Associação de alterações anatomopatológicas com o Helicobacter spp. por meio de análise imuno-histoquímica em estômagos de leitões

O objetivo deste trabalho foi relacionar os achados anatomopatológicos das lesões gástricas subclínicas de ocorrência natural em leitões com a presença, ou não, de Helicobacter spp. por meio da imuno-histoquímica. Foram utilizados 48 leitões de linhagem genética comercial. Os animais foram adquiridos em uma granja comercial, com peso médio de 34 Kg e idade média de 79 dias; após o abate, seus estômagos foram coletados e avaliados. Avaliações histopatológicas e imuno-histoquímicas foram realizadas em amostras das regiões anatômicas aglandular e glandular. Macroscopicamente, 34 (70,83%) leitões apresentaram lesões na região aglandular, enquanto que em 14 animais (29,17%) não foram encontradas alterações nesta região. Dos estômagos com lesão, 14 foram classificados como grau 1, seis como grau 2 e 14 como grau 3. Microscopicamente, 44 amostras (91,66%) apresentaram paraqueratose. Deste total, 22 apresentaram a forma discreta, 20 a moderada e dois a acentuada. Na avaliação macroscópica da porção glandular, 41 (85,4%) animais apresentaram alteração em pelo menos uma das três regiões, e em somente sete (14,6%) não foram encontradas lesões em nenhuma delas. Em 14 deles, houve aumento da atividade mucípara, em dois, houve erosão e, em cinco, hiperemia.  As lesões na região glandular do estômago foram mais extensas no antro e no cárdia, seguidas do fundo. Em relação à análise imuno

The aim of this study was to relate the anatomopathological findings of naturally occurring subclinical gastric lesions in piglets, with or without Helicobacter spp. through immunohistochemistry. Forty-eight piglets of commercial genetic lineage were used. The animals were acquired in a commercial farm, with an average weight of 34 kg and an average age of 79 days, and after slaughter, their stomachs were collected and evaluated. Samples from the glandular and aglandular anatomical regions were evaluated. Macroscopically, 34 (70.83%) samples had lesions on aglandular region, while 14 (29.17%) nothing had. Of the injured stomachs, 14 were classified as grade 1, six as grade 2 and 14 as grade 3. Microscopically, 44 samples (91.66%) showed parakeratosis. Of these, 22 showed a discreet manner, 20 moderate and two severe. In the glandular region, in 41 (85.4%) samples there was a change in at least one of the three regions, and only seven animals (14.6%) showed no change in any of the three. Fourteen samples showed increased muciparous activity, two showed erosion and five hyperemia. The lesions were higher in antral regions and cardic, followed the fundus. In relation to immunohistochemistry, 21(43.8%) samples were negative in all areas, 24 (50%) were positive in at least one, and none were positive in all. The anatomopathological findings showed a statistical relationship with the bacteria, and its immunostaining, not associated with gastric lesions in certain regions, demonstrates its saprophytic and opportunistic character.

Associação de alterações anatomopatológicas com o Helicobacter spp. por meio de análise imuno-histoquímica em estômagos de leitões / Association of anatomopathological changes with the Helicobacter spp. through immunohistochemical analysis in piglets stomachs

O objetivo deste trabalho foi relacionar os achados anatomopatológicos das lesões gástricas subclínicas de ocorrência natural em leitões com a presença, ou não, de Helicobacter spp. por meio da imuno-histoquímica. Foram utilizados 48 leitões de linhagem genética comercial. Os animais foram adquiridos em uma granja comercial, com peso médio de 34 Kg e idade média de 79 dias; após o abate, seus estômagos foram coletados e avaliados. Avaliações histopatológicas e imuno-histoquímicas foram realizadas em amostras das regiões anatômicas aglandular e glandular. Macroscopicamente, 34 (70,83%) leitões apresentaram lesões na região aglandular, enquanto que em 14 animais (29,17%) não foram encontradas alterações nesta região. Dos estômagos com lesão, 14 foram classificados como grau 1, seis como grau 2 e 14 como grau 3. Microscopicamente, 44 amostras (91,66%) apresentaram paraqueratose. Deste total, 22 apresentaram a forma discreta, 20 a moderada e dois a acentuada. Na avaliação macroscópica da porção glandular, 41 (85,4%) animais apresentaram alteração em pelo menos uma das três regiões, e em somente sete (14,6%) não foram encontradas lesões em nenhuma delas. Em 14 deles, houve aumento da atividade mucípara, em dois, houve erosão e, em cinco, hiperemia. As lesões na região glandular do estômago foram mais extensas no antro e no cárdia, seguidas do fundo. Em relação à análise imuno-histoquímica, 21 (43,8%) amostras tiveram resultados negativos em todas as regiões, e 24 (50%) foram positivas em pelo menos uma delas, sendo que nenhuma foi positiva em todas. Os achados anatomopatológicos demonstraram relação estatística com a bactéria e, sua imunomarcação não associada à lesão em certas regiões gástricas, demonstra seu caráter saprofítico e oportunista.

