الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Helicobacter pylori is a Gram-negative spiral bacterium that inhabits the gastric mucosa of the human stomach in approximately half of the world’s population for a life time. H.pylori infection is widespread in humans; it is present in 20% to 50% of the population in the developed countries and 80% of the population in developing countries. Infection of this unique ecological niche by H.pylori display only asymptomatic gastritis whereas a small proportion may progress into peptic ulcers, persistent infection also increases an individual’s risk for development of gastric adenocarcinoma,and gastric mucosa-associated lymphoid tissue lymphoma. In 1994, H.pylori was declared a group I carcinogen.
Different degrees of bacterial virulence, environmental influences, and host factors are believed to contribute to the differential clinical squeal of the infection. For the bacterium to succeed in its long-term colonization in the human stomach a set of bacterial virulence determinants was developed for initial adhesion; adherence of H.pylori to gastric mucosa is widely assumed to play an important role in the initial colonization and long term persistence; and for the altering gastric physiology. The major virulence factors of H.pylori is vaculating cytotoxin (Vac A), which causes cytoplasmic vacuolization in gastric epithelial cells, another well-characterized virulence factor is cytotoxin associated gene (cag A), which encoded by one of the genes located in the cag pathogenecity island (PAI), and the blood group antigen binding adhesin (BabA), encoded by BabA2 gene has been shown to mediate adherence of H. pylori to human gastric Lewis b blood group antigens on gastric epithelial cells that function as receptors for H.pylori adhesin and mediate its attachment to gastric pit and surface mucosa.
Currently, there are several popular methods for detecting the presence of H.pylori infection, each having its own advantages, disadvantages, and limitations. Several invasive and noninvasive tests are available according to whether or not endoscopic biopsy is necessary. Non invasive tests, based on peripheral samples, such as blood, breath samples, stool, urine, or saliva for detection of antibodies, urease activity (UBT), or bacterial antigens respectively. Invasive tests such histological evaluation, culture, polymerase chain reaction (PCR), and rapid urease tests are typically performed on tissue obtained at endoscopy
The aim of this study is detection of BabA2 gene to estimate its prevalence in our H.pylori infected patients as a one of the major virulence factors associated with H.pylori and contribute to the different clinical outcomes, and to evaluate the efficiency of using conventional PCR technique as a powerful technique for its molecular detection in gastric tissue biopsies obtained during endoscopy from patients infected by H.pylori, evaluation of the clinical outcomes due to infection by BabA2 positive strains, detection of the Lewis b expression status, and its relation to H.pylori colonization density and subsequent disease outcome.
Fifty untreated patients suffering from dyspeptic complaints were consecutively enrolled in this study and underwent upper gastro-duodenal endoscopy. The following investigations were carried out for all cases under study:
Routine upper gastro-duodenal endoscopy with recording of all pathological signs found and 6-8 gastric tissue biopsies were taken.
Detection of H.pylori infection in gastric tissue biopsy using:
i. Biochemical analysis to detect the presence of H. pylori urease activity using rapid urease test (CLO test).
ii. Histological examination, gastric biopsies were fixed in 10% formalin ,embedded in paraffin wax; sections from tissue blocks were examined using routine H&E Stain , determining H.pylori colonization densities (if present),and scoring of H. pylori infection as a gold standard.
Histopathological examination of H&E Stained sections, for examining gastric (-duodenal) pathology to detect severity of gastritis, atrophy, ulceration, or intestinal metaplasia.
The paraffin sections from tissue blocks were stained using immunohistochemical analysis for Lewis b, and scoring of Lewis b expression in gastric tissue biopsy.
Molecular detection of BabA2 gene of H.pylori using conventional PCR technique in DNA samples which isolated from gastric tissue biopsy.
The results of these investigations revealed that:
• Routine upper gastro-duodenal endoscopy:
Superficial gastritis was observed in most of the cases (62%), gastro-duodenitis (16%), and Hiatal Hernia (10%). while atrophic gastritis and gastric ulcers were observed in only 2% and 4% of cases, respectively. It was noted that most of the cases showed more than one endoscopic finding during examination.
• Biochemical investigation to detect the presence of H.pylori urease activity (RUT/CLO Test):
Biochemical findings of rapid urease test, it was found that 37 out of 50 cases are positive for RUT while 13 cases were negative.
• Histological grading of H.pylori infection as a gold standard.
It was found that out of 50 studied cases, 9 (18%) of patients showed grade 0 infection (negative cases) in histological examination, while the remaining 41 (82%) of patients showed grade 1 (42%), grade 2 (28%) and grade 3 (12%).
Rapid urease test ,when compared to histological grading of H.pylori infection ,it was found that out of 41 positive cases by histological examination, only 36 (87.8 %) were also positive by the rapid urease test. On the other hand, out of 9 that were negative by histology 8 (88.9%) were negative by rapid urease test. this results showed that RUT had sensitivity of 87.8%, specificity of 88.9%.
This study was enrolled on 50 subjects (30 males and 20 females). Patient’s age ranged from 25 to 68 years. Study of age and gender distributions of H.pylori infection in studied cases revealed that, Although higher percentage of H.pylori infection were detected in middle age groups with slight predominance in men, the statistical analysis showed that , there was no significant difference between neither age groups nor both sex groups.
• Histopathological examination of H&E Stained sections:
It was found that within the 50 patients studied, most of the cases (48%) positive for H.pylori infection were accompanied by superficial gastritis. Moreover, 34% of cases representing incomplete intestinal metaplasia (IM),10% with complete IM, atrophic gastritis and chronic gastric ulcers were recorded in only 2% and 4% of cases respectively .While all cases (18%) negative for H.pylori infection representing mild superficial gastritis.
• Immunohistochemical analysis for Lewis b, and scoring of Lewis b expression in gastric tissue biopsy:
Immunohistochemical scoring of Lewis b expression in gastric tissue biopsy of studied cases showed that (24%) of studied cases showed grade 0 (negative expression), while the (42%) of cases showed grade 1, grade 2 (26%) and grade 3 (8%). Comparing the grades of Le b expression with the histological scoring of H.pylori infection showed that, there is a significant association (p< 0.001*) between the intensity of Le b expression and colonization density of H.pylori.
• Molecular detection of BabA2 gene of H.pylori using conventional PCR technique:
BabA2 gene was detected using conventional PCR technique in tested DNA samples which extracted from gastric tissue biopsies of all studied cases. The results revealed that, Out of 41 positive cases by histological examination, 39(95.12%) were positive by the PCR test for BabA2 gene. On the other hand, the 9 negative cases histological examination were also negative by PCR. Based on these results, the sensitivity and specificity of PCR were determined and were found 95.1% and 100% respectively. Studying the relation between BabA2 gene according to PCR results and disease outcomes regarding to histopathological findings revealed that, severity of disease outcome and BabA2 gene was found to be statistically significant (p = 0.001*).Relation between BabA2 gene and colonization density regarding to the Lewis b expression detected using immunohistochemistry, was found to be statistically significant (p <0.001*).