Aliment Pharmacol Ther 2002; 16: 1449–1456.
Helicobacter pylori eradication therapy improves atrophic gastritis
and intestinal metaplasia: a 5-year prospective study of patients
with atrophic gastritis
M. ITO* ,  , K. H ARUMAà, T. KAM ADAà, M . MIHA RA§, S. K IM*, Y. K ITADA I*, M. SUMII*,
S. TA NAKA*, M. YOSHIHARA   & K . CHAYAM A*
*Department of Medicine and Molecular Science, Graduate School of Biomedical Science, Hiroshima University, Hiroshima,
Japan;  Health Service Center, Hiroshima University, Higashi-Hiroshima, Japan; àGastroenterology II, Department
of Internal Medicine, Kawasaki Medical School, Kurashiki, Japan; §First Department of Internal Medicine, JR Hiroshima
Hospital, Hiroshima, Japan
Accepted for publication 19 April 2002
SUMMARY
Aim: To investigate the effect of the eradication of
Helicobacter pylori on histological gastritis.
Methods: Twenty-six patients with moderate to severe
atrophy received successful eradication therapy of
H. pylori. Four patients dropped out and 22 were
followed up prospectively for 5 years. The grades of
gastritis were estimated from gastric biopsy specimens.
The grade of intestinal metaplasia was also evaluated by
dye-endoscopy using methylene blue (methylthioni-
nium chloride). The serum levels of pepsinogen, gastrin
and anti-parietal cell antibody were also determined.
Results: The grades of atrophy decreased in patients
with successful eradication therapy in the gastric corpus
INTRODUCTION
Helicobacter pylori plays an important role in the
promotion of atrophic gastritis.1 Long-term infection
with H. pylori results in glandular atrophy and intes-
tinal metaplasia. It has been accepted that there is a
strong association between H. pylori-associated gastritis
Correspondence to: Dr M. Ito, Department of Medicine and Molecular
Science, Graduate School of Biomedical Science, Hiroshima University,
1-2-3 Kasumi Minami-ku, Hiroshima 734-8551, Japan.
E-mail: maito@hiroshima-u.ac.jp

2002 Blackwell Science Ltd
(before vs. 5 years after eradication, 2.09 ± 0.15 vs.
0.91 ± 0.17; P < 0.01) and in the antrum (2.14 ±
0.17 vs. 1.36 ± 0.17; P < 0.01). The levels of intestinal
metaplasia were also decreased in the corpus (0.91 ±
0.24 vs. 0.50 ± 0.16; P < 0.05) and in the antrum
(1.41 ± 0.20 vs. 1.00 ± 0.16; P < 0.05), which was
also demonstrated by the methylene blue (methylthio-
ninium chloride) staining method (33.4 ± 8.2% vs.
23.0 ± 6.5%; P < 0.05). The improvement of corpus
atrophy correlated well with the high serum level of
pepsinogen I (P ¼ 0.005), but showed no correlation
with the levels of anti-parietal cell antibody.
Conclusions: These results suggest that gastric atrophy
and intestinal metaplasia are reversible events in some
patients.
and gastric diseases, including peptic ulcer and gastric
cancer.2–5 Severe gastric atrophy induced by H. pylori is
suggested to be an important risk factor in the
development of gastric carcinoma. Therefore, it is
presumed that the control of histological gastritis is
linked to the control of gastric cancer developments.
Indeed, Uemura et al. reported that the eradication of
H. pylori decreased the occurrence of gastric cancer in
patients with early cancer treated by endoscopic
resection.6 We also confirmed a lower prevalence
of a Ki-67 labelling index of gastric cancer cells
in H. pylori-negative gastric cancer tissue than in
1449
1450 M. ITO et al.
H. pylori-positive tissue, suggesting a growth-promoting
role of H. pylori on gastric cancer cells.7
Another major factor in the induction of gastritis is an
autoimmune factor. It has been reported that sera from
patients with pernicious anaemia contain autoantibod-
ies for both the a and b subunits of H+,K+-ATPase.8, 9
H+,K+-ATPase is a well-known major autoantigen in
patients diagnosed with autoimmune gastritis and
pernicious anaemia.10 Although it is presumed that
autoantibodies against H+,K+-ATPase are important for
the pathogenesis of autoimmune gastritis, Claeys et al.
have found that H+,K+-ATPase is an autoantigen in
H. pylori-associated gastritis.11 The level of anti-parietal
cell antibody (APCA) expression, estimated by immu-
nohistochemical analysis, is associated with the histo-
logical degree of atrophy, the level of functional acid
secretion and the serum levels of pepsinogen and
gastrin.12, 13 Previously, we developed a system to
evaluate the levels of APCA using an enzyme-linked
immunoabsorbent assay. We demonstrated that the
expression of APCA is an indicator of atrophic change in
the corpus after H. pylori infection.14 The APCA level is
closely associated with the development of gastric
diseases, and is similar in both Japanese and Western
patients.15
Human gastritis could be modified using gastro-pro-
tecting agents.16 However, the most direct and simple
method to control atrophic gastritis is the eradication of
H. pylori. In Japan, autoimmune gastritis is a rare disease
and it has been suggested that the most important factor
in human gastritis is H. pylori infection.17 Many reports
have demonstrated an improvement in neutrophil ⁄ lym-
phocyte infiltration after H. pylori eradication therapy.
However, no consensus has been obtained as to the
improvement of glandular atrophy or intestinal metapl-
asia after eradication. In this study, we followed up
patients with atrophic gastritis after H. pylori eradication
therapy, prospectively for 5 years, and examined the
effect of therapy on the improvement of glandular
atrophy and intestinal metaplasia. Moreover, we exam-
ined the prognostic factors for the improvement of
corpus atrophy.
PATIENTS AND METHODS
Patients
Thirty-six patients with atrophic gastritis (11 with
gastric cancer, five with gastric adenoma, three with