The aim of this study was to relate the anatomopathological findings of naturally occurring subclinical gastric lesions in piglets, with or without Helicobacter spp. through immunohistochemistry. Forty-eight piglets of commercial genetic lineage were used. The animals were acquired in a commercial farm, with an average weight of 34 kg and an average age of 79 days, and after slaughter, their stomachs were collected and evaluated. Samples from the glandular and aglandular anatomical regions were evaluated. Macroscopically, 34 (70.83%) samples had lesions on aglandular region, while 14 (29.17%) nothing had. Of the injured stomachs, 14 were classified as grade 1, six as grade 2 and 14 as grade 3. Microscopically, 44 samples (91.66%) showed parakeratosis. Of these, 22 showed a discreet manner, 20 moderate and two severe. In the glandular region, in 41 (85.4%) samples there was a change in at least one of the three regions, and only seven animals (14.6%) showed no change in any of the three. Fourteen samples showed increased muciparous activity, two showed erosion and five hyperemia. The lesions were higher in antral regions and cardic, followed the fundus. In relation to immunohistochemistry, 21(43.8%) samples were negative in all areas, 24 (50%) were positive in at least one, and none were positive in all. The anatomopathological findings showed a statistical relationship with the bacteria, and its immunostaining, not associated with gastric lesions in certain regions, demonstrates its saprophytic and opportunistic character.

La microbiota según la topografía gástrica en pacientes con bajo y con alto riesgo de cáncer gástrico en Nariño, Colombia / Microbiota according to gastric topography in patients with low or high risk of gastric cancer in Nariño, Colombia

Resumen Introducción. La inflamación del antro gástrico por Helicobacter pylori aumenta el riesgo de úlcera duodenal, y la del cuerpo gástrico puede producir gastritis atrófica e incrementar la probabilidad de cáncer gástrico. Estas reacciones inflamatorias diferenciadas según su localización, podrían explicarse por la composición de la microbiota gástrica asociada con H. pylori. Objetivo. Identificar y comparar la microbiota del antro y del cuerpo del estómago en individuos de dos poblaciones: una con alto riesgo y otra con bajo riesgo de cáncer gástrico en Nariño, Colombia. Materiales y métodos. Se incluyeron biopsias del cuerpo y el antro gástrico de pacientes con gastritis no atrófica o con gastritis atrófica y metaplasia. La microbiota se definió por secuenciación de la región V3-V4 del gen 16S del ARNr de H. pylori (illumina-MiSeq™). Las unidades taxonómicas operativas se clasificaron utilizando las bases de datos BLASTn y RDPII. Las diferencias entre las poblaciones microbianas del antro y del cuerpo gástrico se evaluaron mediante el análisis de varianza multivariado con base en permutaciones (Permutational Multivariate Analysis of Variance, PERMANOVA) y análisis multivariados. Resultados. La clase Epsilonproteobacteria representada por H. pylori fue más abundante en las biopsias del antro y del cuerpo de los individuos con gastritis no atrófica (>50 %), en tanto que, en los individuos con gastritis no atrófica, esta clase correspondió al 20 % con una mayor diversidad metagenómica. La infección por H. pylori disminuyó significativamente la diversidad metagenómica del antro (p=0,005), en comparación con la del cuerpo gástrico. Conclusiones. Los grupos bacterianos involucrados en la disbacteriosis pueden colonizar ambas regiones topográficas del estómago, independientemente de las reacciones sectorizadas de inflamación. La infección por H. pylori asociada con la microbiota gástrica está relacionada con su localización en el estómago, el tipo de lesión y el mayor o menor riesgo de cáncer gástrico, lo que sugiere su importancia en la disbacteriosis y la de esta en la enfermedad gástrica.

Abstract Introduction: Inflammation in the gastric antrum caused by Helicobacter pylori increases the risk of duodenal ulcer while inflammation in the body generates atrophic gastritis and increased risk of gastric cancer. These inflammatory responses according to gastric topography could be explained by the composition of the gastric microbiota associated with H. pylori. Objective: To identify and compare the microbiota of the gastric antrum and body of individuals from two populations, one with high risk and one with low risk of gastric cancer from Nariño, Colombia. Materials and methods: Biopsies of the gastric antrum and body of patients with non-atrophic gastritis or metaplastic atrophic gastritis were included. The microbiota was defined by sequencing the 16S rRNA gene, V3-V4 region, (illumina-MiSeq™). The operational taxonomic units were classified using the BLASTn and RDPII databases. The differences among microbial populations were evaluated with the PERMANOVA and multivariate analyses. Results: The Epsilonproteobacteria class represented by H. pylori was more abundant in the antrum and body biopsies of individuals with metaplastic atrophic gastritis (>50%) while in individuals with non-atrophic gastritis it was 20 % and had greater metagenomic diversity. Helicobacter pylori infection significantly decreases the metagenomic diversity of the gastric antrum (p=0.005) compared to that of the body. Conclusions: The bacterial groups involved in the dysbiosis can colonize both topographic regions of the stomach, regardless of the sectorized inflammation responses. Helicobacter pylori infection associated with the gastric microbiota is related to its localization in the stomach, the type of lesion, and the population at risk of gastric cancer, which suggests its importance in microbial dysbiosis and gastric disease.

Development and validation of a whole-cell ELISA for serologically diagnosing Helicobacter pylori infection in Brazilian children and adults: a diagnostic accuracy study

