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BACTERIOLOGY RESEARCH DEVELOPMENTS
HELICOBACTER PYLORI
DETECTION METHODS, DISEASES
AND HEALTH IMPLICATIONS
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DIGESTIVE DISEASES - RESEARCH

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HELICOBACTER PYLORI
DETECTION METHODS, DISEASES
AND HEALTH IMPLICATIONS
MARCO MANFREDI, M.D.

AND
GIAN LUIGI DE'ANGELIS
EDITORS
New York

Copyright © 2013 by Nova Science Publishers, Inc.
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to our families
for their endless support

Contents
Preface xi
Acknowledgment xiii
Detection Methods 1
Non-Invasive Procedures 3
Chapter 1: Common Techniques: Serological, Stool Antigen,
and Urea Breath Tests 5
Rosalia Aloe, Chiara Bonaguri, Marco Manfredi,
Fabiola Fornaroli, Pierpacifico Gismondi
and Gian Luigi de'Angelis
Chapter 2: Unusual Techniques: Stool and Dental Plaque
PCR, and Urinary Antibodies 19
Tessa Horemans, Gaëlle Boulet, Peter Delputte,
Louis Maes and Paul Cos
Invasive Procedures 39
Chapter 3: Common Techniques: Endoscopy, Histological
Examination and Rapid Urease Test 41
Pellegrino Crafa,, Marco Manfredi, Elisabetta Manzali,
Barbara Bizzarri and Gian Luigi de’Angelis
Chapter 4: Unusual Techniques: Identification of H. pylori
from Biopsies: Culture, PCR and FISH 69
Carina Almeida, Nuno F. Azevedo and Maria João Vieira
Chapter 5: New Optical Techniques Helping in Diagnoses 83
Noriya Uedo, Hyung Hun Kim, Rapat Pittayanon
and Ryu Ishihara

viii Contents
Diseases and Health Implications 99

Chapter 6: Epidemiology, Transmission Routes, and Recurrence
of Infection 101
Marco Manfredi, Valentina Maffini and Gian Luigi de'Angelis
Helicobacter pylori Infection and Gastrointestinal Diseases – Gastric Pathology 113
Chapter 7: Epithelial Dysfunction in the Pathogenesis
of Helicobacter pylori Infection 115
Tamia K. Lapointe and Andre G. Buret
Chapter 8: Clinical Aspects and Complications of Helicobacter
pylori Infection 145
Marino Venerito and Peter Malfertheiner
Helicobacter pylori Infection and Associated Extra-Gastric Diseases 163
Chapter 9: Esophageal and Colon Diseases 165
Sergei F. Tatishchev and Hanlin L. Wang
Chapter 10: Biliary and Liver Diseases 185
Peng Zhu, Xun Li, Yuming Wang and Liang Qiao
Helicobacter pylori Infection and Associated Extra-Gastrointestinal Diseases 199
Chapter 11: Neurological Disorders and Helicobacter pylori Infection 201
Georgia Deretzi, Christos Zavos, Stergios A. Polyzos,
Evangelia Giartza-Taxidou, Emmanouel Gavalas,
Iakovos Tsiptsios, Theocharis Tsironis, Marina Boziki
and Jannis Kountouras
Chapter 12: Helicobacter pylori Infection and Autoimmune
and Rheumatological Disorders 223
Sarfaraz Ahmed Hasni
Chapter 13: The Impact of Helicobacter pylori Colonization
on Peptides Regulating Appetite and Satiety: Ghrelin,
Leptin, and Obestatin 241
Víctor Rafael Coria Jiménez, Carolina Romo González
and Patricia Chico Aldama
Chapter 14: Role of Helicobacter pylori Infection
in Hematological Disorders 269
Wajid Ali, Zhong Wu, Jin Zhou and Bing Peng
Chapter 15: Helicobacter pylori Infection and Cardiovascular Disorders 283
Benjamin Longo-Mbenza Jacqueline Nsenga Nkondi,
Marco Manfredi and Bertrand Tchana
Chapter 16: Helicobacter pylori and Respiratory Tract Diseases 297
Qiang Wang, Chaoran Yu, Gian Luigi de’Angelis
and Vincenzo Di Comite

Contents ix
Chapter 17: Helicobacter pylori Infection and Gynecological

and Reproductive System Diseases 307
Ghada M. Mansour and Marco Manfredi
Chapter 18: Helicobacter pylori Infection and Skin Diseases 323
Zekayi Kutlubay, Ozge Karakus, Burhan Engin,
Server Serdaroğlu and Yalçın Tüzün
Chapter 19: Helicobacter pylori Infection in Children 337
Stefano Pontone, Laura Petrarca and Raffaella Nenna
Chapter 20: Therapy of Helicobacter pylori Infection:
Many Drugs for Which Association? 347
Ping-I Hsu and Sung-Shuo Kao
Chapter 21: The Biosynthesis of Isoprenoid Precursors as a New Target
for the Development of Specific Antibiotics for the
Treatment of Helicobacter pylori-Associated Diseases 363
Jordi Perez-Gil, Joan Miralles, Maria Bergua,
Albert Boronat, Jaime Rubio-Martinez and Santiago Imperial
Chapter 22: Helicobacter pylori Infection: The Vaccine Development 379
Irina V. Pinchuk and Ellen J. Beswick
Chapter 23: Helicobacter pylori Infection: Eradication or Preservation? 397
Sun-Young Lee and Soo-Heon Park
Index 415

Preface
Since its discovery way back in 1983 by Warren and Marshall, infection by Helicobacter
pylori continues to be a challenge for all gastroenterologists, for children as well as adults. Its
discovery revolutionised the field of gastroenterology and its eradication is one of the greatest
clinical successes, since it can lead to curing many common diseases such as chronic gastritis
and peptic ulcer. H. pylori was classified by the WHO among Type I carcinogens, i.e.
substances having certain carcinogenic risk in humans: this infection is, in fact, at the origin
of the inflammatory process that leads to the formation of adenocarcinoma and gastric
MALT-lymphoma. Its eradication has allowed significant reduction of the incidence of these
serious diseases.
However, infection by H. pylori continues to be one of the biggest challenges, since the
exact route of transmission, pathogenesis and its complete cause-effect relation on the entire
human organism are not exactly known; a ubiquitously valid safe and effective eradication
treatment is also currently not available.
Recently, infection by H. pylori has been correlated to other gastrointestinal diseases;
from diseases of the hepatobiliary system to oesophageal and colic diseases. Moreover, in
recent years, there is increasing awareness that H. pylori might have an important role in the
genesis or evolution of extra-intestinal diseases (of the cardiovascular, respiratory and
endocrinological system, and in certain skin diseases, rheumatic and neurological diseases).
In this book, various international experts from all over the world have illustrated the
current knowledge regarding the various diagnostic methods, the different diseases caused
and the consequent clinical implications on human health in relation to infection caused by H.
pylori.
Although the international guidelines still recommend the triple therapy regimen as the
first-line treatment for eradication, this treatment regimen currently shows a high percentage
of failure linked mainly to increased resistance to the antibiotics commonly used, especially
clarithromycin and metronidazole. In this regard, the various first-line and second line
therapeutic regimens and the recommendations advised as rescue therapy used so far are
listed.
Moreover, the authors have summed up the knowledge and studies currently under way
regarding the development of a vaccine in the preventive as well as therapeutic context,
probably the only way to ensure a widely safe and effective treatment.
Considering the various correlations between infection by H. pylori and various human
diseases, the advantages and disadvantages linked to its eradication were discussed, also

xii Marco Manfredi and Gian Luigi de'Angelis
taking into consideration its possible role of commensal and non-pathogenicity on the human
organism.
About 30 years after its discovery, Helicobacter pylori continues to be a field of zealous
research not only by gastroenterologists; with the passage of time and increased scientific
knowledge on the matter, it is seen how H. pylori seems to be involved in different diseases,
including extra-gastrointestinal disorders, for which there seems to be no clear causal
relationship yet, nor a complete unambiguity regarding the potential benefits acquired by
eradication of the infection.

Acknowledgment
We would like to thank the Italian non-profit organization SNUPI ONLUS

(www.snupi.it) for their constant help to us and mainly to our patients.

Detection Methods

Non-Invasive Procedures

In: Helicobacter pylori ISBN: 978-1-62808-749-9
Editors: Marco Manfredi and Gian Luigi de'Angelis © 2013 Nova Science Publishers, Inc.
Chapter 1
Common Techniques:
Serological, Stool Antigen,
and Urea Breath Tests
Rosalia Aloe1,, Chiara Bonaguri1, Marco Manfredi2,3,

Fabiola Fornaroli2, Pierpacifico Gismondi3
and Gian Luigi de'Angelis2,3
1
Department of Pathology and Laboratory Medicine,
Azienda Ospedaliero-Universitaria, Parma University Hospital, Parma, Italy
2
Gastroenterology and Endoscopy Unit, Azienda Ospedaliero-Universitaria,
Parma University Hospital, Parma, Italy
3
Department of Pediatrics, “Pietro Barilla” Children's Hospital,
Azienda Ospedaliero-Universitaria, Parma University Hospital, Parma, Italy
Abstract
Usually the first detection methods for diagnosing Helicobacter pylori (H. pylori)
infection are the non-invasive tests. Among these, the most used are serological, stool
antigen and urea breath tests.
This chapter summarizes the main characteristics and methods of each test.
Antibodies anti-H. pylori are not recommended in low prevalence population and
they do not show an ongoing infection but they only prove a contact with the bacterium.
They can also persist long time after the eradication of infection, then they should not be
used to verify the success of therapy.
Instead stool antigen and urea breath tests are useful both in diagnosis and during
follow-up after eradication treatment. The stool antigen test is cheaper than Urea breath
test with similar sensitivity and specificity.
Non-invasive tests are not able to diagnose the associated complications to H. pylori
infection.
 Corresponding author: Rosalia Aloe, MD. Department of Pathology and Laboratory Medicine, Azienda
Ospedaliero-Universitaria, University Hospital, Via Gramsci 14, 43126 Parma, Italy. E-mail: raloe@ao.pr.it.

6 Rosalia Aloe, Chiara Bonaguri, Marco Manfredi et al.
Keywords: Helicobacter pylori, Urea breath test, Antibodies, Stool antigen, Eradication
Introduction
Helicobacter pylori (H. pylori) colonizes the human stomach during childhood [1] and
survives in the human stomach for the lifetime of the carrier [2]. In most of the individuals H.
pylori infection may be asymptomatic, causing chronic gastritis. Around 20 to 30% of the
infected individuals may develop peptic ulcer disease, [3] and less than 2% may develop
gastric cancer [4]. Therefore, testing for H. pylori infection has become a very important part
of the diagnostic process for gastric and duodenal inflammatory disease, since the presence or
absence of infection determines the type of treatment to be applied. Testing is also useful for
monitoring the effectiveness of anti-microbial treatment.
A number of different invasive and non-invasive diagnostic methods are currently
available [1-3]. Invasive testing is considered to be highly specific, particularly histological
examination, but its sensitivity is partly dependent on the accuracy of the biopsy procedure.
Moreover, both histological examination and culture of biopsy samples are time-consuming
and require specialized laboratory facilities with highly trained staff. For these reasons,
several techniques for H. pylori non-invasive diagnostic tests have been developed and they
are in widespread use. Laboratory serologic assay, Urea breath test and fecal antigen test are
the common non-endoscopic diagnostic methods for H. pylori infection.
There is no single test that can be considered the gold standard for the diagnosis of H.
pylori. Rather, a variety of factors including the need of endoscopy, pre-test probability of
infection, local availability, an understanding of the performance characteristics (sensitivity,
specificity, positive and negative predictive value) and cost of the individual tests influences
choice of evaluation in a given patient [2].
Laboratory Serological Assay

Serological testing is the most widely available non-invasive test for diagnosing of H.
pylori infection. Moreover serology is the only test that is not affected by local changes in the
stomach that could lead to false-negative results in other tests. Furthermore these tests are
rapid and cheap, and may be helpful in screening populations or in confirming the presence of
H. pylori infection in case of equivocal results of the other diagnostic methods due to
bleeding ulcers, antibiotic and/or antisecretory treatment [5].
People infected with H. pylori generally exhibit in samples of blood specific circulating
antibodies (IgG, IgA and IgM) and these can be detected by specific serological tests. At
present a number of commercially available tests have been developed, mostly based on IgG
detection, which is considered slightly more sensitive and specific, while serology for IgM
and IgA actually has limited clinical utility.
Infection by CagA (cytotoxin-associated gene A) positive strains (type I) is generally
more likely to be associated with more serious gastroduodenal disease, as MALT lymphoma
and gastric adenocarcinoma, compared with negative (type II) strains. The detection of a

Common Techniques: Serological, Stool Antigen, and Urea Breath Tests 7
serological response to CagA antigen could, therefore, give clinically useful information
about the infecting strain. This association, however, is not seen in all countries [6].
Several different techniques are available for serum antibody detection, that is
enzyme-linked immunosorbent assay (ELISA), latex agglutination techniques or
immunochromatography [5, 7].
Quantitative Immunoassays
Autoantibody detection is made usually by ELISA methods presented in a microplate

format, although in the last years fully automated immunoassay systems have also been
developed as enzyme-linked fluorescence assay (ELFA) for IgG.
At present several ELISA kits are available. The ELISA test is based on sandwich
enzyme immunoassay technique with purified H. pylori bacterial antigen adsorbed on micro
well plate and detection antibody labelled with an enzyme (i.e. horse radish peroxidase). In
the final step, a solution containing the enzyme's substrate is added. The subsequent reaction
produces a detectable signal, most commonly a colour change in the micro well that is read
using a spectrophotometer.
Rapid Serological Tests
Rapid tests are also available. They can be applied at the point-of-care, either based on
latex agglutination or on immunochromatographic technology.
Latex Agglutination Assay

The latex tests contain latex particles sensitized with H. pylori antigens. H. pylori if
present in the serum samples will react with the sensitized latex resulting in visual detectable
clumps. This assay is still used as a rapid test even if its interpretation (positive/negative
result) is highly subjective. Latex agglutination tests are most suitable as near patient tests
because they are technically simple to perform and provide a result within minutes rather than
the hour or two for ELISA tests.
Immunochromatography
The assay employs a combination of anti-human immunoglobulin dye conjugate and
highly purified H. pylori proteins.
As the sample flows through the adsorbent device, the anti-human immunoglobulin dyed
conjugate bind to the human IgG antibodies present in positive sample forming an antigen
antibody complex. This complex binds to H. pylori proteins fixed in the adsorbent device and
produces a coloured band.
At present, this test is little used as laboratory serological assay for H. pylori.

Performance Characteristics of Serological Assays
A meta-analysis evaluated the performance characteristics of several commercially

available quantitative serological assays and found their overall sensitivity and specificity to
be 85% and 79%, respectively, with no significant differences between the different assays
[4]. Three of the qualitative whole blood antibody kits were compared in another study
demonstrating sensitivities ranging from 76 to 84% and specificities from 79 to 90% [5]. In
general, performance characteristics for the qualitative near patient tests have been more
variable than those detected for the quantitative tests, which are more, standardized.
Serology H. Pylori Limitations
Several factors limit the usefulness of antibody testing in clinical practice.

First, serological testing cannot be used to monitor the progress of antimicrobial therapy,
since patients whose infection has been eradicated may continue to carry serum antibodies
specific to H. pylori for several months after successful treatment.
Really, qualitative test remain positive for up 3 years after successful treatment and
quantitative antibodies levels do not decline significantly for 6 to 12 months after treatment.
For these reasons, antibody tests are of little benefit in documenting eradication [7].
Further, false positive serology tests are more common in low prevalence population,
since the positive-predictive-value of antibody testing is greatly influenced by the prevalence
of H. pylori infection [6]. Generally, the prevalence of raised IgG in the population tends to
be higher in developing countries than in developed ones [8].
The American College of Gastroenterology does not recommend use of serology in low
prevalence populations. If you do choose to utilize a serological test for H. pylori in low
prevalence populations, positive results should be confirmed with a non invasive test that
identifies active H. pylori infection, such as the urea breath test or the fecal antigen test [2].
Finally, antibody tests developed using antigens from one region of the world may not
perform well when applied to patients in another part of the world suggesting that local
validation may be necessary [9, 10].
H. pylori Stool Antigen Tests (HpSA)

The evidence that H. pylori is present in stool was the prerequisite for the development of
non-invasive diagnostic immunoassay tests using mono or polyclonal antibodies, based on the
direct identification of the bacterium antigen in the stool [15, 16]. Unlike other tests normally
used for the diagnosis of H. pylori infection, H. pylori stool antigen test (HpSA) detects the
antigen of the bacterium and not the antibodies. HpSA is able to diagnose an ongoing
infection, while the serological tests are limited to diagnose a contact with the bacterium,
which can be current or lifetime.
It is in fact well known that the antibodies remain for a long time in serum, even after
overcoming an infection from the clinical side.

The stool antigen test has many positive aspects: non-invasive, quick, good sensitivity,
specificity and reliability (good replication standards).
These tests can be used both for diagnosis of the infection and for monitoring therapy
effectiveness, already four weeks after the end of treatment.
Low cost, easy use and sample collection at home have increasingly widespread the use
of this method.
HpSA ELISA Test
The method uses polyclonal or, more recently, monoclonal antibodies for anti-H. pylori
adsorbed in micro-wells (HpSA ELISA) (Figure 1.1) [17-19].
Method
A small amount of stool sample is diluted with a diluent ready for use. The diluted fecal
sample is centrifuged for 5 minutes at 5000 rpm. The supernatant and the monoclonal
antibody conjugate with peroxidase is then added to the wells and incubated for one hour at
room temperature on horizontal shaker, and then the wells are washed to remove unbound
material.
The substrate is then added and the wells are incubated for 10 minutes at room
temperature. In the presence of H. pylori antigen a color react is developed. After addition of
the stop solution, the results are read in a spectrophotometer (450 nm or 450/630 nm) or
visually.
The fecal sample can be stored at 2-8° C for up to three days or at -20 ° C until the time
of the test. Samples may be frozen and thawed twice.
Figure 1.1. HpSA ELISA test.

Features of the Test

On the basis of numerous studies and International Consensus Report, Urea Breath Test
(UBT) and the stool antigen research test are considered the first line diagnostic methods with
sensitivity and specificity above 90%.
In the infection diagnosis, HpSA presents values of sensitivity and specificity only
modestly lower than UBT, 93.3% and 93.2% respectively (review of 89 studies) [15, 20-22].
Limitations of the Test

However, it should be underlined the importance of a proper collection and storage of the
sample, considering that the test sensitivity drops to 69% if the sample is kept at room
temperature for 48-72 hours. The method should not be applied on watery stools or diarrhea.
The method loses in sensitivity in the early stage post-eradication therapy for the
presence of false-positive responses, with the sensitivity varying from 88 to 92% and the
specificity from 87 to 88%.
It has been reported that the presence of a relatively higher number of false positives,
after eradication therapy, can limit the use in this field for a poor positive predictive value
(69% vs. 95% than UBT).
It is hypothesized that the presence of false positives immediately after the eradication
therapy could be linked to the physiological elimination of gastric cells containing the H.
pylori without real infectious capacity, but recognized positive at the antigenic research
[23, 24].
Rapid HpSA Test
Recently, a rapid, mono-phase test for the detection of H. pylori bacteria in the stool has
been placed on the market. It is a rapid, high quality and easy to perform method, able to
determine the presence of antigens of H. pylori in human feces, in conditions of hygienic
safety [25].
Method
H. pylori card is a totally non-invasive sandwich-type immunochromatographic assay
method, only used for a qualitative detection of the antigen of H. pylori, simple to perform,
quick and very precise in the result.
The reactive support ("card") is ready to use, and is able to reveal, in a specific and
sensitive way, the presence of H. pylori antigens in the stool sample, utilizing an
immunochromatographic technology on nitrocellulose membrane (immunoassay with lateral
flow of the sample). On the membrane, in correspondence of the region containing the test
line (T), the second anti-antibody immunocomplex is allocated.
Also this test is based on the use of monoclonal antibodies whose accuracy has been
certified by the third scientific journal in the field of gastroenterology, the American Journal
of Gastroenterology, both in the diagnosis of the infection and in verifying whether or not the
treatment has been effective [23].
During execution of the test, the sample, appropriately diluted and inoculated into the
sample well, comes into contact with monoclonal antibodies anti-H. pylori conjugated with

colloidal gold (first antibody): chemically, it is a lateral migration of chromatographic

support, for capillary diffusion, in which the diluted sample, if containing human hemoglobin,
binds to the first mouse antibody anti-H. pylori (conjugate). This immune complex continues
to spread laterally to bind to the area coated with the second antibody (goat) against anti-
mouse antibodies to form a colored line in wine red, localized to the region marked T (test
line). The presence of this colored in red reaction indicates a positive result (the presence of
antigens of H. pylori), while the absence indicates a negative result. The simultaneous
development of further red colored band in the region labeled C (control line), is used as a
procedural control: its presence indicates that the correct amount of sample was inoculated
and that the migration took place appropriately (see Figure 1.2).
The card for the antigen of H. pylori mono-phase test in the stool should not be used as
the sole criterion for the diagnosis of H. pylori.
Features of the Test

To evaluate the performance of the test, the results were compared with the diagnosis of
H. pylori infection by the reference tests, UBT and by means of a true gold standard of
reference, represented by 500 patients who underwent gastroscopy with multiple biopsies.
The Quick test on stool is not only accurate, but it works the same way in all European
countries, when compared to the true gold standard (i.e., 500 patients underwent gastroscopy
with multiple biopsies), and also it is not only accurate for the diagnosis of H. pylori, but also
to determine whether the therapy was effective or not. Also in this case, all the European
countries have been involved. Four weeks after discontinuation of treatment, all patients
underwent gastroscopy again to have a true gold standard of reference [26, 27].
In addition, the fecal test was found accurate, unlike the UBT, for the evaluation of the
possible ineffectiveness of eradication therapy. Studies published in the prestigious journal of
Internal Medicine, "Annals of Internal Medicine" show that, after 7 days of treatment, the
majority of patients continue to experience symptoms and in theory have to wait the standard
4 weeks before performing the fecal test or the UBT (continuing to suffer for the symptoms).
In contrast, if the rapid fecal test made after 7 days of therapy were positive, it would not be
clinically appropriate to wait for the standard 4 weeks, and therefore the patient may either be
submitted to another eradication therapy or be subjected to gastroscopy for antibiogram [28,
29].
Figure 1.2. HpSA card.

Table 1.1. Diagnostic accuracy of Immuno-Card STAT!
Overall population Patients with strongly

(n = 303) coloured test line only
(n = 294)
Sensitivity (95% CI) 91.3% (85.6–94.8) 90.9% (85.1–96.4)
Specificity (95% CI) 93.5% (88.5–96.4) 95.4% (90.7–97.7)
LR + (95% CI) 14 (7–25.6) 19.6 (9.49–40.5)
LR - (95% CI) 0.093 (0.05–0.15) 0.095 (0.6–0.16)
HpSA before treatment: (CI, confidence interval; LR +, likelihood ratio for a positive test; LR -,
likelihood ratio for a negative test).
Table 1.2. Diagnostic accuracy of Immuno-Card STAT!
Overall population Patients with strongly

(n = 121) coloured test line only
(n = 118)
Sensitivity (95% CI) 92% (78–97) 91% (76–97)
Specificity (95% CI) 100% (96–100) 100% (96–100)
LR + (95% CI) Infinite (21.2–∞) Infinite (21.01–∞)
LR - (95% CI) 0.08 (infinite–∞) 0.09 (infinite–∞)
HpSA after treatment: (CI, confidence interval; LR +, likelihood ratio for a positive test; LR -,
likelihood ratio for a negative test) [25].
Subsequent studies have shown the effectiveness of the HpSA Immuno-Card test both
before and after eradication therapy (Tables 1.1, and 1.2) [25].
Limitations of the Test

As with all diagnostic tests, all results should be interpreted in the light of the overall
clinical picture of the patient. If the test result is negative and clinical symptoms persist, it is
recommended to perform additional clinical evaluation. A negative result does not exclude
the possibility of H. pylori infection.
As for the UBT, the positivity for HpSA can be affected by ongoing bleeding of the
gastrointestinal tract, by the presence of atrophic gastritis or use of proton-pump inhibitor
(PPI) drugs, antibiotics, and preparations of bismuth that inhibit the growth of H. pylori, for
which the sample collection must be performed not earlier than two weeks after the intake of
inhibitors and/or preparations of bismuth and, in the case of taking antibiotics, four weeks
after the end of treatment.
The test can be used, 30 days after the completion of eradication therapy, to evaluate the
outcome.
It should be noted that the search of the fecal antigens does not allow any screening in
depth about the heterogeneity of the strains, and their related different pathogenicity,
considering that target antigens of the tests in use are common to all H. pylori subtypes.

Comparison between HpSA ELISA and Rapid HpSA
Therefore, compared with other non-invasive methods, Rapid HpSA has been proven
simple to perform, being used, in particular, for those who have difficulty to undergo the
breath test such as children and elderly, patients with asthma, gastrectomy, acloridia [28].
Table 1.3 shows and compares the performance characteristics of Rapid and ELISA HpSA
tests.
Scientific and technological progress provides rapid tests with increasing levels of
"quality" and "effectiveness", combined with ease of use and interpretation of the results.
Rapid HpSA is therefore an indispensable diagnostic tool, in the presence of dyspeptic
symptoms as "stomach acidity", “stomach pain”, heartburn gastro-esophageal reflux,
postprandial bloating, digestion slow and laborious, post-prandial drowsiness, nausea and so
on, symptoms often associated with H. pylori infection. These tests seem to be a valuable aid,
immediate and precise, in guiding the diagnostic process managed by the physician.
Table 1.3. comparison of the performance characteristics

between Rapid HpSA and ELISA HpSA
PERFORMANCE
CHARACTERISTICS
Rapid HpSA ELISA HpSA
Sensitivity 94.6% 94.6%
Specificity 98.4% 96.1%
Positive predictive value 94.6% 87.5%
Negative predictive value 98.4% 98.4%
Urea Breath Test

Principle
Urea Breath Test (UBT) is a widely available test with higher sensitivity and specificity
(from 90 to 100%) for diagnosing H. pylori infection. Moreover its non-invasiveness, the
simplicity of execution and safety, make it elective in the suspicion of infection in adulthood,
childhood, and in pregnancy [30-32].
However, the test specificity of UBT decreases in young children (< 6 year old) because
it requires active cooperation of the patient [33, 34].
H. pylori is the only one bacterium capable to resist to the gastric acidity, to such an
extent as to hide within the gastric mucosa and replicate therein. This characteristic is given
by the distinct ability to produce urease, an enzyme that breaks down the urea in the stomach
releasing carbonic acid and ammonia. Then, the urease neutralizes gastric acid, creating a
micro-environment conducive to replication of the bacterium.
UBT uses such high urease activity to diagnose the infection. It is in fact based on the
administration of urea labeled with a carbon isotope (13C or 14C) which, once ingested, is
hydrolyzed by the urease produced by the bacterium into ammonia and carbon dioxide, that is
subsequently absorbed across the lining of the stomach and into the blood. It then travels in
the blood to the lungs where it is excreted in the breath. Samples of exhaled breath are
collected, and the isotopic carbon in the exhaled carbon dioxide is measured.
Urease activity is missing in stomach of healthy subjects, therefore urea administered is
absorbed and eliminated by urine. Instead, in H. pylori-infected subjects an amount of
labelled carbon dioxide will be exhaled by the patients few minutes after the ingestion. This
indicates the presence of H. pylori in the stomach.
Exam Preparation and Execution
UBT is carried out after a fasting period of at least six hours. Firstly, a baseline sampling
of exhaled air is collected, after administration to the patient of a dose of sodium citrate. Then
the patient takes a deep inspiration and subsequent full expiration blowing the air through a
drinking straw in a vial which is immediately closed. A second breath sample is collected.
Then, the patient drinks a solution of 13C-labeled urea and after thirty minutes avoiding to
drink, eat, and smoke he blows again through a drinking straw in a first vial (labelled as time
30 minutes). The same procedure is repeated a second time. The exhaled breath is analysed
by a mass spectrometer which compares the amount of 13 C-carbon dioxide than to total
amount of carbon dioxide exhaled. The amount of exhaled C13-labelled C-carbon dioxide
will be superior in H. pylori-positive than H. pylori-negative patients. Usually the 13C
isotope is preferred as urease-marker because of its stability, non-radioactivity and safety,
than the 14C isotope which presents slight radioactivity and then the repetition of tests is not
recommended. The safety of stable isotopes allows to perform this test both in children and in
pregnant women. This test can give false negative results when performed shortly after drug
therapies such as PPI which inhibit metabolic and ureasic activities of bacterium. In this case
the negative result of the UBT could mean only a temporary inhibition of the H. pylori’s
clearance and not the complete and definitive eradication.
Therefore the execution of UBT should be performed at least 4 weeks after the use of
antibiotics (the use of single antibiotic seems not to interfere on the exam) and 2 weeks after
the suspension of drugs such as PPIs and H2-receptor antagonists and sucralfate.
If the contact time of urea with the gastric mucosa is short, then the hydrolisis doesn’t
occur and false negative results will be obtained. For this reason several meals have been
proposed to administer together with the the labeled urea trying to delay the gastric emptying.
Therefore, the oral administration of citric acid 10 minutes before the urea administration
seems to be the best procedure, until now. Most studies evaluating the need for citric acid in
UBT showed higher delta values with citric acid when compared with other tests [35-38].
UBT is an accurate test for diagnosing H. pylori infection in patients with an intact stomach,
but the sensitivity and specificity of the UBT in subjects underwent to partial gastrectomy are
variable because of the lower bacterial load [39].
Conclusion
Several non-invasive H. pylori tests are established in clinical routine, but at present,
there is no single method, among the non-invasive tests, that can be considered as the gold

standard for the diagnosis of H. pylori infection. Clinical circumstances, availability and costs
should be taken in consideration in choosing the test more suitable to perform. Serological
testing is the most available test used in diagnosing H. pylori infection. In patients treated
with PPIs, if it not possible to stop them for at least 2 weeks a validated IgG serology test
may be used. Antibodies against H. pylori and especially against its most specific antigen
CagA, remain elevated despite transient decreases of the bacterial load and even for long
periods (months, even years) after the disappearance of H. pylori from the stomach. H. pylori
serology combined with serum pepsinogen I/II ratio may constitute a non-invasive method to
detect premalignant conditions, although it has a limited sensitivity [40]. During recent years
new formats of the HpSA using monoclonal antibodies instead of polyclonal antibodies,
which lead to a constant quality of the reagents have been developed. A systematic review by
Leal et al. established that stool antigen test using monoclonal antibodies is an efficient non-
invasive test for the diagnosis of H. pylori infection in children, too [41].
UBT is similar to gastroscopy and biopsies for diagnosing the H. pylori infection, but it is
not able to show the associated complications such as gastroduodenal ulcers / erosions, gastric
intestinal metaplasia nor neoplastic lesions. The 13C-UBT has a high accuracy, is easy to
perform, and remains the best test to diagnose H. pylori infection although it has shown a
variable level of accuracy in the pediatric population mainly in young children (< 6 years of
age) as confirmed in a meta-analysis by Leal and colleagues [42].
In special cases such as gastric ulcer or gastric MALT lymphoma, follow-up is necessary
with upper digestive endoscopy and then biopsy-based tests should be performed for
confirmation of H. pylori eradication. In other situations, a non-invasive test is used.
As the H. pylori antibodies remain present for months after suppression and even
eradication of H. pylori infection, serology is not recommended in follow-up. However,
stopping PPIs 2 weeks before testing allows the bacteria to repopulate the stomach and the
tests previously negative (UBT, HpSA, rapid urease test, histology, culture) can once again
become positive. Furthermore, no study has evaluated the washout period necessary after
long-term PPI treatment. For UBT a study claimed that the use of a more acidic test meal
would overcome the problem of false-negative tests. Anti-H2 drugs may also lead to some
false-negative results but to a much lesser extent [43, 44] and the panel did not find it
necessary to stop them before testing if using citric acid. The monoclonal HpSA tests are
suitable and widely available methods for the primary as well as for post-treatment diagnosis
of H. pylori infection [40], but there is now overwhelming evidence that UBT is an excellent
test for follow-up after H. pylori eradication. The proposed period of 4 weeks has therefore
been questioned and proposals have been made to extend it to 6 or 8 weeks. In fact the
guidelines of the European group of H. pylori recommend the UBT as the ideal method to
confirm the eradication of infection and to ascertain the infection state in patients with
recurrent symptoms after eradication treatment [45].
References
[1] Parente JM, da Silva BB, Palha-Dias MP, Zaterka S, Nishimura NF, Zeitune JM,
(2006). Helicobacter pylori infection in children of low and high socioeconomic status
in northeastern Brazil. Am. J. Trop. Med. Hyg., 75(3): 509–12.

