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  
J. Pathol. 188: 24–29 (1999)
TWO- TO FOUR-YEAR HISTOLOGICAL FOLLOW-UP OF
GASTRIC MUCOSA AFTER HELICOBACTER PYLORI
ERADICATION
 š1*,  ̌̌2,  . 3,  4,  4,  4   ̌5
1
Medical Center Rogaška, Rogaška Slatina, Slovenia
Department of Pathology, Hospital Celje, Celje, Slovenia
3
Institute for Biomedical Statistics, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia
4
Institute for Microbiology and Immunology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia
5
University Department of Internal Medicine, Department of Gastroenterology, Ljubljana, Slovenia
2
SUMMARY
In a 2- to 4-year prospective study, the reversibility of gastritis after Helicobacter pylori eradication was analysed. Sixty-three
H. pylori-positive, chronic duodenal ulcer patients were studied after the successful eradication of bacteria in the period from 1990 to
1993. H. pylori eradication was obtained by triple antimicrobial regimens (colloidal bismuth subcitrate, amoxycillin, and metronidazole)
applied for at least 14 days. The criteria for eradication were the absence of bacteria from two antral and two body of stomach biopsies
stained with haematoxylin, eosin, and Warthin Starry, and a negative antral biopsy culture. The same diagnostic procedures were
repeated, at regular follow-up endoscopies, each year for up to 4 years. Neutrophil-granulocyte infiltration of gastric mucosa
disappeared in 2 months after bacterial eradication. Mononuclear cellular infiltration was disappearing with statistical significance up
to the second year and normal mucosa was observed in the majority of patients in the fourth year of follow-up. Degeneratively changed
lymphoid aggregates were also present in the fourth year in the antrum (12·5 per cent of patients) and in the body of stomach (14 per
cent of patients). There was no significant change in antral intestinal metaplasia during the 4 years of follow-up. Antral atrophy declined
significantly in the period from 1 to 3 years of follow-up. In conclusion, 3–4 years are needed for gastric mucosa to become normal after
H. pylori eradication, although some residual lymphoid aggregates persist even after that period. Copyright 1999 John Wiley & Sons,
Ltd.
KEY WORDS—Helicobacter
pylori eradication; reversibility of gastritis; duodenal ulcer
INTRODUCTION
Up to the early 1990s, the increased incidence of
gastritis with age was supposed to be unavoidable. After
the age of 40, more than half of the population in the
developed world used to have non-atrophic chronic
gastritis that usually progressed over the years to gland
atrophy and intestinal metaplasia.1,2 The discovery of
Helicobacter pylori was a significant break-through in
our understanding of the aetiology of stomach and
duodenal diseases.3 At the 1990 World Congress
of Gastroenterology in Sydney, a new classification of
gastritis was accepted and antimicrobial therapy of
H. pylori-positive patients with severe or complicated
duodenal ulcer (DU) was recommended.4 In the same
year, we started a prospective, 4-year histological
follow-up in DU patients after successful H. pylori
eradication. The aim of our study was to evaluate the
reversibility of gastritis, especially changes in atrophy
and intestinal metaplasia, after H. pylori eradication.
PATIENTS AND METHODS
Inclusion criteria and follow-up policy
We included in the prospective study patients with
severe or complicated DU disease after successful H.
*Correspondence to: Bojan Tepeš, MD, PhD, Medical Center
Rogaška, Zdraviliška Trg 9, 3250 Rogaška Slatina, Slovenia.
CCC 0022–3417/99/060024–06$17.50
Copyright 1999 John Wiley & Sons, Ltd.
pylori eradication in the period from October 1990 to
October 1993. Eradication was achieved in 11 patients
with colloidal bismuth subcitrate (CBS, 120 mg qid) and
amoxycillin (AMO, 375 mg tid), both for 4 weeks, and
metronidazole (MET, 250 mg qid) for 2 weeks. In 52
patients, H. pylori eradication was achieved by CBS
(120 mg qid), AMO (500 mg qid), and MET (500 mg
qid) for 2 weeks. Upper gastrointestinal endoscopies
were performed before starting therapy, after 2 months,
and yearly. According to the Sydney recommendations,
two biopsies were taken from the antrum and two
from the body of stomach. At the first endoscopy,
we took one additional biopsy from the antrum for a
rapid urease test (Yatrox H.p. Test, Röhm Pharma,
Weiterstadt, Germany). At the subsequent endoscopies,
an additional biopsy from the antrum was taken for
culture. The patients were assumed to be cured of H.