2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456
peptic ulcer and 17 with dyspepsia) were included in
this study, and all gave informed consent. All 16
patients with tumours (11 with gastric cancer and five
with adenoma) received endoscopic mucosal resection
before entering the study. In these patients, no recur-
rence was found after endoscopic mucosal resection and
no additional treatment was carried out for the
tumours. All 36 patients had histological gastritis in
both the corpus and antrum, and their histological
features were diagnosed as pan-gastritis. We also
confirmed that all sections revealed moderate to severe
atrophy by histological examination and were positive
for H. pylori as determined by urease test, Giemsa
staining, 13C-urea breath test or serum immunoglobulin
G antibodies against H. pylori (AMRAD, Australia). No
patient who had undergone a gastrectomy was included
in the study. After the diagnosis of H. pylori infection,
the 36 patients received eradication therapy, consisting
of a proton pump inhibitor, amoxicillin and clarithro-
mycin for 1 or 2 weeks, and eradication therapy was
successful in 26 patients. Prior to and every 12 months
after treatment, routine endoscopic examination was
performed for each patient. We excluded three patients
who dropped out from the study. A further patient also
dropped out because he received a gastrectomy for
gastric cancer (poorly differentiated adenocarcinoma in
the gastric corpus) at 36 months. At the time of the
operation, H. pylori infection was not detected by
histological examination. We followed up the remain-
ing 22 patients (15 men, seven women; range,
39–76 years; mean age, 59.3 years) prospectively for
5 years. The 10 non-eradicated patients were also
followed up for 5 years in the same way. No patient
received long-term antibiotics, which might have affec-
ted the status of H. pylori infection. We selected 22 age-
and gender-matched patients with atrophic gastritis
(moderate to severe atrophy with H. pylori infection) as
controls. They were also followed up prospectively for
5 years without eradication therapy. Patients were non-
randomized to treatment.
Histological examination
Four biopsy specimens (two from the lesser curvature of
the antrum and two from the anterior and posterior wall
of the corpus) were obtained from each patient. The
degree of gastritis was determined from haematoxylin
and eosin-stained sections and scored on a scale of 0–3
according to the updated Sydney System.18 Two experts
 H. PYLORI ERADICATION IN ATROPHIC GASTRITIS
(MI and KH) assessed the histological gastritis inde-
pendently without clinical information on the patients.
Dye-endoscopy using methylene blue (methylthioninium
chloride)
Eleven patients received dye-endoscopy using methy-
lene blue (methylthioninium chloride) (MB) prior to and
5 years after eradication therapy. After removing excess
gastric mucous in routine gastroendoscopy, 0.2% MB
solution was spread over the gastric antrum. Endoscopic
images were saved as a pict-file and the stained area
was calculated from the digital photoimage of each
examination using NIH Image Pro software (National
Institutes of Health, USA).
Serum gastrin and pepsinogens
Fasting serum was collected from all patients at entry
into the study. The samples were centrifuged immedi-
ately at 4 C and stored at )20 C until use. The serum
concentrations of gastrin and pepsinogen were deter-
mined by a modified radioimmunoassay.19
Preparation of H+,K+-ATPase
Fresh pig stomachs were obtained immediately after
slaughter. After the surface of the stomach had been
washed, the mucosal layer was scraped off and the
microsomal fraction was prepared. Vesicles containing
H+,K+-ATPase (G1) from the microsomal fraction were
purified as described previously.20 The purified G1 pre-
parations were lyophilized and stored in 250 mm sucrose
containing 1 mm ethylenediaminetetra-acetic acid–Tris,
pH 7.4. The protein was determined by the method of
Bradford using bovine serum albumin as the standard.21
Enzyme-linked immunoabsorbent assay
Enzyme-linked immunoabsorbent assay was performed
as described previously with some modifications.22
Briefly, 100 lL of antigen (porcine H+,K+-ATPase
membrane fraction) was incubated in a 96-well immu-
noplate (MaxiSorp, Nunc, Denmark) overnight at 4 C.
After the antigen had been removed, the plates were
washed with phosphate-buffered saline (pH 7.2)
)0.05% Tween 20. Phosphate-buffered saline )1%
bovine serum albumin was added for 1 h at room
temperature to block non-specific binding. The diluted

