Reviewer's report
Title:Diffuse duodenal nodular lymphoid hyperplasia: A large cohort of patients
etiologically related to helicobacter pylori infection.
Version: 3Date:4 March 2011
Reviewer: vatsala misra
Reviewer's report:
Thanks for sending the manuscript by Khuroo et al for re review. I regret the
delay in review process due to difficulty in downloading the mainfile of the
manuscript. For the concern expressed by the authors, I would like to state that
any comment by the reviewers are for the modification and improvement of the
manuscript so that all the aspects become more clear to the unbiased reader and
the manuscript may suite to the stature of the journal. But, in present case
instead of understanding the questions and answering and incorporating the
changes in the manuscript, authors have felt offended and showed concern for
the knowledge of the reviewer and tried to prove that every query is irrelevant. It
should not be encouraged.
I’m again sending the point wise comment for many answers and how those
details would have helped in improving the manuscript.
Q 2 Which classification was used to classify gastritis and duodenitis on
histology?
Please give method of doing RUT.
Ans: Both these have been addressed in Material and methods. We have used
Modified Sydney classification for grading of gastritis and H pylori infection.
Relevant references are included. (For RUT, 2 forceps punch biopsies were
taken from gastric incisura and embedded in agar gel urea-rich medium (HP
test™, Allied Marketing Corporation, Kolkata, India) and read as per
manufacturer’s instructions. Multiple gastric biopsies (two from antrum; two from
body and additional specimens from any visible endoscopic visible lesions, if
needed) were taken and stained with Hematoxylin & Eosin to type and grade
gastritis; Alcian blue to detect intestinal metaplasia and Giemsa stain for H. pylori
detection and density.25,26)
My comments –
At no place in the material and methods they have mentioned the name of
Sydney System, neither the reference for it has been quoted. The reference for
Sydney system is by Mesiwicz J J et al,1990.This system describes 4-5
parameters Viz.Inflammation, atrophy, activity, intestinal metaplasia and H,Pylori
Separately. Lymphoid aggregates and Lymphoid proliferation if present should
also be mentioned. It does not take superficial or full thickness in account but the
area involved like antrum predominant, corpus predominant or Pan gastritis.By
the terminology used by them it appears they have classified according to
whitehead classification.
Q3. It appears to be quite unusual that despite 100% biopsies showing presence
Of H. pylori (heavy in 34 patients), only 2 patients had MALToma and rest others
had no evidence of lymphoid hyperplasia in stomach but all the cases had
nodules in the duodenum.
Ans: The reviewer is mixing up the entity of low grade MALT lymphoma and
Mucosa Associated lymphoid tissue. Low grade MALT lymphoma is a malignant
disease of stomach related to H. pylori infection and potentially reversible
disease after H. pylori eradication as seen in 2 of our patients. Low grade MALT
lymphoma can evolve in to high grade gastric lymphoma with local and distant
spread. Mucosa Associated Lymphoid Tissue (MALT) is a very common
association of H. pylori gastritis. In fact most of patients with H. pylori gastritis (in
our study as well) have lymphoid follicles in the gastric mucosa and this is a
hallmark of gastritis caused by H. pylori.
My comments:
I’m not at all mixing the two entities. Lymphoid follicles (MALT) are not seen in
normal gastric mucosa. (Owen et al, 1986 and Genta et al human Pathology
1993), and commonly seen with the H.pylori infection. When the histology of the
gastric biopsies was described in detail they should have mentioned about the
lymphoid proliferation which may vary from Chronic infiltrate to few lymphoid
aggragates to well-formed lymphoid follicles without any lymphoepithelial lesions.
I just wanted to give them the no (%) of biopsies showing the L. aggregate/L.
follicles out of 38 (If they had observed it), as the study is mainly concerned with
the lymphoid proliferation and correlation with duodenal changes would have
helped in explaining the pathogenesis. In a scientific paper, we need to have
exact numbers for any measurable parameter and terms like ‘most’ ,’sparse’ and
‘infrequent’ should not be used.
.
Q4. How many duodenal biopsies had evidence of gastric metaplasia and
Evidence of H.pylori?
Ans: This has been dealt with in the Results section. In fact duodenal mucosal
changes have been dealt with in detail in this section. H. pylori was rarely seen in
The section of duodenal biopsies and was very sparse.
