Scandinavian Journal of Gastroenterology
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
Disappearance of Gastritis after Eradication of Helicobacter pylori: A Morphometric Study J. Valle, K. Seppälä, P. Sipponen & T. Kosunen To cite this article: J. Valle, K. Seppälä, P. Sipponen & T. Kosunen (1991) Disappearance of Gastritis after Eradication of Helicobacter pylori: A Morphometric Study, Scandinavian Journal of Gastroenterology, 26:10, 1057-1065, DOI: 10.3109/00365529109003956 To link to this article: http://dx.doi.org/10.3109/00365529109003956
Published online: 08 Jul 2009.
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Disappearance of Gastritis after Eradication of Helico bacter p ylori A Morphometric Study
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J. VALLE, K. SEPPALA, P. SIPPONEN & T. KOSUNEN Second Dept. of Medicine and Depts. of Pathology and Bacteriology, University of Helsinki, Helsinki, and Dept. of Pathology, Jorvi Hospital, Espoo, Finland
Valle J . Seppala K, Sipponen P, Kosunen T. Disappearance of gastritis after eradication of Helicobacter pylori. A morphometric study. Scand J Gastroenterol 1991, 26, 1057- 1065 Helicobacter pylori infection is strongly associated with and is considered a common cause of gastritis. To study the relationship between H. pylori and gastritis, we examined whether a reduction occurs in acute granulocytic and chronic mononuclear inflammation of gastric mucosa after eradication therapy. The examination is based on morphometric counting and on semiquantitative estimation of the density of the inflammatory cells in endoscopic biopsy specimens from antrum and corpus. The series consisted of 23 consecutive outpatients with H . pylori-associated gastritis who received a 2-week course of triple therapy with colloidal bismuth subcitrate, amoxicillin, and metronidazole and who underwent an endoscopic follow-up for 6 weeks, 6 months (23 patients). and 12 months (21 patients). The eradication was successful in 20 patients ('responders'), who also remained H. pylori-free for 6 months, and in 18 examined patients for 12 months but was unsuccessful in 3 patients ('non-responders'). Both acute and chronic inflammation decreased significantly in intensity in responders in the follow-up. The acute inflammation had already disappeared at a 6-week control. The reduction in chronic inflammation was slower and occurred gradually within the study. At the 12-month follow-up, the corpus muscosa was interpreted as normal in all 18 patients studied, and the antral mucosa was interpreted as normal in 10 (56%) patients. No significant reduction in intensity of gastritis was found in the 3 non-responders or the 23 matched, untreated controls. We conclude that the eradication of H . pylori results in a disappearance of both acute and chronic gastritis. This supports the view that H. pylori plays a causal role in the pathogenesis of chronic gastritis. Key words: Acute gastritis; chronic gastritis; Helicobacrer pvlori; treatment Prof. Kari Seppala, M . D. Gastroenterological Unit. Second Dept. of Medicine, University of Helsinki, SF-00290 Helsinki, Finland I
Colonization of gastric surface epithelium by Helicobacter pylori is commonly associated with chronic gastritis, and in a great majority of cases chronic gastritis is considered an inflammatory response against this organism (1-1 1). If H . pylori and gastritis are causally related, a reduction and disappearance of gastritis would be expected to occur after eradication of the bacterium. Few studies on this aspect have, however, been published (5, 12, 13), and none of them are morphometric.
The aim of our study was to examine histologic and morphometric findings on gastritis after H . pylori eradication therapy at a 1-year follow-up study. MATERIALS AND METHODS Patient group (series) Twenty-three patients (15 men, 8 women; mean ages, 47 and 53years, respectively) were consecutively selected from among outpatients
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subjected to diagnostic upper gastrointestinal endoscopy in the Gastroenterological Unit of the Second Dept. of Medicine, University of Helsinki, Helsinki, in 1989. The selection criterion was that a colonization of the gastric mucosa by H . pylori could be demonstrated by histology (Giemsa staining) and by culture. At the initial endoscopy six patients had duodenal ulcer (DU), six had endoscopic duodenitis, and two had gastric ulcer (GU), and at histology, everyone had chronic gastritis with or without active (acute) inflammation. Control group The control group of 23 outpatients (15 men, 8 women; mean ages, 48 and 51 years, respectively) was matched by age (+-3year-s) and sex with a previous H . pylori-infected patient group who were asked to undergo follow-up gastroscopy within 3 years (mean, 25 months) after the initial gastroscopy. At the initial endoscopy six control patients had DU, five had endoscopic duodenitis, and two had antral erosions, and all also had chronic gastritis. Another control group of nine patients (mean age, 53 years) with histologically normal stomachs (both antrum and corpus mucosa had normal ordinary histologic findings) was also selected. This group was used at morphometry to count the ‘basal’density of inflammatory cells in a ‘normal’ stomach.