[2] Carroll IM, Ahmed N, Beesley SM, Khan AA, Ghousunnissa S, Morain CA, et al.
(2004). Microevolution between paired antral and paired antrum and corpus
Helicobacter pylori isolates recovered from individual patients. J. Med. Microbiol., 53:
669–77.
[3] Marques SB. Sao Paoulo: University of Sao Paoulo; 2009. Prevalence of H. pylori
infection associated with clinical disorders diagnosed by upper gastrointestinal
endoscopies, retrospective analysis of 1478 cases.
[4] Kusters JG, van Vliet AH, Kuipers EJ, (2006). Pathogenesis of Helicobacter pylori
infection. Clin. Microbiol. Rev. 19(3): 449–90.
[5] Kazu H, Tatsuhiro M, Sachiko N et al. (2003). Current consensus on the diagnosis and
treatment of H. pylori-associated gastroduodenal disease. Keio. J. Med., 52(3): 163-73.
[6] Vaira D, Holton J, Menegatti M et al. (1999). New immunological assays for the
diagnosis of Helicobacter pylori infection. Gut., 45(1): 123-7.
[7] Chey WD and Wong BCY, (2007). American College of Gastroenterology Guideline
on the Management of Helicobacter pylori Infection. Am. J. Gastroenterol., 1808-1825.
[8] Loy CT, Irwig LM, Katelaris PH et al. (1996). Do commercial serological kits for
Helicobacter pylori infection differ in accuracy? A meta-analysis. Am. J.
Gastroenterol., 91(6): 1138-44.
[9] Chey WD, Murthy U, Shaw S et al. (1999). A comparison of three finger stick, whole
blood antibody tests for Helicobacter pylori Infection: a Unite States multicenter trial.
Gastroenterol., 94: 1512-6.
[10] Ho B and Marshall BJ, (2000). Accurate diagnosis of Helicobacter pylori. Serologic
testing. Gastroenterol. Clin. N. Am., 29: 853-62.
[11] Nurgalieva ZZ and Graham DY, (2003). Pearls and pitfalls of assessing Helicobacter
pylori status. Dig. Liver Dis., 35: 375-7.
[12] Marshall BJ, Howat AJ, Wright PA, (1999). Oral fluid antibody detection in the
diagnosis of Helicobacter pylori infection. J. Med. Microbiol., 48: 1043-6.
[13] Makristathis A,Hirschl AM, Lehourst P et al. (2004). Diagnosis of Helicobacter pylori
infection. Helicobacter., 9: 7-14.
[14] Hoang TT, Wheeldon TU, Bengtsson C et al. (2004). Enzyme-linked immunosorbent
assay for Helicobacter pylori needs adjustment for the population investigated. J. Clin.
Microbiol., 42: 627–30.
[15] Gisbert JP, Pajares JM, (2004). Stool antigen test for the diagnosis of Helicobacter
pylori infection: a systematic review. Helicobacter., 9: 347-68.
[16] Gatta L, Ricci C, Tampieri A, Vaira D, (2003). Non-invasive techniques for the
diagnosis of Helicobacter pylori infection. Clin. Microbiol. Infect., 9: 489-96.
[17] Asfeldt AM, Lochen ML, Sstraume B, Steigen SE, Florholmen J, Goll R, Nestergard O,
Paulssen EJ, (2004). Accuracy of a monoclonal antibody-based stool antigen test in the
diagnosis of Helicobacter pylori infection. Scand. J. Gastroenterol., 39: 1073-7.
[18] Veijola L, Oksanen A, Lofgren T, Sipponen P, Karvonen AL, Rautelin H, (2005).
Comparison of three stool antigen tests in confirming Helicobacter pylori eradication in
adults. Scand. J. Gastroenterol., 40: 395-401.
[19] Manes G, Zanetti MV, Piccirillo MM, Lombardi G, Balzano A, Pieramico O, (2005).
Accuracy of a new monoclonal stool antigen test in post-eradication assessment of
Helicobacter pylori infection: comparison with the polyclonal stool antigen test and
urea breath test. Dig. Liver Dis., 37(10): 751-5.

[20] Kazemi S, Tavakkoli H, Habizadeh MR, Emami MH, (2011). Diagnostic values of
Helicobacter pylori diagnostic tests: stool antigen test, urea breath test, rapid urease
test, serology and histology. J. Res. Med. Sci., 16(9): 1097–1104.
[21] Choi J, Kim CH, Kim D, Chung SJ, Song JH, Kang JM, et al. (2011). Prospective
evaluation of a new stool antigen test for the detection of Helicobacter pylori, in
comparison with histology, rapid urease test, (13)C-urea breath test, and serology. J.
Gastroenterol. Hepatol., 26(6): 1053–9.
[22] Peng NJ, Lai KH, Lo GH, Hsu PI, (2009). Comparison of noninvasive diagnostic tests
for Helicobacter pylori infection. Med. Princ. Pract., 18(1): 57–61.
[23] Gisbert JP, de la Morena F, Abraira V, (2006). Accuracy of Monoclonal Stool Antigen
Test for the Diagnosis of H. pylori Infection: A Systematic Review and Meta-Analysis.
Am. J. Gastroenterol., 1922-1930.
[24] Dominguez J, Fornè M, Blanco S, Prat C, Galì N, Latorre I, Viver JM, Ausina V,
(2006). Comparison of a monoclonal with a polyclonal antibody-based enzyme
immunoassay stool test in diagnosing Helicobacter pylori infection before and after
eradication therapy. Aliment. Pharmacol. Ther., 15;23(12): 1735-40.
[25] Gatta L, Perna F, Ricci C, Osborn J.F, Tampieri A, Bernabucci V, Miglioli M and Vaira
D, (2004). A rapid immunochromatographic assay for Helicobacter pylori in stool
before and after treatment. Aliment Pharmacol. Ther., 20: 469-474.
[26] Vaira D, Malfertheiner P, Megraud F, Axon AT, Deltenre M, Hirschl AM, Gasbarrini
G, O’Morain C, Garcia JM, Quina M, Tytgat GN, (1999). Diagnosis of Helicobacter
pylori infection with a new non-invasive antigen-based assay. HpSA European study
group. Lancet., 3;354(9172): 30-3.
[27] Vaira D, Malfertheinner P, Megraud F, Axon AT, Deltenre M, Gasbarrini G, O’Morain
C, Pajares-Garcia JM, Quina M, Tytgat GN, (2000). Noninvasive antigen-based assay
for assessing Helicobacter pylori eradication: a European multicenter study. The
European Helicobacter pylori HpSA Study Group. Am. J. Gastroenterol., 95(4): 925-9.
[28] Yang HR, Seo JK, (2008). Helicobacter pylori Stool Antigen (HpSA) Tests in Children
Before and After Eradication Therapy: Comparison of Rapid Immunochromatographic
Assay and HpSA ELISA. Dig. Dis. Sci., 53: 2053-2058.
[29] Vaira D, Vakil N, Menegatti M, van’t Hoff B, Ricci C, Gatta L, et al. (2002). The Stool
Antigen Test for Detection of Helicobacter pylori after Eradication Therapy. Ann..
Intern. Med., 136, 280-287.
[30] Calvet X, Sanchez-Delgado J, Montserrat A, Lario S, Ramirez-Lazaro MJ, Quesada M,
et al. (2009). Accuracy of diagnostic tests for Helicobacter pylori: a reappraisal. Clin.
Infect. Dis., 48(10): 1385–91.
[31] Vaira D, Gatta L, Ricci C, Miglioli M, (2002). Review article: diagnosis of
Helicobacter pylori infection. Aliment Pharmacol. Ther, 16(Suppl 1): 16–23.
[32] Jonaitis LV, Kiudelis G, Kupcinskas L, (2007). Evaluation of a novel 14C-urea breath
test "Heliprobe" in diagnosis of Helicobacter pylori infection. Medicina (Kaunas).,
43(1): 32–5.
[33] Imrie C, Rowland M, Bourke B, et al. (2001). Limitations to carbon13-labeled urea
breath testing for Helicobacter pylori in infants. J. Pediatr., 139: 734–7.
[34] Hino B, Eliakim R, Levine A, Sprecher H, Berkowitz D, Hartman C, Eshach-Adiv O,
Shamir R, (2004). Comparison of invasive and non-invasive tests diagnosis and

monitoring of Helicobacter pylori infection in children. J. Pediatr. Gastroenterol.

Nutr., 39(5): 519-23.
[35] Dominguez-Munoz JE, Leodolter A, Sauerbruch T, Malfertheiner P, (1997). A citric
acid solution is an optimal test drink in the 13C-urea breath test for the diagnosis of
Helicobacter pylori infection. Gut., 40(4): 459–62.
[36] Kopacova M, Bures J, Vorisek V, Konstacky M, Rejchrt S, Zivny P, et al. (2005).
Comparison of different protocols for 13C-urea breath test for the diagnosis of
Helicobacter pylori infection in healthy volunteers. Scand. J. Clin. Lab. Invest., 65(6):
491–8.
[37] Gisbert JP, Vazquez MA, Jimenez I, Cruzado AI, Carpio D, Del CE, et al. (2000).
13C-urea breath test for the diagnosis of Helicobacter pylori infection before treatment:
is citric acid necessary? Dig. Liver Dis., 32(1): 20–4.
[38] Leodolter A, Dominguez-Munoz JE, Von AU, Malfertheiner P, (1999). Citric acid or
orange juice for the 13C-urea breath test: the impact of pH and gastric emptying.
Aliment. Pharmacol. Ther. 13(8): 1057–62.
[39] Tonkic A, Tonkic M, Lehours P, Megraud F, (2012). Epidemiology and diagnosis of
Helicobacter pylori infection. Helicobacter., 17 (suppl.1): 1-8.
[40] Malfertheiner P, Megraud F, O'Morain CA, Atherton J, Axon AT; Bazzoli F et al.
(2012). Management of Helicobacter pylori infection-the Maastricht IV/Florence
Consensus Report. Gut., 61: 646-64.
[41] Leal YA, Cedillo-Rivera R, Simon JA, Velazquez JR, Flores LL; Torres J, (2011).
Utility of stool sample-based tests for the diagnosis of Helicobacter pylori infection in
children. J. Pediatr. Gastroenterol. Nutr., 52: 718-28.
[42] Leal YA, Flores LL, Fuentes-Pananà EM, Cedillo-Rivera R, Torres J, (2011). 13C-Urea
Breath Test for the Diagnosis of Helicobacter pylori Infection in Children: A
Systematic Review and Meta-Analysis. Helicobacter., 16: 327-337.
[43] Gisbert JP, Pajares JM, (2005).13C-urea breath test in the management of Helicobacter
pylori infection. Dig. Liver Dis. 37: 899-906.
[44] Graham DY, Opekun AR, Jogi M, et al. (2004). False negative urea breath tests with
H2-receptor antagonists: interactions between Helicobacter pylori density and pH.
Helicobacter., 9: 17-27.
[45] Pellicano R, Fagoonee S, Palestro G, Rizzetto M, Figura N, Ponzetto A, (2004). The
diagnosis of Helicobacter pylori infection: guidelines from the Maastricht 2-2000
Consensus Report. Minerva. Gastroenterol. Dietol., 50(2): 125-33.

Chapter 2
Unusual Techniques: Stool and Dental

Plaque PCR, and Urinary Antibodies
Tessa Horemans, Gaëlle Boulet,

Peter Delputte, Louis Maes, and Paul Cos*
Laboratory of Microbiology, Parasitology and Hygiene (LMPH),
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences,
University of Antwerp, Antwerp, Belgium
Abstract
A wide array of techniques is available for diagnosis of Helicobacter pylori
infection. Non-invasive procedures are generally preferred as first-line to decrease both
the risks for the patient and limit health care costs. Well-established assays such as
serology, the stool antigen test and the urea breath test can be complemented with
polymerase chain reaction (PCR) analysis of stool and dental plaque and/or by detection
of anti-H. pylori antibodies in urine. The use of stool PCR assays is currently gaining
importance in clinical settings because it allows simultaneous assessment of antimicrobial
susceptibility. The immuno-chromatographic analysis of urine samples can be performed
in the doctor’s office, and allows a ‘see-and-treat’ strategy.
Keywords: Helicobacter pylori, Diagnosis, Polymerase chain reaction (PCR), Antimicrobial

Susceptibility Testing, Antibody, Stool, Dental Plaque, Urine
Introduction
Since its discovery in 1983, reliable detection of Helicobacter pylori (H. pylori) is a topic
of widespread interest [1]. Widely used techniques for the detection of H. pylori consist of a
*
Corresponding author: Paul Cos. University of Antwerp, Laboratory of Microbiology, Parasitology and Hygiene
(LMPH), Universiteitsplein 1, B-2610 Wilrijk, Belgium. E-mail: paul.cos@ua.ac.be.

20 Tessa Horemans, Gaëlle Boulet, Peter Delputte et al.
gastric biopsy and the subsequent cultivation of the bacterium, the rapid urease test and
histological analysis [2].
Gastric sampling however is invasive, expensive, time-consuming and implies risks such
as gastric bleeding or perforation [3].
Hence, non-invasive techniques such as the urea breath test (UBT) and stool antigen test
are preferred when endoscopy is not absolutely required. These alternative techniques have
proven their diagnostic accuracy in several studies [2, 4, 5].
Recently, the detection of H. pylori DNA in clinical samples such as feces, saliva and
dental plaque has been reported [4]. The polymerase chain reaction (PCR) can be used as a
rapid, sensitive and specific technique for the diagnosis of infection. Besides the detection of
the bacterium, this assay may provide useful information about virulence factors and
antimicrobial susceptibility through genotyping or DNA sequencing of the PCR products [4].
Another non-invasive approach is the detection of IgG H. pylori antibodies in urine
samples. This might prove a good alternative to blood-based antibody tests and has the major
benefit that it can be easily implemented in the doctor's office [5].
Adapted from Guatelli et al. 1989.
Figure 2.1. Principle of the PCR reaction. Double-stranded target DNA melts by increasing the
temperature, primers anneal and Taq-polymerase enzyme rebuilds double- stranded chains. Exponential
amplification of the target region is obtained after 30-40 cycles.

Unusual Techniques: Stool and Dental Plaque PCR … 21
Stool and Dental Plaque PCR

PCR As a Diagnostic Tool
Principle
The polymerase chain reaction was first described by Kary Mullis in the early 1980’s [6].
In essence, the reaction is based on an exponential amplification of a target DNA sequence
which is selected by adding ‘template-specific’ primers (Figure 2.1). In each of the
amplification steps, the double-stranded DNA is first denatured by heating to yield single-
stranded chains of nucleotides.
The reaction temperature is then decreased to allow annealing of the primers and again
increased to the optimum activity temperature of the Taq-polymerase enzyme, which starts to
build a new DNA strand complementary to the DNA template. This elongation step leads to
the formation of two double-stranded DNA chains identical to the original template and thus,
doubling the amount of DNA present in the sample.
This process is then repeated, resulting again in a doubling of the amount of template
DNA. After 30-40 rounds of amplification, a billion fold amplification of the template
sequence can be achieved. These products can be detected by numerous methods, from
classical agarose gel electrophoresis to quantitative real-time detection using fluorescent
probes during the amplification steps.
Assets and Points of Interest

PCR is the most sensitive technique to demonstrate the presence of microbial pathogens
in clinical samples by detecting their DNA. Since the growth of H. pylori in vitro is complex
and requires specific culture conditions, PCR is a logical and practical diagnostic alternative.
Additionally, PCR analysis requires only a couple of hours and implies a significant gain in
time when compared to the long cultivation period in vitro [7].
Unfortunately, the equipment and consumables to perform PCR reactions are quite
expensive and limit the use in small laboratories.
When implementing PCR as a diagnostic tool, quality control is a point of particular
interest. The risk of false-positive results is due to the extreme high sensitivity of the
technique.
By implementing negative controls in each run, these errors can easily be traced.
However, a specialized laboratory environment with separate facilities for each stage of the
procedure is required to prevent contamination. Rigorous staff training, decent primer design
and the use of the carry-over prevention system [8] included in most of the commercial PCR
kits all contribute to avoid false-positive results.
On the other hand, false-negative results may occur by failure of the extraction method to
remove all inhibitory molecules present in the clinical samples. Even when high amounts of
target DNA are present, no positive signal will be obtained.
To avoid false-negative results due to technical factors (e.g. variability of reagents, errors
in DNA extraction, malfunction of the thermal cycler, correct primer design and annealing
temperatures), it is recommended to use an internal control to check the validity of the PCR
reaction [9, 10].

PCR Detection of H. pylori in Feces
Sampling and Storage

Stool samples for DNA analysis should be as fresh as possible. To prevent DNA
degradation, stabilization of the target molecules is crucial [11]. Higher amounts of DNA can
be recovered when feces are stored in RNAlater® (Qiagen, Germany) or Paxgene® (Qiagen,
Germany) or when they are dried over silica gel or on Whatman FTA cards® [12]. Reducing
the storage temperature below -20°C also prevents degradation [13].
Sample Preparation
Fecal specimens are highly complex as they contain large amounts of various
microorganisms, inorganic compounds (mostly calcium and phosphates), bile salts,
polysaccharides, undigested plant fibres and insoluble products of the gastrointestinal tract
[14]. Many of these components can interfere directly with the DNA polymerases or interact
with the DNA present in the sample, preventing amplification of target DNA [10]. DNA
polymerases require cofactors, such as magnesium that can also be the target of inhibition.
Agents that reduce Mg2+ availability or interfere with the binding of Mg2+ to the DNA
polymerase can therefore inhibit PCR [15].
The sample preparation aims to exclude PCR-inhibitory substances and increase the
concentration of target DNA to produce a homogeneous sample and ensure the
reproducibility and repeatability of the test [16]. If no specific concentration step is
performed, the detection limit of any routine PCR is >10 copies of target DNA per microgram
total DNA because of the loss of material during extraction and the large amount of non-
specific DNA [17]. The most frequently used methods may be divided into four categories:
(a) biochemical, (b) immunological, (c) physical, and (d) physiological methods [14].
Biochemical methods are the most widely employed because of the availability of many
different commercial kits, such as QIAamp® DNA Stool Mini Kit (Qiagen, Germany),
AccuPrep® Stool DNA Extraction Kit (Bioneer, Korea) and FastDNA® SPIN Kit for Feces
(MP Biomedicals, US). In general, these kits bind the DNA to silica beads or glass fibers and
remove the contaminants by several washing steps. After purification, the DNA is eluted in a
Tris-buffer and ready-to-use.
Immunological methods are mainly based on the use of magnetic beads coated with
antibodies [18-20]. For example, magnetic beads pre-coated with sheep anti-rabbit IgG are
incubated with rabbit anti-H. pylori IgG. These beads are then added to the sample and bind
the bacteria to form aggregates, which are drawn to the side of the test tube by a magnet.
Changing the medium removes the inhibitory factors. The isolated bacteria are lysed and their
DNA is released into the storage buffer [18]. This method is relatively expensive, laborious
and time-consuming [21].
Physical methods can be subdivided in diffusion in agarose [22] or filtration over a
polypropylene [23] or a microcon 100 filter [24] to remove large polysaccharide molecules.
By diluting the samples, the amount of inhibitory molecules can be decreased to a level that
doesn’t influence the Taq polymerases [25]. Treatment with an aqueous two-phase system,
consisting of polyethylene glycol (PEG) and dextran, can be used to separate inhibitors from
bacterial cells [14, 26].
Alternatively, streptavidin-coated magnetic beads can also be used to capture specific H.
pylori DNA (Sequence capture PCR, figure 2.2). Cells and bacteria are lysed and biotin-
labelled H. pylori DNA probes are added to the mixture to bind the target DNA. When the
streptavidin-coated beads are added, the H. pylori DNA binds to the beads and potential PCR
inhibitors can be removed by washing the beads. DNA can be isolated using a magnet and
detached from the beads by a simple heating step [27].
Physiological methods are most appropriate for samples that contain low amounts of PCR
inhibitors and are based on e.g. bacterial growth to obtain detectable concentrations of viable
bacteria prior to PCR. Since fecal samples contain high amounts of inhibitory molecules and
H. pylori is difficult to culture from this matrix [28, 29], physiological methods are not
recommended for DNA extraction from stool samples.
DNA Amplification
The optimization of the reaction conditions is of pivotal importance for the detection of
H. pylori DNA in stools. Suboptimal reaction conditions may arise for a number of reasons:
inappropriate primers, improper time or temperature conditions, variable polymerase quality
and incorrect Mg2+ concentration.
These factors should all be optimized using positive controls before starting the analysis
of stool samples [16]. Optimization of PCR reactions falls outside the scope of this chapter,
but a review by Sachse can be consulted for more information about the different PCR
techniques and the possible optimization strategies [30].
Different types of PCR can be used to amplify the extracted DNA from stool samples:
conventional, quantitative real-time, nested and restriction fragment length polymorphism
(RFLP)-PCR [31-33].
Various primer sets targeting highly conserved areas on the 16s or 23s rRNA gene,
general housekeeping genes, virulence factors or mutations related to antimicrobial resistance
have been described in tables A, B and C (see addendum). All of the primers mentioned have
been checked for accuracy by Primer-BLAST and are often described in H. pylori literature.
Figure 2.2. Scheme of the selective hybridization technique. DNA is liberated from biological samples by
treatment with proteinase K and lysis buffer. Biotin-labeled probes are added to hybridize with H. pylori
DNA in the sample. After adding streptavidin-coated magnetic beads, H. pylori DNA is selectively isolated
by placing samples in a magnetic block. Interfering DNA and PCR inhibitors are removed by several
washing steps, allowing the immediate amplification of purified DNA [27].

Applications
An important advantage of using molecular techniques is that additional information

regarding the genotype of the bacterium can be gathered; hence, PCR techniques can be used
to study routes of transmission [34] and risk factors [35]. Momtaz et al. found a high degree
of similarity in the genotype of H. pylori isolates from saliva, stomach and stool, supporting
the idea that fecal-oral is the main route of transmission [34]. Applications of greater interest
with implications for treatment are the detection of virulence markers [36] and antimicrobial
susceptibility [37] which will be discussed separately.
Detection of Virulence Markers

The detection of H. pylori virulence genes, such as CagA, VacA and urease, in stool
samples facilitates epidemiological studies. However, many studies of virulence markers are
biased mainly because only symptomatic patients are subjected to endoscopy whereby the
more virulent strains are selected. Stool samples can be easily collected and allow the analysis
of large cohorts [38]. Through the characterization of bacterial strains in asymptomatic
individuals, the disease onset in the general population may be predicted [39]. The
identification of H. pylori virulence markers in stool samples may enhance the future
possibility of large-scale screening of people at risk of gastric cancer [40].
Antimicrobial Susceptibility Testing

A major advantage of molecular diagnostic H. pylori tests is the possibility of obtaining
information about the antimicrobial susceptibility of a specific isolate without laborious and
time-consuming standard susceptibility tests, such as the E-test, agar dilution and disk
diffusion.
Since H. pylori in stool is often transformed to the uncultivable coccoid form, molecular
analysis remains the only non-invasive method to check antibiotic resistance. Obviously, this
approach is only feasible when the resistance mechanism for the antibiotic is known. Point
mutations in genes leading to antibiotic resistance have been identified for several drugs used
in H. pylori eradication therapy (table 2.1) [41].
Clarithromycin resistance is due to a mutation in the 23S rRNA subunit of the 50S
ribosome. The main 23S rRNA mutations are an adenine-to-guanine/cytosine transition at
positions 2142 (A2142G/C), an adenine-to-guanine transition at position 2143 (A2143G) and
a thymine-to-cytosine transition at position 2717 (T2717C) [42].
Table 2.1. H. pylori genes involved in the development of antibiotic resistance
Antibiotic(s) Resistance gene(s)

Macrolides* rrn 23S
Quinolones* gyrA
Tetracycline* rrn 16S
Rifampins rpoB
Amoxicillin plp1
Metronidazole rdxA, frxA
*
Molecular susceptibility test available.
Adapted from Megraud and Lehours, 2007.

These single point mutations also generate specific restriction sites (MboII, BsaI and
HhaI respectively) that can be used for RFLP analysis and thus for the rapid screening of
clarithromycin resistance [43].
A biprobe real-time PCR protocol in combination with a melting point analysis can also
be used to demonstrate point mutations (figure 2.3). Point mutations cause a deviation in the
melting curve after DNA amplification and allow the identification of clarithromycin
resistance. This technique was first published by Schabereiter-Gurtner in 2004 [37] and has
been commercialized as the H. pylori ClariRes Assay (Ingenetix GmbH, Vienna, Austria). A
high sensitivity and specificity can be obtained when the appropriate pre-analytic and
laboratory practices are applied [44].
Several clinical surveys have evaluated the use of this assay in routine testing. The
performance of the ClariRes assay is presented in table 2.2.
In general, the ClariRes assay demonstrates 100% specificity in both adults and children,
but with a relatively low sensitivity. One study attributed the limited sensitivity of 63% in
children to the use of frozen stool samples or to the difference between the gastrointestinal
tracts of children and adults (e.g. composition of fecal microbiota and shorter gastrointestinal
passage time in children) [45].
This illustrates the importance of the correct storage and transportation of stool samples
and the need for in duplo analysis to increase sensitivity [44]. Follow-up studies using the
correct sampling methods showed improved sensitivity rates in children and suggested that
stool PCR was as effective as gastric biopsy in determining the H. pylori eradication therapy
in children [31, 46].
Figure 2.3. Melt curve analysis in the H. pylori ClariRes assay. Point mutations in H. pylori DNA cause
differences in melting temperatures (Tm) of the amplified DNA from H. pylori strains with varying
clarithromycin sensitivity (Schabereiter-Gurtner, personal communication).

Table 2.2. Performance of the H. pylori ClariRes assay in routine clinical setting,
compared to the E-test method as standard
Number of Sensitivity Specificity PPV NPV

Population Reference, year
samples (%) (%) (%) (%)
Schabereiter-
Adults (19-90) 45 73 100 100 92 Gurtner et al.
2004
Lottspeich et al.
Children (0,3-18) 100 63 100 100 76,1
2007
Children (2-18) 67 89,2 100 100 88,2 Vécsei et al. 2010
Skaletsky et al.
Children (1-18) 217 83,3 100 - -
2011
Sensitivity: proportion of positive samples identified as such; Specificity: proportion of negative
samples identified as such; PPV: positive predictive value, probability that a positive test means
infection with a H. pylori clarithromycin resistant strain; NPV: negative predictive value,
probability that a negative test means infection with a H. pylori clarithromycin sensitive strain.
Ciprofloxacin (Cipr) resistance is caused by point mutations in the quinolone resistance-

determining region (QRDR) of the gyrA gene, mainly involving amino acid substitutions at
amino acid 87 (aa87, Asn to Lys) and amino acid 91 (aa91, Asp to Gly/Asn/Tyr). Glocker et
al. developed two pairs of hybridization probes to detect these mutations by Fluorescence
Resonance Energy Transfer (FRET)-based Real-Time PCR [47]. In their hands, all Cipr H.
pylori strains could be distinguished from the Cips strains.
Tetracycline binds to the 30S ribosomal subunit and blocks the protein synthesis, thereby
inhibiting the growth of the bacterium. In H. pylori, resistance is induced by mutations in the
16s rRNA genes that affect the binding sites of the antibiotic [48, 49]. High level tetracycline
resistance is caused by a triple-base-pair substitution: AGA926-928  TTC in both copies of the
16s rRNA genes [48]. Ribeiro et al. developed a PCR-RFLP method using the restriction
enzyme HinfI, allowing a rapid and reproducible identification of high-level TetR strains [49].
For metronidazole, many mutations occur in the rdxA gene. Since some of these are not
related to the development of resistant strains, molecular methods to detect resistance are
unavailable [41].
Detection of H. pylori in Dental Plaque by PCR
The detection of H. pylori in the oral cavity was first reported in 1989 when Banatvala et
al. isolated the bacterium from dental plaque of a patient with gastric disease [50]. Since then,
several reports on the presence of H. pylori in the oral cavity, both in dental plaque and saliva,
have been published [34, 51-53]. The relevance of this finding remains controversial.
Whereas most authors suggest that the oral cavity plays a role as a potential reservoir and a
possible source of reinfection, a recent study by Al-Ahmad et al. could not detect H. pylori in
the oral cavity of any of their H. pylori positive patients.
Samples were taken from supragingival and subgingival plaque, saliva, periapical
exudates and tongue swabs. This observation challenges the concept that the bacterium
resides in the mouth [53].


Dental plaque can be collected from supra- and subgingival dental sites, using sterile
curettes or dental floss and dissolving the plaque in isotonic saline solution [34, 54]. In this
aqueous environment, DNA degradation can be prevented by storage at -70°C without
additional stabilization.
Sample Preparation
In contrast to the laborious sample preparation needed for DNA extraction from stool
samples, DNA from dental plaque can be amplified after a straightforward extraction
procedure such as boiling [55], phenol-chloroform extraction [56] or using commercial DNA
isolation kits [57].
DNA Amplification
Bacterial DNA isolated from dental plaque can be amplified by conventional, nested or
quantitative real-time PCR [52, 56, 57]. Due to the presence of taxonomically related species
and the risk of PCR cross-reactivity, Chaudry et al. reported that the detection of H. pylori is
more reliable when two genes of the bacterium are simultaneously amplified [57, 58]. The use
of an inhibition control plasmid may also reduce the risk of false-negative results caused by
PCR inhibition [53].
Applications
The research on the presence of H. pylori in dental plaque mainly focuses on the
association between gastro-esophagal disease and oral colonization [55]. Unfortunately, these
studies show contradictory results with a wide range of variation in detection [41, 53, 55]. A
recent meta-analysis by Navibi et al. found that co-infection of gastric H. pylori and dental
plaque is reported by half of the studies published from 2000 to 2011 [59]. Dissimilarities in
these reports can be due to incorrect sampling, use of unspecific and insensitive primers and
the targeted study populations. Therefore, initiation of eradication treatment based on H.
pylori detection in the oral cavity is not recommended [41].
Urinary Antibodies
Antibodies to H. pylori are present in body fluids other than serum, such as saliva, gastric
secretions and urine [60, 61]. Despite the less invasive nature of serologic analysis compared
to the traditional bioptic methods, blood samples still have to be taken from the patient to
confirm presence of the bacterium. Consequently, detection of antibodies by totally non-
invasive procedures is preferred for children and elderly. Two urine-based commercial tests
have been developed: a standard enzyme-linked immunosorbent assay (ELISA) (Urinelisa®,
Otsuka, Tokyo, Japan) and a rapid immunochromatography test (Rapirun®, Otsuka, Tokyo,
Japan) [62, 63].

