidity ELISA was used, 15 of 19 (78.9%) recently infected women had low-avidity antibodies, and 20 of 22 (90.9%) women with chronic infection displayed high-avidity antibodies. The results suggested better performance of the GRA6 avidity ELISA than the Euroimmun avidity ELISA for exclusion of a recent infection occurring less than 4 months previously. Similarly, all 35 Iranian women with acute Toxoplasma infection had low-avidity antibodies against GRA6, whereas all 34 women with chronic infection displayed IgG antibodies of high avidity, indicating the value of GRA6 avidity testing for ruling out a recent infection. Avidity tests based on lysed whole-cell Toxoplasma gondii antigen are currently used to exclude recently acquired infections; however, the use of recombinant antigen(s) might improve the diagnostic performance of avidity tests and facilitate the development of more standardized assays.
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Recombinant allergens combined with biological markers in the diagnosis of allergic bronchopulmonary aspergillosis in cystic fibrosis patients.
Clin. Vaccine Immunol.
PUBLISHED: 07-14-2010
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Allergic bronchopulmonary aspergillosis (ABPA) is a frequent complication in cystic fibrosis patients. The diagnosis remains difficult and requires a combination of clinical, radiological, biological, and mycological criteria. The aim of this study was to analyze the added value of two recombinant antigens, rAspf4 and rAspf6, associated with the detection of specific IgG; precipitins; total IgE; and Aspergillus fumigatus in sputum for the diagnosis of ABPA. In a retrospective study, we determined the specific IgE responses to these recombinants in 133 sera of 65 cystic fibrosis patients. We selected an average of five serum samples from each of the 17 patients with ABPA (13 proven and 4 probable ABPA) and from 3 patients with Aspergillus bronchitis and rhinosinusitis. One serum sample for the 45 patients without ABPA was tested. The sensitivity of specific IgE detection against rAspf4 calculated per patient (92.3%) was significantly higher (P < 0.05) than that of rAspf6 (53.8%). When rAspf4 IgE detection was associated with anti-Aspergillus IgG enzyme-linked immunosorbent assay (ELISA) and precipitin detection, the sensitivity rose to 100%. The specificities of rAspf4 and rAspf6 IgE detection were 93.7% and 91.6%, respectively. Other diagnostic criteria had slightly lower specificities (87.5% for anti-Aspergillus IgG ELISA, 89.6% for precipitins, 84.4% for total IgE, and 85.0% for positive A. fumigatus culture in sputum). In conclusion, this retrospective study showed the relevance of rAspf4 IgE detection, in combination with other biological markers (Aspergillus IgG ELISA, precipitins, and total IgE), for improving the biological diagnosis of ABPA.
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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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Contribution of anti-Hsp70.1 IgG antibody levels to the diagnostic certainty of clinically suspected ocular toxoplasmosis.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 06-16-2010
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Laboratory diagnosis of ocular toxoplasmosis, the major cause of posterior uveitis worldwide, can be improved. Heat shock protein (Hsp) 70 is involved in cellular infection by Toxoplasma gondii but also in the immune response to this parasite. The authors postulate that infected patients may exhibit serum IgG anti-Hsp70.1 antibodies and that determining the presence of these antibodies could improve the diagnosis of suspected ocular toxoplasmosis.
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Therapeutic impact and diagnostic performance of multiplex PCR in patients with malignancies and suspected sepsis.
J. Infect.
PUBLISHED: 04-08-2010
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New molecular methods allow rapid pathogen detection in patients with sepsis, but their impact on treatment decisions remains to be established. We evaluated the therapeutic usefulness of multiplex PCR testing in patients with cancer and sepsis.
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Why prevent, diagnose and treat congenital toxoplasmosis?
Mem. Inst. Oswaldo Cruz
PUBLISHED: 05-12-2009
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Evidence that prevention, diagnosis and treatment of toxoplasmosis is beneficial developed as follows: anti-parasitic agents abrogate Toxoplasma gondii tachyzoite growth, preventing destruction of infected, cultured, mammalian cells and cure active infections in experimental animals, including primates. They treat active infections in persons who are immune-compromised, limit destruction of retina by replicating parasites and thereby treat ocular toxoplasmosis and treat active infection in the fetus and infant. Outcomes of untreated congenital toxoplasmosis include adverse ocular and neurologic sequelae described in different countries and decades. Better outcomes are associated with treatment of infected infants throughout their first year of life. Shorter intervals between diagnosis and treatment in utero improve outcomes. A French approach for diagnosis and treatment of congenital toxoplasmosis in the fetus and infant can prevent toxoplasmosis and limit adverse sequelae. In addition, new data demonstrate that this French approach results in favorable outcomes with some early gestation infections. A standardized approach to diagnosis and treatment during gestation has not yet been applied generally in the USA. Nonetheless, a small, similar experience confirms that this French approach is feasible, safe, and results in favorable outcomes in the National Collaborative Chicago-based Congenital Toxoplasmosis Study cohort. Prompt diagnosis, prevention and treatment reduce adverse sequelae of congenital toxoplasmosis.