The study protocol was accepted by the ethical committee of the department. All patients were informed of the study protocol before their voluntary enrollment in the study. Eradication of H. pylori In the series all patients received (1 week after the initial endoscopy and the verification of the H. pylori colonization) a 2-week course of 4 x 200 mg colloidal bismuth subcitrate (De-NoP), 4 x 500 mg amoxicillin, and 3 x 500 mg metronidazole. H . pylori was eradicated (as determined by culture and histology at the 6-week follow-up) in 20 (87%) of the 23 patients (2 patients did not participate in the 12-month follow-up), and this subgroup of the series was considered ‘responders’. The three patients without eradication of H . pylori were considered ‘non-responders’. Endoscopy and biopsy procedure In the patient series follow-up endoscopies were performed 6 weeks and 6 and 12 moths after the initial endoscopy. In 23 matched controls a follow-up gastroscopy was carried out 25 months (mean) after the first endoscopy. In all subjects of both patient and control groups, two random biopsy specimens were taken from the antrum (anterior wall, at least 2 cm from the pylorus) and corpus (anterior and posterior wall of the middle
Table I. Prevalence of acute inflammation before and 6 weeks and 6 and 12 months after the treatment of Helicobacter pylori (responders; 20 patients) After eradication Grade of mucosal inflammation
Before eradication
6 weeks
6 months
12 months
n
%
n
%
n
%
n
%
4 13 3 0
20 65 15 0
20 0 0 0
100 0 0 0
20 0 0 0
100 0 0 0
18 0 0 0
100 0 0 0
10 10 0 0
50 50 0 0
20 0 0 0
100 0 0 0
20 0 0 0
100 0 0 0
18 0 0 0
100 0 0 0
Antral mucosa None
Mild Moderate Severe
Corpus mucosa None Mild Moderate
Severe
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Gastritis after H. pylori Eradication
corpus), in addition to the specimens from endoscopic lesions. Extra specimens were taken in the series from the antral and corpus mucosa for the culture of H . pylori. Histologic biopsy specimens were fixed overnight in neutral-buffered 10% formalin and were embedded in paraffin. Tissue sections were stained with hematoxylin-eosin (HE) and Alcian blue (pH 2.5)-periodic acid-Schiff (PAS) methods. In addition, a set of sections was immunohistochemically stained with a monoclonal antiserum (MAC-387, Dakopatts) raised against myeloid/histiocyte antigen t o demonstrate tissue granulocytes (15). Histologic colonization of H . pylori was interpreted from sections stained with a modified Giemsa method, Culture of H. pylori
Antral biopsy specimens were seeded on chocolate plates under microaerophilic conditions (57% oxygen) within 2-3 h of the collection. After F6 days of culture a t 35”C, H. pylori was identified as small translucent colonies of curved gramnegative rods, which were oxidase-, catalase-, and urease-positive and did not ferment glucose or grow at 42°C.
Serniquantitative assessment of gastritis Gastritis was considered to be present if mononuclear inflammatory cells (lymphocytes, plasma
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cells) and/or granulocytes were seen in the lamina propria, using the criteria and suggestions in the Sydney system (16). Gastritis was determined separately in specimens from antrum and corpus, being expressed as ‘antral gastritis’, ‘corpus gastritis’, or ‘pangastritis’, depending o n whether gastritis occurred in the antrum, corpus, or in both, respectively. In the semiquantitative assessment, acute inflammation was graded as follows: absence of granulocytes (none); presence of granulocytes focally in the lamina propria with some possible focal intraepithelial erosions (mild); involvement of the mucosa and the epithelium by granulocytes in several neighboring glands (moderate); and massive, continuous, and confluent granulocytic inflammation with foveolar abscesses and erosions (severe). Chronic inflammation was graded as follows: at most a minimal number of lymphocytes and plasma cells in the lamina propria (none); mild, inconsistent inflammation in the upper third of the mucosa (mild); diffuse, moderate inflammation occupying the subepithelial layer in the corpus mucosa (corpus gastritis) or predominantly upper half of the antral mucosa (antral gastritis) (moderate); and a strong and dense inflammation of a thick, continuous, confluent subepithelial layer of lymphocytes and plasma cells in the corpus mucosa, or a diffuse, continuous, confluent in-
Table 11. Prevalence of chronic inflammation before and 6 and 12 months after the treatment of Helicobacrer pylori (responders; 20 patients) ~
~
~~~
After eradication 6 months
Before eradication Grade of mucosal inflammation
n
%
%
n ~
Antral mucosa None Mild Moderate Severe Corpus mucosa None Mild Moderate Severe
12 months
~
n
%
~~~
0 7 12 1
0 35 60 5
3 17 0 0
15 85 0 0
10 8 0 0
56 44 0 0
0 10 9 1
0 50
17 3 0 0
85 15 0 0
19 0 0 0
100 0 0 0
45 5
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volvement of mononuclear inflammatory cells in the whole thickness of the gastric mucosa (severe). The presence of lymphoid aggregates in the lamina propria was recorded separately. All semiquantitative interpretations were done blindly by one pathologist (P. Sipponen).
estimated by counting the number of intercepts (points) of the graticular squares falling on the nuclear cells and dividing by the total number of points falling on the mucosa (17,18). To obtain unbiased estimates in counting, the rules of Gundersen (19) were used. In each case, 10 appropriate random fields were counted, and the cell densities (in arbitrary units) were expressed as means (LSD) of these counts. All counting was done blindly by one pathologist (J. Valle). Mononuclear cells were assessed from HE-stained sections and granulocytes from MAC-387-stained sections.
Morphometry The density of granulocytes and mononuclear inflammatory cells was estimated morphometrically by determining the ‘areal fraction’ of cells in the subepithelial layer of the mucosa (14). The counting of cells was done microscopically at high magnification (objective, 4 0 ;~ Statistics The Wilcoxon rank test was used in calculations ocular, lox) by using an ocular grid composed of 100 small squares. The areal fraction of cells was of differences in the morphometric data, and the chi-square test was used in calculations of differences in the semiquantative data.
Fig. 1 . Antral mucosa before and after the eradication of Helicobacier pyfori. Before (left) and 12 months after (right). (Hematoxylin and eosin; magnification, ~ 2 5 0 . )
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Fig. 2. Corpus mucosa before and after the eradication of Helicobacter pylori. Before (left) and 12 months after (right). (Hematoxylin and eosin; magnification, ~ 2 5 0 . )
RESULTS Semiquantitative assessment of gastritis The prevalence and grade of acute and chronic gastritis in the antrum and corpus in the initial and follow-up endoscopic biopsy specimens in responders ( H . pylori bacterium was eradicated; see Materials and Methods) of the series are presented in Tables I and 11, and an example of gastritis before and after the treatment is given in Figs. 1 and 2. In these patients a significant decrease in the grade of both chronic and acute inflammation occurred in the course of time, in the same manner in both the antrum and corpus mucosa. Acute inflammation disappeared rapidly and was not seen in any of the subjects in the follow-up at 6weeks. The resolution of chronic gastritis was slower than that of the acute inflammation, and
the antral gastric mucosa was graded as normal in 3 (15%) of 20 patients in the follow-up at 6 months, and in 10 (56%) of 18 subjects at the 12month follow-up. On the other hand, the corpus mucosa was graded as normal in 17 (85%) of 20 subjects already at the 6-month follow-up. No significant changes occurred in the grade of acute or chronic gastritis in the 3 non-responders or in the 23 controls within the follow-up period (data not shown). The number of cases of lymphoid aggregates in the gastric mucosa decreased in the follow-up among the responders. Lymphoid aggregates in antral biopsies were seen in 12 subjects (63%) in the initial endoscopy but only in 3 (16%) subjects in the 6 month follow-up, and in none in the 12month follow-up. No corresponding decrease in the prevalence of lymphoid aggregates was seen in the controls (data not shown).