2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456
1451
sera (1 : 100 with phosphate-buffered saline) were
inoculated in each well for 2 h at room temperature.
Peroxidase-conjugated antihuman immunoglobulin G
sheep antibody (diluted 1 : 200) was used as a secon-
dary antibody. After the colour had developed, the
absorbance was read at 492 nm.
Non-specific binding was calculated from the enzyme-
linked immunoabsorbent assay using control serum
without antigen and subtracted from the absorbance
reading (A492).14 All reactions were performed in
duplicate.
Statistics
The results are reported as the mean ± standard error.
Analysis was performed by the Wilcoxon signed rank test,
paired t-test, Yates’ corrected chi-squared test and
Fisher’s protected least significant difference test with
StatView software (SAS Institute Inc., Cary, NC, USA).
P < 0.05 was considered to be significant.
RESULTS
Clinical features of the patients
Of the 36 patients enrolled, eradication therapy was
successful in 26 patients and 22 were followed up for
5 years. We examined the clinicopathological features
of 22 patients with successful eradication (group A) and
10 with failed eradication (group B). We selected 22
control patients who were followed up for 5 years
without eradication therapy (group C). No difference
was demonstrated between the three groups (Table 1).
Changes in histological gastritis after H. pylori eradication
The 54 patients were followed up prospectively for
5 years. Changes in the histological features are sum-
marized in Table 2. In group A, the degrees of atrophy
and intestinal metaplasia significantly decreased 5 years
after H. pylori eradication. These findings were similar
in both the gastric corpus and antrum. However, no
significant difference was detected in groups B or C. We
subclassified all patients into two groups (H. pylori-
positive and H. pylori-negative) by final H. pylori status
and compared the histological changes between the two
groups. Histological recovery was detected only in
patients without H. pylori infection at the end-point of
the study (Table 3).
1452 M. ITO et al.

