Does colonization of Helicobacter pylori in the heterotopic gastric mucosa play a role in bleeding of Meckel's diverticulum?

Does Colonization of Helicobacter pylori in the Heterotopic Gastric Mucosa Play a Role in Bleeding of Meckel’s Diverticulum? By Orkan Ergu¨n, Ahmet C¸elik, Ulus S. Akarca, Teoman S¸en, Murat Alkanat, and Ata Erdener I˙zmir, Turkey

Background/Purpose: Helicobacter pylori is a microorganism known to colonize in gastric type of mucosa and is associated with gastritis and peptic ulceration. The aim of the study was to determine whether colonization of H pylori in heterotopic gastric mucosa plays a role in bleeding of Meckel’s diverticulum. Methods: Histopathologic slides of patients who had undergone resection of Meckel’s diverticulum in recent 5 years were reexamined for the presence of H pylori in heterotopic gastric mucosa. Polimerase chain reaction (PCR) test was used to trace the genetic material of urease gene and 16s rDNA amplifications for H pylori.

patients presented with acute bleeding of the diverticula, whereas 3 of them were asymptomatic. None of the 13 gastric mucosa bearing diverticula were colonized with H pylori. PCR was unable to show any trace of genetic material for H pylori. Conclusion: Although the role of H pylori is well established in the gastric mucosal ulceration, its presence is not essentially required to induce “heterotopic gastritis” that may result in bleeding of the Meckel’s diverticulum. J Pediatr Surg 37:1540-1542. Copyright 2002, Elsevier Science (USA). All rights reserved.

Results: Thirteen of the 30 histopathologic slides of Meckel’s diverticula had heterotopic gastric mucosa. Ten of the 13

INDEX WORDS: Helicobacter pylori, Meckel’s diverticulum, heterotopic gastric mucosa, polimerase chain reaction.

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colonization of H pylori by an experienced pathologist in a blinded fashion. The results of the current histopathologic review were compared with previous reports for each specimen to provide a double blind control. Possibility of bacterial disintegration either promptly owing to inadequate formaline fixation of the specimen or over the past time was considered. In such case, H pylori, with its typical morphologic appearance, would not be detected in the histologic examination; however, the genetic material would not be lost, and therefore could be traced by PCR study. Paraffin blocks of the samples bearing heterotopic gastric mucosa preserved for pathologic analysis were recut into 5-␮m cross-sectional slices for each patient. These slices were deparaffinized by 300 ␮L of toluene, and the remaining tissue was homogenized, and DNA extraction was performed using EZ-DNA kit (Biological Industries, Israel). The homogenate was treated by 100 ␮L of lysis solution (guanidinum isothiocyanate) for 5 minutes and then centrifuged in 10,000g for 10 minutes to precipitate cellular debris. The supernatant was separated and treated by 100 ␮L of cold absolute alcohol for 3 minutes to precipitate the DNA. After 5 minutes of centrifugation in 5,000g, the supernatant was discharged, and the precipitate was treated by 50 ␮L of absolute alcohol plus 50 ␮L lysis solution. This compound then was centrifuged for one minute in 1,000g, and the supernatant was discharged. Absolute alcohol was added onto the precipitate to preserve until PCR study.

ECKEL’S DIVERTICULUM (MD) is the most common developmental anomaly of the gastrointestinal (GI) tract. Gastric heterotopia is present in almost half of the cases,1,2 and bleeding often results from the ulceration of adjacent ileal mucosa. The association of Helicobacter pylori in chronic gastritis and peptic ulceration is well established.3,4 To determine whether bleeding from the MD is associated with H pylori–induced gastritis, we undertook detailed histopathologic review of the specimen. We also incorporated polimerase chain reaction (PCR) to strengthen the evidence of any possible genetic material of H pylori in heterotopic gastric mucosa in MD. MATERIALS AND METHODS Patients who had undergone resection of MD in the last 5 years were identified form the hospital registry, and the charts were reviewed. The symptoms of presentation, sex, and age were recorded. Histopathologic slides were retrieved from the department of pathology, and all the slides were available for reexamination. To increase the accuracy, 5 to 8 additional slides for each specimen were obtained from the paraffin blocs reserved in the archives of the department of pathology. The slides were reviewed for the presence of heterotopic mucosa and