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Morphometry The mean densities of granulocytes and mononuclear inflammatory cells in the biopsy specimens from the initial and follow-up endoscopies in the patient series and controls are presented in Tables 111and IV and in Figs. 3 and 4. The tables also show the ‘basal’ density of inflammatory cells in nine healthy controls who showed ‘normal’ stomach (both antrum and corpus mucosa are normal) at ordinary histology. In the patient series with a successful eradi-
cation of H . pylori (responders) a significant timerelated decrease occurred in the density of both granulocytes and mononuclear cells in the subepithelial layer of both the antral and corpus mucosa. This decrease was rapid with regard to granulocytes but slower with regard to mononuclear cells. No decrease in the density of mononuclear inflammatory cells was found in the three non-responders at the 1-month control. No changes in the cell densities were seen in the controls between the initial and follow-up endoscopies.
Table 111. Density (mean ? SD) of granulocytes in the lamina propria in responders and non-responders before and after the treatment of Helicobacter pylori After eradication Before eradication
6 weeks
Responders ( a = 20) Antrum Corpus
2.6 t 0.5 1.3 t 0.4
0.15 t 0** 0.1 +o*
Non-responders ( n = 3) Antrum Corpus
4.7 t 3.6 0.1 t 0
2.2 t 1.7 0.1 t 1.0
6 months
12 months
0 t o*** 0 t 0***
O t O*** ( n = 18) 0 t 0** ( n = 18) 2.3 t 0.5 1.5 2 0.1
2.4 t 1.0 2.4 t 1.0
Patients with normal stomach ( n = 9)t Antrum 0.0 t 0.0 Corpus 0.0 2 0.0
* p < 0.05; **p < 0.01; ‘ * * p < 0.001 differences compared with values before the eradication (Wilcoxon). tA selected group of patients with normal stomach (both antrum and corpus are normal; see Materials and Methods).
Table IV. Density (mean ? SD) of mononuclear inflammatory cells in the lamina propria in responders (20 patients) and non-responders (3 patients) before and after the treatment of Helicobacter pylori After eradication Before eradication Responders ( n = 20) Antrum Corpus
10.3 0.7 9.7 t 0.9
Non-responders (n = 3) Antrum Corpus
15.3 ? 1.7 10.3 ? 3.7
*
6 weeks
8.2 t 0.4** 6.6 t 0.6** 10.4 ? 1.0 7.9 t 2.5
6 months
5.9 t 0.5*** 4.3 t 0.4*** 11.8 t 1 12.4 t 1
12 months 3.6 t 1.7*** (n = 18) 3 . 3 ? 1.7***( n = 18)
*
11.0 2.5 10.0 t 1.7
Patients with normal stomach (n = 9)t Antrum 1.5 t 1.3 Corpus 2.1 ? 2.0 * p < 0.05; * * p< 0.01; * * * p < 0.001 differences compared with values before the eradication (Wilcoxon). ?A selected group of patients with normal stomach (both antrum and corpus are normal; see Materials and Methods).
Gastritis after H. pylori Eradication
ANTRUM
CORPUS
4 10
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5
ttt
I
O
l
o
w
2
0
MONTHS
0
6 12 MONTHS
Fig. 3 . Mean density (?SD, arbitrary units) of mononuclear inflammatory cells in the lamina propria before and after the eradication of Helicobacter pylori in responders.
DISCUSSION Helicobacter pylori is considered a cause of chronic gastritis in a long list of studies of different kinds (see Ref. 20). Spontaneous healing of chronic gastritis is a rather rare phenomenon in
CORPUS
ANTRUM 15
v)
I
l5
10
z Lu cs A A
Lu
0
5
5
0 -0 0
MONTHS
26
0
MONTHS
26
Fig. 4. Mean density (kSD, arbitrary units) of mononuclear inflammatory cells in the lamina propria in controls (patients with Helicobacter pylori-associated gastritis, no treatment).
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adult subjects, and gastritis is generally a longlasting, obviously life-long progressive disease that tends t o lead gradually to atrophy of the underlying mucosa, at least in several affected subjects (21,22). From this viewpoint of gastritis itself, there is evidence that the cumulative risk of peptic ulcer has been shown to be high in patients with gastritis but very low in those with normal gastric mucosa (23,24). The role of chronic gastritis is therefore very important in clinical practice. Colloidal bismuth with certain antimicrobial drugs eradicates H . pylori effectively in a great number of patients (25-27) and also decreases the recurrence rate of duodenal ulcer (25,28). This eradication treatment makes it possible to test the hypothesis concerning the interrelationship between H . pylori and gastritis. A causality between H . pylori and gastritis implies that a significant healing in gastritis ought to occur after the clearance or eradication of the organism. Earlier studies support this view (5, 12, 13) but are handicapped by the fact that they score both acute and chronic inflammatory changes together. There is also a lack of follow-up studies, and, in addition, there is no evidence in the literature that the healing process of chronic inflammation in gastritis is slow after H . pylori eradication. This gave us a reason to examine separately the fate of acute and chronic gastritis after H. pyfori therapy. The healing of chronic gastritis after a successful H . pylori therapy is indicated in our study. There was no healing of gastritis in three nonresponders and no spontaneous healing of chronic gastritis during the follow-up time (mean, 25 months) in matched controls. The present observations indicate that the acute granulocytic inflammation disappears rapidly-at the latest, 6 weeks after eradication. The results suggest that the rapid disappearance of the acute inflammation after treatment is such a prominent feature in the responders that this phenomenon can be considered a good practical indicator of a successful eradication of H . pylori. Correspondingly, this resolution of acute inflammation seems to be also a predictor of later resolution of the chronic inflammation, and the final healing of the gastric mucosa. The chronic mononuclear inflammation