ABSTRACT BACKGROUND: Serological tests are practical, with low cost, but no noninvasive tests are available for diagnosing Helicobacter pylori (H. pylori) infection in Brazil. The aim here was to develop and validate enzyme-linked immunosorbent assay (ELISA) serological tests to detect anti-H. pylori immunoglobulin G antibodies, based on cultured strains from Brazilian patients. DESIGN AND SETTING: Cross-sectional, diagnostic accuracy study comparing a locally developed and validated ELISA and invasive tests among dyspeptic patients at two public hospitals in São Paulo, Brazil. METHODS: An ELISA test was prepared using whole-cell antigen from 56 strains. After genotypic characterization, it was standardized and optical density (OD) cutoffs were determined based on the serum antibody response of 100 H. pylori-negative samples, compared with 82 H. pylori-positive samples. Validation was performed on 174 symptomatic patients. RESULTS: The optimal OD cutoffs established (for monoclonal and polyclonal tests, respectively) were 0.167 and 0.164; overall ELISA sensitivity: 84.3%, 78.9%; specificity: 88.6%, 90.6%; positive predictive value (PPV): 75.4%, 80%; negative predictive value (NPV): 93.1%, 81.8%; accuracy: 87.3%, 86.2%; child and adolescent ELISA sensitivity: 74.2%, 81.8%; specificity: 90.8%, 86.7%; PPV: 66.6%, 84.3%; NPV: 95.8%, 84.8%; accuracy: 88.5%, 84.6; adult ELISA sensitivity: 84.4%, 75%; specificity: 86.9%, 93%; PPV: 81.8%, 78.3%; NPV: 88.9%, 91.8%; accuracy: 85.9%, 88.5%. CONCLUSION: The polyclonal serological test developed using local strains presented better diagnostic performance among children and adolescents, while the monoclonal test was better among adults. The results from both tests suggest that these in-house serological tests could be used to detect anti-H. pylori antibodies in our population, for screening purposes.

Asociación clínica, patológica y microbiológica de Helicobacter pylori en biopsias gástricas en el departamento de Caldas-Colombia / Clinical, pathological and microbiological association of Helicobacter pylori in gastric biopsies in the department of Caldas-Colombia

Objectivo: Establecer la asociación clínica, patológica y microbiológica de Helicobacter pylori en biopsias gástricas en una población del departamento de Caldas (Colombia). Materiales y método: Se incluyeron 72 pacientes que fueron remitidos para endoscopia digestiva a la clínica San Marcel de la ciudad de Manizales durante el segundo semestre de 2015; a quienes se les tomaron dos biopsias del antro y dos del cuerpo del estómago para estudio histopatológico y cultivo microbiológico. Los datos fueron sometidos a procedimientos estadísticos descriptivos y relacionales. Resultados: se encontró una proporción de H. pylori del 47,2% por análisis histológico y del 26,4% por cultivo microbiológico, con predominio del sexo femenino. Siendo la epigastralgia el principal motivo de consulta asociado a la infección por H. pylori. Conclusión: se encontró asociación entre motivo de consulta, sexo femenino, cultivo microbiológico y presencia histológica de H. pylori. La edad se asoció al diagnóstico endoscópico establecido. El cultivo presentó frente al hallazgo histopatológico una especificidad elevada (84,2%) y una baja sensibilidad (38,2%).

Objective: To establish the clinical, pathological and microbiological association of Helicobacter pylori in gastric biopsies in a population of the department of Caldas (Colombia). Materials and methods: We included 72 patients, who were referred for digestive endoscopy to the San Marcel clinic in the city of Manizales during the second half of 2015; two biopsies of the antrum and two of the body of the stomach were taken for histopathological study and microbiological culture. Data were submitted to descriptive and relational statistical procedures. Results: H. pylori was found in 47.2% by histological analysis and 26.4% by microbiological culture, with predominance in the female sex. Epigastralgia is the main reason for consultation associated with H. pylori infection. Conclusion: we found an association between the reason for consultation, female sex, microbiological culture and histological presence of H. pylori. Age was associated with established endoscopic diagnosis. The culture presented a high specificity (84.2%) and a low sensitivity (38.2%), compared to the histopathological finding.

Sensibilidad a los antimicrobianos de aislamientos de Helicobacter pylori aislados de lesiones gástricas / Antimicrobial susceptibility of Helicobacter pylori strains isolated from gastric lesions

Se evaluó la sensibilidad a los antimicrobianos de 30 aislamientos de Helicobacter pylori aislados de biopsias gástricas mediante los métodos de difusión por discos y tiras de E-test. Los antimicrobianos evaluados fueron amoxicilina, claritromicina, metronidazol y ciprofloxacina. No se encontraron cepas resistentes a amoxicilina, el 17% (5/30) fueron resistentes a claritromicina, el 20% (6/30) a ciprofloxacina por ambos métodos, y el 37% (11/30) a metronidazol por E-test. Si bien el número de cepas estudiadas fue reducido, hubo una sola discrepancia en la interpretación de la sensibilidad cuando se compararon ambos métodos: el metronidazol fue categorizado como sensible por E-test e intermedio por el método de difusión por discos. No pudo determinarse una asociación estadísticamente significativa entre el tipo de lesión histológica y el patrón de resistencia encontrado.

Antimicrobial susceptibility was evaluated by two diffusion methods: E-test strips to determine minimum inhibitory concentration (MIC) and disk diffusion for amoxicillin, clarithromycin, metronidazole and ciprofloxacin in 30 Helicobacter pylori strains isolated from gastric biopsies. No strains were resistant to amoxicillin, 17% (5/30) were resistant to clarithromycin, 20% (6/30) ciprofloxacin by both methods, and 37% (11/30) to metronidazole by the E-test. Although the number of strains studied was reduced, there was a single mismatch in interpreting susceptibility when both methods were compared; the same mismatch was observed for metronidazole, being categorized as sensitive by the E-test and as intermediate by disk diffusion. No association between the histological type of lesion and the resistance pattern found could be determined.