My comments:
In result section they have not mentioned about the areas of gastric metaplasia
with the description of duodenal histology. these could have been easily
identified in the sections stained with the PAS stain (which they have done
according to the material and method).H.pylori is highly selective for its
microenvironment and is not seen in duodenal mucosa except in areas of gastric
metaplasia.They are not even seen in the areas of atrophy and intestinal mucosa
in gastric mucosa( very rarely reported in low density).Authors are reporting them
in duodenal mucosa without metaplasia and 100% positivity for H.pylori in gastric
mucosa in moderate and high density despite 14 cases showing chronic atrophic
gastritis with intestinal metaplasia (?).Here also I just wanted them to give the
no(%) of duodenal biopsies showing gastric metaplasia and how many of them
had H.pylori that would have helped them in establishing the H.pylori as
etiological agent for DDNLH which they are trying to prove and publish. .
Q5. How the H.pylori present in the stomach led to nodular lymphoid hyperplasia
In the duodenum without producing any change in the gastric mucosa – please
explain
Ans: H pylori can cause lot of extragastric manifestations such as Idiopathic
thrombocytopenic purpura (ITP), iron deficiency anemia etc. The reviewers
comments that there were no changes in the stomach is incorrect. Refer to
endoscopic and histologic findings of gastric mucosa in results section which
reads as follows < Examination of stomach showed no endoscopic abnormality in
8 patients; linear erythematous antral gastritis in 20 patients; exudative fundic
gastritis in 6 patients and atrophic gastritis in 4 patients. Two patients had diffuse
ulcerative nodular lesions limited to the antrum. None of the patient had pyloric or
duodenal ulcer. RUT was positive in all patients and H. pylori were seen in
gastric biopsies in all patients. Density of H. pylori was moderate in 6 patients
and heavy in 34 patients. Histology of gastric biopsies revealed chronic
superficial gastritis in 24 patients, and chronic atrophic gastritis with intestinal
metaplasia in 14 patients. Two patients with diffuse ulcerative nodular disease of
antrum showed histologic features of low grade MALT lymphoma>
My comments:
If they would have noted the lymphoid hyperplasia in the gastric biopsies and
correlated it with the corresponding duodenal biopsies, they could have easily
explained it as follows ‘ though the lymphoid hyperplasia was present in both the
gastric and duodenal biopsies the immune response was more marked in the
duodenum leading to focal oedema (as can be seen from the picture in
lowpower) and nodularity”
Q6. Diagnosis of H.pylori was made by RUT and histology but after treatment
Only Breath test was used
It seems that reviewer is not aware of diagnostic protocol of H. pylori
infection. Prior to antibiotic therapy H. pylori infection uniformly affects antrum
(not patchy) and grows up to body, and fundus. In such a situation endoscopic
forceps biopsies are very useful for diagnosis of H. pylori (histology and RUT).
Both tests need to be done for diagnosis and we have meticulously followed the
protocol for taking correct site and number of biopsies. However, after antibiotic
therapy H. pylori infection persists (in resistant cases) in small pockets and is
scattered as small islands of infection in gastric epithelium. Thus forceps biopsies
cannot be used to diagnose H. pylori eradication. In such a situation we need a
global test for H. pylori eradication and these are either 14C-UBT or fecal H.
pylori antigen. In this study we have employed 14C-UBT to check for H. pylori
eradication.
Q7. How can you compare the three different methods for showing eradication.
A pre and post Urea breath test should have been done to show the eradication.
Ans: It seems that reviewer is not aware of diagnostic protocol of H. pylori
infection. Prior to antibiotic therapy H. pylori infection affects antrum and grows
up to body, and fundus. In such a situation endoscopic forceps biopsies are very
useful for diagnosis of H. pylori (histology and RUT). Both tests need to be done
for diagnosis and we have meticulously followed the protocol for taking correct
site and number of biopsies. However, after antibiotic therapy H. pylori infection
persists (in resistant cases) in small pockets and islands in gastric epithelium.
Thus forceps biopsies cannot be used to diagnose H. pylori eradication. In such
a situation we need a global test for H. pylori eradication and these are either
14C-UBT or fecal H. pylori antigen. In this study we have employed 14C-UBT to
check for H. pylori eradication. The reviewers contention of comparing diagnostic
tests for H.pylori infection and eradication is grossly misconceived as she needs
to review proper protocol for diagnosis of H.pylori infection and for checking
eradication of H pylori infection.
My comments:
I’m very well aware of the diagnostic protpcol for diagnosis and showing
eradication while treating an individual patient, In that case 14CUBT is done post
therapy to show eradication and avoid unnecessary endoscopy and biopsy,
where chances of finding the organism are less even in expert hands but while
doing the scientific projects, sometimes you have to deviate or add some extra
tests to existing protocol to prove your hypothesis by comparing pre and post
treatment findings by statistical methods,(as you have done in the present
study.Do you routinely perform all these tests on a patient coming for UGI
endoscopy including IHC for T and B lymphocyte or serological estimation of
immunoglobulin?)
