Browsing by Author "Mégraud, Francis"
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- Complex Polysaccharides as PCR Inhibitors in Feces: Helicobacter pylori ModelPublication . Monteiro, Lurdes; Bonnemaison, Dominique; Vekris, Antoine; Petry, Klaus G.; Bonnet, Jacques; Vidal, Rui; Cabrita, José; Mégraud, FrancisA model was developed to study inhibitors present in feces which prevent the use of PCR for the detection of Heticobacterpylori. A DNA fragment amplified with the same primers as H. pytori was used to spike samples before extraction by a modified QIAamp tissue method. Inhibitors, separated on an Ultrogel AcA44 column, were characterized. Inhibitors in feces are complex polysaccharides possibly originating from vegetable material in the diet.
- Detection of Helicobacterpylori DNA in human feces by PCR: DNA stability and removal of inhibitorsPublication . Monteiro, Lurdes; Gras, Nathalie; Vidal, Rui; Cabrita, José; Mégraud, FrancisIn this study, the stability of Helicobacter pylori DNA in human feces and the effect of a diet lacking in plant material, the suspected source of PCR inhibitors in human feces, were investigated. In addition, a method to remove these inhibitors was developed. Stools inoculated with H. pylori were used as a model. For this purpose, a H. pylori suspension (108 CFU/ml) was used to spike stool samples obtained from four healthy adults known to be H. pylori negative. The evaluation of the stability of H. pylori DNA in feces showed that DNA was degraded after 3 days of contact with fecal material at 37°C. A 2-day diet completely free of plant material was sufficient to eliminate PCR inhibitors from human feces. However, inhibitors were detected 48 h after a normal diet was resumed. A new technique consisting of agarose blocks containing embedded DNA as a template for PCR amplification was used for removal of inhibitors, following DNA extraction by a modified QIAamp tissue method (Qiagen, Hilden, Germany). When this method was applied to inhibiting stool samples known to have an inhibitory effect and spiked with H. pylori (5.108 CFU/g), a positive PCR was obtained showing that inhibitors present in the original DNA samples were completely removed. The agarose embedded DNA block method is highly efficient and provides clean, high quality template DNA for PCR purposes avoiding long and fastidious conventional extraction methods. In conclusion, this study confirms that H. pylori DNA degrades with time in stools. A diet free of plant material or a special DNA preparation can be used to remove inhibitors and to allow the detection of H. pylori.
- Evaluation of Performances of Three DNA Enzyme Immunoassays for Detection of Helicobacter pylori PCR Products from Biopsy SpecimensPublication . Monteiro, Lurdes; Cabrita, José; Mégraud, FrancisPCR is recognized as a promising method for the detection ofHeticobacterpytori in gastric biopsy specimens. However, detection of PCR products by gel electrophoresis is difficult to implement in routine clinical laboratories. The aim of this study was to compare three new DNA enzyme immunoassays with the standard method in their ability to detect PCR products. lhe three assays were based on Lhe amplification of a fragment of the ureC gene of H. pytori and a colorimetric hybridization assay. lhe first assay (GEN-ElI-K DNA enzyme immunoassay; Sorin, Sailugia, ltaly) was based on the hybridization of amplified DNA with a probe bound in microtiter wells and detected with labelled anti-DNA antibody. lhe second assay (Pylori-prob; Biocode, Sclessin, Belgium) comprised a solid-phase sandwich hybridization system with a specific biotinylated probe being used for detection. Finally, the third assay (PCR enzyme-linked immunosorbent assay; Boehringer, Mannheim, Germany) was based on the hybridization of amplified DNA Iabelled with digoxigenin as a probe (used as a coating in microtiter wells) and detected with antidigoxigenin-peroxidase as conjugate. The sensitivity of the colorimetric assay was evaluated by using amplification products from PCR assays performed on several 10-fotd dilutions of DNA from H. pylori CIP 101260, and the specificitv was assessed with dilferent urease-positive bacteria. Biopsy specimens from 199 patients were tested; 106 were classified as H. pylori positive, and 93 were classified as II. pylori negative by culture and/or histological examination as Lhe ‘goId standard.” lhe receiving operating characteristic curve was used to determine the best cutotf point for each assay. The detection of PCR products by colorimetric hybridization increases the sensitivit up to 100-told compared to that with gel electrophoresis. The results are rapid (4 h) and easy to interpret and can be automated.
- Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migrationPublication . Vale, Filipa; Mégraud, Francis; Vítor, Jorge M. B.Background Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M) systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations.
- Helicobacter pylori: métodos de diagnósticoPublication . Monteiro, Lurdes; Cabrita, José; Mégraud, FrancisO H. pylori é considerado actualmente como o principal agente etiológico das doenças gastroduodenais. A sua prevalência a nível da população portuguesa é bastante elevada atingindo cerca de 80% em indivíduos com idade superior a 15 anos. O diagnóstico laboratorial da infecção por H. pylori é complexo, podendo-se recorrer a métodos de diagnóstico directos e indirectos mantendo-se como método de referência o exame cultural, o qual requer uma biópsia obtida por endoscopia. A biópsia permite a execução de outros testes, quer de natureza morfológica (histologia), quer de natureza bioquímica (detecção da actividade ureásica) ou ainda de biologia molecular como seja a PCR. Dentro dos métodos indirectos são largamente utilizados o teste respiratório com ureia marcada e a serologia. Actualmente, encontram-se em fase de estudo métodos que utilizando outros produtos biológicos (fezes, urina, saliva) têm como objectivo o diagnóstico da infecção por H. pylori de forma não invasiva. A utilização dos diversos métodos existentes depende essencialmente do estudo a efectuar ser um estudo clínico ou um estudo epidemiológico. No presente artigo procede-se a uma análise comparativa da sensibilidade, especificidade e simplicidade de execução dos diferentes métodos de diagnóstico da infecção por H. pylori, em diferentes produtos biológicos, utilizados na rotina e na investigação.