Created from Katsuragi et al. 1998.
Figure 2.4. Indirect Elisa used in Urinelisa®. Anti-H. pylori antibodies present in urine bind to the antigen
(Hp AG) present on the test plate. After incubation with HRP-labeled anti-human IgG, the substrate TMB is
added which is oxidized to the blue TMB-diimine (TMB-dI).
An important difference between DNA- and antibody-based diagnosis is the ability of the
DNA-tests to identify an active infection. Antibodies remain present for a long time after
exposure to H. pylori and therefore indicate past infections, while the bacterial DNA
disappears when the infection is cured.
Enzyme-Linked Immunosorbent Assay
The principle of ELISA was first described in the 1970’s [64]. Nowadays it is a popular
technique used for detection of antigens or antibodies in all kinds of biological samples [65].
Urinelisa® is an indirect ELISA used for the detection of anti-H. pylori antibodies in urine
and is based on the principle described in figure 2.4. H. pylori antigens are extracted from H.
pylori strain OHPC-040 and coated on the wells of microtiter plates. Antibodies present in the
patient sample bind to these antigens. After addition of horseradish peroxidase (HRP)-
conjugated anti-human IgG, the substrate 3,3’,5,5’-tetramethylbenzidine (TMB) is added and
oxidized to its blue derivative, 3,3’,5,5’-tetramethylbenzidine diimine (TMB-dI) [66, 67].
Immunochromatography
Rapid immunochromatography tests or line assays are often used to detect antibodies,
hormones or drugs in urine [63, 68, 69]. These tests are very simple, require no skills or
laboratory equipment and can be easily performed in the doctor’s office [70]. Using a
disposable syringe, the urine is diluted in sample diluent and dropped into the sample window
(S) (figure 2.5).

Created from Graham et al. 2001.
Figure 2.5. Rapid immunochromatography test, Rapirun®. Antibodies present in a urine sample are bound by
anti-human IgG coated gold particles. These complexes diffuse to the evaluation window and can be trapped
by H. pylori antigen in the test line (T) and/or anti-human IgG antibodies in the control line (C).
The antibodies present in the urine sample diffuse through the nitrocellulose membrane
and bind to the anti-human IgG coated gold particles. The resulting complexes migrate to the
evaluation zone and can be trapped by H. pylori antigens present in the test line (T) and/or the
anti-human IgG antibodies in the control line (C). After 10-20 minutes of incubation, the
results can be interpreted. A patient is considered positive for H. pylori when both the test and
the control line appear. The urine of an uninfected patient will only result in a control line.
When no lines or only the test line appear, the test is considered inconclusive and diagnosis
should be performed using another method [63, 70].
In 2011, a paper on the evaluation of a second chromatography based diagnostic kit,
ODK-072, was published by Murakami et al. [71]. This assay combines the chromatographic
properties of the Rapirun® assay with the dipstick method. High rates of agreement between
the ODK-072 and the control kits Rapirun® and Urinelisa® were found. The authors
concluded that ODK-072 allows testing within 15 minutes and can be clinically useful in the
diagnosis of H. pylori [71].
Detection of Anti-H. pylori Antibodies

Sample collection for urinary antibody tests is very simple. Antibodies present in the
sample are stable for 60 days at 4°C and for 3 days at 37°C, allowing routine transportation of
urine samples to the laboratory [67]. Freezing of the samples is not recommended. Repeated
freezing and thawing of the specimens may lead to protein precipitation, which significantly
reduces both the sensitivity and specificity of the tests [72].
Sensitivity, Specificity and Predictive Value

Thorough evaluation of the sensitivity and specificity of the urinary antibody tests has
preceded the commercialization of both Rapirun® and Urinelisa® [63, 67]. Several research
groups have evaluated the use of both tests in clinical practice. Data published before 2004
are summarized in a paper by Megraud et al. [41], while more recent statistics are presented
in table 2.3. Considering the combined data sets, the use of Rapirun® and Urinelisa® is
justified in the general healthy population.

However, caution is required when using the tests in young children. Muhsen et al. found
a sensitivity of 34.2% using Urinelisa® when analyzing urine samples of 159 children aged 3
to 5 years old [73].
These results were in accordance with a previous report by Megraud et al. demonstrating
a direct association between the detection of H. pylori antibodies and the children’s age [74].
Indeed, previous research has demonstrated less activation of CD4+ cells, resulting in a lower
level of serum anti-H. pylori IgG in children under the age of 10 [75].
On the other hand, high levels of total IgG present in the urine of young children can
cause false-positive reactions [76]. Therefore, the urea breath test (UBT) is recommended for
diagnosis of H. pylori infection in young children [77].
In adults, the intake of too much water before urination should be avoided, because it can
lead to the production of urine with a very low concentration of IgG. Patients presenting with
proteinuria should be examined using other tests, since urinary proteins can contribute to
false-positive results [61].
Investigations by Yamamoto et al. showed that the use in patients with abnormal urinary
analysis (treatment with rifampicin for Mycobacterium tuberculosis infection or proteinuria)
doesn’t affect the sensitivity or specificity of the tests [78, 79].
Table 2.3. Detection of H. pylori antibodies in urine using Urinelisa® (U) and Rapirun®
(R) commercial kits
Number of Reference Sensitivity Specificity PPV NPV Reference,

Population Test
samples method (%) (%) (%) (%) year
Wu et al.
Adults U 170 Serum ELISA 93,46 92,06 95,24 89,23
2004
UBT, RUT,
Kuo et al.
Adults U 317 Culture, 91,7 90,8 96,4 80,6
2005
Histology
U 316 UBT, HpSA, 63,2 97,3 94,4 78,4 Megraud et
Children
R 272 Serum ELISA 30,2 98,7 94,7 64,5 al. 2005
Children Muhsen et
U 159 HpSA 34,2 96,3 90 60
(3-5) al. 2006
Adults + U 61 93,1 65,6 - - Yamamoto
Serum ELISA
TB R 61 86,2 93,7 - - et al. 2005
Adults + Yamamoto
R 59 Serum ELISA 92,3 100 - -
Proteinuria et al. 2006
Culture,
Nguyen et
Adults R 148 Histology, 79,5 90,7 84,5 -
al. 2010
Serum ELISA
Children U 101 UBT, HpSA 91,9 96,9 - - Okuda et
(2-15) R 101 UBT, HpSA 78,4 100 - - al. 2013
UBT: Urea Breath test; RUT: Rapid Urease Test; HpSA: H. pylori stool antigen test; TB: infection with
Mycobacterium tuberculosis; Sensitivity: Proportion of positive samples identified as such;
Specificity: Proportion of negative samples identified as such; PPV: Positive predictive value,
probability that a positive test means infection with H. pylori; NPV: Negative predictive value,
probability that a negative test means no infection with H. pylori.

Applications
The non-invasive nature of antibody detection in urine makes this technique an ideal
candidate for epidemiological studies [80]. Urinelisa® and Rapirun® are inexpensive, reliable
and easy-to-use as a first-line screening tool in healthy adults and adolescents. Rapirun®
enables a diagnosis within 20 minutes, allowing patients to rapidly proceed to the next step in
medical examination and treatment on the same day as testing [63].
Conclusion
PCR is an elegant tool to detect active H. pylori infections both in gastric biopsy and
stool samples. The main advantage of using this technique is the possibility of simultaneous
acquisition of information on antimicrobial susceptibility. Several genes involved in antibiotic
resistance have been identified, but only one assay for the detection of clarithromycin
resistant strains has been commercialized to date.
Additional research on assays for other first- or second-line antibiotics might further
improve the benefits of PCR for diagnosis of H. pylori. The importance of H. pylori detection
in dental plaque is still controversial. Several groups have found the bacterium in the oral
cavity and associated the presence with gastric malignancies, whereas others could not find
any H. pylori DNA in oral samples. Further investigations are required to clarify these
observations.
Although the two currently commercialized antibody detection kits for urine samples are
often used, improvements are still possible. For example, increase of sensitivity would allow
more reliable diagnosis in small children. Additionally, since both tests use Japanese H.
pylori strains for the extraction of antigens, supplementation with proteins extracted from
European H. pylori strains might improve the accuracy of the test in Europe.
Addendum
Table A. Primer sequences used to detect H. pylori using polymerase chain reaction
Housekeeping Size Reference,

Primer Sequence 5' - 3'
gene (bp) year
FW 5'-TGC GAA GTG GAG CCA ATC TT-3' 119 Yamazaki
16S rRNA*
REV 5'-GGA ACG TAT TCA CCG CAA CA-3' et al. 2005
HP23S1 5'-GGA GCT GTC TCA ACC AGA GAT TC-' 132 Lascols et
23S rRNA*
HP23S2 5'-CGC ATG ATA TTC CC[AG] TTA GCA G-3' al. 2003
FW 5'-GGA TAT GGG CGA TCA GCA-3' 164 Puz et al.
RecA*
REV 5'-CGG TTG TGG TCT CTG GAC TC-3' 2008
5′-AAG GCA TGC AAT TTG ATA GAG GCT-
HSP1 609
3′
HSP60 Singh et al.
HSP2 5′-CTT TTT TCT CTT TCA TTT CCA CTT-3'
(nested PCR) 2008
HSPN1 5′-TTG ATA GAG GCT ACC TCT CC-3′ 501
HSPN2 5′-TGT CAT AAT CGC TTG TCG TGC-3′
*
Primers suited for quantitative real-time PCR.

Table B. Primer sequences used to detect virulence factors of H. pylori using

polymerase chain reaction
Virulence Reference,
Primer Sequence 5' - 3' Size (bp)
factor year
5'-GGA ACC CTA GTC AGT AAT GGG TT-
FW 473 Hirai et al.
CagA 3'
2009
REV 5'-AAT TCT TGT TCC CTT GAA AGC CC-3'
CagA FW 5'-AGG CAT GAT AAA GTT GAT GAT-3' 92 Yamazaki
western* REV 5'-AAA GGT CCG CCG AGA TCA T-3' et al. 2005
CagA FW 5'-AAA GGA GTG GGC GGT TTC A-3' 92
Yamazaki
East-
REV 5'-CCT GCT TGA TTT GCC TCA TCA-3' et al. 2005
Asian*
VacA VA1-F 5'-ATG GAA ATA CAA CAA ACA CAC-3'
Signal 259 (s1), Atherton et
Region VA1-R 5'-CTG CTT GAA TGC GCC AAA C-3' 286 (s2) al. 1995
(s1/s2)
VacA VA3-F 5'-GGT CAA AAT GCG GTC ATG G-3' 290
Middle Atherton et
Region VA3-R 5'-CCA TTG GTA CCT GTA GAA AC-3' al. 1995
(m1)
VacA VA4-F 5'-GGA GCC CCA GGA AAC ATT G-3' 352
Middle Atherton et
Region VA4-R 5'-CAT AAC TAG CGC CTT GCA C-3' al. 1995
(m2)
UreA-F 5'-CGTGGCAAGCATGATCCAT-3' 77 Schabereit
UreA er-Gurtner
UreA-R 5'-GGGTATGCACGGTTACGAGTTT-3'
et al. 2004
HPU1 5′-GCCAATGGTAAATTAGTT-3′ 411 Clayton et
UreA
HPU2 5′-CTCCTTAATTGTTTTTAC-3′ al. 1992
GlmM FW 5'-TCT AAA AAC GCC CTT TCT TCT CA-3' 130 Puz et al.
(ureC)* REV 5'-ATT CGC TCA CAA ACT TAT CCC C-3' 2008
*
Primers suited for quantitative real-time PCR.
Table C. Primer sequences and probes used to antimicrobial resistant H. pylori strains
using polymerase chain reaction
Antimicrobial
Size Reference,
susceptibility Primer Sequence 5' - 3'
(bp) year
testing
HP1-FW 5'-CCACAGCGATGTGGTCTCA-3' 993
Clarithromycin
HP2-REV 5'-TGTGTAGCTACCCAGCGATGCTC-3' Fontana et
(nested PCR-
HP4-FW 5'-GTCGGTTAAATACCGACCTG3' 783 al. 2003
RFLP)
HP2-REV 5'-TGTGTAGCTACCCAGCGATGCTC-3'
Clarithromycin 23S-F 5'-AGA TGG GAG CTG TCT CAA CCA G-3' 121 Schabereit
(melting curve er-Gurtner
23S-R 5'-TCC TGC GCA TGA TAT TCC C-3'
analysis)* et al. 2004

Antimicrobial
Size Reference,
susceptibility Primer Sequence 5' - 3'
(bp) year
testing
5'-GCT AGT CTA AGG GCG TAG ATT
23S-F 1379
GGA-3'
Clarithromycin
5'-GTA GTA GTC AGC TAA ACG CAT Rimbara et
(nested PCR- 23S-R
TAC TGC-3' al. 2005
RFLP)
23S-F' 5'-TGT GGT CTC AGC AAA GAG T-3' 463
23S-R' 5'-AGG CCA GTT AGC TAA CAG AA-3'
23S
5'-GGT CTC AGC AAA GAG TCC CT-3' 493
1835F1
23S
Clarithromycin 5'-CCC ACC AAG CAT TGT CCT-3'
2327R1 Noguchi et
(nested PCR-
23S al. 2007
sequencing) 5'-AGG ATG CGT CAG TCG CAA GAT-3' 367
1942F2
23S
5'-CCT GTG GAT AAC ACA GGC CAG T-5'
2308R2
5'-AAT TAG GCC TTA CTT CCA AAG TCG
GyrHPfor 238
CTT ACA-3'
5'-TCT TCA CTC GCC TTA GTC ATT CTG
GyrHPrev
GC-3'
Ciprofloxacin
5'-AAA AAT CTT GCG CCA TTC TCA CTA Glocker et
(FRET, melting A probe87
GCG C-3' al. 2004
curve analysis)
M probe87 5'-ATA AAC GGC GTT ATC GCC A-3'
5'-AAA AAT CTT GCG CCA TTC TCA CTA
A probe91
GCG-3'
M probe91 5'-ACC ATA AAC GGC ATT ATC GCC A-3'
*
Primers suited for quantitative real-time PCR, A: Anchor, M: Mutation.
References
[1] Marshall, B. J., Warren, J. R., (1984). Unidentified curved bacilli in the stomach of
patients with gastritis and peptic ulceration. Lancet.,16;1(8390):1311-5.
[2] Atherton, J. C., Spiller, R. C., (1994). The urea breath test for Helicobacter pylori. Gut.,
35(6):723-5.
[3] Quine, M. A., Bell, G. D., McCloy, R. F., Charlton, J. E., Devlin, H. B., Hopkins, A.,
(1995). Prospective audit of upper gastrointestinal endoscopy in two regions of
England: safety, staffing, and sedation methods. Gut., 36(3):462-7.
[4] Rimbara, E., Sasatsu, M., Graham, D. Y., (2013). PCR detection of Helicobacter pylori
in clinical samples. Methods Mol. Biol., 943:279-87.
[5] Leodolter, A., Vaira, D., Bazzoli, F., Schutze, K., Hirschl, A., Megraud, F., et al.
(2003). European multicentre validation trial of two new non-invasive tests for the
detection of Helicobacter pylori antibodies: urine-based ELISA and rapid urine test.
Aliment Pharmacol. Ther., 1;18(9):927-31.

[6] Mullis, K. B., (1990). The unusual origin of the polymerase chain reaction. Sci. Am.,
262(4):56-5.
[7] Yamamoto, Y., (2002). PCR in diagnosis of infection: detection of bacteria in
cerebrospinal fluids. Clin. Diagn. Lab. Immunol., 9(3):508-14.
[8] Longo, M. C., Berninger, M. S., Hartley, J. L., 1990). Use of uracil DNA glycosylase to
control carry-over contamination in polymerase chain reactions. Gene., 1;93(1):125-8.
[9] Monteiro, L., Bonnemaison, D., Vekris, A., Petry, K. G., Bonnet, J., Vidal, R., et al.
(1997). Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori
model. J. Clin. Microbiol., 35(4):995-8.
[10] Lisby, G., (1999). Application of nucleic acid amplification in clinical microbiology.
Mol. Biotechnol., 12(1):75-99.
[11] Olson, J., Whitney, D. H., Durkee, K., Shuber, A. P., (2005). DNA stabilization is
critical for maximizing performance of fecal DNA-based colorectal cancer tests. Diagn.
Mol. Pathol., 4(3):183-91.
[12] Nechvatal, J. M., Ram, J. L., Basson, M. D., Namprachan, P., Niec, S. R., Badsha, K.
Z., et al. (2008). Fecal collection, ambient preservation, and DNA extraction for PCR
amplification of bacterial and human markers from human feces. J. Microbiol.
Methods., 72(2):124-32.
[13] Sambrook, J., Fritsch, E. F., Maniatis, T., (1989). Molecular Cloning. A Laboratory
Manual. 2nd ed. Cold Spring Harbor Laboratory Press.
[14] Kabir, S., (2004). Detection of Helicobacter pylori DNA in feces and saliva by
polymerase chain reaction: a review. Helicobacter., 9(2):115-23.
[15] Wilson, I. G., (1997). Inhibition and facilitation of nucleic acid amplification. Appl.
Environ. Microbiol., 63(10):3741-51.
[16] Radstrom, P., Knutsson, R., Wolffs, P., Lovenklev, M., Lofstrom, C., (2004). Pre-PCR
processing: strategies to generate PCR-compatible samples. Mol. Biotechnol., 26(2):
133-46.
[17] Meltzer, S. J., (1998). Methods in Molecular Biology. PCR in bioanalysis. New Jersey,
US: Humana Press
[18] Enroth, H., Engstrand, L., (1995). Immunomagnetic separation and PCR for detection
of Helicobacter pylori in water and stool specimens. J. Clin. Microbiol., 33(8):2162-5.
[19] Monteiro, L., Gras, N., Megraud, F., (2001). Magnetic immuno-PCR assay with
inhibitor removal for direct detection of Helicobacter pylori in human feces. J. Clin.
Microbiol., 39(10):3778-80.
[20] Watanabe, T., Tomita, S., Kudo, M., Kurokawa, M., Orino, A., Todo, A., et al. (1998).
Detection of Helicobacter pylori gene by means of immunomagnetic separation-based
polymerase chain reaction in feces. Scand. J. Gastroenterol., 33(11):1140-3.
[21] Radstrom, P., Knutsson, R., Wolffs, P., Lovenklev, M., Lofstrom, C., 2004). Pre-PCR
processing: strategies to generate PCR-compatible samples. Mol. Biotechnol., 26(2):
133-46.
[22] Moreira, D., (1998). Efficient removal of PCR inhibitors using agarose-embedded DNA
preparations. Nucleic Acids Res., 1;26(13):3309-10.
[23] Cavallini, A., Notarnicola, M., Berloco, P., Lippolis, A., De, L. A., (2000). Use of
macroporous polypropylene filter to allow identification of bacteria by PCR in human
fecal samples. J. Microbiol. Methods., 39(3):265-70.

[24] Makristathis, A., Pasching, E., Schutze, K., Wimmer, M., Rotter, M. L., Hirschl, A. M.,
(1998). Detection of Helicobacter pylori in stool specimens by PCR and antigen
enzyme immunoassay. J. Clin. Microbiol., 36(9):2772-4.
[25] Mapstone, N. P., (1997). The Detection of H. pylori by the Polymerase Chain Reaction.
Methods Mol. Med., 8:31-6.
[26] Lantz, P. G., Matsson, M., Wadström, T., Rådström, P., (1997). Removal of PCR
inhibitors from human faecal samples through the use of an aqueous two-phase system
for sample preparation prior to PCR. J. Microbiol. Methods., 1;28(3):159-67.
[27] Horemans, T., Deschacht, M., Clais, S., Van, C. J., de, R. P., Holvoet, J., et al. (2011).
An alternative, sensitive method to detect Helicobacter pylori DNA in feces.
Helicobacter., 16(2):113-8.
[28] Falsafi, T., Favaedi, R., Mahjoub, F., Najafi, M., (2009). Application of stool-PCR test
for diagnosis of Helicobacter pylori infection in children. World J. Gastroenterol., 28;
15(4):484-8.
[29] Cellini, L., Allocati, N., Angelucci, D., Iezzi, T., Di, C. E., Marzio, L., et al. (1994).
Coccoid Helicobacter pylori not culturable in vitro reverts in mice. Microbiol.
Immunol., 38(11):843-50.
[30] Sachse, K., (2004). Specificity and performance of PCR detection assays for microbial
pathogens. Mol. Biotechnol., 26(1):61-80.
[31] Vecsei, A., Innerhofer, A., Binder, C., Gizci, H., Hammer, K., Bruckdorfer, A., et al.
(2010). Stool polymerase chain reaction for Helicobacter pylori detection and
clarithromycin susceptibility testing in children. Clin. Gastroenterol. Hepatol., 8(3):
309-12.
[32] Rimbara, E., Noguchi, N., Yamaguchi, T., Narui, K., Kawai, T., Sasatsu, M., (2005).
Development of a highly sensitive method for detection of clarithromycin-resistant
Helicobacter pylori from human feces. Curr. Microbiol., 51(1):1-5.
[33] Atherton, J. C., Cao, P., Peek, R. M., Jr., Tummuru, M. K., Blaser, M. J., Cover, T. L.,
(1995). Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association
of specific vacA types with cytotoxin production and peptic ulceration. J. Biol. Chem.,
28;270(30):17771-7.
[34] Momtaz, H., Souod, N., Dabiri, H., Sarshar, M., (2012). Study of Helicobacter pylori
genotype status in saliva, dental plaques, stool and gastric biopsy samples. World J.
Gastroenterol., 7;18(17):2105-11.
[35] MacKay, W. G., Williams, C. L., McMillan, M., Ndip, R. N., Shepherd, A. J., Weaver,
L. T., (2003). Evaluation of protocol using gene capture and PCR for detection of
Helicobacter pylori DNA in feces. J. Clin. Microbiol., 41(10):4589-93.
[36] Sicinschi, L. A., Correa, P., Bravo, L. E., Peek, R. M., Jr., Wilson, K. T., Loh, J. T., et
al. (2012). Non-invasive genotyping of Helicobacter pylori cagA, vacA, and hopQ from
asymptomatic children. Helicobacter., 17(2):96-106.
[37] Schabereiter-Gurtner, C., Hirschl, A. M., Dragosics, B., Hufnagl, P., Puz, S., Kovach,
Z., et al. (2004). Novel real-time PCR assay for detection of Helicobacter pylori
infection and simultaneous clarithromycin susceptibility testing of stool and biopsy
specimens. J. Clin. Microbiol., 42(10):4512-8.
[38] Hirai, I., Sasaki, T., Kimoto, A., Fujimoto, S., Moriyama, T., Yamamoto, Y., (2009).
Assessment of East Asian-type cagA-positive Helicobacter pylori using stool

specimens from asymptomatic healthy Japanese individuals. J. Med. Microbiol., 58 (Pt

9):1149-53.
[39] Sicinschi, L. A., Correa, P., Bravo, L. E., Peek, R. M., Jr., Wilson, K. T., Loh, J. T., et
al. (2012). Non-invasive genotyping of Helicobacter pylori cagA, vacA, and hopQ from
asymptomatic children. Helicobacter., 17(2):96-106.
[40] Megraud, F. The most important diagnostic modalities for Helicobacter pylori, now and
in the future. Eur. J. Gastroenterol. Hepatol. 2012 Apr.;9 Suppl. 1:S13-S15.
[41] Megraud, F., Lehours, P., (2007). Helicobacter pylori detection and antimicrobial
susceptibility testing. Clin. Microbiol. Rev., 20(2):280-322.
[42] Versalovic, J., Osato, M. S., Spakovsky, K., Dore, M. P., Reddy, R., Stone, G. G., et al.
(1997). Point mutations in the 23S rRNA gene of Helicobacter pylori associated with
different levels of clarithromycin resistance. J. Antimicrob. Chemother., 40(2):283-6.
[43] Fontana, C., Favaro, M., Pietroiusti, A., Pistoia, E. S., Galante, A., Favalli, C., (2003).
Detection of clarithromycin-resistant Helicobacter pylori in stool samples. J. Clin.
Microbiol., 41(8):3636-40.
[44] Makristathis, A., Hirschl, A. M., Russmann, H., Koletzko, S., (2007). Detection and
clarithromycin susceptibility testing of Helicobacter pylori in stool specimens by real-
time PCR: how to get accurate test results. J. Clin. Microbiol., 45(8):2756-7.
[45] Lottspeich, C., Schwarzer, A., Panthel, K., Koletzko, S., Russmann, H., (2007).
Evaluation of the novel Helicobacter pylori ClariRes real-time PCR assay for detection
and clarithromycin susceptibility testing of H. pylori in stool specimens from
symptomatic children. J. Clin. Microbiol., 45(6):1718-22.
[46] Scaletsky, I. C., Aranda, K. R., Garcia, G. T., Goncalves, M. E., Cardoso, S. R., Iriya,
K., et al. (2011). Application of real-time PCR stool assay for Helicobacter pylori
detection and clarithromycin susceptibility testing in Brazilian children. Helicobacter.,
16(4):311-5.
[47] Glocker, E., Kist, M., (2004). Rapid detection of point mutations in the gyrA gene of
Helicobacter pylori conferring resistance to ciprofloxacin by a fluorescence resonance
energy transfer-based real-time PCR approach. J. Clin. Microbiol., 42(5):2241-6.
[48] Gerrits, M. M., de Zoete, M. R., Arents, N. L., Kuipers, E. J., Kusters, J. G., (2002).
16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori.
Antimicrob. Agents Chemother., 46(9):2996-3000.
[49] Ribeiro, M. L., Gerrits, M. M., Benvengo, Y. H., Berning, M., Godoy, A. P., Kuipers,
E. J., et al. (2004). Detection of high-level tetracycline resistance in clinical isolates of
Helicobacter pylori using PCR-RFLP. FEMS Immunol. Med. Microbiol., 15;40(1):57-
61.
[50] Banatvala, N., Lopez, C. R., Owen, R., Abdi, Y., Davies, G., Hardie, J., et al. (1993).
Helicobacter pylori in dental plaque. Lancet., 6;341(8841):380.
[51] Agarwal, S., Jithendra, K. D., (2012). Presence of Helicobacter pylori in subgingival
plaque of periodontitis patients with and without dyspepsia, detected by polymerase
chain reaction and culture. J. Indian Soc. Periodontol., 16(3):398-403.
[52] Ou, Z., Xiong, L., Li, D. Y., Geng, L., Li, L., Chen, P., et al. (2013). Evaluation of a
new fluorescence quantitative PCR test for diagnosing Helicobacter pylori infection in
children. BMC Gastroenterol., 14;13(1):7.

[53] Al-Ahmad, A., Kurschner, A., Weckesser, S., Wittmer, A., Rauberger, H., Jakob, T., et
al. (2012). Is Helicobacter pylori resident or transient in the human oral cavity? J. Med.
Microbiol., 61(Pt 8):1146-52.
[54] Leszczynska, K., Namiot, D. B., Namiot, Z., Leszczynska, J. K., Jakoniuk, P., Kemona,
A., (2009). Application of immunoassay for detection of Helicobacter pylori antigens
in the dental plaque. Adv. Med. Sci., 54(2):194-8.
[55] Morales-Espinosa, R., Fernandez-Presas, A., Gonzalez-Valencia, G., Flores-Hernandez,
S., Delgado-Sapien, G., Mendez-Sanchez, J. L., et al. (2009). Helicobacter pylori in the
oral cavity is associated with gastroesophageal disease. Oral Microbiol. Immunol.,
24(6):464-8.
[56] Assumpcao, M. B., Martins, L. C., Melo Barbosa, H. P., Barile, K. A., de Almeida, S.
S., Assumpcao, P. P., et al. (2010). Helicobacter pylori in dental plaque and stomach of
patients from Northern Brazil. World J. Gastroenterol., 28;16(24):3033-9.
[57] Chaudhry, S., Idrees, M., Izhar, M., Butt, A. K., Khan, A. A., (2011). Simultaneous
amplification of two bacterial genes: more reliable method of Helicobacter pylori
detection in microbial rich dental plaque samples. Curr. Microbiol., 62(1):78-83.
[58] Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E., (2005). Defining the
normal bacterial flora of the oral cavity. J. Clin. Microbiol., 43(11):5721-32.
[59] Navabi, N., Aramon, M., Mirzazadeh, A., (2011). Does the presence of the
Helicobacter pylori in the dental plaque associate with its gastric infection? A meta-
analysis and systematic review. Dent. Res. J. (Isfahan)., 8(4):178-82.
[60] Wirth, H. P., Vogt, P., Ammann, R., Altorfer, J., (1993). [IgA-antibodies against
Helicobacter pylori in gastric secretions: gastric secretory immune response or salivary
contamination?]. Schweiz Med. Wochenschr., 29;123(21):1106-10.
[61] Alemohammad, M. M., Foley, T. J., Cohen, H., (1993). Detection of immunoglobulin
G antibodies to Helicobacter pylori in urine by an enzyme immunoassay method. J.
Clin. Microbiol., 31(8):2174-7.
[62] Katsuragi, K., Noda, A., Tachikawa, T., Azuma, A., Mukai, F., Murakami, K., et al.
(1998). Highly sensitive urine-based enzyme-linked immunosorbent assay for detection
of antibody to Helicobacter pylori. Helicobacter., 3(4):289-95.
[63] Fujisawa, T., Kaneko, T., Kumagai, T., Akamatsu, T., Katsuyama, T., Kiyosawa, K., et
al. (2001). Evaluation of urinary rapid test for Helicobacter pylori in general practice. J.
Clin. Lab. Anal., 5(3):154-9.
[64] Engvall, E., Perlmann, P., (1971). Enzyme-linked immunosorbent assay (ELISA).
Quantitative assay of immunoglobulin G. Immunochemistry., 8(9):871-4.
[65] Lequin, R. M., (2005). Enzyme immunoassay (EIA)/enzyme-linked immunosorbent
assay (ELISA). Clin. Chem., 51(12):2415-8.
[66] Martin, T. L., Mufson, E. J., Mesulam, M. M., (1984). The light side of horseradish
peroxidase histochemistry. J. Histochem. Cytochem., 32(7):793.
[67] Katsuragi, K., Noda, A., Tachikawa, T., Azuma, A., Mukai, F., Murakami, K., et al.
(1998). Highly sensitive urine-based enzyme-linked immunosorbent assay for detection
of antibody to Helicobacter pylori. Helicobacter., 3(4):289-95.
[68] Dehghannezhad, A., Paknejad, M., Rasaee, M. J., Omidfar, K., Seyyed Ebrahimi, S. S.,
Ghahremani, H., (2012). Development of a nanogold-based immunochromatographic
assay for detection of morphine in urine using the Amor-HK16 monoclonal antibody.
Hybridoma (Larchmt)., 31(6):411-6.