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disappears slowly, and, particularly in the antral mucosa, the final healing of the mucosa will take longer than previously has been assumed. Chronic inflammation was healed (both antrum and corpus are normal) in only 3 of 20 patients 6 months and in 10 of 18patients 12months after the eradication. We conclude that a successful eradication of H . pylori will lead to a significant improvement in both acute and chronic gastritis. The resolution of chronic inflammation is a rather slow process, and a complete healing of the mucosa will obviously take several months, perhaps even a few years. Many still unresolved problems of other elements of chronic gastritis need to be studied further in a much longer follow-up. The role of H. pylori in gastric cancer is open. Whether the atrophic changes also are reversible in chronic gastritis or will develop into severe atrophy in all H . pylori-infected patients or only in genetically determined persons is not known. REFERENCES 1. Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983, 1, 1273-1275 2. Stolte M, Eidt S, Ritter M, Bethke B. Campylobacter pylori und Gastritis: Assoziation oder Induktion? Pathologe 1989, 10, 21-26 3. Dooley CP, Cohen H, Fitzgibbons PL, et al. Prevalence of Helicobacter pylori infection and histologic gastritis in asymptomatic persons. N Engl J Med 1989, 321, 1562-1566 4. Musgrove C, Bolton FJ, Krypczyk AM, et al. Campylobacter pylori: clinical, histological and serological studies. J Clin Pathol 1988, 41, 1316-1321 5. Rauws EAJ, Langenberg W, Houthoff HJ, Zanen HC, Tytgat GNJ. Campylobacter pyloridis-associated chronic active antral gastritis. A prospective study of its prevalence and the effects of antibacterial and antiulcer treatment. Gastroenterology 1988, 94, 3 H 0 6. Peterson WL, Lee F, Feldman M. Relationship between Campylobacter pylori and gastritis in healthy humans after administration of placebo or indornethacin. Gastroenterology 1988, 95, 11851197 7. Langenberg ML, Tytgat GNJ, Schipper MEI, Rietra PJGM, Zanen HC. Campylobacter like organisms in the stomach of patients and healthy individuals. Lancet 1984, 1, 1348 8. Goodwin CS, Armstrong JA, Marshall BJ. Campylobacterpyloridis, gastritis, and peptic ulceration. J Clin Pathol 1986, 39, 353-365
9. Siurala M, Sipponen P, Kekki M. Campylobacter pylori in a sample of Finnish population: relations to morphology and functions of the gastric mucosa. Gut 1988, 29, 909-915 10. Barthel JS, Westblom TU, Havey AD, Gonzalez F, Everett ED. Gastritis and Campylobacter pylori in healthy, asymptomatic volunteers. Arch Intern Med 1988, 148, 1149-1151 11. Sipponen P, Varis K, Cederberg A, et al. Campylobacter pylori is associated with chronic gastritis but not with active peptic ulcer disease. APMIS 1988, 96, 84-88 12. Morgan D, Kraft W, Bender M, Pearson A. The Gastrointestinal Physiology Working Group of Cayetano Heredia and the Johns Hopkins Universities. Nitrofurans in the treatment of gastritis associated with Campylobacter pylori. Gastroenterology 1988, 95, 1178-1184 13. Hislop I, Glancy R, Armstrong J. Histological irnprovement of active chronic gastritis in patients treated with De-Nol. Aust NZ J Med 1984,14(suppl 4). 907 14. Valle J, Seppala K, Sipponen P, Kosunen T. Resolution of gastritis following Helicobacter pylori eradication: a morphometric study. The 9th World Congress of Gastroenterology, Sydney, 1990 15. Brandtzaeg P, Jones DB, Flavell DJ, Fagerhol MK. Mac 387 antibody and detection of formalin resistant myelomonocytic L1 antigen. J Clin Path 1988, 41, 963-970 16. Misiewicz JJ, Tytgat GNJ, Goodwin CS, et al. The Sydney system: a new classification of gastritis. Working Party Reports 1990, 1-10 17. Aherne WA, Dunnill MS. Morphometry. E. Arnold, London, 1982, 46-59 18. Gundersen HJG, Bendtsen TF, Korbo L, et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988, 96, 379-394 19. Gundersen HJG. Notes on the estimation of the numerical density of arbitrary profiles. The edge effect. J Microsc 1977, 111, 219-223 20. Graham DY. Campylobacterpylori and peptic ulcer disease. Gastroenterology 1989, 96, 615-625 21. Kekki M, Siurala M, Varis K, Sipponen P, Sistonen P, Nevanlinna HR. Classification principles and genetics of chronic gastritis. Scand J Gastroenterol 1987, SUPPI PI 141), 1-28 22. Siurala M, Sipponen P, Kekki M. Chronic gastritis: dynamic and clinical aspects. Scand J Gastroenterol 1985, 20(suppl 109), 69-76 23. Sipponen P, Seppala K, Aarynen M, Helske T, Kettunen P. Chronic gastritis and gastroduodenal ulcer: a case control study on risk of coexisting duodenal or gastric ulcer in patients with gastritis. Gut 1989, 30, 922-929 24. Sipponen P, Varis K, Fraki 0, Korri U-M, Seppala K , Siurala M. Cumulative 10-year risk of symptomatic duodenal and gastric ulcer in patients with or without chronic gastritis. A clinical follow-up study of 454 outpatients. Scand J Gastroenterol 1990, 25, 966-973
Gastritis after H. pylori Eradication
25. Marshall BJ, Goodwin CS, Warren JR, et al. Prospective double-blind trial of duodenal ulcer relapse after eradication of Campylobacter pylori. Lancet 1988, 2, 1437-1442 26. Rauws EAJ, Tytgat GNJ. Cure of duodenal ulcer associated with eradication of H . pyfori. Lancet 1990, 225, 1233-1235
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Received 28 January 1991 Accepted 6 May 1991