pylori only when all four biopsies and the culture were
negative. Specimens for histology were fixed in 10 per
cent formalin, embedded in paraffin, and stained with
haematoxylin and eosin and Warthin Starry. Specimens
for culture were put in Portagerm pylori transport
medium (Bio Mérieux, Marcy l’Etoile, France) and
cultured on 1·5 per cent brain heart infusion agar with
1 per cent yeast extract enriched with 10 per cent horse
blood, 20 per cent horse serum with 1 per cent Biovitex
(Biolife), and 2 mg/l amphotericin B and 10 mg/l
vancomycin for 14 days in microaerophilic conditions. Specimens for histology were assessed by one
Received 17 February 1998
Revised 29 July 1998
Accepted 21 December 1998
REVERSIBILITY OF GASTRITIS AFTER H. PYLORI ERADICATION
experienced gastrointestinal pathologist, who was blind
for clinical data, according to the Sydney recommendations.5 The activity of inflammation (neutrophil polymorphs), chronic inflammation (lymphocytes and
plasma cells), H. pylori, atrophy, and intestinal metaplasia were rated on a scale ranging from zero to three:
0=absent; 1=mild; 2=moderate, and 3=severe. For
non-gradable variables we used the following criteria:
foveolar hyperplasia was diagnosed if the length and
tortuosity of the foveolae were increased to at least 25
per cent above normal. Normal gastric mucoid cells are
cuboidal cells with mucus vacuoles which occupy the
apical two-thirds of the cell. If the volume of mucus in
the cell is reduced, the cell becomes flat and this was
interpreted as an effect of inflammation injury. When we
found H. pylori reinfection, we tried to differentiate
reinfection from relapse by restriction fragment length
polymorphism (RFLP) analysis of the urea-B segment
amplified by polymerase chain reaction (PCR) from
paraffin-embedded specimens. A 933 bp segment of the
urea-B gene was amplified using HPU50 and HPU25
primers and analysed using RFLP analysis, as described
previously.6 Immunoblots from patients’ serum were
performed (Helicoblot 2.0 Genalab, Singapore) to assess
whether H. pylori reinfection was cag A- or vac
A-positive.
Ethics
The study was approved by the Ethical Committee of
the Medical Faculty, University of Ljubljana, and was
carried out in accordance with the Declaration of
Helsinki. All patients gave their written, informed
consent.
Statistics
Statistical analysis was undertaken using the NCSS
and BMDP statistical packages. The following tests
25
were used: Wilcoxon signed-rank test; Mann–Whitney
test; Friedman two-way analysis of variance by ranks;
Wilcoxon signed-rank test for paired comparison with
Bonferroni correction following Friedman two-way
analysis of variance by ranks; chi-squared test and
McNemar test. The results were considered significant at
p<0·05 in all tests except in paired comparisons following the Friedman test, where the level of significance was
0·05, divided by the number of comparisons.
RESULTS
Sixty-three patients were included in the study (18
women and 45 men) with a median age of 45 years
(range 19–78 years). Patients had a mean history of
duodenal ulcer (DU) of 12·8 years (range 2–30 years).
Seventeen patients (26·9 per cent) had altogether 43
episodes of DU bleeding and seven (11 per cent) patients
had DU perforations. Thirty out of 63 (47·6 per cent)
were smokers and 53/63 (84·1 per cent) had an active
DU a year before their inclusion in the study. Sixty-three
patients had at least a 2-year follow-up, 35 patients had
a 3-year follow-up, while 11 patients had a 4-year
follow-up. Two patients were lost to follow-up in the
third year. H. pylori was present in the antrum and body
in 62/63 patients (98·4 per cent) and only one had H.
pylori only in the antrum before entering the study.