Therefore adding either pre-treatment 14CUBT or post treatment biopsy
examination (patients already had upper G.I. endoscopy) along with comparison
of results and demonstration of disappearance of lymphoid hyperplasia along
with disappearance of nodularity would have further helped in proving your
hypothesis and supporting the manuscript that DDNLH is associated with
H.pylori.
Q8. Discussion –
Authors try to state that the nodules in the duodenum were probably due to
immune stimulation secondary to heavy H.pylori infection. If it was so, then how
the stomach, small intestine and colon were spared
Ans: Pathogenesis of H. pylori induced nodular lymphoid hyperplasia of the
duodenum need further studies. In fact selective involvement of the duodenum is
characteristic and pathognomonic of this entity. This can only be answered by
doing studies similar to those done on H. pylori induced ITP. This we are
convinced will define the cause of selective involvement of the duodenum.
My comments:
Lymphoid proliferation was seen in both stomach and duodenum but the
response was more exaggerated in duodenum which was associated with other
changes like oedema leading to nodularity. Besides due to lesser thickness of
duodenal mucosa as compared to gastric mucosa, even single large l.follicle lead
to nodularity,(as seen in figure).
Q9. References – Most of the references are quite old. some new references
should
be added.
Ans: We have added 2 references of the reviewer in the list.
Q10. Photographs
Histology photograph is not good. Getting a single lymphoid follicle is not
unusual. A scanner or low magnification (10x) showing multiple large follicles
should be given
Ans: Reviewer is confusing this entity with nodular gastritis induced by H. pylori.
In H. pylori induced gastritis numerous lymphoid follicles are seen in the gastric
mucosa in the low power view. However, in DDNLH we have striking nodular
lesions of 2 to 5 mm size and endoscopic view of these lesions is the basis of
this entity. Forceps biopsy with a size of 2 to 3 mm can pick up one nodule of
same or larger size. Characteristic of these biopsies is the large lymphoid follicles
which cause these characteristic elevated lesions. It is possible to see numerous
nodular lymphoid lesions only in surgically resected specimens and we have not
surgically resected any of these lesions. We are enclosing a low power view of
the duodenal biopsy to address this issue. In the low power view of the duodenal
biopsy (around 3 mm in size) only one lymphoid follicle can be seen, the cause of
the elevated nodular lesion. I hope this point is clear.
My comments:
Again there is no question of confusion. The size of the biopsy forceps used and
the tissue obtained is same for the stomach and duodenum and therefore if a
gastric biopsy can show multiple lymphoid follicles(as told by the authors in their
answer), a duodenal biopsy can also show if it is there. Even in the figure
provided two more follicles of the same size can easily come if present. Anyway
that was not an issue. I just wanted a better photograph to improve the
manuscript. If authors did’nt had it they would have simply stated that ‘ none of
the biopsies obtained had more than one lymphoid follicle per section” rather
than explaining the size of the biopsy and labelling me confused.
My recommendations:
Re write the material and methods and results with separate headings for
endoscopic and histological changes in gastric mucosa and duodenal mucosa
along with the changes suggested to make it more clear for the readers.
Modify the discussion as suggested.
Reviewer's report
Title:Diffuse duodenal nodular lymphoid hyperplasia: A large cohort of patients
etiologically related to helicobacter pylori infection.
Version: 3Date:4 March 2011
Reviewer: vatsala misra
Reviewer's report:
Thanks for sending the manuscript by Khuroo et al for re review. I regret the
delay in review process due to difficulty in downloading the mainfile of the
manuscript. For the concern expressed by the authors, I would like to state that
any comment by the reviewers are for the modification and improvement of the
manuscript so that all the aspects become more clear to the unbiased reader and
the manuscript may suite to the stature of the journal. But, in present case
instead of understanding the questions and answering and incorporating the
changes in the manuscript, authors have felt offended and showed concern for
the knowledge of the reviewer and tried to prove that every query is irrelevant. It
should not be encouraged.
I’m again sending the point wise comment for many answers and how those
details would have helped in improving the manuscript.
Q 2 Which classification was used to classify gastritis and duodenitis on
histology?
Please give method of doing RUT.
Ans: Both these have been addressed in Material and methods. We have used
Modified Sydney classification for grading of gastritis and H pylori infection.