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Comparative evaluation of the ARCHITECT Toxo IgG, IgM, and IgG Avidity assays for anti-Toxoplasma antibodies detection in pregnant women sera.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 05-07-2009
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We assessed the performance of the ARCHITECT Toxo IgG, IgM, and IgG Avidity assays against corresponding assays on AxSYM and Vidas using 730 sera from pregnant women. The ARCHITECT Toxo IgG and IgM assays showed a relative sensitivity of 97.5% and 89.9% and a relative specificity of 99.1% and 99.8%, respectively. If IgM sensitivity is calculated only for sera drawn less than 4 months after infection, the relative sensitivity rises to 98.1%. Correlation between the ARCHITECT and Vidas Avidity assays was 0.87 (n = 103). Testing 86 IgG-positive specimens from recent infection (<4 months), we never obtained high avidity results, but 2 specimens were in the gray zone, whereas sera from past infections (>4 months) exhibited high avidity results in 72.5% (137/189) of cases. The method can be used reliably to exclude recent infections in sera with concurrently positive results for IgM and IgG (IgG, >3 IU/mL).
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Mobile air-decontamination unit and filamentous fungal load in the hematology ward: how efficient at the low-activity mode?
Am J Infect Control
PUBLISHED: 04-10-2009
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Air treatment with a mobile Plasmair air-decontamination unit significantly reduces the fungal spore load in hematology wards. We report that this system used at a low aspiration flow does not perform total biodecontamination against filamentous fungi. Moreover, the filamentous fungus load remaining in rooms equipped with this mobile air-decontamination unit is lowest in wards in which other preventive measures against nosocomial filamentous fungal infections are implemented.
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Severe filamentous fungal infections after widespread tissue damage due to traumatic injury: six cases and review of the literature.
Scand. J. Infect. Dis.
PUBLISHED: 04-09-2009
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We describe 6 cases of severe filamentous fungal infections after widespread tissue damage due to traumatic injury in previously healthy people. Additionally, we report 69 cases from an exhaustive 20-y review of the literature to investigate the epidemiological and clinical features, the prognosis and the therapeutic management of these post-traumatic severe filamentous fungal infections. Traffic (41%) and farm accidents (25%) were the main causes of injury, which involved either the limbs only (41%) or multiple sites (41%). Necrosis was the main symptom (60%) and Mucorales (72%) and Aspergillus (11%) were the 2 most frequent fungi causing infection. These infections required substantial surgical debridement or amputation (96%) associated with aggressive antifungal therapy (81%), depending on the responsible fungi. This study underlines the need for early, repeated and systematic mycological wound samples to guide and adapt surgical and antifungal management in these filamentous fungal infections.
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Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites.
J. Exp. Med.
PUBLISHED: 04-06-2009
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Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although little is yet known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. We investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase inhibitor (HDACi). We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACis such as trichostatin A and the clinically relevant compound pyrimethamine. We identify T. gondii HDAC3 (TgHDAC3) as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (nonreplicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of approximately 370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDACi of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and, likely, other Apicomplexa.
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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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Influence of internal and outdoor factors on filamentous fungal flora in hematology wards.
Am J Infect Control
PUBLISHED: 02-09-2009
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Nosocomial invasive filamentous fungi infections could result from inhalation of filamentous fungi conidia present in hospital environment.
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Diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of DNA detection and serological techniques.
Clin. Infect. Dis.
PUBLISHED: 01-24-2009
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The biological diagnosis of toxoplasmosis after allogeneic hematopoietic stem cell transplantation (HSCT) is based on the detection of Toxoplasma gondii DNA in blood specimens or other samples. Serological testing is used mainly to define the immunity status of the patient before HSCT. The aim of our study was to examine the performance of polymerase chain reaction (PCR) and serological techniques in the diagnosis of toxoplasmosis after HSCT.
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Comparative assessment of a commercial kit and two laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis.
J. Clin. Microbiol.
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Toxoplasmosis is a worldwide infection that may cause severe disease and is regarded as a serious health problem in France. Detection of the parasite by molecular methods is crucial for diagnosing the disease. The extreme diversity of methods and performances of Toxoplasma PCR assays makes the use of commercial PCR kits an attractive alternative, as they offer a chance for standardization. We compared the performances of three molecular methods for the detection of Toxoplasma gondii DNA in amniotic fluid: a commercial method using nested PCR and two laboratory-developed methods, one using conventional PCR and the other one real-time PCR. This evaluation was based upon a T. gondii DNA serial dilution assay, three amniotic fluid samples spiked with T. gondii at different concentrations, and a clinical cohort of 33 amniotic fluid samples. The T. gondii DNA serial dilution assay showed a much lower sensitivity for the commercial kit than for the laboratory-developed methods. Moreover, out of 12 proven congenital toxoplasmosis cases, 91.7% were detected by the laboratory-developed assays, whereas only 50% were detected by the commercial kit. A lack of sensitivity of the method, partly due to the presence of PCR inhibitors, was the main drawback of the commercial method. This study emphasizes that commercial PCR diagnostic kits do not systematically perform better than carefully optimized laboratory-developed methods. There is a need for thorough evaluation of such kits by proficient groups, as well as for performance standards that commercial kits can be tested against to improve confidence in those selected by health care providers.