[69] Moraes, G. S., Amaral, C. R., Savaris, R. F., (2011). Comparative analysis of the
accuracy of urinary hCG tests in vitro. Rev. Assoc. Med. Bras., 57(5):516-22.
[70] Graham, D. Y., Reddy, S., (2001). Rapid detection of anti-Helicobacter pylori IgG in
urine using immunochromatography. Aliment Pharmacol. Ther., 15(5):699-702.
[71] Murakami, K., Kamada, T., Ishikawa, H., Imamura, H., Matsumoto, H., Fujita, M., et
al. (2011). An evaluation of the performance of a novel stick-type kit for rapid detection
of Helicobacter pylori antibodies in urine. Clin. Lab., 57(7-8):481-7.
[72] Adachi, K., Kawamura, A., Ono, M., Masuzaki, K., Takashima, T., Yuki, M., et al.
(2002). Comparative evaluation of urine-based and other minimally invasive methods
for the diagnosis of Helicobacter pylori infection. J. Gastroenterol., 37(9):703-8.
[73] Muhsen, K., Athamna, A., Athamna, M., Spungin-Bialik, A., Cohen, D., (2006).
Evaluation of a urine-based enzyme-linked immunosorbent assay test for the detection
of Helicobacter pylori infection among 3- to 5-year-old Israeli Arab healthy children. J.
Pediatr. Gastroenterol. Nutr., 43(3):398-401.
[74] Megraud, F., (2005). Comparison of non-invasive tests to detect Helicobacter pylori
infection in children and adolescents: results of a multicenter European study. J.
Pediatr., 146(2):198-203.
[75] Soares, T. F., Rocha, G. A., Rocha, A. M., Correa-Oliveira, R., Martins-Filho, O. A.,
Carvalho, A. S., et al. (2005). Phenotypic study of peripheral blood lymphocytes and
humoral immune response in Helicobacter pylori infection according to age. Scand. J.
Immunol., 62(1):63-70.
[76] Shimizu, T., Yarita, Y., Haruna, H., Kaneko, K., Yamashiro, Y., Gupta, R., et al.
(2003). Urine-based enzyme-linked immunosorbent assay for the detection of
Helicobacter pylori antibodies in children. J. Paediatr. Child Health., 39(8):606-10.
[77] Megraud, F., (2005). Comparison of non-invasive tests to detect Helicobacter pylori
infection in children and adolescents: results of a multicenter European study. J.
Pediatr., 146(2):198-203.
[78] Yamamoto, T., Kojima, K., Sanaka, M., Ishii, T., Osaki, Y., Tsutsumi, H., et al. (2006).
Reliability of rapid urinary test for antibody to Helicobacter pylori in adult patients with
proteinuria. Diagn. Microbiol. Infect. Dis., 54(2):105-8.
[79] Yamamoto, T., Ishii, T., Kawakami, T., Sase, Y., Horikawa, C., Aoki, N., et al. (2005).
Reliability of urinary tests for antibody to Helicobacter pylori in patients with
pulmonary tuberculosis. World J. Gastroenterol., 21;11(3):412-4.
[80] Tamura, T., Morita, E., Kondo, T., Ueyama, J., Tanaka, T., Kida, Y., et al. (2012).
Prevalence of Helicobacter pylori infection measured with urinary antibody in an urban
area of Japan, 2008-2010. Nagoya J. Med. Sci. Feb.;74(1-2):63-70.

Invasive Procedures

Chapter 3
Common Techniques: Endoscopy,

Histological Examination
and Rapid Urease Test
Pellegrino Crafa,1, Marco Manfredi,2,3 Elisabetta Manzali,2 Barbara

Bizzarri,2 and Gian Luigi de’Angelis2,3
1
Pathology Unit, Department of Biomedical, Biotechnological and Transalational
Science, Azienda Ospedaliero-Universitaria, University Hospital, Parma, Italy
2
University Hospital, Parma, Italy
3
Departments of Pediatrics, “Pietro Barilla” Children's Hospital, Azienda
Ospedaliero-Universitaria, University Hospital, Parma, Italy
Abstract
Helicobacter pylori (H. pylori) is one of the most common worldwide human
infections and is associated with several important gastrointestinal disorders including
chronic gastritis, peptic ulcer disease, and gastric malignancy.
The methods of diagnostic testing for H. pylori can be divided into those that do and
those that do not require endoscopy, but currently, there is no single test that can be
considered the gold standard.
However, histology has been considered, by some, to be the gold standard for
detection of H. pylori; it is also able to highlight the first pre-neoplastic changes. But
unfortunately, histology is an imperfect gold standard, by others, because the detection of
H. pylori is dependent on several issues including the site, number, and size of gastric
biopsies, method of staining, and the level of experience of the examining pathologist.
Rapid urease test is a simple, inexpensive, rapid and reliable method for detected
urease activity in gastric specimens, but it can rarely be used as a unique test to detect H.
pylori infection.

Corresponding author: Pellegrino Crafa MD, Associate Professor, Pathology Unit, Department of Biomedical,
Biotechnological and Transalational Science, Azienda Ospedaliero-Universitaria, University Hospital, Via
Gramsci 14, 43100 Parma, Italy. E-mail: pellegrino.crafa@unipr.it.

42 Pellegrino Crafa, Marco Manfredi, Elisabetta Manzali et al.
Introduction
Probably the first description of Helicobacter pylori (H. pylori) date back in 1892 when
spiral bacteria in the dogs' stomach were shown by Giulio Bizzozero, a pathologist at the
University of Turin. H. pylori has been identified to have a main role in many diseases, such
as chronic atrophic gastritis which is considered to be the first step of sequential mucosal
changes leading to gastric cancer.
H. pylori is a fascinating and malicious bacterium with a selective tropism for the gastric
mucosa, able to survive in a hostile environment for colonization of organisms other than
itself, able to develop strategies for survival, causing inflammatory changes, that vary from
asymptomatic mild gastritis to more severe injury such as peptic ulcer as well as premalignant
lesions and malignant tumors.
This is the reason why its diagnosis is necessary in many different circumstances and
therefore numerous reliable invasive and non-invasive diagnostic tests have been developed
[1].
Each test has advantages and disadvantages, which will make it more or less appropriate
depending on the clinical situation [2].
The methods to diagnose H. pylori can be divided into those that do and those that do not
require endoscopy. The most recent recommendation by the American College of
Gastroenterology states that if endoscopy is required depending on the patient’s presentation,
biopsy-based endoscopic tests are the most appropriate tool to diagnose H. pylori infection
[3]. Moreover the role of biopsies in the recognition of epithelial and lymphoid neoplasia and
of morphological features preceding them is also important.
Among endoscopic tests there are histology, rapid urease test (RUT), and culture.
Sensitivity and specificity of RUT is 80-95% and 90-100% respectively; histological
examination has a sensitivity of 83-95% and a specificity of 90-100%; for culture, the
reported sensitivity and specificity is 80-90% and 95-100% respectively [4].
The pattern and distribution of gastritis correlate strongly with the sequelae of infection.
It explains thus the importance of gastric biopsies in the precise definition of the disease and
how important is the grading of gastritis according to the updated Sydney system or the
assessment of the risk of evolution of metaplastic lesion in agreement with OLGA system or
the recognition of intraepithelial lesions of both low and high grade.
Upper Gastrointestinal Endoscopy: When and Why

When?
As shown by recent guidelines, the test-and-treat strategy is appropriate in patients with a

low risk of having gastric cancer. This means dyspeptic patients below a determined age cut-
off point, which depend on local incidence of gastric cancer in different age groups, and
without so-called “alarm” symptoms or signs. These include weight loss, dysphagia, overt GI
bleeding, abdominal mass, iron deficiency anaemia, odynophagia, persistent vomiting, a
palpable mass, lymphoadenopathy and a family history of upper GI malignancy which are
associated with an increased risk of gastric cancer [1, 5].

Common Techniques: Endoscopy, Histological Examination … 43
On the other side, in patients with “alarm” symptoms a test-and-treat strategy is not
recommended but it is preferred a strategy of “endoscope and treat” [6]. However, many
patients with early stage malignancy do not have any “alarm” symptoms or signs. Therefore if
a clinical suspicion of malignancy is supposed an endoscopy should be considered even in the
absence of “alarm features” [7].
It should also be noted that the “alarm features” mentioned previously have limited
predictive value for the identification of underlying peptic ulcer disease (PUD), which is one
of the most common disease detected in patients with dyspepsia [5, 7]. The most common
cause of duodenal and gastric ulcer has been shown to be H. pylori infection, being present in
about 75% of the cases of PUD [8].
Therefore, upper endoscopy is advisable for any patient with persistent dyspeptic
symptoms or in patients non-responders to prolonged PPI therapy to exclude structural
disease. In fact, early endoscopy in dyspeptic patients has the advantage to establish a specific
diagnosis, such as PUD and moreover it has the advantage to decrease anxiety and increase
patient satisfaction if it is negative.
Recent studies evaluate the comparison between endoscopic approach and “test-and-
treat” strategy: this last one is less expensive and 60% of dyspeptic patients in the “test-and-
treat” group did not need a further endoscopy, but 12% of these patients did not state
satisfaction, while only 4% were dissatisfied in the endoscopic approach group [7].
During endoscopy in all patient with PUD, testing the presence of H. pylori should be
performed, despite some studies report that in the setting of an active gastrointestinal bleeding
false-negative results for the presence of H. pylori are increased [9].
Moreover in elderly non-invasive tests are less accurate [10] considering that older
populations are at highest risk for H. pylori-related conditions including gastric cancer and in
these patients a significantly higher prevalence of peptic ulcers was observed in H. pylori-
positive than in H. pylori-negative subjects [11].
Endoscopy is useful because it permits a direct visualization of gastric cavity and it
allows to obtain specimens from gastric mucosa to diagnose H. pylori infection; furthermore
it make it possible to perform therapeutic intervention for instance in the case of an active
bleeding due to a PUD. Even before prescription of the first-line treatment, upper endoscopy
should be performed in a region or in a population of high clarithromycin resistance or in
patients with hypersensitivity that reduce the choice of antibiotic. This because endoscopy
allows to perform culture and standard susceptibility testing to antimicrobial agents. In fact,
the range of success of the clarithromycin is between 10 and 30% in region with high
clarithromycin resistance. The approach of performing culture showed to be economically
and ecologically preferred in countries of high clarithromycin resistance or in specific
population, because it permits to set a specific therapy for each patient. Furthermore, if an
endoscopy is carried out for any reasons, during the procedure culture should be obtained in
all patients before of second-line treatment because the possibility of having a resistant
organism ranges between 60-70% for clarithromycin.
Endoscopy with culture has to be performed also in all patients who have received two
courses of antibiotic treatment and remain H. pylori positive before administering a third line
treatment.
Molecular tests such as Fluorescence In Situ Hybridisation and Polymerase Chain
Reaction can replace culture, if this last one is not possible, because they can detect H. pylori
and antibiotic-resistance in gastric biopsies [1].

Unfortunately, most diagnostic methods used for detecting H. pylori, including RUT,
histology, and culture, are negatively affected by drugs such as bismuth, antibiotics, or proton
pump inhibitors (PPIs) which suppress the bacterial population. Many patients undergo upper
endoscopy while taking acid-suppressive agents or other drugs such as antibiotics or bismuth,
or after a too short period of time from suspension of them. In those cases it is proper to take
anyway biopsies for histology with or without RUT or to plan Urea Breath Test (UBT) or
fecal antigen test after interrupting therapy for an appropriate period of time [3].
There is no doubt that H. pylori infection is responsible for the majority of cases of
gastritis [12] and that chronic gastritis due to H. pylori infection may progress to intestinal
metaplasia (IM) [13]. By now chronic atrophic gastritis and IM constitute the background in
which dysplasia and intestinal-type gastric adenocarcinoma develop and they are considered
to be precancerous conditions. Therefore patients with atrophic gastritis or IM need an
endoscopic surveillance because of the increased risk of developing gastric cancer. Even
though, the optimal timing for the follow up is still not known [1]. European guidelines
advise that patients with extensive atrophy and/or IM should receive follow-up every 3 years
after diagnosis, while in patients with mild to moderate atrophy/IM restricted to the antrum
there is no evidence to recommend surveillance [14].
Since overall, gastric cancer risk is too low (0-1.8 %, and 0-10% per year respectively for
atrophic gastritis and IM) to justify endoscopic surveillance in all patients with these
histological findings. Therefore, it has been take into consideration the extent of gastric
lesions, since there is a correlation between extensive intragastric lesions and an increased
gastric cancer risk [15, 16]. If gastric dysplasia is observed, an endoscopic surveillance is
mandatory. In fact patients with low grade dysplasia seem to show a risk to progress to
invasive carcinoma of 7% [17].
The timing of endoscopic surveillance in patients with low grade dysplasia in the absence
of an endoscopically defined lesion is within 1 year after diagnosis. If dysplasia is confirmed
by repeated endoscopy, surveillance need to be continued; on the other hand if there is no
confirmation of low grade dysplasia during endoscopic follow-up, it is still not clear when
surveillance need to finish.
If an endoscopically defined lesion is found (depressed or elevated lesions, minute or flat
lesions, isolated or multifocal lesions) endoscopic resection should be considered, to obtain a
more accurate histological diagnosis [14]. In fact, endoscopic resection of a lesion may
upgrade dysplasia from low grade to high grade or adenocarcinoma. In a Korean study 19%
of patients with low grade dysplasia on biopsy forceps specimens were upgraded after
endoscopic mucosal resection [18]. In patients with high grade dysplasia in the absence of
endoscopically defined lesions, immediate endoscopic reassessment with extensive biopsy
sampling and surveillance at 6-month to 1–year intervals is indicated, while
endoscopic/surgical resection has to be considered in the case of endoscopically defined
lesions [14].
Some other pre-neoplastic conditions have to be considered for endoscopic surveillance:
firm diagnosis of pernicious anaemia with histological confirmation of type A autoimmune
atrophic gastritis; histological and serological signs of subtotal or total atrophic gastritis with
hypo-achlorhydria; in case of diagnosis/removal of gastric adenoma [1].
Regarding the demonstration of the H. pylori eradication, usually a non-invasive test is
recommended.

Only in peculiar cases, such as a previous diagnosis of gastric ulcer or gastric MALT
lymphoma, endoscopy should be performed because of its high cost [1]. If endoscopy is
performed, histology with or without RUT should be obtained, since RUT alone has a low
sensitivity [19].
Any procedure to determine H. pylori eradication should generally be performed at least
4 weeks after the completion of treatment.
Why?
We already reported that endoscopy can permit a clear and direct visualization of gastric
mucosa. Although a very clear visualization of gastric mucosa by the new endoscope, the
majority of H. pylori-infected individuals show no obvious abnormalities, or only limited
abnormalities. With the new development of endoscopic techniques, such as high resolution
or magnification endoscopy, however subtle changes may be seen [20].
In 1990 on the basis of the new etiological facts on gastritis such as H. pylori a new
system of classification was presented at the World Congress of Gastroenterology held in
Sydney, Australia [21]. This system comprises pathology and endoscopy sections and after
some criticism to the original system, an update of the Sydney system was worked out at the
H. pylori Congress held in 1994 in Houston, US.
Endoscopy carried out to investigate gastroenterological diseases should always include
taking biopsies specimens and for this purpose, the update Sydney system considered to
perform the standard two biopsies each from the antrum and corpus, and an additional biopsy
from the angulus [12].
Diffuse and multiple biopsies, made with standard biopsy forceps, are important since
atrophic change due to H. pylori infection is reported to expand from antrum to fundus
because of mucosal inflammation [22] and topological prevalence of chronic active gastritis
evaluated by endoscopy is useful to predict future risk for gastric cancer.
Considering the Sydney system, the topography of the gastritis was considered the core
of the classification: gastritis was classified restricted to the antrum, restricted to the corpus,
or a pangastritis [23].
Although many investigators have attempted to categorize the endoscopic findings
characteristic of a H. pylori-infected stomach, no consensus has been reach about whether
gastritis H. pylori-related can be diagnosed from other macroscopic changes in the gastric
mucosa not H. pylori-related and the diagnosis of H. pylori infection in gastric mucosa by
endoscopic features has not yet been established [26, 27]. The endoscopic findings of H.
pylori infection are erythema, erosions, antral nodularity, thickened gastric folds, visible
submucosal vessels [28] and the most common change due to H. pylori infection is
erythematous/exudative gastritis (up to 75% of patients) [27, 28]. However, these findings are
not a reliable method of diagnosis because of their low sensitivity and specificity, in fact
antral nodularity had a poor sensitivity (32%) but a high specificity (96%) [30]; while
abnormal antral surface texture, a mamillated corpus surface, and white antral erosions have a
sensitivity and specificity of 75% and 63%, respectively.
Additionally, other endoscopic findings such as erythema, erosions, and the presence of
visible vessels had sensitivity for H. pylori infection of 56% [30].

Considering antral nodularity, it is rather rare in adults, in contrast to children. It is

observed especially in the antrum [27], and it showed a high specificity (98.5%) and positive
predictive value (91.7%) for the diagnosis of H. pylori infection in children Therefore antral
nodularity is considered a common and specific finding in paediatric patients with H. pylori
infection [31].
Recently other endoscopic findings were studied. A study from Japan in 2002 found that
a “regular arrangement of collecting venules” (RAC), seen as numerous minute red dots in
the gastric corpus, was a characteristic finding in the normal stomach without H. pylori
infection. On the other hand, H. pylori-infected mucosa was defined as the disappearance of
both RAC and the network of capillaries surrounding the gastric pits. The RAC pattern had
high sensitivity and specificity for predicting the absence of H. pylori infection in adults [32].
Recently, a study from Taiwan showed that a close-up observation between the
endoscopic tip and the gastric mucosa can show the “mosaic pattern” with or without spotty
erythema in the corpus mucosa. This method revealed a sensitivity of 100% and a specificity
of 86% for prediction of gastric H. pylori infection using two categories: normal RAC and
abnormal mosaic pattern [33]. More recently a new study from Korea used four categories of
endoscopic findings: a normal RAC and three abnormal patterns including mosaic-like
appearance (type A), diffuse homogenous redness (type B), and untypical pattern (type C) to
predict a H. pylori-infected stomach.
The sensitivity, specificity, and positive and negative predictive values of all abnormal
patterns for prediction of H. pylori infection were 93.3%, 89.1%, 92.3%, and 90.6%,
respectively with an overall accuracy of 91.6% [26]. It has been showed however that a
combination of more findings can improve diagnostic accuracy and sensitivity of endoscopic
examination but there were no single specific endoscopic findings for inflammation and
activity [34].
Moreover it is still uncertain what endoscopic changes are characteristic of gastritis in
subjects infected with H. pylori and it was found that this disease could not be diagnosed
from the endoscopic appearance alone [28]. Some studies have evaluated if conventional
white light endoscopy can reliably distinguish H. pylori gastritis and gastric preneoplastic
lesions from normal mucosa.
Poor correlation between endoscopy and histology was found in the diagnosis of H.
pylori gastritis, atrophy, or intestinal metaplasia [30, 31]. Endoscopic findings linked to
eradication of H. pylori are a decrease in spotty redness while diffuse redness of fundic
mucosa, non-transparency of gastric juice, enlarged fold, flat erosion of stomach were not
specific changes in cases of successful eradication. By now the change after H. pylori
eradication on conventional endoscopy has not been yet clarified [25].
Histologic Examination
Gastritis is a histological term that defines an inflammation of the gastric mucosa. It
needs biopsy(ies) to be confirmed.
Retrospective studies comparing endoscopic and pathologic diagnoses of gastritis showed
a discordance between endoscopy and histology, in about 34% of cases. These data included
14% of patients with normal endoscopy and abnormal histology and 20% of patients with

abnormal endoscopy and normal histology. In addiction, 3,5% of patients had normal
histology, although erosions were endoscopically diagnosed. These findings showed that
standard endoscopy alone is a poor predictor of gastric pathological changes [35, 36].
The gastric biopsy, when correctly performed, is an extreme sensitive method [37] to
diagnose H. pylori infection, to characterize the inflammatory damage and to quantify the
mucosal alterations. It also allows the evaluation of the effectiveness of eradication therapy
and helps in differential diagnoses between various type of gastric inflammation.
Biopsy Protocol
Sidney system recommends at least five biopsy specimens to assure the correct diagnosis
of gastritis.
Most gastric diseases occur with an irregular topographic distribution. As a consequence,
multiple specimens are also necessary to determine disease distribution within the mucosa.
Multiple specimens from: a) antrum (two) from the lesser and the greater curvature, both
within 2 to 3 cm from the pylorus; b) antral-body transizional zone (one) from the incisura
angularis; and c) corpus (two) from the lesser curvature about 4 cm proximal to the angulus
and from the middle portion of the greater curvature, approximately 8 cm from the cardia.
The biopsies should be properly identified and should be submitted in separate containers to
the pathology laboratory. In addition, biopsies from any macroscopically lesion should be
taken (ulcer, erosion, or depressed area detected etc.).
Corpus biopsies are particularly valuable for yielding positive results after treatment,
especially if proton pump inhibitors have been used. Under these circumstances, H. pylori
organisms may become rare or disappear from the antrum and remain in the oxyntic mucosa;
in this area cystic dilatations and hypertrophy of the parietal cells may also develop [38]. The
sample from incisura angularis is important because in this area metaplastic and dysplastic
lesions are consistently found. It should generally be treated as an additional antral specimen
and its scores averaged with the antral scores.
Sampling orientation is critical for optimal histologic evaluation: fragments shall be
deposited with the uneven, rough surface, to adhere on paper blotting and then into the
fixative for allowing a proper orientation of the biopsy. The gastritis characterization is
possible whereas each biopsy includes the muscularis mucosae, being completely represented
the full thickness of the mucosa. Assessments of the degree of atrophy will be reliable if the
sample includes at least 15-20 foveols.
The Sidney system uses a numeric or descriptive value in order to describe the presence
of H. pylori, neutrophilic and mononuclear cells, intestinal metaplasia and the loss of proper
glands in antrum and corpus. Value 0 represents absent, 1 is mild, 2 is moderate, and 3 is
marked (or severe) [38].
The degree of inflammation in the antrum and corpus allows to determine whether the
inflammation is similar in intensity (i.e., pangastritis) or more severe in either the antrum
(antrum-predominant gastritis) or the corpus (corpus-predominant gastritis). The last
step in the Sidney classification is to decide whether focal or diffuse atrophy is present
(metaplastic or nonmetaplastic) [39, 40].
The Sidney system can be applied only when a full set of biopsy specimens is available.
When less than two antral and two corpus biopsies are available, the use of the Sidney system

is not recommended whereas is more appropriated a descriptive diagnosis. Sometimes the

knowledge of the "normal" distribution of endocrine cells in the various gastric compartments
can be exploited to differentiate, through the use of immunostaining, the sampling error from
true metaplastic features. In this case, the presence of gastrin-positive cells indicates that the
biopsy was taken from the antrum, whereas rare or none gastrin-positive cells are more
probably taken from a metaplastic area of the corpus.
An international group of gastroenterologists and pathologists (the Operative Link for
Gastritis Assessment [OLGA]) has proposed a system for reporting gastritis in terms of stage
(the OLGA Staging System), which arranges the histological phenotypes of gastritis along a
scale of progressively increasing gastric cancer risk, from the lowest (OLGA stage 0) to the
highest (OLGA stage IV) [39].
In the OLGA system gastric atrophy is a lesion that indicates disease progression.
Topography of Chronic Gastritis
The microscopic characteristics of the inflammatory infiltrate, the topographical

distribution of the lesions, the presence/absence of IM characterize different form of gastritis.
The majority of subjects with H. pylori infection but without histological demonstration of
IM, have a slightly more pronounced inflammatory infiltrate in the antrum compared with the
corpus (one grade higher).
The most common type of gastritis, antral-predominant gastritis, formerly called
diffuse superficial gastritis, is defined as chronic non atrophic gastritis. It is characterized by
the presence of a moderate to severe inflammatory infiltrate (two or more grade higher than in
the corpus), which presents an uneven distribution among the antral mucosecreting glands in
the lamina propria. At the same time, in the gastric body an inflammatory infiltrate (presence
of polymorphonuclear neutrophils) of lesser intensity than in the antrum is observed. A subset
of these patients are likely to develop duodenal ulcer. The definition of chronic not atrophic
antral gastritis implies the complete absence of loss of appropriate glandular units or
metaplastic lesions.
Likewise, as a mirror image, corpus-predominant gastritis is a chronic non atrophic
gastritis with a more severe inflammatory infiltrate in the oxyntic mucosa compared with the
antral region. Infrequently, some H. pylori-infected individuals show a corpus-predominant
pattern that overlaps with autoimmune gastritis. Inflammation restricted to the oxyntic
mucosa in H. pylori-negative subjects is associated with diffuse glandular atrophy in the
corpus and is more characteristic of autoimmune gastritis. It remains possible, however, that
the pernicious anemia phenotype could still be a long-term consequence of H. pylori
infection. Corpus predominance is also the usual pattern in lymphocytic gastritis and in
proton pump inhibitors long term users [38]. Chronic non atrophic pangastritis, as can be
deduced from the name, is a type of gastritis in which the inflammatory infiltrate, with only
slight topographical differences, is dispersed, in all gastric compartments. The chronic non
atrophic pangastritis represents the step immediately prior to the loss of the glandular
component.

Atrophic gastritis is defined by the presence of atrophy or IM.

Atrophy and IM can be focal, diffuse or multifocal. The latter is recognized if there is
fibrosis of the lamina propria due to (multi)focal glandular loss, and IM is alternated with
normal appearing areas with an almost normal heritage of proper glands.
Multifocal atrophic gastritis is associated with gastric ulcer disease and, in some
populations, with the risk for gastric carcinoma. Usually diffuse chronic gastritis and
multifocal atrophy is associated with H. pylori infection, but this pattern may be encountered
in H. pylori negative cases. These patients could have been affected by a previous H. pylori
infection, but atrophy can result from other injurious factors, such as bile reflux, nonsteroidal
anti-inflammatory drugs (NSAIDs), and dietary injury. These and other factors may act
independently, or together with H. pylori to determine multifocal atrophy [38].
Multifocal chronic atrophic gastritis has an inflammatory infiltrate with an uneven
distribution both in antrum and body.
In more advanced stages, a chronic atrophic pangastritis can develop with similar
findings in all topographical compartments. After this stage complications such as ulceration
or, intraepithelial neoplasia (NIN; formerly known as dysplasia) or finally gastric
adenocarcinoma can occur.
Morphological Variables in Update Sidney System

Characteristic lesions allow to distinguish the topographic types of H. pylori-induced
chronic gastritis, metaplastic or not, from other subtypes of gastritis due to different etiologic
agents.
Polymorphonuclear Neutrophil Activity
Under normal conditions, in the lamina propria of the gastric mucosa it is possible to
recognize only a few scattered neutrophils, thus a more prominent polymorphonuclear
neutrophil harvested in a background of mononuclear infiltration is a marker of active
gastritis. Although host defense against microbial pathogens is indispensable, misdirected
acute activation of neutrophils presents the potential hazard of tissue damage.
Polymorphonuclear neutrophils (PMNs) are programmed to release reactive oxygen
species, proteinases, and eicosanoids/prostanoids immediately on exposure to pro-
inflammatory stimuli. Chronic inflammation in the absence of neutrophils actives cytotoxic
T-lymphocytes and other cell effectors which may play a role in tissue damage, and operate in
glandular destruction in some patterns of gastritis [38]. If a sufficient number of biopsy
specimens from antrum and corpus are examined, neutrophils are detected in the lamina
propria and/or in the region of the glandular neck and as pit abscesses within the foveolar
lumen, in virtually all subjects with H. pylori infection.
The mucosal damage is directly connected to the initial bacterial load and the related
number of neutrophils [41]. Neutrophils are a very sensitive indicator of the presence or
absence of H. pylori and they disappear some days after therapy.

If neutrophilic polymorphs are seen in a post-treatment biopsy but organisms are not
apparent, a careful search for Helicobacter using one of the special stains or immunostains
should be carried out [38].
Although H. pylori plays a key role in promoting the migration of PMNs to the mucosa, it
is able to survive in this hostile environment by manipulating phagocytosis and the
subsequent oxidative burst [44].
Variable number of eosinophils infiltrates the lamina propria in most types of gastritis,
but their pathogenetic role is unknown. Therefore, routine grading of eosinophils is not
required. Increased number of eosinophils is frequently seen in biopsy specimens from
patients successfully treated for H. pylori gastritis; however, their density rarely approaches to
that found in eosinophilic gastritis.
Chronic Inflammation
Normally in a healthy individual, there are slight differences about the presence of
mononuclear inflammatory cells in the lamina propria in the gastric antrum and body. In the
antrum few lymphocytes and plasma cells are expected to represent a normal finding whereas
normal corpus mucosa contains virtually none. Sidney system stated that the normal number
of mononuclear leukocytes in the lamina propria of gastric mucosa ranges from 2 to 5
lymphocytes, plasmacells and macrophages per highpower (×40 objective) microscopic field
or, by another approach, two or three lymphocytes or plasma cells between foveolae (the area
in which chronic inflammatory cells are more often found). Plasmacells are sparse or absent
in the stomach of healthy persons; so their presence is an important indicator of a chronic
inflammatory response. Some observers consider chronic inflammation even when there are
as few as one or two plasma cells per high-power field. Occasional lymphocytes may also be
observed in the epithelium of the normal stomach, especially in the surface (up to about 5 per
100 epithelial nuclei); but when the number is higher it can be considered a marker of chronic
inflammation. In these instances the term lymphocytic gastritis is appropriate and it is
suggestive (but not diagnostic) of an immune-mediated component of the inflammatory
disease [41].
In chronic gastritis, elicitated by H. pylori action, the inflammatory infiltrate consists
mainly of effectors of the immune response, including CD4+ and CD8+ T-lymphocytes, B-
lymphocytes, plasma cells, monocytes, mast cells, and eosinophils [38].
Lymphocytes can be dispersed among the glands or organized in follicular or nodular
structures occupying space, thereby it is difficult to assess the loss of glandular units.
Lymphoepithelial lesions are remnants of glandular structures destroyed by lymphocytic
infiltrate and they are almost pathognomonic of primary gastric lymphomas (almost always
associated with H. pylori).
Gastric lymphocyte populations from H. pylori infected patients contain increased IFN-γ-
producing T cells, with a Th1 cytokine response. Also H. pylori-specific T cell clones derived
from gastric mucosa have a Th1-profile in patients with peptic ulcer disease. Other T-cell
populations are important. Interleukin (IL)-17 has been linked to chemokine-mediated
neutrophil infiltration, and IL-17 levels are increased in H. pylori infected gastric tissues.
Recent data suggest that the IL-17/Th17 response may thus be defective in a normal host,
thereby contributing to chronic persistence of the bacterium.