PCR Technique From the Departments of Pediatric Surgery, Gastroenterology, and Pathology, Ege University Faculty of Medicine, I˙zmir, Turkey. Address reprint requests to Orkan Ergu¨n, MD, Assistant Professor of Pediatric Surgery, Department of Pediatric Surgery, Ege University Faculty of Medicine, 35100, I˙zmir, Turkey. Copyright 2002, Elsevier Science (USA). All rights reserved. 0022-3468/02/3711-0005$35.00/0 doi:10.1053/jpsu.2002.36180 1540

Nested PCR test was used for H pylori urease gene amplification. DNA precipitate was dissolved in 50 ␮L TE buffer solution. First-step PCR was performed using 50 ␮L solution that consisted of 10 ␮L extracted DNA, 10 pmol external primers, 2.5 U Taq polimerase, 50 mmol/L KCl, 10 mmol/L TrisHCl, 1.5 mmol/L MgCl2, and 0.25 ␮mol/L dNTP (dATP, dCTP, dGTP, dTTP). The primers used for the first step were H3: 5⬘-GCCAATGGTAAATTAGTTCC-3⬘ and H4: 5⬘-TTACTCCTTAATTGTTTTTAC-3⬘. This solution was denatured

Journal of Pediatric Surgery, Vol 37, No 11 (November), 2002: pp 1540-1542

HELICOBACTER PYLORI IN MECKEL’S DIVERTICULUM

at 94°C for 3 minutes, and then amplification was performed in 35 cycles at 94°C for 1 minute, 50°C for 1 minute, and 70°C for 1 minute. At the last stage, elongation was performed at 70°C in 5 minutes. Ten microliters of first-step PCR product was subjected to secondstep PCR under same conditions. The primers used in the second step were H5: and H6: 5⬘-ATAGTTGTCATCGCTTTTAGC-3⬘. Ten microliters of the second-step PCR product was processed in 1.5% agar for one hour, and examined under ultraviolet light to determine whether the expected 258 base couples–long band was visible. Positive and negative controls were used for each PCR groups. Gastric mucosal biopsy samples that were known to be H pylori positive were used as positive controls. The sensitivity of this PCR process was determined to be 5 to 10 copies. Extracted DNA also was used for 16s rDNA (DNA of 16s ribosomal RNA that is known to be common for all Helicobacter species) amplification. The primers used for this process were; HS1: 5⬘-AACGATGAAGCTTCTAGCTTGCTAG-3⬘ and HS2: 5⬘-GTGCTTATTCGTTAGATACCGTCAT-3⬘. The solution was denatured at 94°C for 3 minutes. Forty cycles of PCR amplification were performed at 94°C for 1 minute, 60°C for 1.5 minutes, and 72°C for 1 minute cycles. Lastly, elongation was performed at 72°C for 5 minutes. Negative and positive controls were used for each PCR group. Gastric mucosa biopsy samples that were known to be H pylori positive were used as positive controls. PCR product was processed in 1.5% agar for one hour, and examined under ultraviolet light to determine whether the expected 399 base couples–long band was visible. The sensitivity of this PCR process was determined to be 50 to 100 copies.

RESULTS

There were 23 boys and 7 girls with a mean age of 6.6 ⫾ 5.1 years (range, 4 months to 18 years). Thirty specimens were examined. Ten patients were symptomatic (bleeding), whereas 20 patients were asymptomatic (incidental). The latter group were identified during abdominal operations for other causes (appendicitis in 18, volvulus in 2). None of the symptomatic patients required immediate surgery. Bleeding was controlled by conservative measures. Surgery was planned on an elective basis, and no prophylactic antibiotics were used preoperatively. Histopathologic reports indicated the presence of heterotopic gastric mucosa in 13 of the specimens (43.3%). There were pancretic heterotopia in 2 MD (6.6%). No H pylori–like organisms were identified in the ectopic gastric mucosa of MD. Features of chronic gastritis or peptic ulceration in ectopic gastric mucosa were not evaluated in histopathologic examination, because all of the symptomatic patients received H2 receptor blocker prophylaxis preoperatively that would suppress the findings. The remaining 3 patients with ectopic gastric mucosa were asymptomatic. Current histopathologic reports were consistent with previous reports in our records. PCR study for H pylori was negative for all heterotopic gastric mucosa– bearing specimens (Figs 1 and 2). DISCUSSION

