Quantitative real-time PCR for the clinical detection of Helicobacter pylori

Accurate diagnosis of Helicobacter pylori infection is very important in both clinical practice and research. We evaluated the sensitivity of real-time PCR (RT-PCR) for the detection and quantification of Helicobacter pylori using DNA from 91 human gastric biopsy samples divided into three groups: 46 biopsies from untreated patients who according to the references methods were considered H. pylori-negative (group A); 35 biopsies from patients previously treated against H. pylori and considered to be cured by "gold standard" tests (group B); and 10 biopsies from patients H. pylori-positive by all available methods (group C). The sensitivity of the RT-PCR assay was higher than that of standard methods. Of the 81 patients considered to be uninfected according to the references methods, 16 were H. pylori-positive by PCR, 10 of which were patients who had received H. pylori eradication therapy and 6 were untreated patients. Based on these findings we recommend that RT-PCR should be use in addition to standard methods in clinical studies to monitor the results of H. pylori eradication therapy

Solução para o Cálculo de Indicadores-Chave de Desempenho nas Redes Móveis de Comunicação

The work described in this report was developed in the context of the masters in Software Engineering and was carried out at Celfinet company. Telecommunications are a fundamental foundation of our society. A fast, robust and accessible telecommunications network for everyone represents a strategic advantage for companies and countries. Their importance has rapidly grown in recent years. However, as they become increasingly complex, communication networks need to be prepared for intensive and efficient use. Therefore, their monitoring is essential to detect problems and allow corrective measures or avoid their occurrence. Thus, how to rapidly calculate Key Performance Indicators (KPIs) deserved special attention in the initial study of telecommunications concepts. The requirements were gathered as generic to abstract the functionalities and allow the customization to several enterprises. A generic solution was designed to be easily adapted to different operators. The developed application can import and process raw data to calculate KPIs, based on the Microservices architecture style. In summary, it is extensible and flexible and can be included in different ecosystems without complexity.O trabalho descrito neste relatório foi desenvolvido no âmbito do mestrado em Engenharia de Software e foi realizado na empresa Celfinet. As telecomunicações são a base fundamental da nossa sociedade. Uma rede de telecomunicações rápida, robusta e acessível para todos, representa uma vantagem estratégica para empresas e países. A sua importância cresceu rapidamente nos últimos anos. No entanto, à medida que se tornam cada vez mais complexas, as redes de comunicação necessitam de ser preparadas para o uso intensivo e eficiente. Portanto, o seu monitoramento é fundamental para detetar problemas e permitir medidas corretivas ou evitar a ocorrência de erros. Assim, como calcular rapidamente indicadores chave de desempenho mereceu atenção especial no estudo inicial de conceitos de telecomunicações. Os requisitos foram abstraídos de forma a permitir a customização para várias empresas. Uma solução genérica foi projetada para ser facilmente adaptada a diferentes operadores. A aplicação desenvolvida pode importar e processar dados brutos de medidas de desempenho para calcular os indicadores chaves, baseado no estilo de arquitetura de microserviços. Em resumo, é extensível e flexível e pode ser incluído em diferentes ecossistemas sem complexidade

The influence of endoscopic procedures upon the contamination of Helicobacter pylori cultures A influência dos procedimentos endoscópicos na contaminação de culturas de Helicobacter pylori

BACKGROUND: Among the various diagnostic methods for the detection of Helicobacter pylori infection, histological examination and microbiological processing of gastric biopsy samples are assumed to be the gold standard techniques. AIMS: Since H. pylori culture can be affected by the presence of non-H. pylori bacteria, we evaluated the efficacy of endoscope disinfection and the influence of endoscopic procedures on culture contamination. PATIENTS AND METHODS: The procedures used during the first two routine endoscopies were evaluated during 28 consecutive days. Endoscopy room, forceps and endoscopic channel were analyzed before and after the beginning of normal procedures. After disinfection, a biopsy simulation was performed to verify the gastric bacteria. RESULTS: Endoscope disinfection removed all organisms from forceps and endoscopic channel with 100% efficacy. The most frequent non-H. pylori bacteria detected were Streptococcus bovis, Enterobacter hormaechei, and Staphylococcus aureus. The sensibility of the H. pylori culture was affected by the presence of non-H. pylori bacteria. CONCLUSION:The risk of transmission of microorganisms was not detectable when sterilized biopsy forceps and stringent disinfection standards were employed. Whilst S. bovis and E. hormaechei may be common in gastric microbial flora, the presence of P. aeruginosa and S. aureus indicated that the manipulation of biopsies could be responsible for culture contamination with these bacteria.RACIONAL E OBJETIVOS: Dentre os vários métodos diagnósticos empregados na detecção da infecção por Helicobacter pylori, o diagnóstico histológico e a análise microbiológica de biopsia gástrica são consideradas as técnicas mais sensíveis. Entretanto, a sensibilidade da cultura de H. pylori pode ser reduzida pela presença de outras bactérias. Desse modo, avaliou-se a eficácia da desinfecção do endoscópio e a influência dos procedimentos endoscópicos na contaminação da cultura bacteriana. Para tal, as duas primeiras endoscopias durante 28 dias consecutivos foram estudadas. A sala de endoscopia, o fórceps e o canal do endoscópio foram analisados antes e depois do início dos procedimentos endoscópicos rotineiros. Depois da desinfecção, uma simulação de coleta de biopsia foi realizada para verificar a presença das bactérias gástricas. RESULTADOS: A desinfecção do endoscópio foi capaz de remover todos os organismos do fórceps e do canal do endoscópio. As bactérias não-H. pylori mais freqüentemente detectadas foram Streptococcus bovis, Enterobacter hormaechei e Staphylococcus aureus. Em alguns casos a sensibilidade da cultura do H. pylori foi reduzida pela presença de bactérias contaminantes. CONCLUSÃO: Não houve risco de transmissão de microorganismos quando fórceps esterilizados e desinfecção adequada foram empregadas. A presença de S. bovis e E. hormaechei parece ser comum na microflora gástrica; por outro lado, a detecção de P. aeruginosa e S. aureus indica que a manipulação de biopsias pode ser responsável pela contaminação da cultura por essas bactérias