Frequency of periodontal pathogens and Helicobacter pylori in the mouths and stomachs of obese individuals submitted to bariatric surgery: a cross-sectional study

ABSTRACT Objectives This cross-sectional study compared the frequency of oral periodontopathogens and H. pylori in the mouths and stomachs of obese individuals with or without periodontitis submitted to bariatric surgery. Material and Methods One hundred and fifty-four men and women aged 18-65 were conveniently distributed into four groups. Two groups were composed of individuals who underwent bariatric surgery with (BP) (n=40) and without (BNP) (n=39) periodontitis and two obese control groups with (CP) (n=35) and without (CNP) (n=40) periodontitis. The oral pathogens Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Parvimonas micra, Treponema denticola, Tannerella forsythia, Campylobacter rectus, and Helicobacter pylori were detected by a polymerase chain reaction technique using saliva, tongue and stomach biopsy samples. Results Statistical analysis demonstrated that periodontopathogens were highly frequent in the mouth (up to 91.4%). In the bariatric surgically treated group, orally, P. gingivalis, T. denticola and T. forsythia were more frequent in periodontitis, while C. rectus was more frequent in non-periodontitis subjects. Stomach biopsies also revealed the high frequency of five oral species in both candidates for bariatric surgery (91.6%) and the bariatric (83.3%) groups. H. pylori was frequently detected in the mouth (50.0%) and stomach (83.3%). In the stomach, oral species and H. pylori appeared in lower frequency in the bariatric group. Conclusions Obese individuals showed high frequencies of periodontopathogens and H. pylori in their mouths and stomachs. Bariatric surgery showed an inverse microbial effect on oral and stomach environments by revealing higher oral and lower stomach bacterial frequencies.

Dyspeptic symptoms in patients with type 1 diabetes: endoscopic findings, Helicobacter pylori infection, and associations with metabolic control, mood disorders and nutritional factors

Objectives To evaluate, in a group of patients with long-standing type 1 diabetes (DM1), an association of dyspepsia symptoms with: changes in the gastroduodenal mucosa, infection by Helicobacter pylori, glycemic control, and psychological and nutritional factors. Subjects and methods A total of 32 patient with DM1 were studied (age: 38 ± 9 years; females: 25; diabetes duration: 22 ± 5 years). All patients answered a standardized questionnaire for the evaluation of gastrointestinal symptoms and underwent upper gastrointestinal endoscopy, with gastric biopsies for the evaluation of Helicobacter pylori infection. The presence of anxiety and depression was evaluated by the HAD scale. Nutritional parameters were BMI, arm and waist circumference, skinfold measurement, and body fat percentage. Results Upper endoscopy detected lesions in the gastric mucosa in 34.4% of the patients, with similar frequency in those with (n = 21) and without dyspepsia (n = 11). The patients with dyspepsia complaints showed greater frequency of depression (60% vs. 0%; p = 0.001), higher values for HbA1c (9.6 ± 1.7 vs. 8.2 ± 1.3%; p = 0.01) and lower values for BMI (24.3 ± 4.1 vs. 27.2 ± 2.6 kg/m2; p = 0.02), body fat percentage (26.6 ± 6.2 vs. 30.8 ± 7.7%; p = 0.04), and waist circumference (78.7 ± 8 vs. 85.8 ± 8.1 cm; p = 0.02). No association was found between the symptoms and the presence of Helicobacter pylori. Conclusions Dyspepsia symptoms in patients with long-standing DM1 were associated with glycemic control and depression, and they seem to negatively influence the nutritional status of these patients. .

Genotipificación de los factores de virulencia vacA y cagA de Helicobacter pylori en individuos de dos regiones de Colombia con riesgo opuesto de cáncer gástrico / Genotyping of Helicobacter pylori virulence factors vacA and cagA in individuals from two regions in Colombia with opposing risk for gastric cancer

Introducción. La prevalencia de infección por Helicobacter pylori es alta en Colombia; en la zona andina las tasas de cáncer gástrico son altas mientras que en las zonas costeras son bajas. Los genotipos de H. pylori cagA positivo y vacA s1 y m1 se asocian con un mayor riesgo de cáncer gástrico. Objetivo. Determinar las diferencias en las frecuencias de los genotipos de H. pylori asociados a virulencia en dos regiones de Colombia con riesgo opuesto de cáncer gástrico. Materiales y métodos. Se analizaron 401 biopsias del antro gástrico provenientes de 401 individuos con diagnóstico de gastritis no atrófica, gastritis atrófica o metaplasia intestinal; 256 se obtuvieron en la zona de alto riesgo (Tunja y Bogotá) y, 145, en la zona de bajo riesgo (Barranquilla, Santa Marta y Cartagena). La genotipificación de los genes de virulencia cagA y vacA se hizo mediante reacción en cadena de la polimerasa (PCR). Resultados. No se observó diferencia en la frecuencia de infección por H. pylori entre las dos zonas (77,3 Vs . 77,9 %, p=no significativo, ns). La presencia de cagA fue mayor en la zona de bajo riesgo (77,9 Vs . 69,2 %, p=ns). El alelo vacA s1 también fue más prevalente en la zona de bajo riesgo (61,8 Vs . 72,0 %, p=ns). El alelo vacA m1 presentó mayor prevalencia en la zona de alto riesgo (57,2 Vs . 42,8 %, p=ns). La combinación cagA positivo s1m1 también fue más frecuente en la zona de bajo riesgo (48,9 Vs . 38,9 %, p=ns). Conclusiones. Las diferencias en el riesgo de cáncer gástrico en estas dos zonas no pueden explicarse por las diferencias en la prevalencia de infección por H. pylori o en la virulencia de las cepas circulantes.