Relevant references are included. (For RUT, 2 forceps punch biopsies were
taken from gastric incisura and embedded in agar gel urea-rich medium (HP
test™, Allied Marketing Corporation, Kolkata, India) and read as per
manufacturer’s instructions. Multiple gastric biopsies (two from antrum; two from
body and additional specimens from any visible endoscopic visible lesions, if
needed) were taken and stained with Hematoxylin & Eosin to type and grade
gastritis; Alcian blue to detect intestinal metaplasia and Giemsa stain for H. pylori
detection and density.25,26)
My comments –
At no place in the material and methods they have mentioned the name of
Sydney System, neither the reference for it has been quoted. The reference for
Sydney system is by Mesiwicz J J et al,1990.This system describes 4-5
parameters Viz.Inflammation, atrophy, activity, intestinal metaplasia and H,Pylori
Separately. Lymphoid aggregates and Lymphoid proliferation if present should
also be mentioned. It does not take superficial or full thickness in account but the
area involved like antrum predominant, corpus predominant or Pan gastritis.By
the terminology used by them it appears they have classified according to
whitehead classification.
Q3. It appears to be quite unusual that despite 100% biopsies showing presence
Of H. pylori (heavy in 34 patients), only 2 patients had MALToma and rest others
had no evidence of lymphoid hyperplasia in stomach but all the cases had
nodules in the duodenum.
Ans: The reviewer is mixing up the entity of low grade MALT lymphoma and
Mucosa Associated lymphoid tissue. Low grade MALT lymphoma is a malignant
disease of stomach related to H. pylori infection and potentially reversible
disease after H. pylori eradication as seen in 2 of our patients. Low grade MALT
lymphoma can evolve in to high grade gastric lymphoma with local and distant
spread. Mucosa Associated Lymphoid Tissue (MALT) is a very common
association of H. pylori gastritis. In fact most of patients with H. pylori gastritis (in
our study as well) have lymphoid follicles in the gastric mucosa and this is a
hallmark of gastritis caused by H. pylori.
My comments:
I’m not at all mixing the two entities. Lymphoid follicles (MALT) are not seen in
normal gastric mucosa. (Owen et al, 1986 and Genta et al human Pathology
1993), and commonly seen with the H.pylori infection. When the histology of the
gastric biopsies was described in detail they should have mentioned about the
lymphoid proliferation which may vary from Chronic infiltrate to few lymphoid
aggragates to well-formed lymphoid follicles without any lymphoepithelial lesions.
I just wanted to give them the no (%) of biopsies showing the L. aggregate/L.
follicles out of 38 (If they had observed it), as the study is mainly concerned with
the lymphoid proliferation and correlation with duodenal changes would have
helped in explaining the pathogenesis. In a scientific paper, we need to have
exact numbers for any measurable parameter and terms like ‘most’ ,’sparse’ and
‘infrequent’ should not be used.
.
Q4. How many duodenal biopsies had evidence of gastric metaplasia and
Evidence of H.pylori?
Ans: This has been dealt with in the Results section. In fact duodenal mucosal
changes have been dealt with in detail in this section. H. pylori was rarely seen in
The section of duodenal biopsies and was very sparse.
My comments:
In result section they have not mentioned about the areas of gastric metaplasia
with the description of duodenal histology. these could have been easily
identified in the sections stained with the PAS stain (which they have done
according to the material and method).H.pylori is highly selective for its
microenvironment and is not seen in duodenal mucosa except in areas of gastric
metaplasia.They are not even seen in the areas of atrophy and intestinal mucosa
in gastric mucosa( very rarely reported in low density).Authors are reporting them
in duodenal mucosa without metaplasia and 100% positivity for H.pylori in gastric
mucosa in moderate and high density despite 14 cases showing chronic atrophic
gastritis with intestinal metaplasia (?).Here also I just wanted them to give the
no(%) of duodenal biopsies showing gastric metaplasia and how many of them
had H.pylori that would have helped them in establishing the H.pylori as
etiological agent for DDNLH which they are trying to prove and publish. .
Q5. How the H.pylori present in the stomach led to nodular lymphoid hyperplasia
In the duodenum without producing any change in the gastric mucosa – please
explain
Ans: H pylori can cause lot of extragastric manifestations such as Idiopathic
thrombocytopenic purpura (ITP), iron deficiency anemia etc. The reviewers
comments that there were no changes in the stomach is incorrect. Refer to
endoscopic and histologic findings of gastric mucosa in results section which
reads as follows < Examination of stomach showed no endoscopic abnormality in
8 patients; linear erythematous antral gastritis in 20 patients; exudative fundic
gastritis in 6 patients and atrophic gastritis in 4 patients. Two patients had diffuse
ulcerative nodular lesions limited to the antrum. None of the patient had pyloric or
duodenal ulcer. RUT was positive in all patients and H. pylori were seen in
gastric biopsies in all patients. Density of H. pylori was moderate in 6 patients
and heavy in 34 patients. Histology of gastric biopsies revealed chronic
superficial gastritis in 24 patients, and chronic atrophic gastritis with intestinal
metaplasia in 14 patients. Two patients with diffuse ulcerative nodular disease of
antrum showed histologic features of low grade MALT lymphoma>
My comments:
If they would have noted the lymphoid hyperplasia in the gastric biopsies and
correlated it with the corresponding duodenal biopsies, they could have easily
explained it as follows ‘ though the lymphoid hyperplasia was present in both the
gastric and duodenal biopsies the immune response was more marked in the
duodenum leading to focal oedema (as can be seen from the picture in
lowpower) and nodularity”
Q6. Diagnosis of H.pylori was made by RUT and histology but after treatment
Only Breath test was used
It seems that reviewer is not aware of diagnostic protocol of H. pylori
infection. Prior to antibiotic therapy H. pylori infection uniformly affects antrum
(not patchy) and grows up to body, and fundus. In such a situation endoscopic
forceps biopsies are very useful for diagnosis of H. pylori (histology and RUT).