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27. Borsch G, Mai U , Opferkuch W. Oral triple therapy (Om)may effectively eradicate Carnpylobacterpylori (CP) in man: a pilot study. Gastroenterology 1988, 94, A248 28. Borody TJ, Cole P, Noonan S, et al. Long-term Cumpylobucter recurrence post eradication. Gastroenterology 1988, 94, A43
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
Disappearance of Gastritis after Eradication of Helicobacter pylori: A Morphometric Study J. Valle, K. Seppälä, P. Sipponen & T. Kosunen To cite this article: J. Valle, K. Seppälä, P. Sipponen & T. Kosunen (1991) Disappearance of Gastritis after Eradication of Helicobacter pylori: A Morphometric Study, Scandinavian Journal of Gastroenterology, 26:10, 1057-1065, DOI: 10.3109/00365529109003956 To link to this article: http://dx.doi.org/10.3109/00365529109003956
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 3
View related articles
Citing articles: 20 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=igas20 Download by: [University of California, San Diego]
Date: 06 March 2016, At: 13:03
Disappearance of Gastritis after Eradication of Helico bacter p ylori A Morphometric Study
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J. VALLE, K. SEPPALA, P. SIPPONEN & T. KOSUNEN Second Dept. of Medicine and Depts. of Pathology and Bacteriology, University of Helsinki, Helsinki, and Dept. of Pathology, Jorvi Hospital, Espoo, Finland
Valle J . Seppala K, Sipponen P, Kosunen T. Disappearance of gastritis after eradication of Helicobacter pylori. A morphometric study. Scand J Gastroenterol 1991, 26, 1057- 1065 Helicobacter pylori infection is strongly associated with and is considered a common cause of gastritis. To study the relationship between H. pylori and gastritis, we examined whether a reduction occurs in acute granulocytic and chronic mononuclear inflammation of gastric mucosa after eradication therapy. The examination is based on morphometric counting and on semiquantitative estimation of the density of the inflammatory cells in endoscopic biopsy specimens from antrum and corpus. The series consisted of 23 consecutive outpatients with H . pylori-associated gastritis who received a 2-week course of triple therapy with colloidal bismuth subcitrate, amoxicillin, and metronidazole and who underwent an endoscopic follow-up for 6 weeks, 6 months (23 patients). and 12 months (21 patients). The eradication was successful in 20 patients ('responders'), who also remained H. pylori-free for 6 months, and in 18 examined patients for 12 months but was unsuccessful in 3 patients ('non-responders'). Both acute and chronic inflammation decreased significantly in intensity in responders in the follow-up. The acute inflammation had already disappeared at a 6-week control. The reduction in chronic inflammation was slower and occurred gradually within the study. At the 12-month follow-up, the corpus muscosa was interpreted as normal in all 18 patients studied, and the antral mucosa was interpreted as normal in 10 (56%) patients. No significant reduction in intensity of gastritis was found in the 3 non-responders or the 23 matched, untreated controls. We conclude that the eradication of H . pylori results in a disappearance of both acute and chronic gastritis. This supports the view that H. pylori plays a causal role in the pathogenesis of chronic gastritis. Key words: Acute gastritis; chronic gastritis; Helicobacrer pvlori; treatment Prof. Kari Seppala, M . D. Gastroenterological Unit. Second Dept. of Medicine, University of Helsinki, SF-00290 Helsinki, Finland I
Colonization of gastric surface epithelium by Helicobacter pylori is commonly associated with chronic gastritis, and in a great majority of cases chronic gastritis is considered an inflammatory response against this organism (1-1 1). If H . pylori and gastritis are causally related, a reduction and disappearance of gastritis would be expected to occur after eradication of the bacterium. Few studies on this aspect have, however, been published (5, 12, 13), and none of them are morphometric.
The aim of our study was to examine histologic and morphometric findings on gastritis after H . pylori eradication therapy at a 1-year follow-up study. MATERIALS AND METHODS Patient group (series) Twenty-three patients (15 men, 8 women; mean ages, 47 and 53years, respectively) were consecutively selected from among outpatients
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subjected to diagnostic upper gastrointestinal endoscopy in the Gastroenterological Unit of the Second Dept. of Medicine, University of Helsinki, Helsinki, in 1989. The selection criterion was that a colonization of the gastric mucosa by H . pylori could be demonstrated by histology (Giemsa staining) and by culture. At the initial endoscopy six patients had duodenal ulcer (DU), six had endoscopic duodenitis, and two had gastric ulcer (GU), and at histology, everyone had chronic gastritis with or without active (acute) inflammation. Control group The control group of 23 outpatients (15 men, 8 women; mean ages, 48 and 51 years, respectively) was matched by age (+-3year-s) and sex with a previous H . pylori-infected patient group who were asked to undergo follow-up gastroscopy within 3 years (mean, 25 months) after the initial gastroscopy. At the initial endoscopy six control patients had DU, five had endoscopic duodenitis, and two had antral erosions, and all also had chronic gastritis. Another control group of nine patients (mean age, 53 years) with histologically normal stomachs (both antrum and corpus mucosa had normal ordinary histologic findings) was also selected. This group was used at morphometry to count the ‘basal’density of inflammatory cells in a ‘normal’ stomach.