Introduction: The overall prevalence of Helicobacter pylori infection is high in Colombia; however, in the country´s Andean region, gastric cancer rates far surpass those in coastal areas. Helicobacter pylori genotypes cagA positive and vacA s1 and m1 are associated with an increased risk of gastric cancer. Objective: To compare the distribution of H. pylori genotypes associated with virulence in two regions in Colombia with opposing risk for gastric cancer. Materials and methods: Four hundred and one gastric antral biopsies were obtained and analyzed from 401 individuals diagnosed with non-atrophic gastritis, atrophic gastritis and intestinal metaplasia: 256 came from the high-risk area cities of Tunja and Bogotá, and 145 from the low-risk area cities of Barranquilla, Santa Marta and Cartagena. Genotyping of virulence genes vacA and cagA was performed by PCR. Results: No difference was observed in the frequency of H. pylori infection between the two areas (77.3% vs 77.9 %, p=non significant, ns). The presence of cagA was higher in the low-risk area (77.9% vs. 69.2 %, p=ns). The vacA s1 allele was also more prevalent in the low-risk area (61.8 % vs 72.0 %, p=ns). The vacA m1 allele was more prevalent in the high-risk area (57.2 % vs 42.8 %, p=ns). The cagA positive s1m1 combination was also more frequent in the low-risk area (48.9% vs 38.9%, p=ns). Conclusions: The differences in the risk of gastric cancer in these two geographic areas cannot be explained by differences in the prevalence of infection by H. pylori or by differences in the virulence of circulating strains.

Metagenomic analysis of the gastric microbiota cultivable from a patient with gastritis concomitant with Barrett´s esophagus / Análisis metagenómico de la microbiota gástrica cultivable a partir de un paciente con gastritis concomitante con esófago de Barrett

Barrett’s esophagus is a distal metaplasia characterized by the transformation of squamous mucosa into columnar mucosa. This esophageal phenotype is a product not only of the chronic reflux of gastric acids, but also by microorganisms that colonize the oral cavity and stomach. Two classes of microbiota can be identified in Barrett’s esophagus; microbiota type I is associated with the normal esophagus and type II with an inflamed esophagus. The present study describes the gastric microbiota of a patient with antral gastritis concomitant with Barrett’s esophagus absent infection with Helicobacter pylori. Gastric biopsies were obtained following the protocol of Sydney and following ethical practices. The isolates were cultivated under microaerophilic conditions on Columbia Agar supplemented with IsoVitaleX™ and 7% sterile blood. Extracted DNA was sequenced using 454-GS and the results analyzed on the MG-RAST server. Gram negative isolates were found and bacteria resistant to levofloxacin, amoxicillin, tetracycline, erythromycin, and clarithromycin. The phyla Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria, the genus Bacteroides and the species group Bacteroides fragilis were most abundant. Functionally, the metabolism of carbohydrates, amino acids, and to a lesser extent, the metabolism of cofactors and vitamins were most dominant, and of which the enzymes β-glucosidase (EC 3.2.1.21), β-galactosidase (EC 3.2.1.23) and β-N-acetylhexosaminidase (EC 3.2.1.52) were most dominant. The findings of this study, because they are of only one case may probably suggest a possible pathogenic role, previously undescribed for Bacteroides fragilis, associated with human gastritis when concomitant esophageal pathology exists.

El esófago de Barrett es una metaplasia distal caracterizada por la transformación de la mucosa escamosa a mucosa columnar. Este fenotipo esofágico es producto no solo de la exposición crónica al reflujo de ácidos gástricos sino también a microbios colonizantes de la cavidad oral y del estómago. El esófago Barrett presenta 2 clases de microbiotas; la microbiota tipo I asociada con esófago normal y la tipo II a fenotipos esofágicos inflamatorios. En el presente estudio se describió la microbiota gástrica de una paciente con gastritis antral concomitante con esófago de Barrett sin infección por Helicobacter pylori y se obtuvieron biopsias gástricas siguiendo el protocolo de Sydney y estándares bioéticos. Los cultivos se hicieron en condiciones microaerofílicas en agar Columbia suplementados con isovitalex y sangre estéril al 7%. El ADN extraído fue sometido a secuenciación empleando 454 GS y las lecturas fueron analizadas en el servidor MG-RAST. Se obtuvieron aislamientos gram-negativos y resistentes a levofloxacina, amoxicilina, tetraciclina, eritromicina y claritromicina. Los Phylum Bacteroidetes, Firmicutes, Fusobacteria y Proteobacteria, el género Bacteroides y las especies de grupo Bacteroides fragilis fueron los más abundantes. Funcionalmente, el metabolismo de carbohidratos, aminoácidos y en menor grado el metabolismo de cofactores y vitaminas fueron los más dominantes; de los cuales las enzimas la β-glicosidasa (EC 3.2.1.21), β-galactosidasa (EC 3.2.1.23) y la β-N-acetilhexosaminidasa (EC 3.2.1.52) fueron las más dominantes. Estos resultados, por ser de un solo caso, solo podrían sugerir un posible papel patogénico no descrito para Bacterioides fragilis asociado con gastritis humana cuando existe patología esofágica concomitante.

Identification of Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum from honey stomach of honeybee

This study aimed to isolate and identify Lactobacillus in the honey stomach of honeybee Apis dorsata. Samples of honeybee were collected from A. dorsata colonies in different bee trees and Lactobacillus bacteria isolated from honey stomachs. Ninety two isolates were Gram-stained and tested for catalase reaction. By using bacterial universal primers, the 16S rDNA gene from DNA of bacterial colonies amplified with polymerase chain reaction (PCR). Forty-nine bacterial 16S rDNA gene were sequenced and entrusted in GenBank. Phylogenetic analysis showed they were different phylotypes of Lactobacillus. Two of them were most closely relevant to the previously described species Lactobacillus plantarum. Other two phylotypes were identified to be closely related to Lactobacillus pentosus. However, only one phylotype was found to be distantly linked to the Lactobacillus fermentum. The outcomes of the present study indicated that L. plantarum, L. pentosus, and L. fermentum were the dominant lactobacilli in the honey stomach of honeybee A. dorsata collected during the dry season from Malaysia forest area - specifically "Melaleuca in Terengganu".

Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR / Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR.

The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.

Detección molecular y genotipificación de Helicobacter pylori en biopsias gástricas de pacientes adultos sintomáticos de la ciudad de Santa Fe, Argentina / [Molecular detection and genotypification of Helicobacter pylori in gastric biopsies from symptomatic adult patients in Santa Fe, Argentina].

Our goals were: a) to detect Helicobacter pylori in gastric biopsies of symptomatic adults by PCR, b) to detect the presence of the cagA gene as well as of the allelic variants of the vacA gene, and c) to correlate genotypes with the endoscopic diagnoses. H. pylori was detected in 81

(39/48) of patients by nested PCR for hsp60. The presence of cagA was detected in 15/22 of samples and vacA s1 - m1 was the most frequent allelic combination (15/22). Gastritis, the most frequent diagnosis, was associated with genotype cagA+ in 10/13 of patients. In this group, 9/13 showed the allelic variant vacA s1- m1. The variant vacA s2 - m2 was detected in 3/3 of gastritis cases by H. pylori with the cagA- genotype. These results are the first reported in our region and provide data of epidemiological interest.

Evaluation of the effect of presence of blood in the stomach on endoscopic diagnostic tests for Helicobacter pylori infection.

Introduction: Presence of blood in the stomach has been thought to affect the performance of diagnostic tests used in detecting Helicobacter pylori (H. pylori) in the stomach. This study evaluated the effect of blood on the efficacy of rapid urease test (RUT) and microscopic appearance of the biopsy after staining with Giemsa stain. Materials and Methods: Patients with bleeding oesophageal varices who met the inclusion criteria were tested for H. pylori by RUT and microscopic examination of the biopsy. A repeat endoscopy, RUT and histology were done one month following initial presentation. The performance of the diagnostic tests was evaluated with and without the presence of intraluminal blood. A combined result of the two tests, RUT and histology, carried out in presence or absence of blood for the diagnosis of H. pylori, when considered together was considered as the gold standard. Results: Thirty six patients included in the study were in the ages ranging between 15-60 years (mean age = 44.14 years ±2.1). The combination of tests at both visits showed 20/36 (55.6%) patients were positive for H. pylori. The decrease in H. pylori positivity in the presence of blood was significant for RUT (8.3% vs. 38.9%; P=0.005) and combined test (19.4% vs. 47.2%; P=0.02) but the decrease in positivity for histology (11.1% vs 30.6%) was not significant (P=0.08). In the presence of blood, the sensitivity of RUT, histology and combined tests were 15%, 20% and 35%, respectively. In the absence of blood, the sensitivity of RUT, histology and combination of tests was 70%, 55% and 85%, respectively. Conclusion: Blood in the stomach significantly decreased the sensitivity of RUT, histology and the combination of both. Negative results of these tests in acute upper gastro intestinal (GI) bleeding should therefore be interpreted carefully.

Clarithromycin-resistant Helicobacter pylori in Recife, Brazil, directly identified from gastric biopsies by polymerase chain reaction / Helicobacter pylori resistente à claritromicina no Recife, Brasil, identificada diretamente de biopsias gástricas pela reação em cadeia de polimerase

CONTEXT: Clarithromycin is the most effective drug used in the eradication of infection by Helicobacter pylori. Due to worldwide increase in resistance, pre-treatment susceptibility testing for clarithromycin is recommended. OBJECTIVES: To evaluate the prevalence of clarithromycin resistance of H. pylori in Recife, a city in Northeast Brazil. METHODS: From January 2006 to December 2007, 114 gastric biopsy samples positive for H. pylori at culture were directly assayed by polymerase chain reaction (PCR) to detect the most frequent point mutations involved in clarithromycin resistance. Results were compared with those obtained by Etests. RESULT: Molecular and phenotypic methods showed 111 (97.4 percent) susceptible or resistant concordant results. PCR detected 3 (2.6 percent) biopsy specimens with H. pylori-resistant genotypes, which were misdiagnosed as susceptible by Etests. In Recife, based on PCR results, primary clarithromycin resistance was found in 15 (16.5 percent) patients, prevalence close to that observed in Southeast Brazil. Resistance increased to 52 percent among previously treated patients. The point mutation A2143G was present in 20 (71.4 percent) of specimens and A2142G, in 8 (28.6 percent) of specimens. A2142C was not found. CONCLUSION: In Recife, the prevalence of primary clarithromycin resistance, 16.5 percent, showed the need for pretreatment susceptibility testing in H. pylori infections.