Another mechanism of immune dysfunction has been demonstrated by the recent report
that VacA can exert immunosuppressive effects on T cells by binding to the β2-integrin
receptor subunit (CD18) and utilizing integrin receptors to cause cellular vacuolization.
Dendritic cells (DCs) are of great interest because they represent a critical bridge between
the innate and adaptive immune responses. They have been identified as primary responders
to stimuli including bacterial products and they have a role as antigen presenting cells
(APCs). DCs can penetrate epithelial monolayers in vitro and the intestinal epithelial barrier
in vivo and can engulf bacteria directly [42].
In patients with chronic gastritis a decrease of the inflammatory infiltrate after
pharmacological treatment is observed. But even several years after the treatment, a greater
number of chronic inflammatory cells than in normal subjects can be seen in the gastric
mucosa, particularly in the antrum. The density of mononuclear cells in the lamina propria
should be graded in areas away from lymphoid follicles and their surrounding marginal zone
of small lymphocytes [38].
Glandular Atrophy
The normal gastric glands differ in cellular composition as they are constitutionally
entrusted to perform different functions. In the antrum and in the cardia there are
mucosecreting glands whereas in the body there are oxyntic glands.
The worldwide recognized and accepted definition of gastric atrophy is loss of
appropriate glands [41] and it is a histologic finding. Whenever gastric mucosa is damaged, it
may either regenerate to normal (restitutio ad integrum) or undergo an adaptive reparative
change that leads to the replacement of native glands with other types of tissue [38]. The loss
of appropriate glands can be defined as the process of collapse of stromal weft followed by
stromal scar due to fibroblast proliferation that replaces the disappeared glandular units
producing at the same time thickening of the lamina propria.
The loss of the glandular compartment can occur as massive process as a result of
erosions or ulcerations or in a fragmented and dispersed way as a result of a long course
inflammatory process.
According with this definition, an international group of gastrointestinal pathologists
reviewed the histological spectrum of atrophic changes into a formal classification:
Nonmetaplastic. Shrinkage or complete disappearance of glandular units, native to the
region from which the biopsy was obtained, replaced by expanded, fibrotic lamina propria.
This situation leads to a substantial reduction of glandular mass in the absence of phenotypic
changes. When a H. pylori-associated severe inflammation obscures the gland’s population, it
is impossible to assess the mucosal atrophy. Therefore it is more appropriate to defer the final
judgment at the time of resolution of inflammation, when it is possible to observe more
carefully the details of the interglandular spaces and the glandular architecture.
Metaplastic. Replacement of the native glands by metaplastic glands featuring a new
commitment. There are two main types of gastric gland metaplasia, intestinal and/or
pseudopyloric metaplasia. Pseudopyloric metaplasia (or spasmolytic polypeptide-expressing
metaplasia [SPEM]) of the oxyntic epithelia is characterized by antral-like mucosa obtained
from what was anatomically corpus mucosa [41]. The total glandular volume can not be
reduced but the volume of the naïve glands is significantly lower having been replaced by

metaplastic glands resulting in fewer glandular structures. Such condition is consistent with
the definition of loss of appropriate glands.
Metaplasia is the transformation of a mature epithelium in another equally mature
epithelium that shares its ontogenetic origin from the same embryonic layer, suggesting loss
of the appropriate glandular population and therefore atrophy. The variation in phenotypic
expression is not due to a genotypic variation but it is induced by a change in environmental
stimulation. The metaplastic process does not produce structural damage, but rather a
functional damage due to the loss of the typical characteristics of that tissue. Excessive
exposure of metaplastic tissue to stress may give rise to alterations in the mitotic replication
with genomic damage such as mutation, deletion.
Chronic inflammation by H. pylori infection, affects differentiation and promotes
metaplasia. Several studies identified cellular and molecular mechanisms in SPEM.
Researchers have also begun to identify signaling pathways and events that take place during
embryonic development that establish the adult stem cells to maintain the specific features
and functions of the stomach [43].
Evidence suggests that mature chief cells in the adult mammalian stomach are post-
mitotic, and they arise via a relatively long-lived progenitor, the mucous neck cell. The
differentiation of chief cells from neck cells does not involve cell division, and the neck cell
has its own distinct pattern of gene expression and putative physiological function. Thus, the
ontogeny of the normal chief cell lineage exemplifies trans-differentiation. Furthermore,
under pathophysiogical loss of acid-secreting parietal cell, the chief cell lineage can itself
transdifferentiate into a mucous cell metaplasia designated SPEM.
Especially in the presence of inflammation, this metaplastic lineage can regain
proliferative capacity and, in humans may also further differentiate into intestinal metaplasia.
The gastric fundic lineages display remarkable plasticity in both physiological ontogeny and
pathophysiological pre-neoplastic metaplasia [44].
Thus, intestinal metaplasia (IM) may arise in native (antral) muco-secreting epithelia or
in previously-antralized oxyntic glands (pseudo-pyloric metaplasia) [41].
Metaplastic intestinal glands are constituted of goblet cells and absorptive cells, the latter
either with or without a brush border. It is important to differentiate mucosa with true atrophy
from that of the antrum-body transition zone, where pyloric-type glands may be mixed with
oxyntic glands at the base of the mucosa, and from normal fundus-cardia transition mucosa,
where mucous-type glands may also be found in association with oxyntic glands.
From here stems the importance of accurate labeling of samples as it allows to recognize
and differentiate a metaplastic condition from an error sampling. In this case,
immunohistochemistry can help because type I pepsinogen is found only in the oxintyc
mucosa and not in the metaplastic one, whereas immunohistochemical staining for gastrin
positive endocrine cells can help identify the mucosa derived from the antrum.
Moreover, the blood levels of pepsinogen I (PGI) become progressively elevated as the
loss of oxyntic glands advances. Pepsinogen II (PGII) is secreted by foveolar glands in the
mucosa of gastric antrum and body. Its secretion is stimulated by inflammation (such as H.
pylori infection) and cellular proliferation, both hyperplastic and neoplastic. PGI/PGII is a
good indicator of atrophy and to some extent of precancerous lesions [45].

Intestinal Metaplasia
Intestinal metaplasia (IM) is a phenotypic change due to the replacement of gastric

mucinous epithelial cells with goblet cells, enterocytes and colonocytes and it is easily
detected in histopathologic analysis, based on the markedly different cellular organization and
histochemical staining patterns of gastric and intestinal epithelia. Intestinal metaplasia is
considered to be an advanced stage of atrophy because the metaplastic glands replace the
original glands and chronologically the metaplastic glands appear after the gastric glands are
lost. Different subtypes of IM have been classified, on the basis of morphology and enzyme
histochemistry into small intestinal and colonic types or complete and incomplete forms and
using mucin histochemistry into three main types according to its morphology and
glycoprotein content.
- type I corresponds to complete metaplasia. Normal small intestinal epithelium
containing goblet cells, that produce sialomucins, are interspersed among absorptive
enterocytes with eosinophilic cytoplasm (expressing the complete set of digestive enzymes
such as sucrase and trehalase) and a ‘brush border’ given by large numbers of apical
microvilli. Paneth cells may also been observed. The change does not appear to be abrupt but
is progressive, as seen in the changing pattern of mucus secretion. The normal mucins of the
stomach, MUC5AC at the surface and MUC6 in deeper glands, are pH neutral, and stained
magenta with the periodic acid Schiff reagent. In IM, acid mucins are observed with Alcian
blue staining at pH 2.5, mostly sialic MUC2, and may be seen in the cytoplasm together with
neutral mucins. Other metaplastic cells express only sialic acid mucins [45]. As the
metaplastic changes advance and cover larger areas of the mucosa, new phenotypes are
observed in some areas.
- in type II, a disorderly mixture of sialomucin-containing goblet cells are scattered
among gastric-type cells containing either neutral mucin or sialomucins.
- type III, is characterized by tortuous and branched crypts lined by tall columnar cells
containing abundant sulfomucins with smaller numbers of goblet cells containing either
sialomucins or sulfomucins [38].
Both type II and type III are classified as incomplete or colonic type metaplasia because
they resemble the large bowel phenotype in morphology and mucin expression, and because
the set of digestive enzymes disappear partially or completely.
Further, some patients may also re-express gastric (neutral) mucins. Incomplete
metaplastic cells, like the normal colon epithelial cells, do not display a brush border and their
mucin droplets are multiple and of variable size and shape. Gastric biopsy specimens with IM
frequently contain foci of both complete and incomplete metaplasia (mixed metaplasia) [45].
Consistent data show that the extent of intestinalization of gastric mucosa and the
histochemical demonstration of type II–III IM (colonic-type metaplasia) are associated with
an increased risk of gastric cancer [41]. However from a practical point of view, the definition
of the precise type of metaplasia in any single individual is limited by the fact that in
extensive sampling are always present, though to differing degrees, both types of complete or
incomplete metaplasia.
The degree of incomplete IM goes together to the extension of IM in general. Thus, there
is a positive correlation between both the degree of incomplete IM, and the degree of IM in
general, and the risk of progression to carcinoma.

The extension of atrophic/metaplastic changes is another determinant of gastric cancer

risk.
The presence and extent of IM can also be easily evaluated with the use of specific mucin
histochemical stains, such as Alcian blue/periodic acid–Schiff stain at pH 2.5. In routine
histology, subtyping IM by applying specific histochemical stains is not recommended and
have been largely replaced by immunohistochemical stains that identify proteins associated
with particular mucin-encoding genes. Although more than 20 such mucin (MUC) genes have
been identified, in practice, only a few (MUC1, MUC2, MUC5AC, and MUC6) are used
routinely, and even those are used mainly in research settings.
Because H. pylori does not normally adhere to intestinal-type epithelium, and because the
organism usually disappears in mucosa with extensive IM and atrophy, one theory is that IM
represents a host defense against H. pylori infection. Furthermore, changes in the composition
of the gastric mucus in intestinalized epithelium may provide an additional source of defense
against H. pylori, or alternatively, it may represent a type of physiologic adaptation to altered
bacterial flora. The clonal nature of glands with IM is debated. A recent study has suggested
that gastric IM is the result of a mutation and the metaplastic glands are spread in the mucosa.
In addition, there is also evidence in support of the clonal origin of gastric dysplasia from
metaplasia [45].
Although IM causes changes in stem and progenitor cells, it is not clear whether native
gastric stem cells are the initial source of the changes and metaplasia results from their
reprogramming into an intestinal type or if differentiated gastric cells first acquire intestinal
properties and then stem cell properties.
The stomach epithelium of mice converts readily into the intestinal type on transgenic
expression of CDX2, a transcription factor that regulates intestinal development and
differentiation. This observation indicates that intestinalization of gastric stem cells might be
the initiating event in intestinal metaplasia [43].
Other Histological Features

(Nongraded Variables)
Surface Epithelial Damage, Mucous Depletion, and Erosions
The presence of erosions or ulcerations may depend on the production of particular type
of H. pylori cytotoxins, which also increase the risk of peptic ulcer disease. The cytotoxins
are also responsible for recall and degranulation of PMNs that are damage effectors and are
directly related to epithelial damage. In cases of slight damage, the gastric foveolar cells show
depletion of the mucus contents that drops out from apical portion of the cells.
As the damage advances, in parallel to the increase of the inflammatory infiltrate, there is
initially the appearance of erosions, not extend beyond the muscularis mucosae, and later
ulcers, as this boundary is exceeded.
At the margins of the erosion there is hyperplastic, regenerative-appearing epithelium,
which should be distinguished from dysplastic changes, covered by a superficial layer of
fibrinoid necrosis containing neutrophils and cellular debris.

Platelet aggregation mediated by an H. pylori interaction with von Willebrand factor

contributes to the pathogenesis of H. pylori-associated peptic ulcer disease through the
formation of thrombi and ensuing alteration of the local microcirculation [46].
Foveolar Hiperplasia
Following the increase of apoptosis and under the response to the increased production of
cytokines or other inflammatory mediators, there is an increased length and tortuosity of the
foveolae combined with expansion of the proliferative compartment and nuclear
hyperchromatism, increase of mitoses of glandular pit and in nuclear size compared to the
mucin-depleted cytoplasm. [38]. An atypical regeneration of the glandular neck and/or
expansion of the glandular proliferative compartment may make it difficult to differentiate
regenerative from dysplastic lesions (the so-called indefinite for non-invasive neoplasia
lesions) [41].
Lymphoid Follicles
The gastric mucosa, which under normal conditions is devoid of lymphoid tissue,
acquires and develops lymphoid tissue, organized in follicles, as a specific response to
infection by H. pylori.
There is sampling error in determining their prevalence, but if sufficient biopsy
specimens are examined, they are found in 100% of H. pylori positive cases, being more
frequent in antral than in corpus specimen [37].
Lymphoid follicles in a Helicobacter-negative case suggest that the organisms have been
missed or that the infection has been cleared. If large or irregularly shaped lymphoid follicles
are noted or large portions of the mucosa are occupied by a dense population of lymphocytes,
the possibility of a mucosa-associated lymphoid tissue (MALT) lymphoma should be
considered [38]
Pseudopyloric Metaplasia
Pseudopyloric metaplasia is the consequence of a gastric corpus protracted damage over

time.
This determines the loss of parietal and chief cells and their transformation in normal
antral appearing glands. It is associated with increased cell proliferation and as consequence
alters the balance between enzyme- and mucus-secreting cells.
Parietal cells are responsible for the secretion of a number of factors, including
amphiregulin, transforming growth factor (TGF)-α, heparin binding-epidermal growth factor-
like growth factor (HB-EGF), and sonic hedgehog (Shh). Loss of parietal cell-derived
signaling molecules disrupts the proper differentiation of other lineages such as the zymogen-
secreting chief cells and may induce the transdifferentiation of mature chief cells into SPEM
[47, 48]. As mentioned above, it is important to distinguish between true antral and

metaplastic glands also with the use of immunohistochemistry especially when the site of
origin of a gastric biopsy is uncertain [38].
Some studies suggest that gastric tumorigenesis might have a stronger correlation with
SPEM than with intestinal metaplasia, although these metaplasias often occur together. Also,
SPEM is by definition a corpus lesion, whereas cancer is believed to arise more commonly in
the antrum or in the transitional area between antrum and corpus.
However, because SPEM resembles the antrum histologically, cancers arising in corpus
tissue that converted to SPEM might appear to pathologists to arise in the antrum; this would
result in overestimation of the prevalence of antral tumors. Parietal cell loss in humans
correlates with SPEM and rapidly expands the proliferative compartment in the isthmus,
including cells that are morphologically indistinguishable from normal granule-free
presumptive stem cells.
It is unclear if this change in stem cell behavior is associated with permanent or transient
changes in gene expression as far as it is unclear too what proportion of the SPEM cells arise
de novo, from altered stem cells, or from trans-differentiation of chief or even mucous neck
cells [43].
Pancreatic (Acinar) Metaplasia
The pancreatic metaplasia is characterized by the presence of pancreatic glands collected

in nests or lobules, sometimes in combination with intestinal metaplasia during chronic
gastritis. The pancreatic acinar-like cells are characterized by abundant cytoplasm, acidophilic
and finely granular in the apical and middle portions and basophilic in the basal compartment
[38].
Endocrine Cell Hiperplasia
Endocrine cell hyperplasia occurs mainly as a consequence of functional changes in

severe chronic gastritis or in autoimmune gastritis. In some populations, H. pylori triggers an
autoimmune gastritis of the corpus mucosa characterized by presence of autoantibodies
against the subunits of the gastric H+/K+-ATPase in the parietal cells. The presence of these
autoantibodies is associated with a higher degree of body gastritis, increased apoptosis in the
glandular epithelium and atrophy of the corpus mucosa.
The following hypochlorhydria or achlorhydria can leads to G-cell hyperplasia in the
antrum and in hyperplasia of histamine-producing enterochromaffin-like (ECL) cells of the
gastric body [44]. The use of immunostains such as for chromogranin, synaptophysin or
specific hormones could help to focus the type of endocrine lesion.
The ECL cell hyperplasia is typically seen as widespread chains of argyrophilic cells
(termed linear hyperplasia) within the epithelium of the oxyntic glands or their atrophic or
metaplastic replacements.
Hyperplastic micronodules (micronodular hyperplasia) of endocrine cells in the lamina
propria can be also associated that are derived from residual endocrine cells within markedly
atrophic glands [38].

The most widely used criteria for the diagnosis and classification of gastric endocrine cell
proliferation are those proposed by Solcia et al. that distinguish simple/diffuse, linear,
micronodular, and adenomatoid hyperplasia, dysplasia, and neoplasia (well-differentiated
endocrine tumors; i.e. Type I carcinoids) [49].
Helicobacter pylori Infection Sequelae

Peptic Ulceration (See Also Chapter 8)
Peptic ulceration is a mucosal break or loss of substance of the mucosa (crater) that
always exceeds the muscularis mucosa, sometimes even reaching the muscularis propria,
prevalent in the stomach or in the duodenal bulb [50]. The H. pylori infection is the main
cause of peptic ulcer disease, gastric and duodenal ulcer. The other major cause are
nonsteroidal anti-inflammatory drugs (NSAIDs) or associated low dose aspirin in subject with
heart disease [51]. The peptic ulcer disease, both gastric and duodenal ulcers, is a chronic
disease whose natural history is characterized by scarring, even spontaneous, and subsequent
recurrence and the occurrence of complications, such as bleeding and perforation.
Patients with gastric and duodenal ulcer have a different pattern of topographic
distribution of chronic gastritis associated with a different alteration of acid secretion. The
duodenal ulcer is associated with antral chronic gastritis without atrophy of the oxyntic acid-
secreting glands and with acid hypersecretion, while the gastric ulcer is mainly associated
with corpus chronic gastritis or multifocal chronic atrophic gastritis with atrophy of the
oxyntic glands resulting in hypo-acid secretion [52].
H. pylori determines an increase in the serum levels of gastrin through a reduction in the
concentration of somatostatin in the antral mucosa. Somatostatin, a hormone produced by D
cells, inhibits, the gastrin release from the gastrin-secreting G cells by paracrine action.
Moreover the urease activity that protect H. pylori from the acid enviroment, is responsible,
in the same time, for the impaired D cells detection of true levels of acidity leading to
inappropriate release of somatostatin and an increase in gastrin, and consequently excess acid
secretion. The trophic effect of hypergastrinaemia induced by H. pylori also induces
hyperplasia of the ECL and acid-secreting parietal cells.
Another determining factor in etiopathogenesis of duodenal ulcer is the reduction of
duodenal secretion of bicarbonate. The reduced secretion of bicarbonate in the duodenal
mucosa is definitely tied to H. pylori infection as the eradication of infection is associated
with a restoration of normal duodenal bicarbonate secretion. How H. pylori alters the
secretion of bicarbonate is still not clear. The combination of gastric acid hypersecretion and
reduced secretion of duodenal bicarbonates determines the development of bulbar gastric
metaplastia as a response to the increased duodenal acid load.
Additional factors with ulcerogenic potential are released, including platelet activating
factor and components of the complement pathway [47].

Non Invasive Neoplasia and Gastric Cancer (See Also Chapter 8)
Non-Invasive Neoplasia (Formerly Known As Dysplasia; Synonym:

Intraepithelial Neoplasia)
Correa in 1975 proposed a model of gastric carcinogenesis. The model was updated in
1988 and 1992. It postulated the sequence normal gastric mucosa, non-atrophic gastritis,
multifocal atrophic gastritis without intestinal metaplasia, intestinal metaplasia of the
complete (small intestine) type, intestinal metaplasia of the incomplete (colonic) type, low-
grade noninvasive neoplasia, high-grade noninvasive neoplasia, and finally invasive
adenocarcinoma of intestinal type after H. pylori infection [53].
Intestinalization of gastric units, called intestinal metaplasia, phenotypic antralization of
fundic units, called SPEM, and the development directly from the stem/progenitor cell zone
are three pathways that have been described for gastric carcinogenesis [54]. Oxyntic atrophy
has a profound effect on the gastric mucosa because parietal cells play an important role in
the differentiation of other gastric lineages.
The second major result of chronic infection is prominent inflammation throughout the
mucosa. Oxyntic atrophy with prominent inflammation appears to be the prerequisites for
progression to metaplasia and gastric adenocarcinoma [55].
At a later stage outbreaks of dysplasia begin to appear, currently called intraepithelial
neoplasia or noninvasive neoplasia. It is characterized by the simultaneous presence of both
cytological and architectural abnormalities. The nuclei are more voluminous than normal, the
nucleus / cytoplasm ratio tends to be reduced in a more significant way with the increase of
the degree of dysplasia. The nuclei assume an oval shape, are hyperchromatic and the nuclear
membrane thickens. Nucleoli appear, abnormal mitosis and glands are getting longer, tortuous
and sometimes have branching or cribriform features. The stroma interposed between the
glands is reduced and the glands appear a back to back.
These changes may display a gradual transformation from well differentiated to poorly
differentiated and have been classified as low grade or high grade, reflecting the cancer risk
of each phenotype. Both low and high grade intraepithelial neoplasm are confined within the
basal membrane. The continuity/ integrity of the basal membrane separates the neoplastic
epithelia from the lamina propria. This topographical separation rules out the stromal invasion
required for any metastatic spread [41].
Several classifications of gastric dysplasia exist.
The Vienna Classification [56] applies the same nomenclature to precancerous lesions
of the entire gastrointestinal tract and is not specific to the stomach. The recognized
categories are: 1, Negative for dysplasia; 2, Indefinite for dysplasia; 3, Non invasive low
grade neoplasia; 4, Non invasive high grade neoplasia, further subdivided in high grade
adenoma/dysplasia - non invasive carcinoma and -suspicion of invasive cancer; 5, Invasive
neoplasia.
The Padua classification [57] is focused on gastric dysplasia and was developed by an
international group of experienced gastrointestinal pathologists after extensive review and
discussion of gastric biopsies from Europe, Canada, the USA and Japan. The Padua
classification recognizes five categories of lesions, utilizing mostly western nomenclature and
grouping them numerically following the prevailing Japanese system: 1, negative for
dysplasia; 2, indefinite for dysplasia; 3, noninvasive neoplasia (sub-classified in low grade or
high grade); 4, suspicious for invasive carcinoma; 5, invasive adenocarcinoma. This
classification was designed to improve communications, especially among pathologists and

clinicians, by clearly explaining and discussing the possible interpretation and management of
each category [53].
The mean differences between these classifications are from a clinical and a biological
point of view.
From a clinical point of view the classification of Padua includes intraepithelial neoplasia
into 2 subclasses which gather both low and high grade NIN, where the classification of the
Vienna considers two distinct entities and keeps them separate.
From a biological point of view the classification of Padua assembles entities of similar
biological behavior while in Vienna classification are reported in a single point 2 different
biological realities as low-grade non-invasive neoplasia and suspicious for invasive
carcinoma.
The final stage of the process is the overcoming of the basement membrane with invasion
of the lamina propria (intramucosal adenocarcinoma) and then by the overrunning beyond the
muscularis mucosae into the submucosa (early gastric cancer). The early gastric carcinoma is
classified both endoscopically and microscopically [58, 59]. The end stage is advanced gastric
carcinoma, an intestinal type, according WHO that infiltrates the next anatomical structures
till to the serosa.
Gastric MALToma (See Also Chapter 8 and 14)
Gastric (Mucosa Associated Lymphoid Tissue) MALT lymphoma is an extranodal

lymphoma originating in the stomach, composed of morphologically heterogeneous small B
cells. These include marginal zone cells of intermediate size with pale cytoplasm and an
irregular nucleus (centrocyte-like cells; CCL), or small lymphocytes with irregular nuclear
contour, nuclear clefting, hyperchromasia, and with scant to fair amount of cytoplasm cells
resembling monocytoid cells, scattered immunoblasts and centroblast-like cells, with
plasmacells. Plasmacells differentiation is typical and may be very prominent. Dutcher bodies
may be identified.
This lymphoma originates in the marginal zone of reactive follicles located in the lamina
propria, and it extends in the interfollicular region and then infiltrates the gastric glandular
epithelium, giving rise to the lymphoepithelial lesions. Lymphoepithelial lesions typical for
MALT lymphoma are defined as infiltration of the glandular epithelium by clusters of
neoplastic lymphoid cells with associated destruction of gland architecture and morphological
changes within the epithelial cells, including increased eosinophilia [58].
Reactive germinal centers, common in the deeper mucosa associated with H. pylori
gastritis, may be colonized by lymphoma cells, with obliteration of mantle zone and the
appearance of so-called “naked” follicles. The atypical lymphoid infiltrate usually expands
the lamina propria or submucosa. Muscularis mucosae infiltration and disruption can be a
useful clue to the diagnosis in small biopsy specimens.
In more extensive cases, the lymphoma can create mucosal ulcers and can infiltrate
through the muscularis propria [60].
The immunophenotype of the CCL cell is similar to that of the marginal zone B-cell.
There is an expression of pan-B-cell antigens such as CD20 and CD79a. They are usually
positive for bcl-2 protein but negative for CD10, CD5, or CD23. They express the surface

cytoplasmic immunoglobulins (usually IgM or IgA, rarely IgG) and show light chain
restriction. Immunostaining with anti-cytokeratin antibodies is useful in demonstrating
lymphoepithelial lesions.
Immunostaining with antibodies that highlights follicular dendritic cells (anti-CD21, anti-
CD23, or anti-CD35) helps to demonstrate underlying follicular dendritic cell networks in
those cases in which the lymphoid follicles have been completely overrun by the lymphoma
[58].
However, the density and the detectability of H. pylori infection decreases as the
lymphoma evolves from chronic gastritis. The frequency of H. pylori positivity is higher in
patients with lymphoma restricted to the mucosa and submucosa, as high as that observed in
patients with chronic active gastritis and peptic ulcer, than in those with lymphoma invading
beyond the submucosa, and is also higher in patients with low grade MALT lymphoma than
in those with high grade tumors [61].
In 1993 Wotherspoon [62] designed a five tiers scale that helps to disentangle
pathologists in the diagnosis of gastric lymphoproliferative disorders. In lymphomas the
lymphoid infiltrate diffusely concerns the most amount of one or more of adequate size
biopsies, involving the full thickness of gastric mucosa, exceeding the muscularis mucosae,
the glandular component largely obscured, distorted and deleted by lymphoid infiltrate. These
histological criteria are commonly used by pathologists for initial diagnoses, but they have
not been adopted uniformly for the post-treatment evaluation of gastric MALT lymphoma.
In 2003, the Groupe d’Etude des Lymphomes de l’Adult (GELA) proposed a new
histological grading system (GELA grade) for post-treatment evaluation. [63]. The GELA
scoring system identifies the morphologic patterns observed after eradication in four
categories:
Complete histological response (CR): Absent or scattered plasma cells and small
lymphoid cells in the lamina propria. Regressive stromal changes may also be present, such as
fibrosis resulting in areas of lamina propria devoid of glands (empty lamina propria).
Probable minimal residual disease (pMRD): Aggregates of lymphoid cells or lymphoid
nodules in the lamina propria/muscolaris mucosae and/or submucosa associated with
regressive stromal changes. The biological significance of this minimal lymphoid population
is uncertain, but it probably has no clinical significance. In some cases the same gene
rearrangement of the original lymphoma has been demonstrated, suggesting that it could
represent a true minimal residual disease [64].
Responding residual disease (rRD): Dense, diffuse or nodular lymphoid infiltrate,
extending around glands in the lamina propria associated with evident regressive stromal
changes. A comparison with the original histology is particularly helpful in this context.
No change (NC): Dense, diffuse or nodular lymphoid infiltrate is present, consistent with
that originally diagnosed, in the absence of stromal changes suggestive of response [65].
The Ann Arbor staging system, developed for and routinely used in nodal non-Hodgkin’s
lymphoma, is not optimal for documentation of the specific relevant features of primary
extranodal lymphoma in the gastrointestinal tract. For this reason, under the auspices of the
European Gastro-Intestinal Lymphoma Study Group (EGILS), the Paris Staging System has
been proposed by a multidisciplinary group of investigators. This staging system, is a peculiar
modification of the TNM, modulated on the biological behavior of lymphoma.
As the pTNM, the Paris Staging System takes into account the depth of tumour
infiltration of the wall of the gastrointestinal organ evaluated and adjacent organs (T), intra-

and extra-abdominal lymph node involvement (N), specific lymphoma spreading, non-
contiguous involvement (M) of separate site in gastrointestinal tract and of other tissues (eg,
peritoneum, pleura) or organs (i.e., tonsils, parotid gland, ocular adnexa, lung, liver, spleen,
kidney, breast etc.). In addiction bone marrow (B) involvement is also evaluated [66].
The diagnosis of gastric MALT lymphoma is frequently difficult for the general
histopathologist. During recent years there have been relevant changes in the therapeutic
approach to gastric MALT lymphoma and our knowledge about its pathogenesis has greatly
improved.
The management of this disease, as well as any H. pylori infection related disease,
actually requires a close cooperation between the histopathologist and the clinicians [64].
Rapid Urease Test

Urease is produced by several bacterial species, including normal flora and
nonpathogens, but also by some species such as H. pylori as a potent virulence factor. In fact,
urease plays a central role for H. pylori metabolism and virulence and it is necessary for its
colonization of the gastric mucosa. H. pylori is intensely antigenic and secretes various
factors beyond urease, like catalase, mucinase, lipase, hemolysin and alkaline phosphatase
that all decrease viscosity of mucus [67].
Scientists studied gastric urease enzyme from the 1920s. Murray Luck and colleagues
studied the urease enzyme of the gastric mucosa of dogs in 1924 and in 1950 Fitzgerald and
Murphy published a thesis about gastric urease in humans. Their data were obtained by
surgical specimen from gastrectomy and they measured urea levels in gastric juice and they
concluded that urea could be used as antiacid.
In the late 50s then Lieber and Lefevre studied the gastric urease of alcoholics and they
hypothesize a bacterial origin of gastric urease. For some years scientists lost interest in
gastric urease until the discovery that H. pylori synthesizes an extraordinary amount of
urease. This made possible the identification of a simple diagnostic test for detecting H. pylori
by showing presence of its urease enzyme in gastric tissue known by now as rapid urease tests
(RUT) [68]. The RUT was developed by Barry Marshall and was made commercially
available by Kimberly-Clark [67].
Once gastric biopsies are obtained, they are placed into an agar gel or on a reaction strip
containing urea, a buffer, and a pH-sensitive indicator (phenol red). In the presence of H.
pylori’s urease, urea is metabolized to ammonia and carbon dioxide leading to raise the pH,
and change the color of the phenol red from yellow to red. A change in color signifies the
presence of active infection [3].
Biopsies for RUT are usually obtained from the gastric antrum or body; it is still unclear
the sensitivity of the RUT to achieve a positive result on separated specimens rather than
combined tissues.
One study found that the diagnostic yield for detecting H. pylori by RUT was not
adversely affected by the size of the biopsy forceps, while one different study showed that
increasing the amount of tissue significantly hastens the development of positive tests [2].
Regarding the sites of biopsies some authors described that the angulus showed the highest
sensitivity for the detection of H. pylori as compared to other gastric sites [69].