The study protocol was accepted by the ethical committee of the department. All patients were informed of the study protocol before their voluntary enrollment in the study. Eradication of H. pylori In the series all patients received (1 week after the initial endoscopy and the verification of the H. pylori colonization) a 2-week course of 4 x 200 mg colloidal bismuth subcitrate (De-NoP), 4 x 500 mg amoxicillin, and 3 x 500 mg metronidazole. H . pylori was eradicated (as determined by culture and histology at the 6-week follow-up) in 20 (87%) of the 23 patients (2 patients did not participate in the 12-month follow-up), and this subgroup of the series was considered ‘responders’. The three patients without eradication of H . pylori were considered ‘non-responders’. Endoscopy and biopsy procedure In the patient series follow-up endoscopies were performed 6 weeks and 6 and 12 moths after the initial endoscopy. In 23 matched controls a follow-up gastroscopy was carried out 25 months (mean) after the first endoscopy. In all subjects of both patient and control groups, two random biopsy specimens were taken from the antrum (anterior wall, at least 2 cm from the pylorus) and corpus (anterior and posterior wall of the middle
Table I. Prevalence of acute inflammation before and 6 weeks and 6 and 12 months after the treatment of Helicobacter pylori (responders; 20 patients) After eradication Grade of mucosal inflammation
Before eradication
6 weeks
6 months
12 months
n
%
n
%
n
%
n
%
4 13 3 0
20 65 15 0
20 0 0 0
100 0 0 0
20 0 0 0
100 0 0 0
18 0 0 0
100 0 0 0
10 10 0 0
50 50 0 0
20 0 0 0
100 0 0 0
20 0 0 0
100 0 0 0
18 0 0 0
100 0 0 0
Antral mucosa None
Mild Moderate Severe
Corpus mucosa None Mild Moderate
Severe
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Gastritis after H. pylori Eradication
corpus), in addition to the specimens from endoscopic lesions. Extra specimens were taken in the series from the antral and corpus mucosa for the culture of H . pylori. Histologic biopsy specimens were fixed overnight in neutral-buffered 10% formalin and were embedded in paraffin. Tissue sections were stained with hematoxylin-eosin (HE) and Alcian blue (pH 2.5)-periodic acid-Schiff (PAS) methods. In addition, a set of sections was immunohistochemically stained with a monoclonal antiserum (MAC-387, Dakopatts) raised against myeloid/histiocyte antigen t o demonstrate tissue granulocytes (15). Histologic colonization of H . pylori was interpreted from sections stained with a modified Giemsa method, Culture of H. pylori
Antral biopsy specimens were seeded on chocolate plates under microaerophilic conditions (57% oxygen) within 2-3 h of the collection. After F6 days of culture a t 35”C, H. pylori was identified as small translucent colonies of curved gramnegative rods, which were oxidase-, catalase-, and urease-positive and did not ferment glucose or grow at 42°C.
Serniquantitative assessment of gastritis Gastritis was considered to be present if mononuclear inflammatory cells (lymphocytes, plasma
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cells) and/or granulocytes were seen in the lamina propria, using the criteria and suggestions in the Sydney system (16). Gastritis was determined separately in specimens from antrum and corpus, being expressed as ‘antral gastritis’, ‘corpus gastritis’, or ‘pangastritis’, depending o n whether gastritis occurred in the antrum, corpus, or in both, respectively. In the semiquantitative assessment, acute inflammation was graded as follows: absence of granulocytes (none); presence of granulocytes focally in the lamina propria with some possible focal intraepithelial erosions (mild); involvement of the mucosa and the epithelium by granulocytes in several neighboring glands (moderate); and massive, continuous, and confluent granulocytic inflammation with foveolar abscesses and erosions (severe). Chronic inflammation was graded as follows: at most a minimal number of lymphocytes and plasma cells in the lamina propria (none); mild, inconsistent inflammation in the upper third of the mucosa (mild); diffuse, moderate inflammation occupying the subepithelial layer in the corpus mucosa (corpus gastritis) or predominantly upper half of the antral mucosa (antral gastritis) (moderate); and a strong and dense inflammation of a thick, continuous, confluent subepithelial layer of lymphocytes and plasma cells in the corpus mucosa, or a diffuse, continuous, confluent in-
Table 11. Prevalence of chronic inflammation before and 6 and 12 months after the treatment of Helicobacrer pylori (responders; 20 patients) ~
~
~~~
After eradication 6 months
Before eradication Grade of mucosal inflammation
n
%
%
n ~
Antral mucosa None Mild Moderate Severe Corpus mucosa None Mild Moderate Severe
12 months
~
n
%
~~~
0 7 12 1
0 35 60 5
3 17 0 0
15 85 0 0
10 8 0 0
56 44 0 0
0 10 9 1
0 50
17 3 0 0
85 15 0 0
19 0 0 0
100 0 0 0
45 5
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volvement of mononuclear inflammatory cells in the whole thickness of the gastric mucosa (severe). The presence of lymphoid aggregates in the lamina propria was recorded separately. All semiquantitative interpretations were done blindly by one pathologist (P. Sipponen).
estimated by counting the number of intercepts (points) of the graticular squares falling on the nuclear cells and dividing by the total number of points falling on the mucosa (17,18). To obtain unbiased estimates in counting, the rules of Gundersen (19) were used. In each case, 10 appropriate random fields were counted, and the cell densities (in arbitrary units) were expressed as means (LSD) of these counts. All counting was done blindly by one pathologist (J. Valle). Mononuclear cells were assessed from HE-stained sections and granulocytes from MAC-387-stained sections.
Morphometry The density of granulocytes and mononuclear inflammatory cells was estimated morphometrically by determining the ‘areal fraction’ of cells in the subepithelial layer of the mucosa (14). The counting of cells was done microscopically at high magnification (objective, 4 0 ;~ Statistics The Wilcoxon rank test was used in calculations ocular, lox) by using an ocular grid composed of 100 small squares. The areal fraction of cells was of differences in the morphometric data, and the chi-square test was used in calculations of differences in the semiquantative data.
Fig. 1 . Antral mucosa before and after the eradication of Helicobacier pyfori. Before (left) and 12 months after (right). (Hematoxylin and eosin; magnification, ~ 2 5 0 . )
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Fig. 2. Corpus mucosa before and after the eradication of Helicobacter pylori. Before (left) and 12 months after (right). (Hematoxylin and eosin; magnification, ~ 2 5 0 . )
RESULTS Semiquantitative assessment of gastritis The prevalence and grade of acute and chronic gastritis in the antrum and corpus in the initial and follow-up endoscopic biopsy specimens in responders ( H . pylori bacterium was eradicated; see Materials and Methods) of the series are presented in Tables I and 11, and an example of gastritis before and after the treatment is given in Figs. 1 and 2. In these patients a significant decrease in the grade of both chronic and acute inflammation occurred in the course of time, in the same manner in both the antrum and corpus mucosa. Acute inflammation disappeared rapidly and was not seen in any of the subjects in the follow-up at 6weeks. The resolution of chronic gastritis was slower than that of the acute inflammation, and
the antral gastric mucosa was graded as normal in 3 (15%) of 20 patients in the follow-up at 6 months, and in 10 (56%) of 18 subjects at the 12month follow-up. On the other hand, the corpus mucosa was graded as normal in 17 (85%) of 20 subjects already at the 6-month follow-up. No significant changes occurred in the grade of acute or chronic gastritis in the 3 non-responders or in the 23 controls within the follow-up period (data not shown). The number of cases of lymphoid aggregates in the gastric mucosa decreased in the follow-up among the responders. Lymphoid aggregates in antral biopsies were seen in 12 subjects (63%) in the initial endoscopy but only in 3 (16%) subjects in the 6 month follow-up, and in none in the 12month follow-up. No corresponding decrease in the prevalence of lymphoid aggregates was seen in the controls (data not shown).
