INTRODUCTION
Inflammatory bowel diseases [IBD] are comprised of Crohn’s disease [CD] and ulcerative colitis [UC], conditions with distinct clinical, histological and endoscopic features[2323. Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002;347:417-29.]. They can, however, share similar genetic, clinical and histopathological features, making it difficult to establish a definitive diagnosis in some individuals. As a result, a subset of patients are labeled as indeterminate colitis or inflammatory bowel disease unclassified [IBDU][1818. Nikolaus S, Schreiber S. Diagnostics of inflammatory bowel disease. Gastroenterology. 2007;133:1670-89., 3333. Telakis E, Tsironi E. Indeterminate colitis - definition, diagnosis, characteristics and management. Ann Gastroenterol. 2008;21:173-9.].
Significant gastroduodenal involvement occurs in between 0.5% to 4% of all patients with CD and is usually associated with distal small bowel or colonic disease[1313. Isaacs KL. Upper gastrointestinal tract endoscopy in inflammatory bowel disease. Gastrointest Endosc Clin N Am. 2002;12:451-62, vii., 2424. Reynolds HL Jr, Stellato TA. Crohn’s disease of the foregut. Surg Clin North Am 2001;81:117-35, viii.]. The most recognized criteria for gastric CD include the presence of histological evidence of gastric non-caseating granulomatous inflammation, after the exclusion of other granulomatous diseases; or the presence of an established intestinal CD diagnosis with diffuse gastric inflammation compatible with CD[1313. Isaacs KL. Upper gastrointestinal tract endoscopy in inflammatory bowel disease. Gastrointest Endosc Clin N Am. 2002;12:451-62, vii., 1919. Nugent FW, Roy MA. Duodenal Crohn’s disease: an analysis of 89 cases. Am J Gastroenterol. 1989;84:249-254.]. It is estimated that up to 50% of all patients with established intestinal CD also have histological alterations in the gastric mucosa despite the absence of symptoms or radiological abnormalities[55. Castellaneta SP, Afzal NA, Greenberg M et al. Diagnostic role of upper gastrointestinal endoscopy in pediatric inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 2004;39:257-61., 2828. Rutgeerts P, Onette E, Vantrappen G, Geboes K, Broeckaert L, Talloen L. Crohn’s disease of the stomach and duodenum: A clinical study with emphasis on the value of endoscopy and endoscopic biopsies. Endoscopy. 1980;12:288-94.].
Endoscopic evaluation with biopsies of the stomach is the gold standard for the diagnosis of gastric CD[2828. Rutgeerts P, Onette E, Vantrappen G, Geboes K, Broeckaert L, Talloen L. Crohn’s disease of the stomach and duodenum: A clinical study with emphasis on the value of endoscopy and endoscopic biopsies. Endoscopy. 1980;12:288-94.]. These endoscopic characteristics, however, are non-specific and usually have a patchy distribution, including mucosal friability, erythema, nodular mucosa [cobblestoning], ulcerations and thickened folds[1414. Kefalas CH. Gastroduodenal Crohn’s disease. Proc [Bayl Univ Med Cent] 2003;16:147-51.]. Even the most characteristic CD histological finding, the non-caseating granuloma, lacks specificity as granulomas can also be found in other granulomatous conditions[3434. van Hogezand RA, Witte AM, Veenendaal RA, Wagtmans MJ, Lamers CB. Proximal Crohn’s disease: review of the clinicopathologic features and therapy. Inflamm Bowel Dis. 2001;7:328-37.]. The most frequent histological abnormality in gastric CD is the presence of H. pylori [Hp]- negative focally enhanced gastritis [FEG] which is found in almost 80% of patients with known intestinal CD[99. Halme L, Karkkainen P, Rautelin H, Kosunen TU, Sipponen P. High frequency of helicobacter negative gastritis in patients with Crohn’s disease. Gut. 1996;38:379-83., 1313. Isaacs KL. Upper gastrointestinal tract endoscopy in inflammatory bowel disease. Gastrointest Endosc Clin N Am. 2002;12:451-62, vii.].