Table 1. Comparison of clinical features between the three groups

Group A (n ¼ 22) Group B (n ¼ 10) Group C (n ¼ 22)

Clinical features
Mean age (range) (years) 59.3 (39–76) 60.8 (38–76) 60.8 (36–76)
Gender (male ⁄ female) 15 ⁄ 7 9⁄1 15 ⁄ 7
Diagnosis (neoplasm ⁄ ulcer ⁄ dyspepsia) 9 ⁄ 2 ⁄ 11 3⁄1⁄6 9 ⁄ 1 ⁄ 12
Histological features (corpus)
Atrophy 2.09 ± 0.15 2.20 ± 0.25 2.09 ± 0.13
Metaplasia 0.91 ± 0.24 1.20 ± 0.42 0.82 ± 0.23
Histological features (antrum)
Atrophy 2.14 ± 0.17 2.20 ± 0.20 2.09 ± 0.15
Metaplasia 1.41 ± 0.20 1.40 ± 0.40 1.41 ± 0.25
Serological features*
Pepsinogen I (ng ⁄ mL) 46.5 ± 5.5 67.6 ± 18.3 42.3 ± 8.5
Pepsinogen II (ng ⁄ mL) 18.3 ± 2.1 22.8 ± 5.5 18.1 ± 2.3
Pepsinogen I ⁄ II 2.78 ± 0.31 2.95 ± 0.60 2.07 ± 0.26
Gastrin (pg ⁄ mL) 211.4 ± 47.1 291.9 ± 99.8 150.6 ± 26.6

Group A, patients with successful eradication therapy; group B, patients with failed eradication therapy; group C, patients without eradication
therapy.
*Mean ± standard error.

Table 2. Changes in histological gastritis

Corpus Antrum
after eradication therapy: comparison
Before After 5 years Before After 5 years between the three groups

Group A (n ¼ 22)
Atrophy 2.09 ± 0.15 0.91 ± 0.17  2.14 ± 0.17 1.36 ± 0.17 
Metaplasia 0.91 ± 0.24 0.50 ± 0.16* 1.41 ± 0.20 1.00 ± 0.16*
Group B (n ¼ 10)
Atrophy 2.20 ± 0.25 1.90 ± 0.35 2.20 ± 0.20 2.20 ± 0.20
Metaplasia 1.20 ± 0.42 1.20 ± 0.44 1.40 ± 0.40 1.30 ± 0.45
Group C (n ¼ 22)
Atrophy 2.09 ± 0.13 1.96 ± 0.14 2.09 ± 0.15 1.96 ± 0.19
Metaplasia 0.82 ± 0.23 0.82 ± 0.25 1.41 ± 0.25 1.23 ± 0.28

Group A, patients with successful eradication therapy; group B, patients with failed eradica-
tion therapy; group C, patients without eradication therapy.
Data are presented as the mean ± standard error.
Significantly different between the two groups (Wilcoxon signed rank test, before and after
5 years): *P < 0.05,  P < 0.01.

Table 3. Changes in histological gastritis

Corpus Antrum
after eradication therapy: comparison
Before After 5 years Before After 5 years between Helicobacter pylori-negative and
H. pylori-positive patients at the end-point
H. pylori-negative (group A, n ¼ 22) of the study
Atrophy 2.09 ± 0.15 0.91 ± 0.17  2.14 ± 0.17 1.36 ± 0.17 
Metaplasia 0.91 ± 0.24 0.50 ± 0.16* 1.41 ± 0.20 1.00 ± 0.16*
H. pylori-positive (groups B and C, n ¼ 32)
Atrophy 2.13 ± 0.12 1.94 ± 0.14 2.13 ± 0.12 2.03 ± 0.15
Metaplasia 0.94 ± 0.21 0.93 ± 0.22 1.41 ± 0.21 1.25 ± 0.23

Group A, patients with successful eradication therapy; group B, patients with failed eradica-
tion therapy; group C, patients without eradication therapy.
Data are presented as mean ± standard error.
Significantly different between the two groups (Wilcoxon signed rank test, before and after
5 years): *P < 0.05,  P < 0.01.