CONTEXTO: Claritromicina é a droga mais eficaz usada na erradicação da infecção pelo H. pylori. Devido ao aumento mundial da resistência, o teste de susceptibilidade à claritromicina pré-tratamento é recomendado. OBJETIVO: Determinar a prevalência de H. pyloriclaritromicina resistente em Recife, PE, Brasil. MÉTODO: De janeiro de 2006 a dezembro de 2007, 114 biopsias gástricas positivas à cultura para H. pylori foram diretamente analisadas pela reação em cadeia da polimerase (PCR), para detectar a frequência das mutações em ponto que envolvem a resistência à claritromicina. Os resultados foram comparados com os obtidos pelos E-testes. RESULTADO: Os métodos molecular e fenótipo fenotípico mostraram 111 (97.4 por cento) resultados concordantes, sensíveis ou resistentes. A PCR detectou três (2.6 por cento) espécimes de H. pyloricom genótipo resistente, diagnosticados erroneamente como sensíveis pelo E-teste. No Recife, baseando-se nos resultados da PCR, a resistência primária à claritromicina foi encontrada em 15 (16.5 por cento) pacientes, esta prevalência também foi observada no sudeste do Brasil. Entre os pacientes previamente tratados, a resistência elevou-se para 52 por cento. A mutação em ponto A2143G foi observada em 20 (71.4 por cento) dos espécimes e a A2142G, em 8 (28.6 por cento). A mutação A2142C não foi encontrada. CONCLUSÃO: No Recife, a prevalência da resistência a claritromicina, 16.5 por cento, mostrou a necessidade de realização dos testes de susceptibilidade prétratamento nas infecções por H. pylori.

Detection of Helicobacter spp. in gastric, fecal and saliva samples from swine affected by gastric ulceration

The aim of this study was to evaluate the presence of Helicobacter (H.) spp. in swine affected by gastric ulceration. Stomachs from 400 regularly slaughtered swine were subjected to gross pathological examination to evaluate the presence of gastric ulcers. Sixty-five samples collected from ulcerated pars esophagea and 15 samples from non-ulcerated pyloric portions were submitted to histopathological and molecular analyses, to detect Helicobacter spp., H. suis and H. pylori by PCR. Feces and saliva swabs were also collected from 25 animals in order to detect in vivo the presence of Helicobacter spp.. Gastric ulcers were detected in 373 cases (93%). The presence of ulcers in association with inflammatory processes was further confirmed by histological examination. Forty-nine percent (32/65) of the ulcerated esophageal portions as well as 53% (8/15) of the non-ulcerated pyloric portions were positive for Helicobacter spp. by PCR. The Helicobacter spp. positive samples were also positive for H. suis, while H. pylori was not detected. These results were confirmed by restriction enzyme analysis. With regard to feces and saliva samples, 15/25 (60%) and 16/25 (64%) were positive for Helicobacter spp. PCR, respectively but all were negative in H. suis and H. pylori specific PCR.

Caracterización del gen de la citotoxina vacuolizante de Helicobacter pylori a partir de biopsias gástricas de pacientes residentes en Tolima, Colombia / Characterization of the Helicobacter pylori vacuolating cytotoxin gene in gastric biopsy specimens from patients living in Tolima, Colombia

Helicobacter pylori es una bacteria que coloniza la mucosa gástrica de los humanos. Este microorganismo produce una citotoxina vacuolizante conocida como VacA y codificada por el gen vacA, el que se considera un factor de virulencia importante. Las cepas de H. pylori con diferentes alelos de vacA exhiben una gran variedad de fenotipos, algunos de los cuales han sido asociados con enfermedades gastroduodenales. El presente estudio pretende aportar datos sobre la prevalencia de H. pylori y de los genotipos de vacA en pacientes residentes en Tolima (Colombia), así como determinar la relación entre estos datos y el desarrollo de diferentes patologías gastroduodenales. Se incluyeron en este análisis 73 pacientes con diferentes patologías gástricas. Con el ADN total extraído de cada biopsia, se determinó la presencia de la bacteria mediante la amplificación de un fragmento específico del gen 16S ADNr. También se realizó la genotipificación del gen vacA por PCR. De las 50 muestras genotipificadas, el 52% mostró el alelo vacA s1m1, el 42% el alelo vacA s2m2, el 4% el s1m2 y el 2% los alelos s1,s2,m1,m2. Se evidenció una mayor sensibilidad en la detección de H. pylori por medio del gen vacA que por el gen 16S ADNr. En la población evaluada no se encontró asociación entre el genotipo de vacA y la presencia de las distintas patologías incluidas en este estudio.

Helicobacter pylori successfully colonizes the gastric niche. These bacteria produce a vacuolating cytotoxin known as VacA, which is codified by the vacA gene. This protein represents an important virulence factor. H. pylori strains have different vacA alleles, which show a variety of phenotypes that have been associated with gastrointestinal diseases. The aim of this study was to generate data about the prevalence of H. pylori and the vacA genotype in Tolima (Colombia) residents, and to evaluate if there exists a relationship between these data and the development of different gastrointestinal pathologies. Seventy three patients with different pathologies were included. The DNA extracted from biopsy specimens was analyzed and the presence of bacteria was determined by amplifying a fragment of the 16 rDNA gene. The vacA genotype was also determined by PCR. Fifty-two percent out of the 50 genotyped samples showed vacA s1m1 allele, 42% vacA s2m2, 4% s1m2, and 2% s1,s2,m1,m2. A higher sensitivity for the detection of H. pylori was evidenced by amplifying the vacA gene rather than the 16S rDNA gene. No association was found between the vacA genotype and the gastrointestinal diseases included in the study.