Wilcoxon’s matched pairs, signed-rank sum test revealed
a highly statistically significant difference (p=0·00001) in
grade of gastritis between antral and body mucosa at the
beginning of the study. The score of gastritis was the
sum of H. pylori, neutrophil granulocyte, and mononuclear cellular infiltration, with values ranging from 0
to 9 (from 0 to 3 for each parameter, according to the
Sydney classification).
At the first control endoscopy, performed at 2
months, the neutrophil infiltration had disappeared
Fig. 1—Grade of chronic gastritis (median, maximum, minimum) during 4-year follow-up after
Helicobacter pylori eradication (patients reinfected with H. pylori or infected with H. heilmannii
are not included). – – –, Body; ——, antrum. pa=p for antrum; pc=p for body (Wilcoxon
signed-rank test). N=number of patients
Copyright 1999 John Wiley & Sons, Ltd.
J. Pathol. 188: 24–29 (1999)
26
B. TEPES
{ ET AL.
Table I—Changes in intestinal metaplasia during 4-year follow-up after Helicobacter pylori eradication
Follow-up period
Characteristic
No. of patients with
intestinal metaplasia
Site
Grade
0
Antrum
0
1
2
3
44/52
7/52
1/52
0
Body
0
1
2
3
51/51
0
0
0
Second
month
First
year
Second
year
Third
year
Fourth
year
46/52
5/52
1/52
0
NS
51/51
0
0
0
NS
43/52
9/52
0
0
NS
50/51
1/51
0
0
NS
43/52
9/52
0
0
NS
51/51
0
0
0
NS
19/25
6/25
0
0
NS
24/24
0
0
0
NS
6/8
2/8
0
0
NS
7/7
0
0
0
NS
NS=not statistically significant.
Table II—Changes in atrophy during 4-year follow-up after Helicobacter pylori eradication
Follow-up period
Characteristic
No. of patients with atrophy
Site
Grade
0
Antrum
0
1
2
3
9/52
26/52
17/52
0
Body
0
1
2
3
45/51
5/51
1/51
0
Second
month
First
year
Second
year
Third
year
Fourth
year
13/52
26/52
13/52
0
NS
49/51
2/51
0
0
NS
6/52
38/52
8/52
0
p=0·019
49/51
2/51
0
0
NS
14/52
36/52
2/5
0
p=0·011
49/51
2/51
0
0
NS
13/25
11/25
1/25
0
p=0·02
24/24
0
0
0
NS
2/8
6/8
0
0
NS
7/7
0
0
0
NS
NS=not statistically significant.
completely and normalization in cellular height and
mucin content was observed. A gradual reduction
in mononuclear cellular infiltration was observed
(p<0·001) during the 4 years of follow-up (Fig. 1). The
difference between measurements was significant when
p<0·0125 (Wilcoxon signed-rank test for paired comparison with Bonferroni correction, following Friedman
two-way analysis of variance by ranks). By paired
comparisons we found that the mononuclear cellular
infiltration decreased significantly between 0 and 2
months in the antrum (p<0·001) and in the body of
stomach (p<0·001). The same was observed between the
second month and the first year in the antrum (p<0·001)
and in the body (p<0·001). A statistically significant
difference was also observed in the antrum (p<0·002)
and in the body (p=0·01) between the first and second
year. There was no statistically significant difference in
mononuclear cellular infiltration between the second
and third year (antrum p=0·68; body p=0·06); the
patient data were too small for analyses between the
third and fourth year of follow-up.
Data for the frequency of intestinal metaplasia,
atrophy, lymphoid aggregates, and foveolar hyperplasia
Copyright 1999 John Wiley & Sons, Ltd.
are shown in Tables I–III. Six patients who were reinfected with H. pylori or Helicobacter heilmannii and
some patients who had at least one inadequate specimen
(e.g. insufficiently deep for the assessment of atrophy)
were excluded from this analysis. For the prospective analysis, we collected antrum specimens from
52 patients and body of stomach specimens from 51
patients. During the 4-year follow-up, we did not find
any statistically significant difference in intestinal metaplasia either in the antrum or in the body, where it was
extremely rare. Atrophy was rare in the body but very
common in the antrum (82·7 per cent at time 0). Patients
with atrophy grade 2 were older than those with atrophy
grade 1 or 0 (p=0·0249). There was no age difference
between patients with atrophy grade 1 and those without. Between the second month and 1 year of follow-up,
a statistically significant increase in atrophy was
observed (p=0·019). Antral atrophy declined significantly (p=0·011) between the first and second year
and also between the second and third year (p=0·02).