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Evaluation of the new Elecsys Toxo IgG avidity assay for toxoplasmosis and new insights into the interpretation of avidity results.
Clin. Vaccine Immunol.
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Detection and treatment of acute toxoplasmosis during pregnancy can avoid severe disease of the fetus. In this context, assessment of anti-Toxoplasma IgG avidity has been shown to exclude recent infection. The Elecsys Toxo IgG and IgM assays (Roche Diagnostics) have been validated for screening pregnant women and a new assay, Elecsys Toxo IgG Avidity, was recently developed. Our aims were to investigate the performance characteristics of this new avidity assay and explore whether additional information can be provided by avidity assays. The Elecsys assay was compared with the Vidas (bioMérieux) and Architect (Abbott) Avidity assays using two sets of serum samples (n = 291 and n = 255). The rate of general agreement between the Elecsys and Vidas assays was 74%, and that between the Elecsys and Architect assays was 83%. For 11% of the serum samples, avidity was high with the Vidas assay and within the gray zone with the Elecsys assay. None of the assays detected high-avidity antibodies in serum taken <4 months after infection. Avidity values of >90% were exclusively reported in sera taken >9 months after infection by the Elecsys and Architect assays. Almost all avidities of <19% with the Elecsys assay and <17% with the Architect assay corresponded to sera taken <3 and <2 months after infection, respectively. The Elecsys IgG Avidity assay can be used to exclude recent infection. New ways of interpreting the avidity result are also suggested: very high or low values could exclude infections within the last 9 months or help to confirm a recent infection, respectively. However, these potential interpretations require further investigation.
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Interleukin 17A as an effective target for anti-inflammatory and antiparasitic treatment of toxoplasmic uveitis.
J. Infect. Dis.
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Toxoplasmosis is the most common cause of posterior uveitis in immunocompetent subjects. The requirement of limiting both parasite multiplication and tissue destruction suggests that the balance between T-helper (Th) 17 and T-regulatory cells is an important factor in toxoplasmosis-induced retinal damage.
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Seven-year surveillance of nosocomial invasive aspergillosis in a French University Hospital.
J. Infect.
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This study aims at describing the evolution of the epidemiology of invasive aspergillosis (IA) in a French University Hospital focussing on nosocomial cases, in order to assess the efficiency of the environmental preventive measures which were implemented.
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Evaluation of the usefulness of six commercial agglutination assays for serologic diagnosis of toxoplasmosis.
Diagn. Microbiol. Infect. Dis.
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Six agglutination tests for detecting Toxoplasma gondii-specific antibodies (immunoglobulin G or M) in serum were performed and compared. In total, 599 sera were examined using direct and indirect agglutination assays. Sensitivity varied from 93.7% to 100% and specificity from 97.1% to 99.2%. In a selected population with interfering diseases, the percentage of false positives ranged from 4.3% to 10.9%. Although an overall agreement of 100% was found for chronic toxoplasmosis, sensitivity for the detection of confirmed acute toxoplasmosis ranged from 86.4% to 97.3%. Regarding the large variability in terms of the performance of the 6 assays, tests based on the hemagglutination principle were found to be better than the other agglutination tests for all the panels evaluated, meaning that they could be used as qualitative or semiquantitative low-cost screening assays.
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Minimum inhibitory concentrations of amphotericin B against Candida krusei isolates from a French teaching hospital laboratory: a retrospective study over 8 years.
Mycoses
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Recent studies have shown decreased susceptibility of Candida krusei to amphotericin B (AmB), in addition to its inherent resistance to fluconazole. The susceptibility of C. krusei to AmB was studied in the Parasitology-Mycology laboratory of Grenoble Teaching Hospital, France. Between 2003 and 2011, we analysed 200 C. krusei isolates from 130 patients. The isolates were mainly collected in intensive care, cardio-thoracic and cancer/haematology units. Minimum inhibitory concentrations (MICs) were determined by the E-test method. The modal MIC was 0.5 ?g ml(-1); the MIC(50) and MIC(90) (MICs encompassing 50% and 90% of all isolates tested, respectively) were 0.5 ?g ml(-1) and 1 ?g ml(-1). The Cuzicks and Kendalls tests showed a significant increase in MIC values between 2003 and 2011 (P = 0.001 and P ? 0.001, respectively), regardless of age or gender. No statistical difference was reached with these tests when the first 100 or 50 data were excluded. Despite the increase observed in the first period of the study, our results confirm the low AmB MICs reported in previous studies. However, some authors have recently reported much higher MICs. This discrepancy cannot be explained by method biases and could reflect C. krusei epidemiological differences among populations.
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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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