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ABSTRACT Introduction: Helicobacter pylori (H. pylori) is a Gram negative bacterium considered to be the etiologic agent of various gastric diseases. The prevalence of bacterial infection varies according to age, geographic location, ethnicity and socioeconomic status. The chronic infection caused by microorganism can favor the development of severe pathologies such as gastric adenocarcinoma. In this sense, early diagnosis is essential for a better prognosis and therapeutic success. Several diagnostic methods performed using invasive and non-invasive techniques, with different sensitivity and specificity, have been used in the detection of H. pylori. Objective: To compare the performance of the molecular and histopathological technique used in the diagnosis of H. pylori infection. Methods: 76 gastric tissue samples were collected from dyspeptic patients who underwent molecular and histopathological diagnosis. Molecular detection was performed using the ribosomal gene (16S rRNA) using the polymerase chain reaction (PCR) technique. Results: The PCR-based molecular diagnostic method detected the bacterium in 63.1% of the samples, while the histopathological test identified the microorganism in only 38.1% of gastric biopsies. The data demonstrated that the PCR technique was about 1.6 times more sensitive than the histopathological technique. Conclusion: The PCR technique was the most efficient diagnostic method for detecting H. pylori and can be implemented in the laboratory routine as a complementary test for the early detection of H. pylori.
RESUMEN Introducción: Helicobacter pylori (H. pylori) es una bacteria Gram negativa considerada agente etiológico de diversas enfermedades gástricas. La prevalencia de la infección bacteriana varía según la edad, la ubicación geográfica, la etnia y el nivel socioeconómico. La infección crónica provocada por esos microorganismos puede favorecer el desarrollo de patologías graves como el adenocarcinoma gástrico. Por esa razón, el diagnóstico precoz es fundamental para un mejor pronóstico y éxito terapéutico. En la detección de H. pylori se han utilizado varios métodos de diagnóstico realizados mediante técnicas invasivas y no invasivas, con diferentes sensibilidad y especificidad. Objetivo: Comparar el desempeño de las técnicas moleculares e histopatológicas utilizadas en el diagnóstico de la infección por H. pylori. Métodos: Se recolectaron 76 muestras de tejido gástrico de pacientes dispépticos y se las sometieron a diagnóstico molecular e histopatológico. La detección molecular se realizó mediante el gen ribosómico (ARNr 16S) mediante la técnica de reacción en cadena de la polimerasa (PCR). Resultados: El método molecular de diagnóstico basado en PCR detectó la bacteria en el 63,1% de las muestras, mientras que la prueba histopatológica identificó el microorganismo en solo el 38,1% de las biopsias gástricas. Los datos demostraron que la técnica de PCR era aproximadamente 1,6 veces más sensible que la técnica histopatológica. Conclusión: La técnica de PCR fue el método diagnóstico más eficaz para la detección de H. pylori y puede implementarse en la rutina del laboratorio como prueba complementaria para la detección precoz de H. pylori.
RESUMO Introdução: Helicobacter pylori (H. pylori) é uma bactéria Gram negativa considerada o agente etiológico de várias doenças gástricas. A prevalência da infecção bacteriana varia de acordo com idade, localização geográfica, etnia e status socioeconômico. A infecção crônica ocasionada por esse microrganismo pode favorecer o desenvolvimento de patologias severas, como o adenocarcinoma gástrico. Nesse sentido, o diagnóstico precoce é essencial para um melhor prognóstico e o sucesso terapêutico. Vários métodos de diagnóstico realizados com técnicas invasivas e não invasivas, com diferentes sensibilidade e especificidade, têm sido utilizados na detecção de H. pylori. Objetivo: Comparar o desempenho das técnicas molecular e histopatológica utilizadas no diagnóstico da infecção por H. pylori. Métodos: Setenta e seis amostras de tecido gástrico foram coletadas de pacientes dispépticos e submetidas ao diagnóstico molecular e histopatológico. A detecção molecular foi realizada utilizando o gene ribossomal (rRNA 16S) por meio da técnica de reação em cadeia da polimerase (PCR). Resultados: O método molecular de diagnóstico com base na PCR detectou a bactéria em 63,1% das amostras, enquanto o teste histopatológico identificou o microrganismo em apenas 38,1% das biópsias gástricas. Os dados demonstram que a técnica de PCR apresentou cerca de 1,6 vezes mais sensibilidade que a técnica histopatológica. Conclusão: A técnica de PCR foi o método de diagnóstico mais eficiente para detecção de H. pylori e pode ser implementada na rotina laboratorial como teste complementar para detecção precoce de H. pylori.
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Gastric cancer (GC) is the fifth most common type of cancer worldwide with high incidences in Asia, Central, and South American countries. This patchy distribution means that GC studies are neglected by large research centers from developed countries. The need for further understanding of this complex disease, including the local importance of epidemiological factors and the rich ancestral admixture found in Brazil, stimulated the implementation of the GE4GAC project. GE4GAC aims to embrace epidemiological, clinical, molecular and microbiological data from Brazilian controls and patients with malignant and pre-malignant gastric disease. In this letter, we summarize the main goals of the project, including subject and sample accrual and current findings
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Humanos , Adulto , Pessoa de Meia-Idade , Idoso , Neoplasias Gástricas/epidemiologia , Brasil , Adenocarcinoma , ProjetosRESUMO
The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a prevalent systemic mycosis in Latin America. The Paracoccidioides lutzii response to oxidative stress is largely unexplored. Thioredoxins (TRX) are involved in the regulation of the redox environment in the cell, responding to oxidative stress in several organisms. In this study, we describe the isolation and characterization of a cDNA encoding a thioredoxin 1 from yeast cells from P. lutzii. The cDNA codes for a 12kDa protein containing the characteristic thioredoxin active site. The thioredoxin 1 gene was expressed in Escherichia coli and the isolated thioredoxin 1 recombinant protein as the native PlTRX1 from yeast cells showed insulin reduction activity in vitro. We also showed by semi-quantitative RT-PCR analysis that the expression of thioredoxin 1 gene was induced in response to H2O2 and may exert an antioxidant activity in vivo. Our results suggest that the thioredoxin 1 may play an important role in controlling the redox status in P. lutzii which may contribute to this organism's virulence.