Both tests need to be done for diagnosis and we have meticulously followed the
protocol for taking correct site and number of biopsies. However, after antibiotic
therapy H. pylori infection persists (in resistant cases) in small pockets and is
scattered as small islands of infection in gastric epithelium. Thus forceps biopsies
cannot be used to diagnose H. pylori eradication. In such a situation we need a
global test for H. pylori eradication and these are either 14C-UBT or fecal H.
pylori antigen. In this study we have employed 14C-UBT to check for H. pylori
eradication.
Q7. How can you compare the three different methods for showing eradication.
A pre and post Urea breath test should have been done to show the eradication.
Ans: It seems that reviewer is not aware of diagnostic protocol of H. pylori
infection. Prior to antibiotic therapy H. pylori infection affects antrum and grows
up to body, and fundus. In such a situation endoscopic forceps biopsies are very
useful for diagnosis of H. pylori (histology and RUT). Both tests need to be done
for diagnosis and we have meticulously followed the protocol for taking correct
site and number of biopsies. However, after antibiotic therapy H. pylori infection
persists (in resistant cases) in small pockets and islands in gastric epithelium.
Thus forceps biopsies cannot be used to diagnose H. pylori eradication. In such
a situation we need a global test for H. pylori eradication and these are either
14C-UBT or fecal H. pylori antigen. In this study we have employed 14C-UBT to
check for H. pylori eradication. The reviewers contention of comparing diagnostic
tests for H.pylori infection and eradication is grossly misconceived as she needs
to review proper protocol for diagnosis of H.pylori infection and for checking
eradication of H pylori infection.
My comments:
I’m very well aware of the diagnostic protpcol for diagnosis and showing
eradication while treating an individual patient, In that case 14CUBT is done post
therapy to show eradication and avoid unnecessary endoscopy and biopsy,
where chances of finding the organism are less even in expert hands but while
doing the scientific projects, sometimes you have to deviate or add some extra
tests to existing protocol to prove your hypothesis by comparing pre and post
treatment findings by statistical methods,(as you have done in the present
study.Do you routinely perform all these tests on a patient coming for UGI
endoscopy including IHC for T and B lymphocyte or serological estimation of
immunoglobulin?)
Therefore adding either pre-treatment 14CUBT or post treatment biopsy
examination (patients already had upper G.I. endoscopy) along with comparison
of results and demonstration of disappearance of lymphoid hyperplasia along
with disappearance of nodularity would have further helped in proving your
hypothesis and supporting the manuscript that DDNLH is associated with
H.pylori.
Q8. Discussion –
Authors try to state that the nodules in the duodenum were probably due to
immune stimulation secondary to heavy H.pylori infection. If it was so, then how
the stomach, small intestine and colon were spared
Ans: Pathogenesis of H. pylori induced nodular lymphoid hyperplasia of the
duodenum need further studies. In fact selective involvement of the duodenum is
characteristic and pathognomonic of this entity. This can only be answered by
doing studies similar to those done on H. pylori induced ITP. This we are
convinced will define the cause of selective involvement of the duodenum.
My comments:
Lymphoid proliferation was seen in both stomach and duodenum but the
response was more exaggerated in duodenum which was associated with other
changes like oedema leading to nodularity. Besides due to lesser thickness of
duodenal mucosa as compared to gastric mucosa, even single large l.follicle lead
to nodularity,(as seen in figure).
Q9. References – Most of the references are quite old. some new references
should
be added.
Ans: We have added 2 references of the reviewer in the list.
Q10. Photographs
Histology photograph is not good. Getting a single lymphoid follicle is not
unusual. A scanner or low magnification (10x) showing multiple large follicles
should be given
Ans: Reviewer is confusing this entity with nodular gastritis induced by H. pylori.