2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456

 H. PYLORI ERADICATION IN ATROPHIC GASTRITIS 1453

Estimation of intestinal metaplasia by methylene blue

(methylthioninium chloride) staining
In an initial experiment, we examined the accu-
racy of the MB staining method for estimating his-
tological metaplasia, and confirmed a good correlation
between the histological features and status of MB
staining.23 In this study, we found a good correlation
between the histological grade of intestinal metaplasia
and the result of MB staining (data not shown). MB
staining of the gastric antrum was performed in 11
patients in group A before and 5 years after eradica-
tion therapy (Figure 1). The stained area calculated
was significantly decreased after H. pylori eradica-
tion therapy (33.4 ± 8.2% vs. 23.0 ± 6.5%; P < 0.05; Figure 2. Comparison of methylene blue (methylthioninium
Figure 2). chloride)-stained area before (left) and after (right) eradication
therapy. Data are given as the percentage stained area (stained
area ⁄ whole area (%)). *P < 0.05, paired t-test.

Prognostic factors for improvement of corpus atrophy

Figure 1. Methylene blue (methylthioninium chloride) solution
was spread over the gastric antrum before (a) and after (b)
eradication therapy. The stained area was calculated using NIH
Image Pro software (National Institutes of Health, USA). The
patient was a 63-year-old female.
after H. pylori eradication
Of the 22 patients with successful H. pylori eradication
(group A), we observed a prominent improvement in
corpus atrophy in seven patients, in whom the atrophic
score decreased by more than or equal to two grades.
We attempted to clarify the host factor that was
prognostic for the improvement of corpus atrophy. A
comparison between these seven patients and the
remaining 15 is summarized in Table 4. There was no
difference in age, sex or clinical diagnosis between the
two groups. However, the serum levels of pepsinogen I
were significantly higher in the group with remarkable
improvement than in the other group (67.1 ± 9.5 vs.
36.2 ± 5.0 ng ⁄ mL; P ¼ 0.005; Table 4), whereas no

Table 4. Prognostic factors for improve-

Improvement of atrophic score
ment of corpus atrophy
‡ 2 (n ¼ 7) < 2 (n ¼ 15) P value

Mean age (range) (years) 59.0 (45–75) 59.4 (39–76) 0.94*

Gender (male ⁄ female) 6⁄1 9⁄6 0.47 
Diagnosis (neoplasm ⁄ non-neoplasm) 2⁄5 7⁄8 0.74 
Pepsinogen I (ng ⁄ mL) 67.1 ± 9.5 36.2 ± 5.0 0.005*
Pepsinogen II (ng ⁄ mL) 22.1 ± 3.0 16.5 ± 2.6 0.21*
Pepsinogen I ⁄ II 3.23 ± 0.46 2.56 ± 0.40 0.32*
Gastrin (pg ⁄ mL) 280.7 ± 125.8 176.8 ± 34.5 0.31*
APCA (A492) 0.97 ± 0.17 0.90 ± 0.09 0.70*

APCA, anti-parietal cell antibody; A492, absorbance at 492 nm.

Data are presented as mean ± standard error.
*Fisher’s protected least significant difference.
 Yates’ corrected chi-squared test.

2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456

1454 M. ITO et al.
significant difference was found in the levels of pepsi-
nogen II, pepsinogen I ⁄ II ratio or gastrin. We could not
find any difference in the levels of APCA determined by
enzyme-linked immunoabsorbent assay.
DISCUSSION
Many gastric diseases are closely associated with
chronic atrophic gastritis. H. pylori plays an important
role in the pathogenesis of gastritis, as atrophic gastritis
is found primarily in patients with H. pylori infection.1 It
is clinically important to cure Ôatrophic gastritisÕ in order
to prevent gastric diseases including gastric cancer.2–5
The eradication of H. pylori is the best way to control the
frequency of atrophic gastritis and thus contribute to
cancer prevention.6
Although many studies have discussed the effect of
H. pylori eradication therapy on histological gastritis, it
is still unclear whether atrophic change is reversible or
not. Annibale et al. have demonstrated that eradication
therapy does not improve mucosal atrophy.24 Sung
et al. reported the results of a large-scale prospective
randomized study and concluded that eradication
therapy prevented the progression of atrophy which
was not reversible.25 However, these studies were
performed over a relatively short period. Moreover, the
basal grades of atrophy were mild in the latter study and
this may be the reason why the reversibility of
glandular atrophy was overlooked. On the other hand,
Ohkusa et al. enrolled patients with atrophic gastritis
and reported the reversibility of atrophy after H. pylori
eradication therapy.26 However, no report has demon-
strated the long-term effects of eradication therapy on
the reversibility of glandular atrophy.
In this study, we followed up 22 patients in whom
H. pylori had been eradicated for 5 years and confirmed
that glandular atrophy was reversible in both the
gastric corpus and antrum. These results support those
reported by Ohkusa et al.26 The first important point of
our study was that we selected patients with atrophic
gastritis. The alteration of atrophic change should be
emphasized in these patients compared with healthy
volunteers or patients with duodenal ulcer. Recently,
Kokkola et al. reported that atrophic change improved
after H. pylori eradication in patients with advanced
atrophic gastritis,27 confirming the results obtained in
this study. The second important point of our study was
the long-term period of follow-up of 5 years. These two
factors are important in the design to examine the