There are many different commercial tests, but the overall performance are similar with
an overall pretreatment sensitivities of >90% and specificities of >95% [3] and usually cost
and availability are the prime determinants of which is used.
Unfortunately, the usefulness of the RUT in routine clinical practice has been
compromised by the widespread use of PPIs. In fact some medications such as bismuth-
containing compounds, antibiotics, or PPIs, reduce the density and/or urease activity of H.
pylori, and therefore they can decrease the sensitivity of the RUT by up to 25% [70].
Therefore RUT can rarely be used as a unique test to detect H. pylori infection, but
usually it is associated with other endoscopic or non-endoscopic diagnostic tests. No studies
have been performed to define the duration of a PPI’s effects on the sensitivity of the RUT.
Data with the urea breath test suggest that PPI therapy can cause false-negative test results for
1–2 weeks [71]. Therefore it is reasonable to suggest that PPIs should be stopped for 1–2
weeks before performance of the RUT. In this case the sensitivity of the RUT is likely
sufficient to justify its use as a single test for H. pylori [3].
In the case of patients with acute ulcer bleeding sensitivity and negative predictive value
of the RUT may decrease. For this reason a positive RUT during acute bleeding indicates the
presence of active H. pylori infection. On the other hand, a negative RUT in this setting needs
to be confirmed with another test [3].
RUT therefore is a simple, inexpensive, rapid and reliable method for detected urease
activity in gastric specimens.
Conclusion
There is no single test that can be considered the gold standard for the diagnosis of H.
pylori. Rather, the most appropriate test for any specific situation will be influenced by the
clinical circumstances, the pretest probability of infection, as well as the availability and costs
of the individual diagnostic tests.
Invasive biopsy-based tests are important in the assessment of H. pylori status pre-
treatment, and endoscopy allows assessment of treatment indications such as ulcer disease.
Histological examination of gastric biopsies performed in accordance with the Sidney
system criteria is an extremely sensitive method that characterizes the inflammatory damage
and identifies the mucosal alterations. Moreover it allows to recognize the different type of
gastric inflammation and as well as the pre-neoplastic lesions.
In the diagnosis of H. pylori infection, upper gastrointestinal endoscopy, when
performed, should always include an adequate number of biopsy specimens for obtaining an
accurate histological diagnosis.
References
[1] Malfertheiner, P., Megraud, F., O’Morain, C. A., et al. (2012). Management of
Helicobacter pylori infection – the Maastricht IV/Florence Consensus Report. Gut., 61:
646-664

[2] Glupczynski, Y., (1998). Microbiological and serological diagnostic tests for
Helicobacter pylori: an overview. Br. Med. Bull., 54(1):175-186
[3] Chey, W. D., Wong, B. C., (2007). Practice parameters committee of the American
College of Gastroenterology guidelines on the management of Helicobacter pylori
infection. Am. J. Gastroenterol., 102:1808-25.
[4] Redèen, S., Petersson, F., Tornkrantz, E., et al. (2011). Reliability of diagnostic tests
for Helicobacter pylori infection, Gastroenterol. Res. Pract., 940650. doi: 10.1155/
2011/940650. Epub. 2011 Aug. 1.
[5] ASGE Guidelines 2010, The role of Endoscopy in the management of patients with
peptic ulcer disease. Gastrintest. Endosc., 71 (4):663-68.
[6] Ikenberry, S. O., Harrison, M. E., Lichtenstein, D., et al. (2007). The role of endoscopy
in dyspepsia. Gastrointest Endosc., 66:1071e5.
[7] ASGE Guidelines, (2007). The role of endoscopy in dyspepsia, Gastrointest. Endosc.,
66 (6): 1071-75.
[8] Uyanikoğlu, A., Danalioğlu, A., Akyüz, F., et al. (2012). Etiological factors of duodenal
and gastric ulcers. Turk. J. Gastroenterol., 23(2):99-103.
[9] Grino, P., Pascual, S., Such, J., et al. (2001). Comparison of diagnostic methods for
Helicobacter pylori infection in patients with upper gastrointestinal bleeding. Scand. J.
Gastroenterol., 36:1254–8.
[10] Niv, Y., Niv, G., Koren, R., (2004). 13C-urea breath test for diagnosis of Helicobacter
pylori infection in the elderly. Dig. Dis. Sci., 49:1840e4.
[11] Pilotto, A., Franceschi, M., Longoa, M. G., et al. (2004). Helicobacter pylori infection
and the prevention of peptic ulcer with proton pump inhibitors in elderly subjects taking
low-dose aspirin. Dig. Liver. Dis., 36(10):666-70.
[12] Dixon, M. F., Genta, R. M., Yardley, J. H., et al. (1996). Classification and grading of
gastritis. The update Sydney system. Am. J. Surg. Pathol., 20: 1161-81.
[13] Wu, M. S., Shun, C. T., Lee, W. C., et al. (1998). Gastric cancer risk in relation to
Helicobacter pylori infection and subtypes of intestinal metaplasia Br. J. Canc., 78(1):
125-128.
[14] Dinis-Ribeiro, M., Areia, M., de Vries, A. C., (2012). Management of precancerous
conditions and lesions in the stomach (MAPS): guideline from the European Society of
Gastrointestinal Endoscopy (ESGE), European Helicobacter Study Group (EHSG),
European Society of Pathology (ESP), and the Sociedade Portuguesa de Endoscopia
Digestiva (SPED). Endoscopy., 44: 74–94.
[15] Vannella, L., Lahner, E., Osborn, J., et al. (2010). Risk factors for progression to gastric
neoplastic lesions in patients with atrophic gastritis. Aliment Pharmacol. Ther., 31:
1042–1050.
[16] Lahner, E., Bordi, C., Cattaruzza, M. S., et al. (2005). Long-term follow-up in atrophic
body gastritis patients: atrophy and intestinal metaplasia are persistent lesions
irrespective of Helicobacter pylori infection. Aliment Pharmacol. Ther., 22: 471–481.
[17] Abraham, S. C., Montgomery, E. A., Singh, V. K., et al. (2000). Gastric adenomas:
intestinal type and gastric type adenomas differ in the risk of adenocarcinoma and
presence of background mucosal pathology. Am. J. Surg. Pathol., 26: 1276–1285.
[18] Kim, Y. J., Park, J. C., Kim, J. H., et al. (2010). Histologic diagnosis based on forceps
biopsy is not adequate for determining endoscopic treatment of gastric adenomatous
lesions. Endoscopy., 42: 620–626.

[19] Laine, L., Sugg, J., Suchower, L., et al. (2000). Endoscopic biopsy requirements for
post-treatment diagnosis of Helicobacter pylori. Gastrointest Endosc., 51:664–9.
[20] Bah, A., Saraga, E., Armstrong, D., et al. (1995). Endoscopic features of Helicobacter
pylori-related gastritis. Endoscopy., 27: 593–6.
[21] Price, A. B., (1991). The Sydney system: histological division. J. Gastroenterol.
Hepatol., 6:209-22.
[22] Satoh, K., Kimura, K., Taniguchi, Y., et al. (1996). Distribution of inflammation and
atrophy in the stomach of Helicobacter pylori-positive and -negative patients with
chronic gastritis. Am. J. Gastroenterol., 91: 963–9.
[23] Sipponen, P., Price, A. B., (2011).The Sydney System for classification of gastritis 20
years ago. J. Gastroenterol. Hepatol., 26(1):31–34.
[24] Glupczynski, Y., Bourdeaux, L., De Prez, C., et al. Prevalence of Helicobacter pylori in
rural Kivu, eastern Zaire: A prospective endoscopic study. Eur. J. Gastroenterol.
Hepatol. 1991;3:449– 55.
[25] Kato, T., Yagi, N., Kamada, T., et al. (2013). Diagnosis of Helicobacter pylori infection
in gastric mucosa by endoscopic features: A multicenter prospective study Dig.
Endosc., Jan. 29. doi: 10.1111/den.12031.
[26] Cho, J. H., Chang, Y. W., Jang, J. Y., et al. (2013). Close observation of gastric
mucosal pattern by standard endoscopy can predict Helicobacter pylori infection status.
J. Gastroenterol. Hepatol., 28:279–284.
[27] Khakoo, S. I., Lobo, A. J., Shepherd, N. A., et al. (1994). Istological assessment of the
Sydney classification of endoscopic gastritis. Gut., 35:1172–5.
[28] Ohkusa, T., Fujiki, K., Takashimizu, I., et al. Endoscopic and Histological Comparison
of Nonulcer Dyspepsia With and Without Helicobacter pylori Infection Evaluated by
the Modified Sydney System AJG 2000;95(9):2195-99.
[29] Laine, L., Cohen, H., Sloane, R., et al. (1995). Interobserver agreement and predictive
value of endoscopic findings for H. pylori and gastritis in normal volunteers.
Gastrointest. Endosc., 42: 420–3.
[30] Redéen, S., Petersson, F., Jönsson, K. A., et al. (2003). Relationship of gastroscopic
features to histological findings in gastritis and Helicobacter pylori infection in a
general population sample. Endoscopy., 35: 946–50.
[31] Da Graça Soares Bahú, M., Reverbel da Silveira, T., Maguilnick, I., et al. (2003).
Endoscopic Nodular Gastritis: An Endoscopic Indicator of High-Grade Bacterial
Colonization and Severe Gastritis in Children With Helicobacter pylori. J. Pediatr.
Gastroenterol. Nutr., 36 (2):217-222.
[32] Yagi, K., Honda, H., Yang, J. M., et al. (2005). Magnifying endoscopy in gastritis of
the corpus. Endoscopy., 37: 660–6.
[33] Yan, S. L., Wu, S. T., Chen, C. H., et al. (2010). Mucosal patterns of Helicobacter
pylori-related gastritis without atrophy in the gastric corpus using standard endoscopy.
World. J. Gastroenterol., 16: 496–500.
[34] Nomura, S., Terao, S., Adachi, K., et al. (2013). Endoscopic diagnosis of gastric
mucosal activity and inflammation. Dig. Endosc., 25: 136–146.
[35] Carr, N. J., Leadbetter, H., Marriott, A., (2012). Correlation between the endoscopic
and histologic diagnosis of gastritis. Ann. Diagn. Pathol., 16(1):13-5.

[36] Carpenter, H. A., Talley, N. J., (1995). Gastroscopy Is Incomplete Without Biopsy:
Clinical Relevance of Distinguishing Gastropathy From Gastritis. Gastroenterology.,
108:917-924.
[37] Genta, R. M., Graham, D. Y., (1994). Comparison of biopsy sites for the
histopathologic diagnosis of Helicobacter pylori: a topographic study of H. pylori
density and distribution. Gastrointest. Endosc., 40(3):342-5.
[38] Dixon, M. F., Genta, R. M., Yardley, J. H., Correa, P., and the Participants international
workshop on the histopatology of gastritis, (1996). Classification and grading of
gastritis. The Updated Sidney System. Am. J. Surg. Pathol., 20(10): 1161-1181.
[39] Rugge, M., Meggio, A., Pennelli, G., Piscioli, F., Giacomelli, L., De Pretis, G.,
Graham, D. Y., (2007). Gastritis staging in clinical practice: the OLGA staging system.
Gut., 56(5):631-6.
[40] Rugge, M., de Boni, M., Pennelli, G., de Bona, M., Giacomelli, L., Fassan, M., Basso,
D., Plebani, M., Graham, D. Y., (2010). Gastritis OLGA-staging and gastric cancer
risk: a twelve-year clinico-pathological follow-up study. Aliment. Pharmacol. Ther., 31
(10):1104-11.
[41] Davies, G. R., Banatvala, N., Collins, C. E., Sheaff, M. T., Abdi, Y., Clements, L.,
Rampton, D. S., (1994). Relationship between infective load of Helicobacter pylori and
reactive oxygen metabolite production in antral mucosa. Scand. J. Gastroenterol., 29
(5):419-24.
[42] Rugge, M., Pennelli, G., Pilozzi, E., Fassan, M., Ingravallo, G., Russo, V. M., Di
Mario, F., (2011). Gastritis: the histology report. Dig. Liver. Dis., 43 Suppl. 4:S373-84.
[43] Peek Jr, R. M., Fiske, C., Wilson, K. T., (2010). Role of Innate Immunity in
Helicobacter pylori-Induced Gastric Malignancy. Physiol. Rev., 90(3): 831–858.
[44] Mills, J. C., Ramesh, A. S., (2011). Gastric Epithelial Stem Cells. Gastroenterology.,
140:412–424.
[45] Goldenring, J. R., Nam, K. T., Mills, J. C., (2011). The origin of pre-neoplastic
metaplasia in the stomach: Chief cells emerge from the Mist. Exp. Cell. Res., 15; 317
(19): 2759–2764.
[46] Correa, P., Piazuelo, M. B., (2012). The gastric precancerous cascade. J. Dig. Dis., 13
(1): 2–9.
[47] Byrne, M. F., Kerrigan, S. W., Corcoran, P. A., Atherton, J. C., Murray, F. E.,
Fitzgerald, D. J., Cox, D. M., (2003). Helicobacter pylori binds von Willebrand factor
and interacts with GPIb to induce platelet aggregation. Gastroenterology., 124(7):1846-
54.
[48] Weis, V. G., Sousa and Goldenring, J. R. (2009). Current understanding of SPEM and
its standing in the preneoplastic process. Gastric. Cancer., 12: 189–197.
[49] Nam, K. T., Lee, H. J., Sousa, J. F., Weis, V. G., O'Neal, R. L., Finke, P. E., Romero-
Gallo, J., Shi, G., Mills, J. C., Peek, R. M. Jr, Konieczny, S. F., Goldenring, J. R.,
(2010). Mature chief cells are cryptic progenitors for metaplasia in the stomach.
Gastroenterology., 139(6):2028-2037.
[50] Solcia, E., Fiocca, R., Villani, L., Luinetti, O., Capella, C., (1995). Hyperplastic,
dysplastic, and neoplastic enterochromaffin-like-cell proliferations of the gastric
mucosa. Classification and histogenesis. Am. J. Surg. Pathol., 19 (Suppl. 1):S1-7.

[51] Iwamoto, J., Saito, Y., Honda, A., Matsuzaki, Y., (2013). Clinical features of
gastroduodenal injury associated with long-term low-dose aspirin therapy. World J.
Gastroenterol. 21;19(11):1673.
[52] Suerbaum, S., Michetti, P., (2002). Helicobacter pylori infection. N Engl. J. Med.; 347
(15): 1175-86.
[53] Correa, P., Piazuelo, M. B., (2008). Natural history of Helicobacter pylori infection.
Dig. Liver. Dis., 40(7):490-6.
[54] Gomceli, I., Demiriz, B., Tez, M., (2012). Gastric carcinogenesis World J.
Gastroenterol. 7; 18(37): 5164-5170
[55] Weis, V. G., Goldenring, J. R., (2009). Current understanding of SPEM and its standing
in the preneoplastic process. Gastric Cancer 12: 189–197
[56] Schlemper, R. J., Riddell, R. H., Kato, Y., Borchard, F., Cooper, H. S., Dawsey, S. M.,
Dixon, M. F., Fenoglio-Preiser, C. M., Fléjou, J.-F., Geboes, K., Hattori, T., Hirota, T.,
Itabashi, M., Iwafuchi, M., Iwashita, A., Kim, Y. I., Kirchner, T., Klimpfinger, M.,
Koike, M., Lauwers, G. Y., Lewin, K. J., Oberhuber, G., Offner, F., Price, A. B., Rubio,
C. A., Shimizu, M., Shimoda, T., Sipponen, P., Solcia, E., Stolte, M., Watanabe, H.,
Yamabe, H., (2000). The Vienna classification of gastrointestinal epithelial neoplasia.
Gut., 47:251–255
[57] Rugge, M., Correa, P., Dixon, M. F., Hattori, T., Leandro, G., Lewin, K., Riddell, R.
H., Sipponen, P., Watanabe, H., (2000). Gastric Dysplasia: The Padova International
Classification. Am. J. Surg. Pathol., 24(2): 167–176
[58] Bosman, F. T., Carneiro, F., Hruban, R. H., Theise, N. D., (2010). WHO Classification
of Tumours of the Digestive System, Fourth Edition, IARC-WHO Classification of
Tumours, No 3
[59] Kodama, Y., Inokuchi, K., Soejima, K., Matsusaka, T., Okamura, T., (1983). Growth
patterns and prognosis in early gastric carcinoma. Superficially spreading and
penetrating growth types. Cancer. 15;51(2):320-6.
[60] Bautista-Quach, M. A., Ake, C. D., Chen, M., Wang, J., (2012). Gastrointestinal
lymphomas: Morphology, immunophenotype and molecular features. J. Gastrointest
Oncol. 3(3):209-25.
[61] Nakamura, S., Yao, T., Aoyagi, K., Iida, M., Fujishima, M., Tsuneyoshi, M., (1997).
Helicobacter pylori and primary gastric lymphoma. A histopathologic and
immunohistochemical analysis of 237 patients. Cancer., 79(1):3-11.
[62] Wotherspoon, A. C., Doglioni, C., Diss, T. C., Pan, L., Moschini, A., de Boni, M.,
Isaacson, P. G., (1993). Regression of primary low-grade B-cell gastric lymphoma of
mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori.
Lancet., 342(8871):575-7.
[63] Shiozawa, E., Norose, T., Kaneko, K., Yamochi-Onizuka, T., Takimoto, M., Imawari,
M., Ota, H., (2009). Clinicopathological comparison of the World Health Organization/
Wotherspoon score to the Groupe d’Etude des Lymphomes de l’Adult grade for the
post-treatment evaluation of gastric mucosa-associated lymphoid tissue lymphoma. J.
Gastroenterol. Hepatol., 24; 307–315
[64] Doglioni, C., Ponzonia, M., Ferreri, A. J. M., Savio, A., (2011). Gastric lymphoma: The
histology report. Dig. Liver Dis., 43 Supp. S310–S318.
[65] Copie-Bergman, C., Wotherspoon, A. C., Capellam, C., Motta, T., Pedrinis, E., Pileri,
S. A., Bertoni, F., Conconi, A., Zucca, E., Ponzoni, M., Ferreri, A. J. M., (2012). Gela

histological scoring system for post-treatment biopsies of patients with gastric MALT
lymphoma is feasible and reliable in routine practice Br. J. Haematol., 160, 47–52
[66] Ruskoné-Fourmestraux, A., Dragosics, B., Morgner, A., et al. (2003). Paris staging
system for primary gastrointestinal lymphomas. Gut., 52:912–3
[67] Siddique, I., Al-Mekhaizem, K., Alateeqi, N., (2008). Diagnosis of Helicobacter pylori:
improving the sensitivity of CLOtest by increasing the number of gastric antral
biopsies. J. Clin. Gastroenterol., 42:356-60.
[68] Mobley, H. L. T., Mendz, G. L., Hazell, S. L., (2001). Helicobacter pylori: physiology
and genetics. ASM Press.
[69] Laine, L., Chun, D., Stein, C., El-Beblawi, I., Sharma, V., Chandrasoma, P., (1996).
The influence of size or number of biopsies on rapid urease test results: a prospective
evaluation. Gastrointest. Endosc., 43: 49-53.
[70] Midolo, P., Marshall, B. J., (2000). Accurate diagnosis of Helicobacter pylori. Urease
tests. Gastroenterol. Clin. N Am., 29:871–8.
[71] Chey, W. D., Woods, M., Scheiman, J. M., et al. (1997). Lansoprazole and ranitidine
affect the accuracy of the 14 C-urea breath test by a pH-dependent mechanism. Am. J.
Gastroenterol., 92:446–50

Chapter 4
Unusual Techniques: Identification

of H. pylori from Biopsies: Culture,
PCR and FISH
Carina Almeida,1,2 Nuno F. Azevedo2 and Maria João Vieira2,

1
IBB, Institute for Biotechnology and Bioengineering, Centre of
Biological Engineering, Universidade do Minho, Braga, Portugal
2
LEPAE, Departamento de Engenharia Química, Faculdade de
Engenharia, Universidade do Porto, Porto, Portugal
Abstract
The detection of Helicobacter pylori (H. pylori) has been a topic of great interest
since the discovery of the bacterium, and, 30 years later, no consensual method has so far
been proposed. Clinically, non-invasive methods are preferred, with urea breath testing
being the current tests of choice; but biopsy-based tests are still considered by several
authors as the gold standard for the detection H. pylori infection. Culture is particularly
important for susceptibility testing, but its time consuming nature and low sensitivity,
have compromised its clinical application. On the other hand, molecular methods applied
to biopsy specimens have provided a valuable alternative for detecting resistance. Among
this molecular alternative, Polymerase chain reaction (PCR) and Fluorescence in situ
hybridization (FISH) are the most preeminent ones. Although they are still unusual
methods in clinical practice, they are gathering the medical community confidence. In
this chapter, three less common biopsy-based methods, culture, PCR and FISH, will be
reviewed. Their advantages, limitations and commercial application will be addressed.
Keywords: Helicobacter pylori diagnosis, FISH, PCR, culture, gastric biopsies

Corresponding author: Maria João Vieira. LEPAE, Departamento de Engenharia Química, Faculdade de
Engenharia, Universidade do Porto, rua Dr. Roberto Frias, 4200-465 Porto, Portugal. E-mail: mjv@deb.
uminho.pt.

70 Carina Almeida, Nuno F. Azevedo and Maria João Vieira
Introduction
There is no single test that can be considered as the gold standard for the diagnosis of
Helicobacter pylori (H. pylori). The appropriate test for any specific situation will be
influenced by the clinical circumstances, the pretest probability of infection, as well as the
availability and costs of the individual diagnostic tests [1]. Non-invasive tests are the most
usual methods for routine H. pylori detection, but they fail to provide complementary
information on the location of H. pylori in the stomach, on the histopathological lesions
underlying the presence of the bacteria and on the antimicrobial profile of the infecting strain.
Because of these limitations, it is generally considered that invasive tests provide a more
complete diagnosis. Gastric biopsy specimens, obtained by upper endoscopy, are commonly
analyzed by using histological and rapid urease tests, but while the first one is a non-specific
test, the second presents a reduced sensitivity in post-treatment settings [1]. Molecular
methods have emerged in recent years and offer now a valid alternative to these traditional
identification techniques. However, it seems that the lack of molecular products cleared by
the regulatory authorities might be hindering this transition. The availability of these
molecular alternatives in hospitals is still very limited, but their use at a research level is
already a reality. In a completely different position we can find the culture methods. Thirty
years after the original isolation of H. pylori [2], some laboratories still use essentially the
same techniques as those devised in 1982. Culture is a highly specific method for H. pylori
detection and is still the primary mean by which antibiotic sensitivities can be determined.
In this chapter we will focus on three unusual biopsy-based methods: culture, that is now
less common on clinical laboratories; PCR and FISH, which are being gradually introduced as
diagnostic tools in biopsy samples.
Culture
The culture of a freshly collected gastric biopsy specimen, taken during endoscopic
examination of the stomach, has been used for decades and some authors have considered it
as the gold standard for H. pylori infection. Culture is considered the most specific method
for diagnosing H. pylori infections [3, 4]. However, culture sensitivity is highly variable (60–
90%) [4], attributable to several methodological factors such as biopsy site, transport
medium, time from sampling to processing, culture medium and incubation conditions [5]. A
detailed description of the culture features and the procedure influence in the method
performed will be briefly reviewed in the next sections.
Collection of the Gastric Specimens
In untreated patients, H. pylori is commonly found in the antral part of human gastric
mucosa. In patients treated with acid-suppressive drugs (such as proton pump inhibitors and
H2 antagonists) the bacterium may be present at higher numbers in the body part of the
stomach [6]. Thus, it is always important to obtain both antral and corpus biopsies, since
patients with symptoms of gastric diseases are commonly treated with these drugs.

Unusual Techniques 71
The estimated number of biopsies necessary to diagnose H. pylori by culture changes

from study to another. It has been estimated that the culture of a single antral biopsy allows
the detection of the H. pylori in more than 90% of the cases, but for optimal results (at least
four) multiple biopsies should be cultured [4, 7]. According to the updated Sydney system [8]
at least one biopsy from antrum and two biopsies from corpus should be taken for culture to
ensure a correct diagnosis. To our knowledge, the collection procedure changes between
hospitals in terms of number of biopsies, but the collection of both antrum and corpus
biopsies is a common practice.
After the biopsies collection, the samples should be placed into a transport medium, a
step with a remarkable influence in the culture sensitivity. It is known that the type of
transport medium can influence the recovery rate of H. pylori [9], but the transportation time
seems to be the more critical. There are a number of transport medium available: skim milk,
Brucella, Brain Heart Infusion, Cysteine-Albimi, Stuart's transport medium, saline solution or
Portagerm pylori are some of the examples [9-15]. Reported data on stability and culture
results of fresh biopsy specimens in the different transport media, vary considerably. For
instance, reported recovery rates ranged from 77 to 94% to biopsy specimens transported in
Portagerm pylori [9, 10]; from 85 to 94% for Stuart's transport medium [11]; 40 to 66% for
saline solution [9]. Despite the importance of the medium, the transport time and temperature
are recognized as critical factors for ensuring high recovery rates. Heep et al. reported
recovery rates of 94, 87 and 77% for biopsy specimens in Portagerm pylori, in cooled
containers, after 1, 2, and 3 days of transport, respectively. In the same study a recovery rate
of 97% was obtained for storage and shipping in glycerol broth at −70°C. Other studies have
obtained recovery rates higher than 93.5% for fresh biopsies and 85 to 46% for those that
were frozen for 5 to 50 days [5, 11]. Similarly, some studies also have shown that a higher
transport temperature (about 20°C) decreases the number of positive cultures significantly
[12, 13].
As soon as the samples arrive to the clinical laboratories they should be processed
promptly. Biopsies are initially ground with a tissue homogenizer and inoculated then onto
selective medium or non-selective medium. The different media available, growth conditions
and identification procedure will be addressed below.
Non-Selective and Selective Media
H. pylori can grow on different solid media containing blood or lysed blood. The most
common ones are the non-selective Brucella or Columbia agar base, supplemented with 5 to
10% (vol/vol) of horse blood.
Some authors have used sheep blood, but it seems that horse blood improves H. pylori
growth [6]. Blood-free agars have also been used by supplementing the media with
cyclodextrin B or using egg yolk emulsion agar [6].
The rich agar media are commonly supplemented to improve the bacterium growth. One
example is the Isovitalex supplement available from Becton Dickinson [11]. Other
supplements may also be added to rich media to become selective. H. pylori-selective
supplement of Dent (commercially available from Oxoid or BD), which contains
combinations of vancomycin, amphotericin B, trimethoprim and cefsulodin, is a well-known
example [14, 15].

Other selective media are also commonly used for the isolation of H. pylori and the most
common ones are probably Egg yolk emulsion agar, Pylori agar, Skirrow’s medium, Dent’s
medium, and modified Thayer-Martin medium; which, apart from the supplements, usually
contain a “cocktail” of antibiotics and/or antifungal agents [14, 16].
It has been shown that the use of both selective and non-selective media in parallel allows
optimal recovery rates for H. pylori [11, 16]. It was also observed that longer incubation
periods could increase the H. pylori isolation rate and that selective media usually require
shorter periods than the non-selective ones [11, 17]. Because of these data, selective and non-
selective agar medium, combined with a 14-days incubation period, are usually applied on the
routine culture procedures to maximize the odes of recovering the bacterium.
Regarding broth medium, since H. pylori grows slowly in liquid media, they are not
routinely used for the evaluation of gastric tissue [6]. When in liquid medium, H. pylori tends
to transform into the coccoid form, which is usually associated to a non-culturable state and
thus cannot be recovered from the culture media [18]. Additionally, contaminating
microorganisms usually grow faster than H. pylori and its distinction becomes impossible.
Besides the media and incubation time, the atmosphere is another important parameter
when dealing with H. pylori. It is usually grown at 37°C in jars with gas-generation systems,
in CO2 or microaerobic incubators. Since this bacterium is commonly considered a
microaerophile, the atmosphere condition are usually set to 5% O2 and 10% of CO2 (vol/vol);
however it is know that it can adapt to other atmospheres [19]. There is no general consensus
about the specific oxygen and carbon dioxide requirements for this pathogen. It has been
demonstrated that H. pylori tolerates well high concentrations of oxygen, but has an absolute
requirement for elevated carbon dioxide concentrations in the growth atmosphere [19, 20].
However, since high O2 concentration seems to induce the conversion to a full coccoid form
(that is non-culturable) [21, 22]; the use of low concentration of O2 is still considered the best
strategy to avoid false negative results.
Phenotypic Identification
When formed on 7% lysed horse blood agar, H. pylori colonies are small (0.5 to 2 mm),
and translucent to yellowish in color, whereas on blood agar colonies are translucent to pale
grayish and have 0.5 to 1 mm in size [6]. No hemolytic activity is readily observed but may
appear after a few days at 4°C [23]. Because of their small size, H. pylori colonies may be
difficult to identify and isolate when there are few colonies and additional contaminating
microflora is present.
Identification of H. pylori is usually performed according to conventional biochemical
tests: cytochrome oxidase, catalase, oxidase, and urease [6, 23, 24]. Cytochrome oxidase is
present in all members of the Epsilonproteobacteria, while catalase is also present in all
Helicobacter species and most members of the Campylobacteraceae [23]. The urease reaction
is a key reaction in identifying Helicobacter species, since these bacteria produce large
amounts of urease to buffer the acidic conditions of the stomach [25]. Other urease positive
species, such as staphylococci and streptococci [26], may colonize the stomach; but their
colonies morphology is quite distinct. Campylobacter lari is a closely-related species and
may also present urease activity, but is not a common gastric colonizer [27].

After the correct identification of H. pylori, it is possible to proceed with further

antimicrobial susceptibility testing. This is an important feature since H. pylori resistance to
different antibiotics has increased in the last years [28], a fact that is compromising the
success of the current therapeutics. Traditionally, the antibiotic susceptibility is determined in
clinical laboratories by the agar dilution methods, as recommended by the National
Committee for Clinical Laboratory Standards (NCCLS) [6]. The European standardisation
has led to the recommendation of a similar protocol to that recommended by NCCLS [29].
The alternative E-test, available from bioMerieux, which consists of a strip with a range of
antibiotic concentrations, is considered to be simpler, cheap and to perform as well as the
NCCLS [30]. Nonetheless, the agar dilution method is still considered the gold standard to
determine antibiotic resistance, but the frequent inability to culture H. pylori and the
elucidation of the mechanism involved in the bacterium resistance [28], have led to the
development of molecular alternatives which are now being implemented in clinical
laboratories.
Polymerase Chain Reaction (PCR) Methods

PCR is considered by several authors as the most sensitive of the existing rapid methods
to detect microbial pathogens in clinical specimens. The applications of PCR for the detection
of H. pylori in gastric biopsies are very common and diverse. PCR followed by sequencing or
reverse hybridization, Real-Time PCR, nested PCR or multiplex PCR, are some examples of
PCR variation applied in the detection of this bacterium [31-36]. This technique has the
advantage of being easily adapted to the detection of any resistance gene or point mutation. In
addition, PCR does not require the samples to be transported under strict conditions and
might be performed on tissue samples subjected to other procedure, such as the standard
urease tests [23].
A number of genes have been proposed as preferential targets for the PCR detection of H.
pylori, including the 16S rRNA, the 26K species-specific antigen, the glmM, the ureA, the
ureB, the vacA, and the cagA genes. [33-35, 37-39]. Nevertheless, controversy remains
regarding which primer pair or sets of primers/probes is the potential “gold standard” for
gastric samples.
Different works generally report good sensitivity and/or specificity of the primer pairs
used, but a systematic study comparing different PCR primer pairs have shown that clinicians
should not rely on PCR results alone to decide the H. pylori status [40]. A study conducted by
Sugimoto and co-workers has evaluated the performance of 26 PCR primer pairs previously
described for detection of H. pylori DNA in clinical samples [40]. They found that none had
100% specificity or sensitivity; all produced false-positive results; only 2 primer pairs have
sensitivities and specificities of >90% in gastric biopsy specimens; and the combinations of
the primer pairs have not improved the results. An important feature to bear in mind when
designing PCR protocols for H. pylori detection is that the gastric mucosa is possibly
colonized by other species of bacteria [26]. This means that primers should be carefully
designed and PCR conditions should be as stringent as possible.
Regarding the determination of H. pylori resistance, the first strategies to emerge
involved the use of fluorescence resonance energy transfer (FRET) technology.