NITRIC OXIDE INTERFERES WITH HYPOXIA SIGNALING DURING COLONIC INFLAMMATION

Context Intestinal inflammation can induce a local reduction in oxygen levels that triggers an adaptive response centered on the expression of hypoxia-inducible factors (HIFs). Nitric oxide, a well-described inflammatory mediator, may interfere with hypoxia signaling. Objectives We aimed to evaluate the role of nitric oxide in hypoxia signaling during colonic inflammation. Methods Colitis was induced by single (acute) or repeated (reactivated colitis) trinitrobenzenosulfonic acid administration in rats. In addition, one group of rats with reactivated colitis was also treated with Nw-Nitro-L-arginine methyl ester hydrochloride to block nitric oxide synthase. Colitis was assessed by macroscopic score and myeloperoxidase activity in the colon samples. Hypoxia was determined using the oxygen-dependent probe, pimonidazole. The expression of HIF-1α and HIF-induced factors (vascular endothelial growth factor - VEGF and apelin) was assessed using Western blotting. Results The single or repeated administration of trinitrobenzenosulfonic acid to rats induced colitis which was characterized by a high macroscopic score and myeloperoxidase activity. Hypoxia was observed with both protocols. During acute colitis, HIF-1α expression was not increased, but VEGF and apelin were increased. HIF-1α expression was inhibited during reactivated colitis, and VEGF and apelin were not increased. Nw-Nitro-L-arginine methyl ester hydrochloride blockade during reactivated colitis restored HIF-1α, VEGF and apelin expression. Conclusions Nitric oxide could interfere with hypoxia signaling during reactivated colitis inflammation modifying the expression of proteins regulated by HIF-1α

MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults

AIM: To evaluate the association between Helicobacter pylori(H. pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI). METHODS: The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSPPCR) in gastric biopsy samples from uninfected or H. pylori -infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ 2 test with Yates continuity correction or Fisher's exact test, and statistical significance for expression analysis was assessed using an unpaired Student's t -test. RESULTS: Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P < 0.05); this region was methylated in 66% of gastric cancer patients, and the difference in the percentage of methylated samples between these patients and those from H. pylori -infected chronic gastritis patients was statistically significant (P < 0.05). MLH1 methylation frequencies among H. pylori -infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori -infected adult chronic gastritis patients (P < 0.001 and P < 0.01, respectively). The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H. pylori -positive chronic gastritis samples (P < 0.05). The levels of MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated (P < 0.01). MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSPPCR) in gastric biopsy samples from uninfected or H. pylori -infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ 2 test with Yates continuity correction or Fisher's exact test, and statistical significance for expression analysis was assessed using an unpaired Student's t -test. RESULTS: Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P < 0.05); this region was methylated in 66% of gastric cancer patients, and the difference in the percentage of methylated samples between these patients and those from H. pylori -infected chronic gastritis patients was statistically significant (P < 0.05). MLH1 methylation frequencies among H. pylori -infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori -infected adult chronic gastritis patients (P < 0.001 and P < 0.01, respectively). The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H. pylori -positive chronic gastritis samples (P < 0.05). The levels of MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated (P < 0.01). CONCLUSION: In summary, MGMT and MLH1 methylation did not occur in earlier-stage H. pylori infections and thus might depend on the duration of infection. © 2013 Baishideng. All rights reserved