FEG consists predominantly of macrophages and lymphocytes associated with macrophage microaggregates [MMs] found in the non-inflamed gastroduodenal mucosa[2020. Oberhuber G, Puspok A, Oesterreicher C et al. Focally enhanced gastritis: a frequent type of gastritis in patients with Crohn’s disease. Gastroenterology. 1997;112:698-706., 2222. Petrolla AA, Katz JA, Xin W. The clinical significance of focal enhanced gastritis in adults with isolated ileitis of the terminal ileum. J Gastroenterol. 2008;43:524-30.]. Nevertheless, the presence of Hp infection can also generate a similar pattern of gastritis[1010. Herz R, Schaube J, Meining A, Stolte M. Gastritis associated with Crohn disease can be masked by Helicobacter pylori gastritis. Scand J Gastroenterol. 1999;34:471-3.]. It is important, therefore, to rule-out the effect of these bacteria if an association is to be established between FEG and CD. The presence of MMs in non-inflamed normal gastric mucosa, however, is characteristic of CD, regardless of the presence of FEG[3737. Yao K, Yao T, Iwashita A, Matsui T, Kamachi S. Microaggregate of immunostained macrophages in noninflamed gastroduodenal mucosa: a new useful histological marker for differentiating Crohn’s colitis from ulcerative colitis. Am J Gastroenterol. 2000;95:1967-73.].
In Brazil, there is a high prevalence of Hp infection[2525. Rocha GA, Queiroz DM, Mendes EN et al. Indirect immunofluorescence determination of the frequency of anti-H. pylori antibodies in Brazilian blood donors. Braz J Med Biol Res. 1992;25:683-9., 3232. Souto FJ, Fontes CJ, Rocha GA, de Oliveira AM, Mendes EN, Queiroz DM. Prevalence of Helicobacter pylori infection in a rural area of the state of Mato Grosso, Brazil. Mem Inst Oswaldo Cruz. 1998;93:171-4.] and since Hp is associated with several of the histological features recognized in gastric CD, its presence could play a confounding role in the histological diagnosis of IBD patients with gastric involvement. The aim of this study was to determine the clinical utility of the diagnosis of FEG and MMs in the gastric mucosa in an IBD population with an expected high prevalence of Hp infection.
METHODS
Ethical considerations
The study protocol was approved by the Ethical Committee of the HUCFF-UFRJ. All subjects gave their informed consent prior to their inclusion in the study.
Study population
Sixty-three consecutive patients with IBD, 37 CD and 26 UC patients were recruited at the gastroenterology outpatient unit of the HUCFF-UFRJ. The diagnosis of IBD was confirmed by standard radiological, endoscopic and histological features. CD and UC patients were classified with respect to disease characteristics according to the Montreal Classification[2929. Satsangi J, Silverberg MS, Vermeire S, Colombel JF. The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut. 2006;55:749-53.]. Inflammation was assessed using the Crohn’s disease activity index [CDAI][22. Best WR, Becktel JM, Singleton JW, Kern F, Jr. Development of a Crohn’s disease activity index. National Cooperative Crohn’s Disease Study. Gastroenterology. 1976;70:439-44.] and the Lichtiger Index[1515. Lichtiger S, Present DH, Kornbluth A et al. Cyclosporine in severe ulcerative colitis refractory to steroid therapy. N Engl J Med. 1994;330:1841-5.].
Thirty consecutive control patients with dyspeptic symptoms and a normal gastrointestinal endoscopy were also enrolled at the Endoscopy Unit. One patient with intestinal metaplasia and atrophic gastritis was excluded as these histological findings are associated with a higher histological score and could be misinterpreted as a more intense inflammation according to the Updated Sydney System[77. Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol. 1996;20:1161-81.]. In addition, gastric metaplasia and atrophy can underestimate the presence of Hp infection[1111. Hirschl AM, Makristathis A. Methods to detect Helicobacter pylori: from culture to molecular biology. Helicobacter. 2007;12 Suppl 2:6-11.].
None of the IBD patients or controls had received any antibiotics, non-steroidal anti-inflammatory drugs, bismuth compounds, H2-receptor antagonists, proton pump inhibitors or chemotherapy for at least 6 months prior to study inclusion. Bleeding disorders, use of anticoagulant agents, and diagnosis of other granulomatous diseases were also considered exclusion criteria.
Endoscopy setting and mucosal specimens
Patients and controls underwent a blinded upper gastrointestinal video-endoscopy and received conscious sedation with midazolam and meperidine. Four gastric biopsies were obtained from the gastric body and antrum with standard biopsy forceps. Hp infection was determined by positive rapid urease test and histological analysis, as demonstrated by previous studies[11. Al-Fadda M, Powe J, Rezeig M, Al NM, Alrajhi AA, Baynton R. Comparison of carbon-14-urea breath test and rapid urease test with gastric biopsy for identification of Helicobacter pylori. Ann Saudi Med. 2000;20:170-2.]. Patients were defined as Hp-negative when both tests were negative. Two controls, three CD and one UC patients with only one of the two methods confirming Hp infection were excluded.
