Antibiotyping and Prevalence of Virulent Genotypes Among Helicobacter Pylori and Their Impact on Response to Therapy

Extraction of DNA, using Wizard® Genomic DNA Purification Kit (Promega-USA), will be done following the manufacturer instructions. For detection of vacuolating cytotoxin A and cytotoxin-associated gene A gene, polymerase chain reaction amplification will be performed with a 9 minutes' initial denaturation at 94˚C, followed by 35 cycles of 1 minute at 94˚C, 45 seconds at 60˚C, and 45 seconds at 72˚C. Final extension will be performed for 5 minutes at 72˚C. For detection of vacuolating cytotoxin A (S1/S2, m1, m2), PCR will be performed with a 9 minutes' initial denaturation at 94˚C, followed by 35 cycles of 1 minute at 94˚C, 45 seconds at 56˚C, and 45 seconds at 72˚C. Final extension will be performed for 5 minutes at 72˚C. Amplified DNA will be analyzed by agarose gel electrophoresis. A positive sample will give bands at DNA fragment 138-bp for cytotoxin-associated gene A, at 259/286-bp for vacuolating cytotoxin A s1/s2, and 290-bp and 352-bp for m1 \& m2, respectively

During endoscopy, three biopsies will be taken from antrum and/or corpus of and will be examined by rapid urease test also known as Campylobacter-like organism test (Kimberly-Clark,USA). Culture of the bacterium on Columbia agar plus 5-7% defibrinated horse or sheep blood and selective Dent supplement under microaerophilic condition at 37degree Celsius for 3-5days.

Histological sections from the antrum and body regions will be stained with Giemsa stain. Giemsa stain working solution was prepared as follows: 40 ml Giemsa stock solution with 60 ml of distilled water. Giemsa stock solution will be prepared as follows: Giemsa powder 4 g, glycerol 250 ml and methanol 250 ml. Histological sections will be examined by a gastrointestinal pathologist to standardize the classification of gastritis. Histopathological classification will be recorded as following (acute gastritis, mild-moderate- severe chronic gastritis, Glandular atrophy or Intestinal metaplasia).

it will be done patient before start empirical therapy and multiple antrum and/or corpus biopsies will be taken . Patient will be fasting at least 8 hours.Upper and findings will be recorded in the report as presence of gastroesophageal reflux disease, diffuse or localised gastric and/or duodenal redness, mucosal swelling, atrophy of mucosa, nodularity, intestinal metaplasia, erosions or ulcers.

Start empirical antibiotics regimens patient triple therapy ((levofloxacin 400 mg once , amoxicillin 1000 mg twice for 2 weeks) and (Proton pump inhibitor twice for 1 month)). Follow up after 2 weeks after finishing regimen by Stool Ag in stool

Any symptomatic patient is above 18 years old with H pylori infection patients diagnosed by positive H pylori Ag in stool will be included. assessment of laboratory response, 2 weeks after finishing empirical therapy.