Value of H and E and giemsa stains in histological identification of helicobacter pylori in gastric biopsies

To determine the frequency of H. Pylori infection and compare the results of H and E and Giemsa stained sections of gastric biopsies. This prospective study conducted in histopathology department at Sheikh Zayed Hospital Lahore from Aug 2008 to Dec 2008 included 88 patients with history of dyspepsia and endoscopic features of gastritis. H and E and Giemsa stained sections of the biopsies were examined microscopically. Giemsa stain was taken as gold standard and the sensitivity, specificity, predictive value [positive and negative] and diagnostic accuracy of H and E were computed. In our study group of 48 males and 40 females [total 88] with ages ranging from 18 to 75 years [Mean +/- SD: 39.13 +/- 11.27], H. Pylori was present in 50 [56.81%] cases and absent in 38 [43.19%] cases on Giemsa stain [gold standard]. H and E stain revealed 42 true positive, 5 were false positive, 33 true negative and 8 false negative cases. H and E stain has sensitivity of 84%, specificity 86.84%, positive predictive value 89.36% and negative predictive value of 80.48%. The overall diagnostic accuracy [efficacy] of H and E stain was 85.22%. H and E stained sections of gastric biopsies reveal tissue morphology and disease status. When combined with Giemsa stain, H and E stain is reliable for diagnosis of H. Pylori infection

Detección de Helicobacter pylori en mucosa gástrica y cavidad oral / Helicobacter pylori detection in gastric and oral mucosa

Helicobacter pylori (H pylori) infection has been related to various gastroduodenal disorders. The objective of this study was to detect H pylori in gastric mucosa and relate it to its presence in the oral cavity. Fifty-four patients with medical indication oi digestive endoscopy from the Gastroenterology Unit of the Regional Hospital of Concepción, Chile, were studied. Gastric samples were obtained from each patient from antrum and corpus through endoscopic biopsies. Oral samples were obtained from dental plaque and saliva swabs from the floor of the mouth and the base of the tongue. Oral and gastric sample were studied by culture. Oral samples from patients with positive gastric cultures for H pylori (n = 21) were studied by culture, conventional PCR and Real Time PCR. Ail cultures from oral samples were negative (0/21) for H pylori. Only one sample of dental plaque was positive with conventional PCR (1/21), while ail samples of saliva were negative. However, samples from ail patients were positive with Real Time PCR (20/21 dental plaque, 21/21 saliva from the floor of the mouth, 20/21 saliva from the base of the tongue). The results suggest that there is a correlation between the presence of H pylori in gastric mucosa and the oral cavity. Also, that Real Time PCR the best technique to detect low number of bacteria in the oral cavity.

La infección por Helicobacter pylori está vinculada a diversas patologías gastroduo-denales. El objetivo de este estudio fue detectar H. pylori en la mucosa gástrica y relacionar su presencia con H. pylori en la cavidad oral. Se estudiaron 54 pacientes de la Unidad de Gastroenterología del Hospital Regional de Concepción, con indicación de endoscopía digestiva alta. Para cada paciente se realizó biopsia de mucosa gástrica (antro y cuerpo), y se obtuvieron 3 muestras orales (placa bacteriana, saliva de la base de la lengua y piso de boca). Las muestras gástricas y orales fueron cultivadas. Las muestras orales de los pacientes que presentaron cultivos de biopsia gástrica positivos para H. pylori (n = 21) fueron sometidas a análisis de detección de la bacteria mediante cultivo, PCR convencional y PCR de Tiempo Real. Los resultados obtenidos para las muestras orales fueron: cultivo 100 por ciento negativos (0/21), con PCR convencional se detectó H. pylori sólo en un paciente (una muestra de las 3 en placa bacteriana) (1/21), mientras que todas las muestras de saliva de piso de boca y base de la lengua fueron negativas. Veintiún pacientes resultaron positivos para la bacteria por la técnica de PCR de Tiempo Real en las muestras orales, 20/21 en muestras de placa bacteriana, 21/21 en saliva de piso de boca y 20/21 en base de lengua. Los resultados sugieren que existe correlación entre la presencia de H. pylori en la mucosa gástrica y en la cavidad oral. Además, que la técnica de PCR de Tiempo Real es la más adecuada para detectar el bajo número de bacterias de H. pylori de la cavidad oral.

Apoptosis in different gastric lesions and gastric cancer: relationship with Helicobacter pylori, overexpression of p53 and aneuploidy

Apoptosis has an essential function in maintaining the integrity of the gastrointestinal mucosa. Its deregulation is associated with the occurrence of lesions such as in atrophic gastritis, peptic ulcers, intestinal metaplasia, and stomach tumorigenesis. Thus, the aim of the present study was to investigate the frequency of apoptotic cells (apoptotic index, AI) by using two different immunohistochemical techniques, TUNEL and anti-activated caspase-3 antibody (CPP32), in gastric dyspepsia [chronic gastritis (CG, N = 34), chronic atrophic gastritis (CAG, N = 11), gastric ulcer (GU, N = 17), and intestinal metaplasia (IM, N = 15)], normal gastric mucosae (NM, N = 8), and gastric adenocarcinoma (GC, N = 12). The relationship was investigated between the AI and Helicobacter pylori infection, diagnosed by PCR, overexpression of p53 protein determined by immunohistochemistry, and aneuploidy by fluorescence in situ hybridization, as performed by our laboratory in previous studies. No significant differences were observed in AI between the different groups, whether by the TUNEL technique (F = 1.60; p = 0.1670) or by CPP32 antibody (F = 1.70; p = 0.1420). Nonetheless, CAG and CG groups had AI statistically higher than those of normal mucosae. These two groups (CAG and CG) also showed a higher frequency of apoptosis-positive cases (TUNEL+ or CPP32+). Generally, there was no correlation between the AI detected by the TUNEL and CPP32 techniques in the groups studied, except in the GC group (r = 0.70). Moreover, there was no significant association between apoptosis and H. pylori infection, overexpression of p53 protein and aneuploidy, but the H. pylori-positive cases only of GU (p = 0.0233) and IM (p = 0.0253) groups displayed a statistically higher AI compared to H. pylori-negative NM, when the CPP32 antibody technique was used. Thus, CG and CAG have increased apoptosis, which may occur independent of an association with H. pylori infection, aneuploidy...