A fluorophore, SYBRGreen, binding specifically to double strand DNA during

amplification, and then a probe labeled with a second fluor dye, Cy5, were used to test H.
pylori in gastric biopsies. Hybridization of the probe to the target sequence leads to an
increase in fluorescence from the Cy5 fluorophore as a result of fluorescent resonance energy
transfer between SYBR Green and Cy5. The emission wavelength of Cy5 is different from
that of SYBR Green and is monitored separately [41]. Similarly to the FISH methodology,
this method has been used to detect the point mutation associated with the clarithromycin
resistance. Later, another FRET approach was used. In addition to the specific primers to the
23S rDNA, two probes were designed: a sensor probe (5′ labeled with LC-Red 640 and 3′
phosphorylated), which hybridizes with the region containing the mutation site and an anchor
probe (3′ labeled with fluorescein) which hybridizes three bases upstream from the former.
When the anchor probe is excited, an energy transfer occurs to the sensor probe and a signal
is emitted. After the amplification, a melting step is performed leading to different melting
points for the wild-type and mutant sequences [30, 42]. This method allows detection of H.
pylori, and the determination of its clarithromycin susceptibility, directly from biopsy
specimens in one reaction which limits the possibility of cross-contamination as the entire
reaction occurs in the same tube. It has proven to be highly sensitive and specific, but since
there was not many 23S rRNA sequences available in the databases at the time that the probes
were designed, specificity has not been extensively tested [28].
PCR have been recently combined with DNA hybridization, in order to simplify the
interpretation of the results. Briefly, the method use a DNA strip coated with different
specific oligonucleotides (DNA probes) designed to hybridize with the sequences of the wild-
type alleles or the mutated alleles [31]. The procedure includes the following sequential steps:
DNA extraction, PCR amplification of the different alleles, hybridization and washing. These
two final steps are performed in the DNA strip, and results are read directly in that strip. This
technology is available as a commercial kit, called GenoType HelicoDR (available from Hain
Lifescience GmbH), and it provides information regarding the presence of the pathogen and
the resistance to macrolides. Reported sensitivity and specificity values are higher than 94%
and 86% for clarithromycin resistance and higher than 83% and 95% for levofloxacin
resistance, respectively [31, 43].
Other commercial PCR kits for the detection of H. pylori in gastric biopsies are already
available in a different format. Most of them are real time PCR kits based on the approaches
described above. AmpliSens® Helicobacter pylori PCR kit (available from Ecoli Ltd.); H.
pylori ClariRes Assay (available from ingenetix) targeting the 23S rRNA; or Helicobacter
pylori real time PCR kit (available from AnDiaTec), also targeting the 23S rRNA; are some
examples of PCR kits that already own the CE marking according to the Directive 98/79/EC
for in vitro medical devices. This means that the adoption of these kits in routine practice can
face now an important progress in Europe. However, until now, none has been officially
cleared by FDA and thus are not allowed in routine medical practice in the US.
Despite the great advantages of these new molecular kits, PCR have also some drawbacks
that have been pointed out by several researchers. Limitations of PCR methods include the
propensity for false-positive results (which alters the specificity) mainly due to the detection
of DNA from non-H. pylori organisms or due to the contamination of the specimen by
exogenous H. pylori DNA. [40, 44]. False-negative results (which affect sensitivity) may also
occur due to a low number of organisms, due to the presence of Taq polymerase inhibitors or
due to sequence polymorphism in the target loci [40, 44, 45].

Fortunately, it is uncommon to find Taq polymerase inhibitors in gastric biopsy

specimens, which simplifies the DNA extraction procedure. The simplest technique consists
of grinding the tissue to release the bacteria and bacterial lysis by boiling at 100°C in a lysis
buffer [23]. Nonetheless, improved DNA extraction procedures have proved to be valuable
for eliminating PCR inhibitors. Phenol/chloroform extraction or the use of commercial
extraction kits (such as QAamp DNA kit from Qiagen), have shown to be superior to simple
boiling [46, 47]. Also, the inclusion of synthetics DNA fragment, that act as internal controls
during DNA amplification, have helped distinguishing true negatives from false negatives due
to PCR inhibition, and true positives from false positives due to contamination with closely-
related species [45, 48]
Fluorescence In Situ Hybridization

(FISH) Methods
Conventional FISH is based on the ability of DNA or RNA molecules to form duplex
strands by providing a complementary sequence, called probe, to a specific target sequence
within a cell. To identify that an hybridization has occurred, the probe is attached to a
fluorescent reporter molecule. FISH is rapidly becoming one of the most well-established
molecular biology techniques, with important applications in pathogen detection in clinical
samples for patient management [49]. When compared to PCR-based methods, the FISH
technique presents some advantages: it is not so easily affected by DNA contamination; it is
not susceptible to inhibitors and allows the direct visualization of bacteria in the gastric
biopsy specimens [24, 28, 50].
The use of FISH methods with rRNA-targeted fluorescence-labeled oligonucleotide
probes specific for H. pylori has already been established [50]. The method was tested in
biopsy samples and compared with culture and rapid urease test. Authors conclude that FISH
is a more sensitive and rapid technique than urease or culturing for the detection of H. pylori
in gastric biopsy specimens [51, 52].
The possibility of detecting antibiotic resistance when using FISH is another important
advantage of this technology. Antibiotic resistance in H. pylori is commonly attributed to the
occurrence of specific point mutations [28], and researchers took advantage of this fact to
develop a method that simultaneously detect the bacterium and its resistance. This is the case
of clarithromycin.
The FISH method developed by Trebesius and colleagues consists of an rRNA based
whole cell in situ hybridization using a set of fluorescent labeled oligonucleotide probes
specific for the species and the 23S point mutations associated with clarithromycin resistance
[50]. Labeling of intact single bacteria is monitored by fluorescence microscopy. This method
allows detection of H. pylori with a 16S rRNA probe labeled with the fluorochrome Cy3 (red)
and of resistant mutants with a 23S rRNA probe labeled with fluorescein (green)
simultaneously. The method proved to be sensitive and specific compared with standard
methods of culture and susceptibility testing. No discrepant result was noted [52]. In another
study, 11 discrepant results were reported among 69 cases. However, these cases
corresponded to mixed infections with susceptible and resistant strains [53], a situation which

culture is not able to discriminate since usually only one phenotype is recovered. Commercial
FISH applications for H. pylori identification already exists.
The BACTfish H. pylori combi kit, available from Izinta, offers a product that allow the
detection of both the bacterium presence and the resistance to clarythromycin; however the
probe dedicated to H. pylori detection is not specific for the species.
Recently, peptide nucleic acid (PNA) probes using FISH have been designed and
optimized for the detection of several bacteria [see[54] for a review]. PNA molecules are
DNA mimics that have the negatively charged sugar-phosphate backbone replaced by an
achiral, neutral polyamide backbone formed by repetitive N-(2-aminoethyl) glycine units [54,
55].
Although PNA lacks pentoses, specific hybridization between the PNA sequences and
nucleic acid complementary sequences still occur according to the Watson-Crick rules. The
neutral PNA molecule characteristic is responsible for a higher thermal stability (high Tm)
between PNA/DNA or PNA/RNA bonding, compared with the traditional DNA probes. Due
to this high affinity, PNA probes normally have sequences relatively smaller than DNA
sequences. Moreover, the PNA molecules present more resistance to nucleases and proteases
than DNA molecules.
PNA-FISH has been applied in a wide range of microbiology fields and has provided
more prompt and robust results in clinical and environmental samples than the traditional
culture methods [54]. In fact, a PNA-FISH method to determine the presence of H. pylori in
gastric biopsy specimens has been already developed in our laboratory, using a specific probe
(Hp769) [56]. Similarly to the DNA-FISH method reported above, the PNA-FISH method
was also adapted to the detection of clarithromycin resistance [24]. Results have shown
sensitivity and specificity values both of 100%. A very recent study has also shown that this
method can accurately estimate the presence of mixed infection with both susceptible and
resistance phenotypes [57]. Actually, a commercial application of this technique
(Probe4pylori kit) is now available from Biomode.
As mentioned above, the FISH methods have the advantage of being independent of
nucleic acid preparations, they are not prone to inhibition such as PCR, are quick, allow the
visualization of the bacteria morphology and can be performed directly on paraffin embedded
biopsy samples [51]. Limitations are usually related with providing an observer dependent
result. Until now, these methods are also limited to the detection of resistance genotypes
associated with point mutations in the rRNA.
Conclusion
H. pylori detection is not an easy task mainly due to the fastidious nature of the bacterium
and the difficulty of assessing the gastric mucosa. Many detection methods for detection of H.
pylori in gastric biopsies have been proposed, each with advantages and disadvantages. The
advent of molecular methods opened the path to the use of culture independent approaches.
Real-time PCR and FISH have become one of the most promising techniques, and the
emergence of commercial detection kits will bring to these technologies the medical
confidence and the required standardization of routine procedures. More importantly, these
technologies also provide additional information regarding macrolide resistance, a valuable

information when establishing the treatment therapy and an important step in the battle
against antimicrobial resistance.
References
[1] Chey, W. D., Wong, B. C., (2007). American College of Gastroenterology guideline on
the management of Helicobacter pylori infection. Am. J. Gastroenterol. 102(8): p.
1808-25.
[2] Marshall, B. J., Royce, H., Annear, D. I., Goodwin, C. S., Pearman, J. W., Warren, J.
R., et al. (1984). Original isolation of Campylobacter pyloridis from human gastric
mucosa. Microbios Lett. 25: p. 83-88.
[3] Grove, D. I., Koutsouridis, G., Cummins, A. G., (1998). Comparison of culture,
histopathology and urease testing for the diagnosis of Helicobacter pylori gastritis and
susceptibility to amoxycillin, clarithromycin, metronidazole and tetracycline.
Pathology. 30(2): p. 183-187.
[4] Thijs, J. C., van Zwet, A. A., Thijs, W. J., Oey, H. B., Karrenbeld, A., Stellaard, F., et
al. (1996). Diagnostic tests for Helicobacter pylori: A prospective evaluation of their
accuracy, without selecting a single test as the gold standard. American Journal of
Gastroenterology. 91(10): p. 2125-2129.
[5] Siu, L. K., Leung, W. K., Cheng, A. F., Sung, J. Y., Ling, T. K., Ling, J. M., et al.
(1998). Evaluation of a selective transport medium for gastric biopsy specimens to be
cultured for Helicobacter pylori. J. Clin. Microbiol. 36(10): p. 3048-50.
[6] Andersen, L. P., Wadström, T., Chapter 4. Basic Bacteriology and Culture, In:
Helicobacter pylori: Physiology and Genetics, M. H. L. T., M. G. L. and H. S. L.,
Editors. 2001, ASM Press: Washington (DC).
[7] Nedenskov-Sorensen, P., Aase, S., Bjorneklett, A., Fausa, O., Bukholm, G., (1991).
Sampling efficiency in the diagnosis of Helicobacter pylori infection and chronic active
gastritis. J. Clin. Microbiol. 29(4): p. 672-5.
[8] Dixon, M. F., Genta, R. M., Yardley, J. H., Correa, P., (1996). Classification and
grading of gastritis. The updated Sydney System. International Workshop on the
Histopathology of Gastritis, Houston 1994. Am. J. Surg. Pathol. 20(10): p. 1161-81.
[9] Cuchi, E., Forne, M., Quintana, S., (2002). Comparison of two transport media and
three culture media for primary isolation of Helicobacter pylori from gastric biopsies.
Clin. Microbiol. Infect. 8(9): p. 609-10.
[10] Heep, M., Scheibl, K., Degrell, A., Lehn, N., (1999). Transport and storage of fresh and
frozen gastric biopsy specimens for optimal recovery of Helicobacter pylori. J. Clin.
Microbiol. 37(11): p. 3764-6.
[11] Boyanova, L., (2003). Influence of transport conditions and media on Helicobacter
pylori isolation. J. Med. Microbiol. 52(Pt 12): p. 1129-30.
[12] Roosendaal, R., Kuipers, E. J., Pena, A. S., de Graaff, J., (1995). Recovery of
Helicobacter pylori from gastric biopsy specimens is not dependent on the transport
medium used. J. Clin. Microbiol. 33(10): p. 2798-800.
[13] Soltesz, V., Zeeberg, B., Wadstrom, T., (1992). Optimal survival of Helicobacter pylori
under various transport conditions. J. Clin. Microbiol. 30(6): p. 1453-6.

[14] Dent, J. C., McNulty, C. A., (1988). Evaluation of a new selective medium for
Campylobacter pylori. Eur. J. Clin. Microbiol. Infect. Dis. 7(4): p. 555-8.
[15] Han, S. W., Flamm, R., Hachem, C. Y., Kim, H. Y., Clarridge, J. E., Evans, D. G., et al.
(1995). Transport and storage of Helicobacter pylori from gastric mucosal biopsies and
clinical isolates. Eur. J. Clin. Microbiol. Infect. Dis. 14(4): p. 349-52.
[16] Piccolomini, R., Di Bonaventura, G., Festi, D., Catamo, G., Laterza, F., Neri, M.,
(1997). Optimal combination of media for primary isolation of Helicobacter pylori
from gastric biopsy specimens. J. Clin. Microbiol. 35(6): p. 1541-4.
[17] Van der Huls, R. W., Verheul, S. B., Weel, J. F., Gerrits, Y., ten Kate, F. J., Dankert, J.,
et al. (1996). Effect of specimen collection techniques, transport media, and incubation
of cultures on the detection rate of Helicobacter pylori. Eur. J. Clin. Microbiol. Infect.
Dis. 15(3): p. 211-5.
[18] Azevedo, N. F., Almeida, C., Cerqueira, L., Dias, S., Keevil, C. W., Vieira, M. J.,
(2007). Coccoid form of Helicobacter pylori as a morphological manifestation of cell
adaptation to the environment. Appl. Environ. Microbiol. 73(10): p. 3423-7.
[19] Bury-Mone, S., Kaakoush, N. O., Asencio, C., Megraud, F., Thibonnier, M., De Reuse,
H., et al. (2006). Is Helicobacter pylori a true microaerophile? Helicobacter. 11(4): p.
296-303.
[20] Park, S. A., Ko, A., Lee, N. G., (2011). Stimulation of growth of the human gastric
pathogen Helicobacter pylori by atmospheric level of oxygen under high carbon
dioxide tension. BMC Microbiol. 11: p. 96.
[21] Donelli, G., Matarrese, P., Fiorentini, C., Dainelli, B., Taraborelli, T., Di Campli, E., et
al. (1998). The effect of oxygen on the growth and cell morphology of Helicobacter
pylori. FEMS Microbiol. Lett. 168(1): p. 9-15.
[22] Tominaga, K., Hamasaki, N., Watanabe, T., Uchida, T., Fujiwara, Y., Takaishi, O., et
al. (1999). Effect of culture conditions on morphological changes of Helicobacter
pylori. J. Gastroenterol. 34 Suppl. 11: p. 28-31.
[23] Megraud, F., Lehours, P., (2007). Helicobacter pylori detection and antimicrobial
susceptibility testing. Clin. Microbiol. Rev. 20(2): p. 280-322.
[24] Cerqueira, L., Fernandes, R. M., Ferreira, R. M., Carneiro, F., Dinis-Ribeiro, M.,
Figueiredo, C., et al. (2011). PNA-FISH as a new diagnostic method for the
determination of clarithromycin resistance of Helicobacter pylori. Bmc Microbiology.
11.
[25] Marshall, B. J., Barrett, L. J., Prakash, C., McCallum, R. W., Guerrant, R. L., (1990).
Urea protects Helicobacter (Campylobacter) pylori from the bactericidal effect of acid.
[26] Bik, E. M., Eckburg, P. B., Gill, S. R., Nelson, K. E., Purdom, E. A., Francois, F., et al.
(2006). Molecular analysis of the bacterial microbiota in the human stomach. Proc.
Natl. Acad. Sci. US 103(3): p. 732-7.
[27] Matsuda, M., Moore, J. E., (2004). Urease-positive thermophilic Campylobacter
species. Applied and Environmental Microbiology. 70(8): p. 4415-4418.
[28] Megraud, F., (2004). H. pylori antibiotic resistance: prevalence, importance, and
advances in testing. Gut. 53(9): p. 1374-1384.
[29] Glupczynsk, Y., Broutet, N., Cantagrel, A., Andersen, L. P., Alarcon, T., Lopez-Brea,
M., et al. (2002). Comparison of the E test and agar dilution method for antimicrobial

suceptibility testing of Helicobacter pylori. European Journal of Clinical Microbiology

and Infectious Diseases. 21(7): p. 549-552.
[30] Osato, M. S., Reddy, R., Reddy, S. G., Penland, R. L., Graham, D. Y., (2001).
Comparison of the Etest and the NCCLS-approved agar dilution method to detect
metronidazole and clarithromycin resistant Helicobacter pylori. International Journal
of Antimicrobial Agents. 17(1): p. 39-44.
[31] Cambau, E., Allerheiligen, V., Coulon, C., Corbel, C., Lascols, C., Deforges, L., et al.
(2009). Evaluation of a New Test, GenoType HelicoDR, for Molecular Detection of
Antibiotic Resistance in Helicobacter pylori. Journal of Clinical Microbiology. 47(11):
p. 3600-3607.
[32] Chattopadhyay, S., Patra, R., Ramamurthy, T., Chowdhury, A., Santra, A., Dhali, G. K.,
et al. (2004). Multiplex PCR assay for rapid detection and genotyping of Helicobacter
pylori directly from biopsy specimens. Journal of Clinical Microbiology. 42(6): p.
2821-2824.
[33] Chisholm, S. A., Owen, R. J., Teare, E. L., Saverymuttu, S., (2001). PCR-Based
diagnosis of Helicobacter pylori infection and real-time determination of
clarithromycin resistance directly from human gastric biopsy samples. Journal of
Clinical Microbiology. 39(4): p. 1217-1220.
[34] Goosen, C., Theron, J., Ntsala, M., Maree, F. F., Olckers, A., Botha, S. J., et al. (2002).
Evaluation of a novel hemi-nested PCR assay based on the phosphoglucosamine mutase
gene for detection of Helicobacter pylori in saliva and dental plaque. Journal of Dental
Research. 81: p. B377-B377.
[35] Koehler, C. I., Mues, M. B., Dienes, H. P., Kriegsmann, J., Schirmacher, P., Odenthal,
M., (2003). Helicobacter pylori genotyping in gastric adenocarcinoma and MALT
lymphoma by multiplex PCR analyses of paraffin wax embedded tissues. Journal of
Clinical Pathology-Molecular Pathology. 56(1): p. 36-42.
[36] Oleastro, M., Menard, A., Santos, A., Lamouliatte, H., Monteiro, L., Barthelemy, P., et
al. (2003). Real-time PCR assay for rapid and accurate detection of point mutations
conferring resistance to clarithromycin in Helicobacter pylori. Journal of Clinical
Microbiology. 41(1): p. 397-402.
[37] Lage, A. P., Godfroid, E., Fauconnier, A., Burette, A., Butzler, J. P., Bollen, A., et al.
(1995). Diagnosis of Helicobacter pylori Infection by Pcr - Comparison with Other
Invasive Techniques and Detection of Caga Gene in Gastric Biopsy Specimens. Journal
of Clinical Microbiology. 33(10): p. 2752-2756.
[38] Park, C. Y., Kwak, M., Gutierrez, O., Graham, D. Y., Yamaoka, Y., (2003).
Comparison of genotyping Helicobacter pylori directly from biopsy specimens and
genotyping from bacterial cultures. Journal of Clinical Microbiology. 41(7): p. 3336-
3338.
[39] Smith, S. I., Oyedeji, K. S., Arigbabu, A. O., Cantet, F., Megraud, F., Ojo, O. O., et al.
(2004). Comparison of three PCR methods for detection of Helicobacter pylori DNA
and detection of cagA gene in gastric biopsy specimens. World Journal of
[40] Sugimoto, M., Wu, J. Y., Abudayyeh, S., Hoffman, J., Brahem, H., Al-Khatib, K., et al.
(2009). Unreliability of Results of PCR Detection of Helicobacter pylori in Clinical or
Environmental Samples. Journal of Clinical Microbiology. 47(3): p. 738-742.

[41] Gibson, J. R., Saunders, N. A., Burke, B., Owen, R. J., (1999). Novel method for rapid
determination of clarithromycin sensitivity in Helicobacter pylori. Journal of Clinical
Microbiology. 37(11): p. 3746-3748.
[42] Lascols, C., Lamarque, D., Costa, J. M., Copie-Bergman, C., Le Glaunec, J. M.,
Deforges, L., et al. (2003). Fast and accurate quantitative detection of Helicobacter
pylori and identification of clarithromycin resistance mutations in H-pylori isolates
from gastric biopsy specimens by real-time PCR. Journal of Clinical Microbiology. 41
(10): p. 4573-4577.
[43] Deyi, V. Y. M., Burette, A., Bentatou, Z., Maaroufi, Y., Bontems, P., Lepage, P., et al.
(2011). Practical use of GenoType (R) HelicoDR, a molecular test for Helicobacter
pylori detection and susceptibility testing. Diagnostic Microbiology and Infectious
Disease. 70(4): p. 557-560.
[44] Lu, J. J., Perng, C. L., Shyu, R. Y., Chen, C. H., Lou, Q. Y., Chong, S. K. F., et al.
(1999). Comparison of five PCR methods for detection of Helicobacter pylori DNA in
gastric tissues. Journal of Clinical Microbiology. 37(3): p. 772-774.
[45] Thoreson, A. C. E., Borre, M., Andersen, L. P., Jorgensen, F., Kiilerich, S., Scheibel, J.,
et al. (1999). Helicobacter pylori detection in human biopsies: a competitive PCR assay
with internal control reveals false results. Fems Immunology and Medical
[46] Roussel, Y., Wilks, M., Harris, A., Mein, C., Tabaqchali, S., (2005). Evaluation of
DNA extraction methods from mouse stomachs for the quantification of H. pylori by
real-time PCR. Journal of Microbiological Methods. 62(1): p. 71-81.
[47] Wang, J. T., Lin, J. T., Sheu, J. C., Yang, J. C., Chen, D. S., Wang, T. H., (1993).
Detection of Helicobacter pylori in Gastric Biopsy-Tissue by Polymerase Chain-
Reaction. European Journal of Clinical Microbiology and Infectious Diseases. 12(5): p.
367-371.
[48] Furuta, T., Kaneko, E., Suzuki, M., Arai, H., Futami, H., (1996). Quantitative study of
Helicobacter pylori in gastric mucus by competitive PCR using synthetic DNA
fragments. Journal of Clinical Microbiology. 34(10): p. 2421-2425.
[49] Barken, K. B., Haagensen, J. A. J., Tolker-Nielsen, T., (2007). Advances in nucleic
acid-based diagnostics of bacterial infections. Clinica Chimica Acta. 384(1-2): p. 1-11.
[50] Trebesius, K., Panthel, K., Strobel, S., Vogt, K., Faller, G., Kirchner, T., et al. (2000).
Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric
tissue by fluorescent in situ hybridisation. Gut. 46(5): p. 608-614.
[51] Russmann, H., Feydt-Schmidt, A., Adler, K., Aust, D., Fischer, A., Koletzko, S.,
(2003). Detection of Helicobacter pylori in paraffin-embedded and in shock-frozen
gastric biopsy samples by fluorescent in situ hybridization. Journal of Clinical
[52] Russmann, H., Kempf, V. A. J., Koletzko, S., Heesemann, J., Autenrieth, I. B., (2001).
Comparison of fluorescent in situ hybridization and conventional culturing for detection
of Helicobacter pylori in gastric biopsy specimens. Journal of Clinical Microbiology.
39(1): p. 304-308.
[53] Feydt-Schmidt, A. K., Russmann, H., Adler, K., Aust, D., Fischer, A. G., Koletzko, S.,
(2002). Fluorescence in situ hybridisation (FISH) for detection of clarithromycin-
resistant H. pylori strains on paraffin-embedded biopsies. Gut. 51: p. A83-A83.

[54] Cerqueira, L., Azevedo, N. F., Almeida, C., Jardim, T., Keevil, C. W., Vieira, M. J.
(2008). DNA mimics for the rapid identification of microorganisms by fluorescence in
situ hybridization (FISH). Int. J. Mol. Sci. 9(10): p. 1944-60.
[55] Stender, H., (2003). PNA FISH: an intelligent stain for rapid diagnosis of infectious
diseases. Expert Review of Molecular Diagnostics. 3(5): p. 649-655.
[56] Guimaraes, N. M., Azevedo, N. F., Figueiredo, C., Keevil, C. W., Vieira, M. J., (2006).
Development and application of a novel peptide nucleic acid (PNA) probe for the
specific detection of Helicobacter pylori. Helicobacter. 11(4): p. 383-383.
[57] Cerqueira, L., Fernandes, R.M., Ferreira, R.M., Oleastro, M., Carneiro, F., Brandão, C.,
Pimentel-Nunes, P., Dinis-Ribeiro, M., Figueiredo, C., Keevil, C.W., Vieira, M.J.,
Azevedo, N.F., (2013). Validation of a PNA-FISH method for the detection of
Helicobacter pylori clarithromycin resistance in gastric biopsies. Journal of Clinical
Microbiology. 51(6):1887-93.

Chapter 5
New Optical Techniques

Helping in Diagnoses
Noriya Uedo1, Hyung Hun Kim2, Rapat Pittayanon3

and Ryu Ishihara1
1
Department of Gastrointestinal Oncology,
Osaka Medical Center for Cancer and Cardiovascular Disease
2
Department of Internal Medicine, Seoul St. Mary's Hospital,
Catholic University of Korea College of Medicine
3
Department of Gastroenterology, King Chulalongkorn Memorial Hospital
Abstract
The diagnosis of H. pylori infection and presence of associated premalignant
condition i.e. atrophy and intestinal metaplasis is improtant in terms of understanding of
pathogenesis of the diseases and identification of individuals at high risk for developing
gastric cancer. With white light endoscopic image, we can suspect H. pylori negative
individuals by presence of regular arrangement of collecting venule, and patients with H.
pylori atrophic gastritis or intestinal metaplasia (IM) by endoscoic finding of atrophic
gastritis (whitish mucosa, loss of gastric fold and visible mucosal vessels). Congo red
chromoendoscpy improve diagnosis of patients with atrophic gastritis according to
impairment of acid secreting function, and methylene blue chromoendocopy enhances
diagnosis of IM as stained areas. Autofluorescence imaging indicate presence and extent
of atrophic gastritis as green mucosa in the gastric corpus. Magnifying narrow band
imaging enables us to suggest H. pylori nfection and to make diagnosis of presence of
atropy and IM according to findings of microsurface structure, microvascular architecture
and presence of the light blue crest sign. Confocal laser endomicroscopy helps
diagnosing atrophy and IM according to cellular and interstitial organization and shows
possibility to identify H. pylori bacterium itself in vivo. Endoscopy with new optical
method can be a useful adjunct to make diagnosis of H. pylori infection and to assess the
risk of gastric cancer.
Keywords: Helicobacter pylori, atrophic gastritis, intestinal metaplasia, magnifying

endoscopy, narrow band imaging, confocal laser endomicroscopy
84 Noriya Uedo, Hyung Hun Kim, Rapat Pittayanon et al.
Introduction
Helicobacter pylori (H. pylori) infection closely associates with incidence and
characteristics of various upper digestive tract diseases including malignancy. It is widely
accepted that gastric cancers, especially differentiated type, evolve through a multistep
process starting with H. pylori-associated superficial gastritis and progressing through
atrophy with intestinal metaplasia (IM), followed by the development of dysplasia and finally
carcinoma [1]. Therefore, diagnosis of H. pylori infection and presence of atrophy and IM is
improtant in terms of understanding of pathogenesis of the diseases and identification of
individuals at high risk for developing gastric cancer. Diagnostic methods for H. pylori
infection includes blood, urine and stool tests to detect H. pylori specific antigen or antibody;
urea breath test and rapid urease test using biopsy specimen to detect urease activity; and
culture of biopsy specimens. Standard method for diagnosing presence of atrophy and IM are
histological examination of biopsy specimens [2]. Recently, serologic test for gastric atrophy
analyzing pepsinogen level becomes popular particularly in Japan [3]. Despite of these
currently available standard methods for H. pylori infection and atrophic gastritis, continuous
attempts have been made to diagnose the diseases macroscopically during gastroscopy.
Advantage of endoscopy to make diagnosis of H. pylori infection and associate gastritis over
other methods is capability of evaluating distribution and quantification of atrophy or IM
precisely. Moreover, it does not need any time to have the test result.
In this chapter, we describe various endoscopic imaging methods to help us diagnosing
H. pylori infection and related mucosal changes such as atrophy or IM that are regarded as
preneoplastic condition of the stomach.
Atrophic Gastritis and Intestinal Metaplasia

as a Pre-Neoplastic Condition
A Japanese cohort study that followed up 1526 patients with peptic ulcers, gastric
hyperplasia, or non-ulcer dyspepsia demonstrated that none of gastric cancer developed from
280 H. pylori negative patients, while gastric cancer developed in 36 (2.9%) of 1246 H. pylori
infected patients during mean period of 7.8 years [4]. Moreover, among H. pylori infected
patients, those with severe gastric atrophy [RR 4.9 (2.8–19.2)], corpus predominant gastritis
[34.5 (7.1–166.7)] and IM [6.4 (2.6–16.1)] were at high risk for developing gastric cancer. A
Dutch nationwide cohort study including more than 90,000 patients indicated that the annual
incidence of gastric cancer was 0.1% for patients with atrophic gastritis, 0.25% for IM, 0.6%
for mild to moderate dysplasia, and 6% for severe dysplasia within 5 years after diagnosis.
The hazard ratio for development of gastric cancer (95% C.I.) in patients with IM, mild-to-
moderate dysplasia and severe dysplasia compared with those with atrophic gastritis was 1.74
(1.5–2.1), 3.93 (3.2– 4.8), and 40.14 (32.2–50.1), respectively [5]. Accordingly, we could
understand that development of gastric cancer from H. pylori naïve patients is extremely rare
and the risk of gastric cancer increased significantly with the presence and severity of pre-
neoplastic condition. Thus, the diagnosis of H. pylori infection and identification of such
precancerous condition and surveying patients in whom they are found could lead to the
diagnosis of gastric cancer at early stage and improve patients’ survival [6]. To detect such

New Optical Techniques Helping in Diagnoses 85
premalignant lesions, biopsies from multiple anatomical sites of the gastric mucosa is
recommended, i.e., the updated Sydney System [2,7], or Operative Link for Gastritis
Assessment [8,9], because the atrophy or IM usually distribute unevenly and there is
possibility of sampling error. For example, the updated Sydney System recommends taking at
least five biopsy specimens: two from the antrum, two from the corpus and one from the
incisura angularis. However, multiple biopsies and histologic examination may be time- and
cost-consuming procedure; thus, they may not be easy to be applied in routine clinical
practices.
Conventional White Light Image of Normal,

Atrophic and Metaplastic Gastric Mucosa
In conventional white light image, H. pylori negative normal gastric corpus musosa looks
homogeneously reddish in color and having longitudinal folds (Figure 1a). With high
definition videoendoscopy, regularly arranged small red spots are observed (Figure 2a).
Under magnifying observation, these red spots are recognized as spider-like venules that is
called “collecting venule” (Figure 2b and c). Yagi et al. indicated that regular arrangement of
these collecting venules was a characteristic finding of H. pylori negative patients [10] and its
diagnostic value was sensitivity of 82% (95% C.I.=71-93%) and specificity of 95% (95%
C.I.=90-100%) [11]. In contrast to the homogeneously reddish normal corpus mucosa with
the gastric folds, mucosa with H. pylori associated atrophic gastritis looks whitish to
yellowish with increased vessel visibility, low mucosal height and absent mucosal folds
(Figure 1b).
Figure 1. Continued on next page.