In H. pylori induced gastritis numerous lymphoid follicles are seen in the gastric
mucosa in the low power view. However, in DDNLH we have striking nodular
lesions of 2 to 5 mm size and endoscopic view of these lesions is the basis of
this entity. Forceps biopsy with a size of 2 to 3 mm can pick up one nodule of
same or larger size. Characteristic of these biopsies is the large lymphoid follicles
which cause these characteristic elevated lesions. It is possible to see numerous
nodular lymphoid lesions only in surgically resected specimens and we have not
surgically resected any of these lesions. We are enclosing a low power view of
the duodenal biopsy to address this issue. In the low power view of the duodenal
biopsy (around 3 mm in size) only one lymphoid follicle can be seen, the cause of
the elevated nodular lesion. I hope this point is clear.
My comments:
Again there is no question of confusion. The size of the biopsy forceps used and
the tissue obtained is same for the stomach and duodenum and therefore if a
gastric biopsy can show multiple lymphoid follicles(as told by the authors in their
answer), a duodenal biopsy can also show if it is there. Even in the figure
provided two more follicles of the same size can easily come if present. Anyway
that was not an issue. I just wanted a better photograph to improve the
manuscript. If authors did’nt had it they would have simply stated that ‘ none of
the biopsies obtained had more than one lymphoid follicle per section” rather
than explaining the size of the biopsy and labelling me confused.
My recommendations:
Re write the material and methods and results with separate headings for
endoscopic and histological changes in gastric mucosa and duodenal mucosa
along with the changes suggested to make it more clear for the readers.
Modify the discussion as suggested.
Please review this paper.
user
Reviewer's report
Title:Diffuse duodenal nodular lymphoid hyperplasia: A large cohort of patients
etiologically related to helicobacter pylori infection.
Version: 3Date:4 March 2011
Reviewer: vatsala misra
Reviewer's report:
Thanks for sending the manuscript by Khuroo et al for re review. I regret the
delay in review process due to difficulty in downloading the mainfile of the
manuscript. For the concern expressed by the authors, I would like to state that
any comment by the reviewers are for the modification and improvement of the
manuscript so that all the aspects become more clear to the unbiased reader and
the manuscript may suite to the stature of the journal. But, in present case
instead of understanding the questions and answering and incorporating the
changes in the manuscript, authors have felt offended and showed concern for
the knowledge of the reviewer and tried to prove that every query is irrelevant. It
should not be encouraged.
I’m again sending the point wise comment for many answers and how those
details would have helped in improving the manuscript.
Q 2 Which classification was used to classify gastritis and duodenitis on
histology?
Please give method of doing RUT.
Ans: Both these have been addressed in Material and methods. We have used
Modified Sydney classification for grading of gastritis and H pylori infection.
Relevant references are included. (For RUT, 2 forceps punch biopsies were
taken from gastric incisura and embedded in agar gel urea-rich medium (HP
test™, Allied Marketing Corporation, Kolkata, India) and read as per
manufacturer’s instructions. Multiple gastric biopsies (two from antrum; two from
body and additional specimens from any visible endoscopic visible lesions, if
needed) were taken and stained with Hematoxylin & Eosin to type and grade
gastritis; Alcian blue to detect intestinal metaplasia and Giemsa stain for H. pylori
detection and density.25,26)
My comments –
At no place in the material and methods they have mentioned the name of
Sydney System, neither the reference for it has been quoted. The reference for
Sydney system is by Mesiwicz J J et al,1990.This system describes 4-5
parameters Viz.Inflammation, atrophy, activity, intestinal metaplasia and H,Pylori
Separately. Lymphoid aggregates and Lymphoid proliferation if present should
also be mentioned. It does not take superficial or full thickness in account but the
area involved like antrum predominant, corpus predominant or Pan gastritis.By
the terminology used by them it appears they have classified according to
whitehead classification.
Q3. It appears to be quite unusual that despite 100% biopsies showing presence
Of H. pylori (heavy in 34 patients), only 2 patients had MALToma and rest others
had no evidence of lymphoid hyperplasia in stomach but all the cases had
nodules in the duodenum.
Ans: The reviewer is mixing up the entity of low grade MALT lymphoma and
Mucosa Associated lymphoid tissue. Low grade MALT lymphoma is a malignant
disease of stomach related to H. pylori infection and potentially reversible
disease after H. pylori eradication as seen in 2 of our patients. Low grade MALT
lymphoma can evolve in to high grade gastric lymphoma with local and distant
spread. Mucosa Associated Lymphoid Tissue (MALT) is a very common
association of H. pylori gastritis. In fact most of patients with H. pylori gastritis (in
our study as well) have lymphoid follicles in the gastric mucosa and this is a
hallmark of gastritis caused by H. pylori.