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Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Insulina/metabolismo , Paracoccidioides/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Proteínas Fúngicas/química , Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Paracoccidioides/efeitos dos fármacos , Filogenia , Tiorredoxinas/químicaRESUMO
ABSTRACT Introduction: Helicobacter pylori is a bacterium found in human epithelial cells of the gastrointestinal tract. Its infection is related to different diseases, such as chronic gastritis, peptic ulcers, gastric lymphoma and adenocarcinoma. The infection by H. pylori is present in more than a half of the world population. Objectives: To detect H. pylori and to compare the diagnostic methods of the rapid urease test (RUT) and polymerase chain reaction (PCR). Materials and methods: The study was conducted between April and July, 2015. For such, three biopsies were collected from each patient. Two were used for PCR and one for RUT. Results: A total of 85 samples were collected from patients undergoing endoscopy, with 56 (65.88%) females and 29 (34.11%) males. From the total samples subjected to RUT, 15 (17.64%) were positive and 70 (82.35%), negative. In PCR for detection of gene 16S ribosomal ribonucleic acid (rRNA) of H. pylori, 66 (77.64%) presented positive results and 19 (22.35%), negative results. For the analysis of the presence of UreA gene in all samples, positive results were found in 70 (82.35%), and negative in 15 (17.64%). According to the results, RUT and the molecular test presented statistical difference. Conclusion: PCR is a useful method in the laboratorial routine to detect the presence of H. pylori in the stomach tissue, due to high sensitivity and specificity, but it requires a more careful analysis and standardization.
RESUMO Introdução: Helicobacter pylori é uma bactéria encontrada nas células epiteliais do trato gastrointestinal humano. Sua infecção relaciona-se com diferentes patologias, como gastrite crônica, úlcera péptica, linfoma gástrico e adenocarcinoma. A infecção por Helicobacter pylori está presente em mais da metade da população mundial. Objetivos: Detectar a presença de H. pylori e comparar os métodos diagnósticos do teste rápido de urease (TRU) e reação em cadeia da polimerase (PCR). Materiais e métodos: No estudo, realizado entre abril e julho de 2015, três biópsias foram coletadas de cada paciente. Duas foram usadas para realizar PCR e uma, para TRU. Resultados: Oitenta e cinco amostras foram coletadas dos pacientes por meio de endoscopia, sendo 56 (65,88%) mulheres e 29 (34,11%) homens. Do total dos indivíduos sujeitos ao TRU, 15 (17,64%) foram positivos e 70 (82,35%), negativos. Na PCR, na detecção do gene 16S ácido ribonucleico ribossômico (rRNA) de H. pylori, 66 (77,64%) apresentaram resultados positivos e 19 (22,35%), negativos. Para a análise da presença do gene UreA em todas as amostras, resultados positivos foram encontrados em 70 (82,35%) e negativos em 15 (17,64%). De acordo com os resultados, o TRU e o teste molecular apresentaram diferenças estatísticas. Conclusão: A PCR é um método útil na rotina laboratorial para detectar H. pylori em tecido de estômago devido à sua alta sensibilidade e especificidade, mas é necessária maior atenção na análise e na padronização.
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Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.
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Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Micélio/metabolismo , Paracoccidioides/metabolismo , Transcrição Gênica , Northern Blotting , DNA Complementar/metabolismo , Regulação para Baixo , Etiquetas de Sequências Expressas , Biblioteca Gênica , Internet , Modelos Biológicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Paracoccidioides/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Regulação para CimaRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. Here we report the sequence and analysis of a novel developmentally regulated gene and cDNA (Pbgadph), encoding a GAPDH homologue (PbGAPDH), of the pathogenic dimorphic fungus Paracoccidioides brasiliensis. We have analyzed the protein, the cDNA and genomic sequences to provide insights into the structure, function, and potential regulation of PbGAPDH. That Pbgapdh encodes PbGAPDH was demonstrated by micro-sequencing of the native protein homologue isolated from the fungus proteome. The deduced amino acid sequence of Pbgapdh showed identity to those of from other species (88-76%). Phylogenetic analysis indicated that GAPDH could be useful for the determination of evolutionary relationships. Expression of the Pbgapdh gene and the cognate protein were developmentally regulated in phases of P. brasiliensis, with a higher expression in the yeast parasitic phase and was induced during the transition from mycelium to yeast and decreased during the reverse process, transition from yeast to mycelium.
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Regulação Fúngica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Paracoccidioides/enzimologia , Paracoccidioides/crescimento & desenvolvimento , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , DNA Complementar , DNA Fúngico/química , DNA Fúngico/isolamento & purificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Íntrons/genética , Dados de Sequência Molecular , Micélio/genética , Paracoccidioides/genética , Filogenia , RNA Fúngico/análise , RNA Fúngico/isolamento & purificação , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência , Transcrição Gênica , Leveduras/genéticaRESUMO
Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus-host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome-PTS-1-targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen-dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis.
