Detection of Helicobacter Pylori infection using the immunohistochemical method

Author: Raiza Roberta VON THORWACHTER Co-authors: Iulian Alexandru CRSTEA MARIAN, Scientific coordinators: Prof. univ. dr. Mia Genevive FINTZESCU ef lucrri univ. drd. Georgian ROMNESCU
Introduction The association between chronic inflammation and cancer is now well established. Helicobacter pylori, which infects 50% of the worlds population and is now known to be responsible for inducing chronic gastric inflammation that progresses to atrophy, metaplasia, dysplasia, and gastric cancer. H. Pylori is a gram negative bacteria, helical shaped, with a length of 3-5 micro-meters and 0.5 micrometers diameter. The bacterium has 4-6 flagella, which allow it to pass through the mucous layer near the gastric epithelial cells. The bacterium produces adhesion molecules which bind with the lypopolizaharides of the cellular membrane, thus creating a better adhesion of the bacteria to the gastric epithelial cell. H. Pylori tests positive for oxidase, catalase, and urease. The virulence of H. Pylori is influenced by multiple factors. The most important are the presence or urease, of flagella and of the protein products of different genes: CagA (cytotoxin-associated gene), VacA (vacuolatingassociated cytotoxin). A problem with current methods for detection is the presence of large numbers of background normal flora.This immunohistochemical staining method may prove extremely helpful in the investigation of the presence of H. pylori, allowing the researcher to observe the morphology of the bacteria as well as its position regarding the gastric epithelium. Material and Methods To identify the bacterial infection of the gastric mucosa we used in this study 16 gastric specimens obtained by gastric biopsy or surgical resection from patients with the clinical supposition of gastritis, gastric ulcer or gastric cancer. The specimens were harvested from patients admitted in the Gastroenterology or Surgery clinics of the Emergency Clinical Hospital of Craiova. The samples were fixed in 10% formalin solution for 3-4 days depending of their size, and were paraffinwax processed. The paraffin blocks were cut using the microtome to 5m thick sections. These sections were stained for the anti-Helicobacter Pylori antibody (Dako, dilution 1:1000) using the Dakos EnVision detection system, and DAB (3,3'-diaminobenzidine) as chromogen substrate. The nuclei were counterstained using Mayer hematoxylin. Results and Discussion All the gastric specimens we used for this study tested positive to the immunohistochemical staining procedure, with different patterns of the bacterial position regarding the gastric mucosa. For most of the patients (10 out of 16), the bacteria was present immediately near the gastric epithelium, in the secreted mucus. The rest of 6 samples presented only some fragments remained from the bacterial degradation. Pathologists prefer immunohistochemistry (IHC) over routine histochemistry for the detection of Helicobacter pylori because it reduces the false-positive rate, being able to actually visualize the characteristic morphology of H pylori in the tissue. Partially treated H. Pylori may change shape, making its detection challenging. This method can evidence the bacterial detritus resulted as a consequence of the partial treatment, as probably was the case of the 6 specimens described earlier. Conclusions - The immunohistochemical method for detection H. Pylori infection is highly specific and sensitive, allowing the investigator to observe the bacterial morphology. - It can also determine the partially treated patients, allowing the clinicians to decide weather the treatment should be continued or not. References
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