My comments:
I’m not at all mixing the two entities. Lymphoid follicles (MALT) are not seen in
normal gastric mucosa. (Owen et al, 1986 and Genta et al human Pathology
1993), and commonly seen with the H.pylori infection. When the histology of the
gastric biopsies was described in detail they should have mentioned about the
lymphoid proliferation which may vary from Chronic infiltrate to few lymphoid
aggragates to well-formed lymphoid follicles without any lymphoepithelial lesions.
I just wanted to give them the no (%) of biopsies showing the L. aggregate/L.
follicles out of 38 (If they had observed it), as the study is mainly concerned with
the lymphoid proliferation and correlation with duodenal changes would have
helped in explaining the pathogenesis. In a scientific paper, we need to have
exact numbers for any measurable parameter and terms like ‘most’ ,’sparse’ and
‘infrequent’ should not be used.
.
Q4. How many duodenal biopsies had evidence of gastric metaplasia and
Evidence of H.pylori?
Ans: This has been dealt with in the Results section. In fact duodenal mucosal
changes have been dealt with in detail in this section. H. pylori was rarely seen in
The section of duodenal biopsies and was very sparse.
My comments:
In result section they have not mentioned about the areas of gastric metaplasia
with the description of duodenal histology. these could have been easily
identified in the sections stained with the PAS stain (which they have done
according to the material and method).H.pylori is highly selective for its
microenvironment and is not seen in duodenal mucosa except in areas of gastric
metaplasia.They are not even seen in the areas of atrophy and intestinal mucosa
in gastric mucosa( very rarely reported in low density).Authors are reporting them
in duodenal mucosa without metaplasia and 100% positivity for H.pylori in gastric
mucosa in moderate and high density despite 14 cases showing chronic atrophic
gastritis with intestinal metaplasia (?).Here also I just wanted them to give the
no(%) of duodenal biopsies showing gastric metaplasia and how many of them
had H.pylori that would have helped them in establishing the H.pylori as
etiological agent for DDNLH which they are trying to prove and publish. .
Q5. How the H.pylori present in the stomach led to nodular lymphoid hyperplasia
In the duodenum without producing any change in the gastric mucosa – please
explain
Ans: H pylori can cause lot of extragastric manifestations such as Idiopathic
thrombocytopenic purpura (ITP), iron deficiency anemia etc. The reviewers
comments that there were no changes in the stomach is incorrect. Refer to
endoscopic and histologic findings of gastric mucosa in results section which
reads as follows < Examination of stomach showed no endoscopic abnormality in
8 patients; linear erythematous antral gastritis in 20 patients; exudative fundic
gastritis in 6 patients and atrophic gastritis in 4 patients. Two patients had diffuse
ulcerative nodular lesions limited to the antrum. None of the patient had pyloric or
duodenal ulcer. RUT was positive in all patients and H. pylori were seen in
gastric biopsies in all patients. Density of H. pylori was moderate in 6 patients
and heavy in 34 patients. Histology of gastric biopsies revealed chronic
superficial gastritis in 24 patients, and chronic atrophic gastritis with intestinal
metaplasia in 14 patients. Two patients with diffuse ulcerative nodular disease of
antrum showed histologic features of low grade MALT lymphoma>
My comments:
If they would have noted the lymphoid hyperplasia in the gastric biopsies and
correlated it with the corresponding duodenal biopsies, they could have easily
explained it as follows ‘ though the lymphoid hyperplasia was present in both the
gastric and duodenal biopsies the immune response was more marked in the
duodenum leading to focal oedema (as can be seen from the picture in
lowpower) and nodularity”
Q6. Diagnosis of H.pylori was made by RUT and histology but after treatment
Only Breath test was used
It seems that reviewer is not aware of diagnostic protocol of H. pylori
infection. Prior to antibiotic therapy H. pylori infection uniformly affects antrum
(not patchy) and grows up to body, and fundus. In such a situation endoscopic
forceps biopsies are very useful for diagnosis of H. pylori (histology and RUT).
Both tests need to be done for diagnosis and we have meticulously followed the
protocol for taking correct site and number of biopsies. However, after antibiotic
therapy H. pylori infection persists (in resistant cases) in small pockets and is
scattered as small islands of infection in gastric epithelium. Thus forceps biopsies
cannot be used to diagnose H. pylori eradication. In such a situation we need a
global test for H. pylori eradication and these are either 14C-UBT or fecal H.
pylori antigen. In this study we have employed 14C-UBT to check for H. pylori
eradication.
Q7. How can you compare the three different methods for showing eradication.
A pre and post Urea breath test should have been done to show the eradication.