H pylori is a pathogen recognized to colonize in the upper gastrointestinal tract selectively and is associated

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Fig 1. Photomicrograph of PCR test for H pylori urease gene amplification Columns (a) sample of a gastric mucosa known to be infected by H pylori (positive patient sample); (b) sample from a gastric mucosa that is not infected by H pylori (negative patient sample); (c) positive control of urease gene amplification; (d) negative control of urease gene amplification; (e) DNA marker of one of the samples in the series that show no amplification of urease gene for H pylori.

with a variety of gastroduodenal diseases including gastritis, gastroduodenal ulcers, and possibly gastric malignancies.1 Although this small Gram-negative bacteria with its typically curved rod-shape appearance could be identified and cultured since 1983,5-8 in 1989 de Cothi et al3 attracted special attention when they first found Helicobacter-like organisms (formerly named Campylobacter-like organisms) identical in appearance, and staining could colonize and inflame the heterotopic gastric mucosa in MD. After this finding, debate has arose regarding the method of its transmission,1 and H pylori has been found in human saliva and heterotopic gastric mucosa in the distal gastrointestinal tract such as MD and rectal heterotopia1,4,5,9-14 suggesting the possibility of a fecal-oral route. MD is the most common congenital anomaly of the gastrointestinal tract. It is caused by a failure of regression of the vitelline duct, a process that normally is present between the fifth and the seventh weeks of fetal life. The ratio of MD between men and women is equal in asymptomatic subjects, but among symptomatic patients, the condition occurs more frequently in men (3 to 4:1).15 One of the most frequent complications of MD in childhood is painless bleeding caused by ulceration of the adjacent ileal mucosa. Many studies have focused on “gastritis” of heterotopic gastric mucosa found in MD and whether this entity could be related to H pylori colonization.1,3,5,9-11 Similarly, our hypothesis was that if H pylori could colonize in heterotopic gastric mucosa, H pylori–induced gastritis could play a role in bleeding of

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Fig 2. Photomicrograph of PCR test for H pylori 16s rDNA (DNA of 16s ribosomal RNA that is known to be common for all Helicobacter species) amplification, Columns (a) positive sample of 16s rDNA; (b) negative sample of 16s rDNA; (c) DNA marker of one of the samples in the series that show no amplification of 16s rDNA gene for H pylori.

MD. Although some reports have failed to show H pylori–like organisms in the heterotopic gastric mucosa of MD,1 those studies that were able to identify H pylori–like organisms in gastric heterotopia of MD are based on standard morphologic findings by histopathologic examinations.3,5,10-14,16 In few studies, further effort to identify the microorganism by transmission electron

microscope and immunohistochemistry-immunoperoxidase methods were made.9,17,18 To our knowledge, this study is the first to incorporate a PCR study to support the histopathologic evidence. In this study, histopathologic examinations of the specimen could not be able to show the presence of H pylori–like organisms in heterotopic gastric mucosa of MD. Presuming that there existed the theoretical possibility that the microorganisms might have been disintegrated after surgical removal until formaline fixation and the paraffin blocs have been prepared, the specimens were processed with PCR to detect any specific genetic trace of H pylori. However, PCR also was unable to detect any genetic material such as urease gene and 16s rDNA that are specific for the microorganism (Figs 1 and 2) The results of the study should not be interpreted as H pylori cannot successfully colonize in the heterotopic gastric mucosa in MD. H pylori is resistant to low pH in human stomach, although it is much less resistant to alkaline contents in the ileum.10,11 However, there is evidence that large numbers of bacteria traverse the length of the bowel and still remain viable.11 In some diverticula, heterotopic gastric mucosa may cover an extensive area and may result in low pH in a selective area that may form an adequate environment for successful colonization of the microorganism. But, the results of the study suggest that the presence of H pylori is not essentially required to induce “heterotopic gastritis” that may result in bleeding of the Meckel’s diverticulum.

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