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Morphometry The mean densities of granulocytes and mononuclear inflammatory cells in the biopsy specimens from the initial and follow-up endoscopies in the patient series and controls are presented in Tables 111and IV and in Figs. 3 and 4. The tables also show the ‘basal’ density of inflammatory cells in nine healthy controls who showed ‘normal’ stomach (both antrum and corpus mucosa are normal) at ordinary histology. In the patient series with a successful eradi-
cation of H . pylori (responders) a significant timerelated decrease occurred in the density of both granulocytes and mononuclear cells in the subepithelial layer of both the antral and corpus mucosa. This decrease was rapid with regard to granulocytes but slower with regard to mononuclear cells. No decrease in the density of mononuclear inflammatory cells was found in the three non-responders at the 1-month control. No changes in the cell densities were seen in the controls between the initial and follow-up endoscopies.
Table 111. Density (mean ? SD) of granulocytes in the lamina propria in responders and non-responders before and after the treatment of Helicobacter pylori After eradication Before eradication
6 weeks
Responders ( a = 20) Antrum Corpus
2.6 t 0.5 1.3 t 0.4
0.15 t 0** 0.1 +o*
Non-responders ( n = 3) Antrum Corpus
4.7 t 3.6 0.1 t 0
2.2 t 1.7 0.1 t 1.0
6 months
12 months
0 t o*** 0 t 0***
O t O*** ( n = 18) 0 t 0** ( n = 18) 2.3 t 0.5 1.5 2 0.1
2.4 t 1.0 2.4 t 1.0
Patients with normal stomach ( n = 9)t Antrum 0.0 t 0.0 Corpus 0.0 2 0.0
* p < 0.05; **p < 0.01; ‘ * * p < 0.001 differences compared with values before the eradication (Wilcoxon). tA selected group of patients with normal stomach (both antrum and corpus are normal; see Materials and Methods).
Table IV. Density (mean ? SD) of mononuclear inflammatory cells in the lamina propria in responders (20 patients) and non-responders (3 patients) before and after the treatment of Helicobacter pylori After eradication Before eradication Responders ( n = 20) Antrum Corpus
10.3 0.7 9.7 t 0.9
Non-responders (n = 3) Antrum Corpus
15.3 ? 1.7 10.3 ? 3.7
*
6 weeks
8.2 t 0.4** 6.6 t 0.6** 10.4 ? 1.0 7.9 t 2.5
6 months
5.9 t 0.5*** 4.3 t 0.4*** 11.8 t 1 12.4 t 1
12 months 3.6 t 1.7*** (n = 18) 3 . 3 ? 1.7***( n = 18)
*
11.0 2.5 10.0 t 1.7
Patients with normal stomach (n = 9)t Antrum 1.5 t 1.3 Corpus 2.1 ? 2.0 * p < 0.05; * * p< 0.01; * * * p < 0.001 differences compared with values before the eradication (Wilcoxon). ?A selected group of patients with normal stomach (both antrum and corpus are normal; see Materials and Methods).
Gastritis after H. pylori Eradication
ANTRUM
CORPUS
4 10
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5
ttt
I
O
l
o
w
2
0
MONTHS
0
6 12 MONTHS
Fig. 3 . Mean density (?SD, arbitrary units) of mononuclear inflammatory cells in the lamina propria before and after the eradication of Helicobacter pylori in responders.
DISCUSSION Helicobacter pylori is considered a cause of chronic gastritis in a long list of studies of different kinds (see Ref. 20). Spontaneous healing of chronic gastritis is a rather rare phenomenon in
CORPUS
ANTRUM 15
v)
I
l5
10
z Lu cs A A
Lu
0
5
5
0 -0 0
MONTHS
26
0
MONTHS
26
Fig. 4. Mean density (kSD, arbitrary units) of mononuclear inflammatory cells in the lamina propria in controls (patients with Helicobacter pylori-associated gastritis, no treatment).
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adult subjects, and gastritis is generally a longlasting, obviously life-long progressive disease that tends t o lead gradually to atrophy of the underlying mucosa, at least in several affected subjects (21,22). From this viewpoint of gastritis itself, there is evidence that the cumulative risk of peptic ulcer has been shown to be high in patients with gastritis but very low in those with normal gastric mucosa (23,24). The role of chronic gastritis is therefore very important in clinical practice. Colloidal bismuth with certain antimicrobial drugs eradicates H . pylori effectively in a great number of patients (25-27) and also decreases the recurrence rate of duodenal ulcer (25,28). This eradication treatment makes it possible to test the hypothesis concerning the interrelationship between H . pylori and gastritis. A causality between H . pylori and gastritis implies that a significant healing in gastritis ought to occur after the clearance or eradication of the organism. Earlier studies support this view (5, 12, 13) but are handicapped by the fact that they score both acute and chronic inflammatory changes together. There is also a lack of follow-up studies, and, in addition, there is no evidence in the literature that the healing process of chronic inflammation in gastritis is slow after H . pylori eradication. This gave us a reason to examine separately the fate of acute and chronic gastritis after H. pyfori therapy. The healing of chronic gastritis after a successful H . pylori therapy is indicated in our study. There was no healing of gastritis in three nonresponders and no spontaneous healing of chronic gastritis during the follow-up time (mean, 25 months) in matched controls. The present observations indicate that the acute granulocytic inflammation disappears rapidly-at the latest, 6 weeks after eradication. The results suggest that the rapid disappearance of the acute inflammation after treatment is such a prominent feature in the responders that this phenomenon can be considered a good practical indicator of a successful eradication of H . pylori. Correspondingly, this resolution of acute inflammation seems to be also a predictor of later resolution of the chronic inflammation, and the final healing of the gastric mucosa. The chronic mononuclear inflammation