Ans: It seems that reviewer is not aware of diagnostic protocol of H. pylori
infection. Prior to antibiotic therapy H. pylori infection affects antrum and grows
up to body, and fundus. In such a situation endoscopic forceps biopsies are very
useful for diagnosis of H. pylori (histology and RUT). Both tests need to be done
for diagnosis and we have meticulously followed the protocol for taking correct
site and number of biopsies. However, after antibiotic therapy H. pylori infection
persists (in resistant cases) in small pockets and islands in gastric epithelium.
Thus forceps biopsies cannot be used to diagnose H. pylori eradication. In such
a situation we need a global test for H. pylori eradication and these are either
14C-UBT or fecal H. pylori antigen. In this study we have employed 14C-UBT to
check for H. pylori eradication. The reviewers contention of comparing diagnostic
tests for H.pylori infection and eradication is grossly misconceived as she needs
to review proper protocol for diagnosis of H.pylori infection and for checking
eradication of H pylori infection.
My comments:
I’m very well aware of the diagnostic protpcol for diagnosis and showing
eradication while treating an individual patient, In that case 14CUBT is done post
therapy to show eradication and avoid unnecessary endoscopy and biopsy,
where chances of finding the organism are less even in expert hands but while
doing the scientific projects, sometimes you have to deviate or add some extra
tests to existing protocol to prove your hypothesis by comparing pre and post
treatment findings by statistical methods,(as you have done in the present
study.Do you routinely perform all these tests on a patient coming for UGI
endoscopy including IHC for T and B lymphocyte or serological estimation of
immunoglobulin?)
Therefore adding either pre-treatment 14CUBT or post treatment biopsy
examination (patients already had upper G.I. endoscopy) along with comparison
of results and demonstration of disappearance of lymphoid hyperplasia along
with disappearance of nodularity would have further helped in proving your
hypothesis and supporting the manuscript that DDNLH is associated with
H.pylori.
Q8. Discussion –
Authors try to state that the nodules in the duodenum were probably due to
immune stimulation secondary to heavy H.pylori infection. If it was so, then how
the stomach, small intestine and colon were spared
Ans: Pathogenesis of H. pylori induced nodular lymphoid hyperplasia of the
duodenum need further studies. In fact selective involvement of the duodenum is
characteristic and pathognomonic of this entity. This can only be answered by
doing studies similar to those done on H. pylori induced ITP. This we are
convinced will define the cause of selective involvement of the duodenum.
My comments:
Lymphoid proliferation was seen in both stomach and duodenum but the
response was more exaggerated in duodenum which was associated with other
changes like oedema leading to nodularity. Besides due to lesser thickness of
duodenal mucosa as compared to gastric mucosa, even single large l.follicle lead
to nodularity,(as seen in figure).
Q9. References – Most of the references are quite old. some new references
should
be added.
Ans: We have added 2 references of the reviewer in the list.
Q10. Photographs
Histology photograph is not good. Getting a single lymphoid follicle is not
unusual. A scanner or low magnification (10x) showing multiple large follicles
should be given
Ans: Reviewer is confusing this entity with nodular gastritis induced by H. pylori.
In H. pylori induced gastritis numerous lymphoid follicles are seen in the gastric
mucosa in the low power view. However, in DDNLH we have striking nodular
lesions of 2 to 5 mm size and endoscopic view of these lesions is the basis of
this entity. Forceps biopsy with a size of 2 to 3 mm can pick up one nodule of
same or larger size. Characteristic of these biopsies is the large lymphoid follicles
which cause these characteristic elevated lesions. It is possible to see numerous
nodular lymphoid lesions only in surgically resected specimens and we have not
surgically resected any of these lesions. We are enclosing a low power view of
the duodenal biopsy to address this issue. In the low power view of the duodenal
biopsy (around 3 mm in size) only one lymphoid follicle can be seen, the cause of
the elevated nodular lesion. I hope this point is clear.
My comments:
Again there is no question of confusion. The size of the biopsy forceps used and
the tissue obtained is same for the stomach and duodenum and therefore if a
gastric biopsy can show multiple lymphoid follicles(as told by the authors in their
answer), a duodenal biopsy can also show if it is there. Even in the figure
provided two more follicles of the same size can easily come if present. Anyway
that was not an issue. I just wanted a better photograph to improve the
manuscript. If authors did’nt had it they would have simply stated that ‘ none of
the biopsies obtained had more than one lymphoid follicle per section” rather
than explaining the size of the biopsy and labelling me confused.
My recommendations:
Re write the material and methods and results with separate headings for
endoscopic and histological changes in gastric mucosa and duodenal mucosa
along with the changes suggested to make it more clear for the readers.
Modify the discussion as suggested.
assistant