Lymphoid follicles are common in H. pylori-positive
gastritis, but because we did not always find a germinal
centre we used the term lymphoid aggregates instead of
J. Pathol. 188: 24–29 (1999)
27
REVERSIBILITY OF GASTRITIS AFTER H. PYLORI ERADICATION
Table III—Changes in lymphoid aggregates and foveolar hyperplasia during 4-year follow-up after
Helicobacter pylori eradication
Follow-up period
Characteristics
No. of patients with
lymphoid aggregates
No of patients with
foveolar hyperplasia
Site
Grade
0
Antrum
0
1
19/52
33/52
Body
0
1
42/51
9/51
Antrum
0
1
49/52
3/52
Body
0
1
49/51
2/51
Second
month
First
year
Second
year
Third
year
Fourth
year
27/52
25/52
NS
45/51
6/51
NS
45/52
7/52
NS
51/51
0
NS
42/52
10/52
p=0·0007
48/51
3/51
NS
43/52
9/52
NS
51/51
0
NS
44/52
8/52
NS
47/51
4/51
NS
42/52
10/52
NS
51/51
0
NS
20/25
5/25
NS
22/24
2/24
NS
16/25
9/25
NS
24/24
0
NS
7/8
1/8
NS
6/7
1/7
NS
6/8
2/8
NS
7/7
0
NS
NS=not statistically significant.
follicles. After H. pylori was eradicated, we observed
degenerative changes in lymphoid aggregates, in the
form of fibrosis and hyalinization. Lymphoid aggregates
were more frequent in the antrum than in the body of
stomach (63 per cent versus 18 per cent; p=0·00005;
Table III) before anti-H. pylori therapy was prescribed.
A statistically significant decrease in lymphoid aggregates in the antrum (p=0·0007) was observed only
between the second and 12th month of follow-up. Lymphoid aggregates were present in 20 per cent of patients
in the antrum and in 8 per cent in the body in the third
year and in 12·5 per cent in the antrum and 14 per cent
in body in the fourth year (Fig. 2). For the duration of
the study, there were no significant changes in foveolar
hyperplasia, which was present almost entirely in the
antrum.
During the follow-up period of 2064 patient-months,
we found three reinfections with H. pylori and three
infections with H. heilmannii, which yielded a 1·7 per
cent (re)infection rate per year for each species. H.
heilmannii (Fig. 3) was confirmed by a second pathologist and electron microscopy. In the first year of
follow-up, we had two H. pylori reinfections, with
another in the third year. Two H. heilmannii infections
were confirmed in the second year and one in the third
year of follow-up. We tried to exclude a relapse of the
original H. pylori strain with RFLP analysis of the
urea-B gene amplified by PCR from paraffin-embedded
Fig. 2—Minimal residual lymphoid aggregate in the basal portion of
the antral mucosa 4 years after eradication of Helicobacter pylori
(haematoxylin and eosin; 400)
Fig. 3—High-power photomicrograph of a biopsy specimen of the
antral mucosa with Helicobacter heilmannii organisms in gastric pits
(Warthin-Starry; 1000)
Copyright 1999 John Wiley & Sons, Ltd.
J. Pathol. 188: 24–29 (1999)
28
B. TEPES
{ ET AL.
Fig. 4—Grade of gastritis after reinfection with Helicobacter pylori. – – –, Antrum; ——, body.
N=number of patients; 0=the year before infection
specimens. The results of the analysis were inconclusive,
most probably because of inproper specimen fixation.