2002 Blackwell Science Ltd, Aliment Pharmacol Ther 16, 1449–1456
reversibility of glandular atrophy. Previously, we dem-
onstrated increased gastric acidity accompanied by an
improvement of glandular atrophy 1 year after eradi-
cation therapy.28 Given our present data, we can
confirm the accuracy of our previous results. We can
also confirm the increase in the pepsinogen I ⁄ II ratio
after successful eradication therapy (data not shown).
We attempted to determine the host factor predicting
the reversibility of glandular atrophy, because all
patients did not show an improvement of glandular
atrophy to the same degree. Atrophic change in the
gastric corpus is closely associated with the risk of
gastric cancer, and is clinically important.5 We have
previously demonstrated that APCA plays an important
role in promoting glandular atrophy after H. pylori
infection.14, 15 Because we have postulated the role of
APCA in the reversibility of atrophic change, we
examined the relation between APCA levels and the
alterations in corpus atrophy after the eradication of
H. pylori. However, we could not confirm any relation
between the level of APCA and reversibility of the fundic
gland. It is probable that APCA does not affect the
destruction of the parietal cell in the absence of H. pylori.
It has recently been observed that the T-cell response is
essential to the mechanism of autoimmune gastritis,
suggesting that this response could be modified by the
eradication of H. pylori.29
We found that high pepsinogen I levels were correlated
with the reversibility of glandular atrophy in the corpus.
The level of pepsinogen I represents a measure of
damage to the fundic gland.30, 31 This suggests that a
certain volume of the fundic gland is necessary for its
reconstruction. In cases with complete disappearance of
the gland, such as gastric adenoma, reconstruction of
the gland may be unlikely. In this study, we enrolled
patients with atrophic gastritis in order to attempt to
demonstrate the reversibility of the atrophic gland in
cases with an appropriate degree of atrophy.
Another major problem is the reversibility of intestinal
metaplasia. Intestinal metaplasia is also evoked mainly
by H. pylori infection and is an important factor in
gastric carcinogenesis.32, 33 As discussed for glandular
atrophy, it is still unclear whether intestinal metaplasia
disappears after H. pylori eradication. Although some
reports have refuted the improvement of intestinal
metaplasia,24, 25, 34 others have suggested that erad-
ication therapy leads to the improvement of intestinal
metaplasia.26, 27 In this study, we evaluated the grade
of intestinal metaplasia as a ÔpointÕ and a ÔfieldÕ.
 H. PYLORI ERADICATION IN ATROPHIC GASTRITIS
Dye-endoscopy using MB allows us to estimate the
degree of intestinal metaplasia as a ÔfieldÕ. The results of
MB staining showed a good correlation with the
histological grading of intestinal metaplasia.23 Our
overall results suggest the reversibility of intestinal
metaplasia on follow-up over long periods. A detailed
classification of intestinal metaplasia has been reported
and it has been discussed that the regenerative ability
may be different between subclasses.35 Further exam-
inations should focus on the characteristics of internal
metaplasia of patients.
In conclusion, we have demonstrated the reversibility
of glandular atrophy and intestinal metaplasia on long-
term follow-up of patients with H. pylori eradication.
Further studies are needed to assess the importance of
these improvements with regard to the clinical out-
come of patients, including the occurrence of gastric
cancer.
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