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disappears slowly, and, particularly in the antral mucosa, the final healing of the mucosa will take longer than previously has been assumed. Chronic inflammation was healed (both antrum and corpus are normal) in only 3 of 20 patients 6 months and in 10 of 18patients 12months after the eradication. We conclude that a successful eradication of H . pylori will lead to a significant improvement in both acute and chronic gastritis. The resolution of chronic inflammation is a rather slow process, and a complete healing of the mucosa will obviously take several months, perhaps even a few years. Many still unresolved problems of other elements of chronic gastritis need to be studied further in a much longer follow-up. The role of H. pylori in gastric cancer is open. Whether the atrophic changes also are reversible in chronic gastritis or will develop into severe atrophy in all H . pylori-infected patients or only in genetically determined persons is not known. REFERENCES 1. Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983, 1, 1273-1275 2. Stolte M, Eidt S, Ritter M, Bethke B. Campylobacter pylori und Gastritis: Assoziation oder Induktion? Pathologe 1989, 10, 21-26 3. Dooley CP, Cohen H, Fitzgibbons PL, et al. Prevalence of Helicobacter pylori infection and histologic gastritis in asymptomatic persons. N Engl J Med 1989, 321, 1562-1566 4. Musgrove C, Bolton FJ, Krypczyk AM, et al. Campylobacter pylori: clinical, histological and serological studies. J Clin Pathol 1988, 41, 1316-1321 5. Rauws EAJ, Langenberg W, Houthoff HJ, Zanen HC, Tytgat GNJ. Campylobacter pyloridis-associated chronic active antral gastritis. A prospective study of its prevalence and the effects of antibacterial and antiulcer treatment. Gastroenterology 1988, 94, 3 H 0 6. Peterson WL, Lee F, Feldman M. Relationship between Campylobacter pylori and gastritis in healthy humans after administration of placebo or indornethacin. Gastroenterology 1988, 95, 11851197 7. Langenberg ML, Tytgat GNJ, Schipper MEI, Rietra PJGM, Zanen HC. Campylobacter like organisms in the stomach of patients and healthy individuals. Lancet 1984, 1, 1348 8. Goodwin CS, Armstrong JA, Marshall BJ. Campylobacterpyloridis, gastritis, and peptic ulceration. J Clin Pathol 1986, 39, 353-365
9. Siurala M, Sipponen P, Kekki M. Campylobacter pylori in a sample of Finnish population: relations to morphology and functions of the gastric mucosa. Gut 1988, 29, 909-915 10. Barthel JS, Westblom TU, Havey AD, Gonzalez F, Everett ED. Gastritis and Campylobacter pylori in healthy, asymptomatic volunteers. Arch Intern Med 1988, 148, 1149-1151 11. Sipponen P, Varis K, Cederberg A, et al. Campylobacter pylori is associated with chronic gastritis but not with active peptic ulcer disease. APMIS 1988, 96, 84-88 12. Morgan D, Kraft W, Bender M, Pearson A. The Gastrointestinal Physiology Working Group of Cayetano Heredia and the Johns Hopkins Universities. Nitrofurans in the treatment of gastritis associated with Campylobacter pylori. Gastroenterology 1988, 95, 1178-1184 13. Hislop I, Glancy R, Armstrong J. Histological irnprovement of active chronic gastritis in patients treated with De-Nol. Aust NZ J Med 1984,14(suppl 4). 907 14. Valle J, Seppala K, Sipponen P, Kosunen T. Resolution of gastritis following Helicobacter pylori eradication: a morphometric study. The 9th World Congress of Gastroenterology, Sydney, 1990 15. Brandtzaeg P, Jones DB, Flavell DJ, Fagerhol MK. Mac 387 antibody and detection of formalin resistant myelomonocytic L1 antigen. J Clin Path 1988, 41, 963-970 16. Misiewicz JJ, Tytgat GNJ, Goodwin CS, et al. The Sydney system: a new classification of gastritis. Working Party Reports 1990, 1-10 17. Aherne WA, Dunnill MS. Morphometry. E. Arnold, London, 1982, 46-59 18. Gundersen HJG, Bendtsen TF, Korbo L, et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988, 96, 379-394 19. Gundersen HJG. Notes on the estimation of the numerical density of arbitrary profiles. The edge effect. J Microsc 1977, 111, 219-223 20. Graham DY. Campylobacterpylori and peptic ulcer disease. Gastroenterology 1989, 96, 615-625 21. Kekki M, Siurala M, Varis K, Sipponen P, Sistonen P, Nevanlinna HR. Classification principles and genetics of chronic gastritis. Scand J Gastroenterol 1987, SUPPI PI 141), 1-28 22. Siurala M, Sipponen P, Kekki M. Chronic gastritis: dynamic and clinical aspects. Scand J Gastroenterol 1985, 20(suppl 109), 69-76 23. Sipponen P, Seppala K, Aarynen M, Helske T, Kettunen P. Chronic gastritis and gastroduodenal ulcer: a case control study on risk of coexisting duodenal or gastric ulcer in patients with gastritis. Gut 1989, 30, 922-929 24. Sipponen P, Varis K, Fraki 0, Korri U-M, Seppala K , Siurala M. Cumulative 10-year risk of symptomatic duodenal and gastric ulcer in patients with or without chronic gastritis. A clinical follow-up study of 454 outpatients. Scand J Gastroenterol 1990, 25, 966-973
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25. Marshall BJ, Goodwin CS, Warren JR, et al. Prospective double-blind trial of duodenal ulcer relapse after eradication of Campylobacter pylori. Lancet 1988, 2, 1437-1442 26. Rauws EAJ, Tytgat GNJ. Cure of duodenal ulcer associated with eradication of H . pyfori. Lancet 1990, 225, 1233-1235
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Received 28 January 1991 Accepted 6 May 1991
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27. Borsch G, Mai U , Opferkuch W. Oral triple therapy (Om)may effectively eradicate Carnpylobacterpylori (CP) in man: a pilot study. Gastroenterology 1988, 94, A248 28. Borody TJ, Cole P, Noonan S, et al. Long-term Cumpylobucter recurrence post eradication. Gastroenterology 1988, 94, A43