We performed the Helicoblot 2.0 test from the serum
of the patients reinfected with H. pylori or infected with
H. heilmannii. All H. pylori-reinfected patients had vac
A- and cag A-positive strains; all H. heilmannii-infected
cases were negative for vac A and cag A. Within the 3
years from H. pylori reinfection, we observed a slow
increase in grade of gastritis (Fig. 4). The grade of
gastritis was much lower in H. heilmannii infection and
gastritis was present in the body of stomach in two out
of three patients. H. heilmannii infection was present
only in the antrum (Fig. 5).
normalization of mononuclear cell infiltration in 56 per
cent of patients compared with controls after 1 year of
follow-up. On the other hand, Genta et al.11 found no
disappearance of chronic inflammation in their patients,
either in the antrum or in the body, after 1 year of
follow-up. The regression of mononuclear cell infiltration was also very slow in an Italian study with 6 months
of follow-up.14 In our study, chronic inflammation
was gradually disappearing up to the fourth year. The
DISCUSSION
To our knowledge, this is the first study to report the
results of up to a 4-year histological follow-up of gastric
mucosa after H. pylori eradication. We observed significant variations in different parameters between antral
and body of stomach mucosa. At the beginning of our
study, the grade of gastritis was significantly higher in
the antrum than in the body (p=0·00001), which is
normal for DU patients.7 Because of the high acid
output in DU patients, the body mucosa was supposed
to be hostile to H. pylori,8 but according to our data,
62/63 (98·4 per cent) patients had H. pylori also in the
body of stomach. Two months after H. pylori eradication, neutrophil granulocyte infiltration had disappeared
and the cellular height and mucus content had returned
to normal; similar data have been reported by others.9–12
All other prospective studies on H. pylori eradication
that have been published so far did not have a follow-up
period longer than 18 months. Valle et al.13 reported
Copyright 1999 John Wiley & Sons, Ltd.
Fig. 5—Grade of gastritis after infection with Helicobacter heilmannii.
– – –, Antrum; ——, body. N=number of patients; 0=the year before
infection
J. Pathol. 188: 24–29 (1999)
REVERSIBILITY OF GASTRITIS AFTER H. PYLORI ERADICATION
process was statistically significant up to the second
year, but only in the fourth year did the majority of
patients have a normal mucosa (chronic inflammation
grade 0). Intestinal metaplasia did not change significantly during the 4-year follow-up, this being in concordance with other reports.11,15–17 The percentage of
atrophy in the antrum increased with statistical significance between the second and 12th month, but a
decrease was observed during the second and third year,
which was also statistically significant. Because of the
patchy distribution of atrophic gastritis with only two
biopsies from the antrum, a sampling bias was possible;
trials with more biopsies would be necessary to prove
the reversibiilty of atrophy. Lymphoid aggregates were
more frequent in the antrum, which confirmed the data
of Genta et al.18 The same authors11 showed that
lymphoid follicles in H. pylori gastritis declined slowly
after H. pylori eradication in the antrum, but this was
not statistically significant and no change was observed
in the body during the first year. On the other hand,
Valle et al.13 reported complete disappearance of lymphoid follicles 1 year after H. pylori eradication. We
noticed that lymphoid aggregates (follicles) significantly
regressed in number between the second month and first
year only in the antrum and slowly, but not significantly
in the body. The disappearance of infection caused
degenerative changes (hyalinization, fibrosis) of lymphoid aggregates, which persisted as the long-lasting
memory of infection even in the fourth year of follow-up
in 12·5 per cent of patients in antrum and in 14 per cent
of patients in body mucosa. Foveolar hyperplasia was
often present in the antrum, most probably as a response
to biliary reflux, which could not be objectively proven.
Reinfection with vac A- and cag A-positive strains
of H. pylori caused a gradual increase in the grade of
gastritis during 2–3 years of follow-up. The grade of
inflammation was higher in the antrum than in the body
and the grade of gastritis became equal to the average
grade before H. pylori eradication therapy after 3 years.
No ulcer recurrence was observed in our infected
patients. Infection with H. heilmannii is a rare phenomenon compared with H. pylori infection and an incidence
Copyright 1999 John Wiley & Sons, Ltd.
29
of 1:1000 was reported by Stolte et al.19 In our
three patients, H. heilmannii was restricted only to
the antrum and the grade of gastritis was lower than in
H. pylori-reinfected patients.
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J. Pathol. 188: 24–29 (1999)
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