Histological analysis
Formalin-fixed gastric sections were stained with hematoxylin and eosin for the evaluation of microscopic lesions and with Giemsa for Hp detection. Histological inflammation was evaluated by two independent blinded pathologists according to the Updated Sydney System[44. Burger-Kentischer A, Goebel H, Seiler R et al. Expression of macrophage migration inhibitory factor in different stages of human atherosclerosis. Circulation. 2002;105:1561-66., 77. Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol. 1996;20:1161-81., 1212. Hoi AY, Iskander MN, Morand EF. Macrophage migration inhibitory factor: a therapeutic target across inflammatory diseases. Inflamm Allergy Drug Targets. 2007;6:183-90.]. In this score, parameters were inflammation [mononuclear leukocyte infiltration], gastritis activity [neutrophil infiltration], Hp density, the presence of atrophy and intestinal metaplasia. All parameters were graded as absent [0], mild [1], moderate [2], or severe [3]. FEG was defined when at least one gland, or foveola, was infiltrated by lymphocytes, monocytes and neutrophils surrounded by normal gastric mucosa, as described by Oberhuber et al.[2020. Oberhuber G, Puspok A, Oesterreicher C et al. Focally enhanced gastritis: a frequent type of gastritis in patients with Crohn’s disease. Gastroenterology. 1997;112:698-706.]. Three patients, one of each group, were excluded, as the gastric samples were inadequate for analysis.
Immunohistochemistry
Tissue samples were immediately embedded in Tissue-Tek O.C.T. compound [Miles Scientific Laboratories Ltd, Naperville, IL] and snap-frozen in isopentane in a liquid nitrogen bath. Samples were then stored at -80 °C until processing, and cut into 6-µm section in a cryostat maintained at -20°C. Tissue sections were air-dried and fixed for 10 min in a 1:1 solution of chloroform-acetone. Immunologic assessment of the intestinal mucosa was made using indirect immunoperoxidase technique using the primary mouse monoclonal antibody anti-CD68 [1:100] [Dako A/S, Glostrup, Denmark]. Briefly, frozen sections were immersed in 0.3% hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity. After being rinsed in phosphate buffered saline [PBS] containing 0.5% Tween 20 for 10 min, tissue sections were incubated with normal horse serum + BSA 0.1% + Triton 0.1% in PBS solution at room temperature in a humidified chamber for 30 min. Subsequently, sections were incubated overnight with the respective primary monoclonal antibody in a humidified chamber at 4oC. Following, the sections were incubated with secondary biotinylated antibody and then with horseradish peroxidase-conjugated streptavidin [LSAB-DAKO® kit] [Dako A/S, Glostrup, Denmark], both for 20 min at room temperature. Sections were then washed in PBS, and developed using 3,3-diaminobenzidine tetrahydrochloride [Dako A/S, Glostrup, Denmark]. Preparations were lightly counterstained in Harris’s hematoxylin, dehydrated, and mounted in Permount [Fisher Scientific, Pittsburgh, PA, USA]. As a negative control, the treatment with the primary antibody was omitted.
Quantitative assessment
Quantitative analysis of tissue sections [under light microscopy at × 400 magnification] and images were captured using a computer-assisted image analyzer [Image-Pro Plus Version 4.1 for Windows, Media Cybernetics, LP, Silver Spring, MD, USA]. Any epithelial and lamina propria cells exhibiting identifiable reactivity distinct from the background were regarded as positive. In the immunoperoxidase studies, percentages of different cell subsets were defined by the number of immunoreactive cells in relation to total cell count [immunoreactive and non-immunoreactive cells] in the lamina propria per millimeter squared [counted in at least five different areas or average sum areas of 0.625 mm² per section]. The presence of five to ten CD68-positive cells in close contact, relatively losing their own limits in subepithelial or lamina propria layers were recorded as macrophage microaggregates, according to Yao et al.[3737. Yao K, Yao T, Iwashita A, Matsui T, Kamachi S. Microaggregate of immunostained macrophages in noninflamed gastroduodenal mucosa: a new useful histological marker for differentiating Crohn’s colitis from ulcerative colitis. Am J Gastroenterol. 2000;95:1967-73.].
Statistical analysis
Statistical analysis was performed using SPSS for windows [version 11.0, SPSS Inc, 1989-1999, USA]. Fisher exact test and Yates’ corrected chi-square were used to test the association between variables. Statistical differences among the experimental groups were evaluated with one-way ANOVA-test in which pair-wise multiple comparisons were carried out using the Dunnett’s T3 test. Correlations between densities of positive cells measured by immunohistochemistry were assessed using Spearman rank correlation coefficient. Values were expressed as means ± SEM. FEG and MM-related sensitivity and specificity with positive and negative predictive values for CD diagnosis were calculated. The level of significance was set at P