Figure 1. In white light endoscopy, the corpus mucosa of a H. pylori naïve patient looks reddish and has
longitudinal gastric folds (a), whereas that in a patient with atrophic gastritis appears whitish, and
shows increased visible vessel and no gastric folds (b). In autofluorescence image of H. pylori naïve
patient, the entire corpus mucosa appears purple (c) while that of atrophic gastritis patients has and has
green mucosa in the lesser curvature (d). Magnifying narrow band image of the corpus mucosa in the
H. pylori naïve patient (representing white squares in a) has regular arrangement of the collecting
venules (white arrow heads in e and g) and its microsurface structure (representing white square in e)
shows regularly arranged round foveola (g). In the patient with atrophic gastritis, the corpus mucosa
(representing white square in b) has ridged microsurface structure (f and h) and the light blue crest sign
(yellow arrow heads in h).
Figure 2. In a H. pylori negative patients, small reddish dots are observed in the corpus mucosa (a).
In magnifying observation at weak magnification, the red spots are visualized as regular arrangement of
collecting venules (white arrow head in b and d). At the maximum magnification level, the spider-like
shape of the collecting venules is well observable (c and e).

Figure 3. Classification of extent of atrophic gastritis according to level of the endoscopic atrophic
border. In H. pylori negative patients, the endoscopic atrophic border is seen at the boundary of the
corpus and the antrum. In patients with atrophic gastritis, diagnosis is made as closed-type when the
endoscopic atrophic border is identified on the lesser curvature, whereas the border no longer exists
along the lesser curvature and observed at the anterior and the posterior walls, it is classified as the
open- type.
Kimura and Takemoto indicated with multiple biopsies across the boundary of the normal
and the atrophic mucosa that the “endoscopic atrophic border” is the boundary of the fundic
type and pyloric type (atrophic) mucosa [12].
The mucosa on the border and the fundic gland side has neutrophil and mononuclear cell
infiltration but neither atrophy nor IM. By contrast, on the pyloric side of the border, the
mucosa shows atrophy of the fundic glands with or without IM [13]. Therefore, the
endoscopic atrophic border marks the furthest front of the extent of atrophic gastritis in the
corpus. Diagnostic value (sensitivity/specificity) of presence of visible vessel and absence of
the gastric folds for severe atrophic gastritis was 90%/84% and 80%/87%, respectively [14].
Moreover, Kimura and Takemoto quantified the extent of atrophic gastritis according to the
location of the endoscopic atrophic border (Figure 3). When the atrophic border remains on
the lesser curvature of the corpus, the diagnosis is closed-type atrophic gastritis (antral
predominant gastritis), whereas when the atrophic border no longer exists on the lesser
curvature and extends along the anterior and posterior walls of the stomach, the diagnosis is
open-type atrophic gastritis (pangastritis or corpus-predominant gastritis). These endoscopic
diagnostic criteria are commonly accepted and are used in practice for the diagnosis of
chronic atrophic gastritis in Japan. An association between the extent of endoscopically
diagnosed atrophic gastritis and the risk of gastric cancer has been demonstrated in a large
cohort study [4,15].
IM in the white light image appears as whitish (ash-colored) nodules (Figure 4a). The
problem of diagnosis of IM by conventional WLI is high inter-observer variability [16] and a
poor correlation with histological findings [17,18].

Figure 4. Endoscopic appearance of elevated type intestinal metaplasia. Whitish nodules are seen on the
lesser curvature of the antrum (a). The lesions become obvious in the narrow band imaging (b).
In magnifying narrow band imaging, brownish subepithelial capillaries are obscured in whitish nodules
(c, representing white square in b). The light blue crest (yellow arrow head) is observable on the surface
of intestinal metaplasia (d, representing white square in c).
Figure 5. Endoscopic finding of flat type intestinal metaplasia. In white light image, an endoscopic
finding is not obvious (a). Whitish patches (white arrow) can be recognized in the narrow band imaging
(b). In magnifying narrow band imaging, brownish subepithelial capillaries are obscured at the whitish
patches (c, representing white square in b). The light blue crest (yellow arrow head) appears on the
surface of micromucosal crests (d, representing white square in c).

Ash-colored nodular change was specific (98–99%) for identifying histological IM, but
sensitivity was extremely low (6–12%). This is because IM also appeared in flat mucosa and
shows few morphologic changes (Figure 5a and 7a).
Chromoendoscopy
Chromoendoscopic evaluation enabled us to evaluate extent of atrophic gastritis and IM,
and contributes identification of high-risk patients for developing gastric cancer. Congo red is
a pH-sensitive indicator that turns its color from red to black in acidic condition. When Congo
red is sprayed over the gastric mucosa in H. pylori negative patients, the entire fundic mucosa
that secretes acid changes into black (Figure 6a), whereas, in patients with H. pylori
associated gastritis, the mucosa that looses acid-secreting function due to atrophy or IM
remains red. Accordingly, the extent of atrophic gastritis of the corpus in H. pylori positive
patients can be observed as red “non-discolored” areas where the Congo red does not change
color (Figure 6b) [19].
Figure 6. Images of the endoscopic Congo red test. In H. pylori negative patients, entire corpus mucosa
turns black due to acid secretion from the oxyntic mucosa (a). In patients with atrophic gastritis,
mucosa in the corpus lesser curvature remains red (b) because of reduced acid secretion due to
atrophic/metaplastic change of the corpus mucosa.
The efficacy of using vital staining with methylene blue to help identifying IM in patients
with H. pylori associated gastritis has been reported in several papers (Figure 7b) [20-23].
Combination use of magnifying endoscopy with methylene blue chromoendoscopy improves
distinction of dysplasia from IM in the stained areas (Figure 7c) [24]. Dinis-Ribeilo et al.
demonstrated magnifying images with methylene blue closely associate with histological
findings and is useful not only for detecting gastric IM, but also for assessing its extent [25].
Although chromoendoscopy has better diagnostic performance than conventional white
light observation, limitations of the chromoendoscopy are complicated and time-consuming
procedure and potential adverse effects of drugs (gastrin, dyes, etc…) [26,27]. Thus,
chromoendoscopy is not likely to be performed in ordinary clinical practice.

Figure 7. Methylene blue chromoendoscopy of flat gastric intestinal metaplasia. In white light image,
an endoscopic finding is not evident (a). After spraying methylene blue onto the gastric mucosa,
intestinal metaplasia is stained as bluish patches (b). In magnifying image, an area with intestinal
metaplasia that has ridged surface structure is specifically stained with methylene blue (c). In narrow
band imaging, the light blue crest is seen at the same area to the methylene blue stained area (d).
Autofluorescence Imaging
In autofluorescence imaging (AFI), the gastric corpus mucosa of H. pylori negative
patients looks dark green to purple (Figure 1c). On the other hands, presence and extent of
corpus atrophic gastritis in patients with H. pylori infection can be diagnosed as bright green
mucosa (Figure 1d). The basic principle of AFI is showing differences of autofluorescence
property between premalignant and normal mucosa by illuminating excitation (390–470 nm)
and green (540-560nm) lights [28]. The reason why the normal gastric corpus shows purple
color is probably because thick fundic mucosa absorbs the illuminated lights and reduces
autofluorescence from the mucosa and the submucosa, whereas the thin atrophic mucosa
permits the illuminated lights penetrating well to produce strong autofluorescence [29].
Because the color in the AFI image depends on the autofluorescence intensity, the extent of
atrophic mucosa in the corpus can be estimated as extent of greenish mucosa in AFI images.
Inoue et al. indicated with AFI that green mucosa in the gastric corpus showed sensitivity of
100% and specificity of 63% for patients with atrophy, and sensitivity of 100% and
specificity of 52% for those with IM [30]. The authors also found that AFI had higher
reproducibility for diagnosis of extent of atrophic gastritis compared with white light image:
intra-observer agreement was substantial (κ=0.67) for AFI but moderate (κ=0.56) for the
white light image.

Magnifying Endoscopy and Narrow Band Imaging

Magnifying endoscopy enables us to evaluate detailed morphological feature i.e. surface
structure and vascular architecture of the superficial gastric mucosa that corresponds to
histological findings [31]. In combination with magnifying observation, application of
chromoendoscopy facilitates assessment of microsurface structure. Narrow band imaging
(NBI) uses the specific narrow banded blue (400-430 nm) and green (525-555 nm) lights that
are well absorbed by hemoglobin instead of conventional white light [32]. As a result, NBI
with magnifying endoscopy enhances evaluation of microvascular architecture as well as the
microsurface structure.
Figure 8. Magnifying narrow band images (NBI) of normal corpus and antrum mucosa. In magnifying
NBI, the normal corpus mucosa is characterized as mucosa with regular round foveolae (gastric pits)
and it looks having dark brown areas that surround light brown areas (foveola-type). Whereas, normal
antrum mucosa is characterized by mucosal ridges that are divided by continuous grooves, and it has a
pattern with light brown areas that surrounds dark brown areas (groove-type). In NBI image, a dark
brown area corresponds to the subepithelial capillary and light brown area corresponds with the
interstitium and the epithelium. The foveola-type mucosa represents tubular epithelial structure, while
the groove type mucosa represents papillary surface structure.

Figure 9. In patients with H. pylori associated atrophic gastritis, the foveola-type mucosa that has low
grade of atrophy/IM and the groove type mucosa that has high grade of atrophy/IM usually distributes
mosaically in the gastric corpus. IM: Intestinal Metaplasia.
In magnifying NBI observation, H. pylori negative normal corpus mucosa looks having
round foveola (gastric pit) that was surrounded by network of brownish subepithelial
capillaries (Foveola-type, Figures 1e, 1g and 8) [11,33]. Moreover, spider-like shaped
collecting venules are regularly arranged. While, in the antrum, normal mucosa has ridged
surface structure that was divided with continuous grooves in which brownish subepithelial
capillaries are situated inside of the light brownish epithelium (Groove-type, Figure 8).
In patients with H. pylori associated atrophic gastritis, the foveola-type mucosa that has
low grade of atrophy/IM and the groove type mucosa that has high grade of atrophy/IM
usually distributes mosaically in the gastric corpus (Figure 9). Kanzaki et al. investigated
association between extent of atrophic gastritis and proportion of faveola- and groove-type
mucosa, and illustrated possible developing pattern of microsurface structure according to the
progression of the atrophic gastritis [34]. They suggested that, as extent of the atrophic
gastritis widens, the groove type mucosa develops focally among the foveola-type corpus
mucosa, increases proportion and eventually replaces all area of mucosa.
Magnifying NBI visualizes specific finding of IM. In non-magnifying NBI image,
mucosa with IM looks whitish patchy area (Figures 4b and 5b). Under magnifying
observation, light-bluish lines of light are observed on the epithelial surface/gyri (Figure 1h,
4c, 4d, 5c, 5d and 7d). This finding is called as the light blue crest (LBC) and correlates with
histological evidence of IM with sensitivity of 89% and specificity of 93% [35]. Authors
speculated that this finding is originated with dense reflection of the shortwave length light at
the ciliated microstructure i.e. brush border on the surface of IM.

Confocal Laser Endomicroscopic Image

Confocal laser endomicroscopy (CLE) is the novel endoscopic imaging device that
enables high-magnification (×1000) observation of glandular and cellular structure of
superficial mucosa [36]. It is expected to acquire a real-time endoscopic analysis for histology
without the need for a biopsy during the endoscopic examination [37,38]. Currently, there are
two types of machines: endoscopy-based confocal laser endomicroscopy (eCLE) and probe-
based confocal laser endomicroscopy (pCLE). Both prefer an intravenous contrast injection
(fluorescein) or a topical dye spray (acriflavine) to enhance the mucosal structures. Injected
fluorescein distributes throughout the mucosa with strong contrast in the capillaries and then
enters into the tissue highlighting the interstitium as a bright object (Figure 10).
Figure 10. Probe type confocal laser endomicroscopy images of gastric mucosa and intestinal
metaplasia. Corpus mucosa looks having dark round tubules that are surrounded with bright interstitium
(a). Antrum mucosa looks having bright interstitium that is surrounded with dark bands consisted of
gastric pits and epithelium (b). Intestinal metaplasia showed large dark goblet cells (red arrow heads) in
the epithelium (c).
In contrast, any structure that has no blood supply, such as nuclei or mucin, does not be
enhanced with fluorescein. Topically used acriflavine strongly labels the superficial epithelial
cells including nuclei. The eCLE has better image quality than pCLE. However, pCLE is
more flexible because it can be used with any endoscopes that accept 10 Fr size accessories.
Moreover, the frame rate of the pCLE system is faster than the current eCLE system (12 vs. ±
1 frames per second) making stream of pCLE images close to video output.

The possibility of in vivo diagnosis of H. pylori bacilli itself with eCLE was
demonstrated in a case study [39]. After topical application of acriflavine onto the gastric
surface, single to accumulated white dots were observed within the gastric mucosa. The
distinct shape and size of the bacteria, including the flagella, were identified. H. pylori
infection was confirmed by histology and culture. Ex vivo examination of the cultures also
showed active uptake of acriflavine with H. pylori.
In CLE image, corpus mucosa looks having dark round regular tubules are surrounded
with bright interstitium (Figure 10a). In the antrum mucosa, bright interstitium are surrounded
with continuous dark bands consisted of gastric pit and foveolar epithelium are seen (Figure
10b) [40]. The sensitivity and specificity of decreased number of dilated gastric pits in eCLE
for atrophy were reported to be 83.6% and 99.6%, respectively [40]. In mucosa with IM,
mucin-containing goblet cells appear large dark dots (Figure 10c) in the epithelium. The
sensitivity and specificity of eCLE for IM diagnosis are excellent at 98% and 95%,
respectively [41]. The use of CLE is may be promising for identifying H. pylori and
associated mucosal changes, whereas the use of CLE for the other gastric findings is still
limited due to poor standardization of the criteria and learning curve required [42].
Conclusion
Usually H. pylori infection is diagnosed with noninvasive and invasive methods
including the urea breath test, stool test, urease testing on endoscopic biopsies, and
serological assays. Despite strong link between H. pylori infection and gastric cancer
development, only H. pylori infection is not enough to select high-risk population. Because
progression of atrophy and IM in the gastric mucosa could cause spontaneous eradication of
H. pylori or underestimation of H. pylori infection, patients with gastric cancer could no
longer have H. pylori detectable, and thus the screening and surveillance of only actively
infected individuals may not be effective. Endoscopic test using new optical technique help us
diagnosing H. pylori infection as well as H. pylori associated preneoplastic mucosal changes
such as atrophy and IM without sampling of test specimens. Another great advantage of
endoscopic test for making diagnosis of H. pylori infection, atrophy and IM is unnecessity of
time until the result to come back. Consequently, endoscopy can be a useful adjunctive
method to assess the risk of gastric cancer. We can make decision for patient management
immediately after the endoscopic procedure. Practical risk stratification and surveillance
strategy based on the endoscopic finding that is relevant to clinical practice warrants to be
established in the future study.
References
[1] Correa P. Human gastric carcinogenesis: a multistep and multifactorial process--First
American Cancer Society Award Lecture on Cancer Epidemiology and Prevention.
Cancer Res. 1992;52:6735-6740.

[2] Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis.
The updated Sydney System. International Workshop on the Histopathology of
Gastritis, Houston 1994. Am. J. Surg Pathol. 1996;20:1161-1181.
[3] Miki K, Morita M, Sasajima M, et al. Usefulness of gastric cancer screening using the
serum pepsinogen test method. Am. J. Gastroenterol. 2003;98:735–9.
[4] Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M,
Taniyama K, Sasaki N, Schlemper RJ. Helicobacter pylori infection and the
development of gastric cancer. N. Engl. J. Med. 2001;345:784-789.
[5] de Vries AC, van Grieken NC, Looman CW, Casparie MK, de Vries E, Meijer GA,
Kuipers EJ. Gastric cancer risk in patients with premalignant gastric lesions: a
nationwide cohort study in the Netherlands. Gastroenterology 2008;134:945-952.
[6] Dinis-Ribeiro M, Areia M, de Vries AC, Marcos-Pinto R, Monteiro-Soares M,
O'Connor A, Pereira C, Pimentel-Nunes P, Correia R, Ensari A, Dumonceau JM,
Machado JC, Macedo G, Malfertheiner P, Matysiak-Budnik T, Megraud F, Miki K,
O'Morain C, Peek RM, Ponchon T, Ristimaki A, Rembacken B, Carneiro F, Kuipers
EJ; European Society of Gastrointestinal Endoscopy; European Helicobacter Study
Group; European Society of Pathology; Sociedade Portuguesa de Endoscopia
Digestiva. Management of precancerous conditions and lesions in the stomach (MAPS):
guideline from the European Society of Gastrointestinal Endoscopy (ESGE), European
Helicobacter Study Group (EHSG), European Society of Pathology (ESP), and the
Sociedade Portuguesa de Endoscopia Digestiva (SPED). Endoscopy. 2012;44:74-94.
[7] Price AB. The Sydney System: histological division. J. Gastroenterol. Hepatol.
1991;6:209-222.
[8] Rugge M, Meggio A, Pennelli G, Piscioli F, Giacomelli L, De Pretis G, Graham DY.
Gastritis staging in clinical practice: the OLGA staging system. Gut 2007;56:631-636.
[9] Capelle LG, de Vries AC, Haringsma J, Ter Borg F, de Vries RA, Bruno MJ, van
Dekken H, Meijer J, van Grieken NC, Kuipers EJ. The staging of gastritis with the
OLGA system by using intestinal metaplasia as an accurate alternative for atrophic
gastritis. Gastrointest Endosc. 2010;71:1150-1158.
[10] K. Yagi, A. Nakamura and A. Sekine. Comparison Between Magnifying Endoscopy and
Histological, Culture and Urease Test Findings from the Gastric Mucosa of the Corpus
Endoscopy 2002;34: 376–381.
[11] Tahara T, Shibata T, Nakamura M, Yoshioka D, Okubo M, Arisawa T, Hirata I Gastric
mucosal pattern by using magnifying narrow-band imaging endoscopy clearly
distinguishes histological and serological severity of chronic gastritis. Gastrointest
Endosc. 2009; 70: 246–253.
[12] Kimura K, Takemoto T. An endoscopic recognition of the atrophic border and its
significance in chronic gastritis. Endoscopy 1969;1:87-97.
[13] Kimura K, Satoh K, Ido K, Taniguchi Y, Takimoto T, Takemoto T. Gastritis in
Japanese stomach. Scand J. Gastroenterol. 1996;31 Suppl 214:17-20.
[14] Redeen S, Petersson F, Jonsson KA, Borch K. Relationship of gastroscopic feature to
histological findings in gastritis and Helicobacter pylori infection in a general
population sample. Endoscopy 2003;35:946–50.
[15] Take S, Mizuno M, Ishiki K, Nagahara Y, Yoshida T, Yokota K, Oguma K. Baseline
gastric mucosal atrophy is a risk factor associated with the development of gastric

cancer after Helicobacter pylori eradication therapy in patients with peptic ulcer
diseases. J. Gastroenterol. 2007;42 Suppl 17:21-7.
[16] Laine L, Cohen H, Sloane R, Marin-Sorensen M, Weinstein WM. Interobserver
agreement and predictive value of endoscopic findings for H. pylori and gastritis in
normal volunteers. Gastrointest Endosc. 1995;42:420-423.
[17] Sauerbruch T, Schreiber MA, Schussler P, Permanetter W. Endoscopy in the diagnosis
of gastritis. Diagnostic value of endoscopic criteria in relation to histological diagnosis.
Endoscopy 1984;16:101-104.
[18] Kamihishi M, Yamaguchi H, Nomura S, Oohara T, Sakai S, Fukutomi H, Nakahara A,
Kashimura H, Oda M, Kitahora T, Ichikawa H, Yabana T, Yagawa Y, Sugiyama T,
Itabashi M, Unakami M, Oguro Y and Sakita T. Endoscopic classification of chronic
gastritis based on pilot study by the research society for gastritis. Dig. Endosc.
2002;14:138-151.
[19] Tatsuta M, Iishi H, Nakaizumi A, Okuda S, Taniguchi H, Hiyama T, Tsukuma H,
Oshima A. Fundal atrophic gastritis as a risk factor for gastric cancer. Int. J. Cancer
1993;53:70-74.
[20] Ida K, Hashimoto Y, Kawai K. In vivo staining of gastric mucosa: its applications to
endoscopic diagnosis of intestinal metaplasia. Endoscopy 1975;7:18-24.
[21] Fennerty MB, Sampliner RE, McGee DL, Hixson LJ, Garewal HS. Intestinal
metaplasia of the stomach: identification by a selective mucosal staining technique.
Gastrointest Endosc. 1992;38:696-698.
[22] Suzuki S, Suzuki H, Endo M, Takemoto T, Kondo T, Nakmakya K. Endoscopic dyeing
method for diagnosis of early cancer and intestinal metaplasia of the stomach.
Endoscopy 1973;5:124-129.
[23] Morales TG, Bhattacharyya A, Camargo E, Johnson C, Sampliner RE. Methylene blue
staining for intestinal metaplasia of the gastric cardia with follow-up for dysplasia.
Gastrointest Endosc. 1998;48:26-31.
[24] Dinis-Ribeiro M, da Costa-Pereira A, Lopes C, Lara-Santos L, Guilherme M, Moreira-
Dias L, Lomba-Viana H, Ribeiro A, Santos C, Soares J, Mesquita N, Silva R, Lomba-
Viana R. Magnification chromoendoscopy for the diagnosis of gastric intestinal
metaplasia and dysplasia. Gastrointest Endosc. 2003;57:498-504.
[25] Areia M, Amaro P, Dinis-Ribeiro M, Cipriano MA, Marinho C, Costa-Pereira A, Lopes
C, Moreira-Dias L, Romãozinho JM, Gouveia H, Freitas D, Leitão MC. External
validation of a classification for methylene blue magnification chromoendoscopy in
premalignant gastric lesions. Gastrointest Endosc. 2008;67:1011-8.
[26] Meneghelli UG, Godoy RA, Oliveira RB, Santos JC, Jr., Dantas RO, Troncon LE.
Effect of pentagastrin on the motor activity of the dilated and nondilated sigmoid and
rectum in Chagas' disease. Digestion 1983;27:152-158.
[27] Olliver JR, Wild CP, Sahay P, Dexter S, Hardie LJ. Chromoendoscopy with methylene
blue and associated DNA damage in Barrett's oesophagus. Lancet 2003;362:373-374.
[28] Uedo N, Iishi H, Tatsuta M, Yamada T, Ogiyama H, Imanaka K, Sugimoto N,
Higashino K, Ishihara R, Narahara H, Ishiguro S. A novel videoendoscopy system by
using autofluorescence and reflectance imaging for diagnosis of esophagogastric
cancers. Gastrointest Endosc. 2005;62:521-8.
[29] Uedo N. What can we see in the different lights? Gut Liver 2008;2:216-217.

[30] Inoue T, Uedo N, Ishihara R, Kawaguchi T, Kawada N, Chatani R, Kizu T, Tamai C,

Takeuchi Y, Higashino K, Iishi H, Tatsuta M, Tomita Y, Tóth E. Autofluorescence
imaging videoendoscopy in the diagnosis of chronic atrophic fundal gastritis. J.
Gastroenterol 2010;45:45-51.
[31] Yao K, Oishi T, Matsui T, Yao T, Iwashita A. Novel magnified endoscopic findings of
microvascular architecture in intramucosal gastric cancer. Gastrointest Endosc.
2002;56:279-284.
[32] Gono K, Obi T, Yamaguchi M, Ohyama N, Machida H, Sano Y, Yoshida S, Hamamoto
Y, Endo T. Appearance of enhanced tissue features in narrow-band endoscopic
imaging. J. Biomed. Opt. 2004;9:568-577.
[33] Bansal A, Ulusarac O, Mathur S, Sharma P. Correlation between narrow band imaging
and nonneoplastic gastric pathology: a pilot feasibility trial. Gastrointest Endosc.
2008;67:210-216.
[34] Kanzaki H, Uedo N, Ishihara R, et al. Comprehensive investigation of areae gastricae
pattern in gastric corpus using magnifying narrow band imaging endoscopy in patients
with chronic atrophic fundic gastritis. Helicobacter 2012;17:224-231.
[35] Uedo N, Ishihara R, Iishi H, et al. A new method of diagnosing gastric intestinal
metaplasia: narrow-band imaging with magnifying endoscopy. Endoscopy
2006;38:819-824.
[36] Kiesslich R, Burg J, Vieth M, Gnaendiger J, Enders M, Delaney P, Polglase A,
McLaren W, Janell D, Thomas S, Nafe B, Galle PR, Neurath MF. Confocal laser
endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo.
Gastroenterology 2004;127:706-713.
[37] Hoffman A, Goetz M, Vieth M, Galle PR, Neurath MF, Kiesslich R. Confocal laser
endomicroscopy: technical status and current indications. Endoscopy 2006;38:1275-
1283.
[38] Nguyen NQ, Leong RW. Current application of confocal endomicroscopy in
gastrointestinal disorders. J. Gastroenterol. Hepatol. 2008;23:1483-1491.
[39] Kiesslich R, Goetz M, Burg J, Stolte M, Siegel E, Maeurer MJ, Thomas S, Strand D,
Galle PR, Neurath MF. Diagnosing H. pylori in vivo by confocal laser endoscopy.
Gastroenterology 2005;128:2119-2123.
[40] Zhang JN, Li YQ, Zhao YA, Yu T, Zhang JP, Guo YT, Liu H. Classification of gastric
pit patterns by confocal endomicroscopy. Gastrointest Endosc. 2008;67:843-853.
[41] Guo YT, Li YQ, Yu T, Zhang TG, Zhang JN, Liu H, Liu FG, Xie XJ, Zhu Q, Zhao YA.
Diagnosis of gastric intestinal metaplasia with confocal laser endomicroscopy in vivo: a
prospective study. Endoscopy. 2008;40:547-53.
[42] Pittayanon R, Rerknimitr R, Wisedopas N, Khemnark S, Thanapirom K,
Thienchanachaiya P, Norrasetwanich N, Charoensuk K, Ridtitid W, Treeprasertsuk S,
Kongkam P, and Kullavanijaya P. The learning curve of gastric intestinal metaplasia
interpretation on the images obtained by probe-based confocal laser endomicroscopy.
Diagnostic and Therapeutic Endoscopy 2012, Article ID 278045, doi:10.1155/
2012/278045.

Diseases and Health Implications

Chapter 6
Epidemiology, Transmission Routes,

and Recurrence of Infection
Marco Manfredi1,2, Valentina Maffini1 and Gian Luigi de'Angelis1,2

1
Azienda Ospedaliero-Universitaria di Parma, University Hospital, Parma, Italy
2
Gastroenterology and Endoscopy Unit, Azienda Ospedaliero-Universitaria di Parma,
Abstract
Helicobacter pylori (H. pylori) infection is one of the most common infection in
humans, affecting more than half of the human population. The prevalence of the
infection is really different in rural developing areas (more than 80%) compared to urban
developed ones (less than 40%), as consequence of different socioeconomic conditions
and hygienic practices. This infection is usually acquired during childhood and the
infected population usually remains asymptomatic, but in about 30% of cases individuals
may developed mild to severe upper gastrointestinal pathologies like gastritis, peptic
ulcer, gastric cancer or MALT lymphoma. The route of transmission is not completely
understood; the person-to-person transmission especially within the same family appears
to be prevalent, but also environmental contamination is possible. The eradication
without a specific therapeutic regimen is very unlikely and the reinfection rate after an
effective eradication therapy is quite rare. The reinfection rate will increase if there are
family members affected.
Keywords: Helicobacter pylori, epidemiology, Prevalence, inter-humans transmission,

reinfection, recurrence

Correspondance author: Marco Manfredi MD, PhD, Department of Pediatrics, “Pietro Barilla” Children's Hospital,
Azienda Ospedaliero-Universitaria di Parma, University Hospital, Via Gramsci 14, 43126 Parma, Italy, email:
marco.manfredi8@gmail.com.

102 Marco Manfredi, Valentina Maffini and Gian Luigi de'Angelis
Introduction
Helicobacter pylori (H. pylori) is an organism that has been intimately associated with
humans for many centuries, even though it was discovered only in the early 1980s [1].
H. pylori infection is a significant cause of morbidity and mortality in humans as it has a
crucial role in the development of chronic gastritis, gastroduodenal ulcer, and gastric cancer
which may seriously affect the quality of life of the patients [2].
Since 1994 H. pylori has been classified in the first group of carcinogenic agents by
WHO [3]. This category means that there is sufficient evidence of carcinogenicity to humans
(see table 6.1).
Table 6.1: classification of carcinogenic agents to humans by WHO [3]
Group 1 = Carcinogenic to humans

Group 2A = Probably carcinogenic to humans
Group 2B = Possibly carcinogenic to humans
Group 3 = Not classifiable as to its carcinogenicity to humans
Group 4 = Probably not carcinogenic to humans
For these reasons, the eradication of H. pylori infection remains a worldwide public
health concern.
All features implicated in the pathogenesis of H. pylori-related disease are not completely
understood and epidemiological data in certain countries are discordant, as in the so-called
“African enigma”. African enigma describes the dissociation between the prevalence of H.
pylori infection and H. pylori-related gastric cancer. Similar observations have now been
made in other geographical areas. As in other part of the developing world, the prevalence of
H. pylori infection in Africa is high, but there is no evidence of the expected high correlation
with related disease. These data are of great interest in relation to the pathogenesis of H.
pylori-related diseases and should lead to a careful examination of host, environmental and H.
pylori virulence [4, 5].
H. pylori infection is predominantly acquired during childhood, usually persists
throughout life without a specific treatment and interpersonal contact seems to be the main
route of infection.
In countries where the socio-economic conditions have been improving, there is evidence
that the prevalence of H. pylori infection is declining. However, large proportions of adult
populations remain infected so the burden of infection manifesting as peptic ulcer disease and
gastric cancer will continue to be an important problem [6].
There is heterogeneity of infection rates even within more developed countries, with
well-defined high-risk groups. These groups include the elderly, those who live in poorer
conditions, migrants from high prevalence areas, the institutionalized and possibly rural
dwellers in some areas.
For these reasons we need of really effective eradication treatments trying to bring down
the risk of complications. Furthermore we also need continuing to improve the socio-
economic conditions of people and checking the intrafamilial members of infected
subjects [7].

Epidemiology, Transmission Routes, and Recurrence of Infection 103
Though the prevalence of this infection appears to be decreasing in many parts of the
world, H. pylori remains an important factor linked to the development of peptic ulcer
disease, gastric malignancy and dyspeptic symptoms.
Epidemiology
The prevalence of H. pylori infection in European studies varies between 7 and 33%,
while in developing countries can exceed 80% [8]. However, as socio-economic level varies
within sub-populations of the same country, the prevalence in these subgroups can be rather
different [9].
Although, once established within the gastric mucosa the H. pylori persists for life [8, 9],
most infected subjects develop no symptoms or peptic ulcer during their life and continue to
live well with only a superficial chronic gastritis [10, 11]. But these subjects are very
important sources of infection therefore they may transmit it to healthy persons which may
become at risk of severe diseases. However, there is a common consensus that the risk of
acquisition and transmission of H. pylori can be reduced and prevented to a large extent by a
better control of intrafamilial spread. High rates of H. pylori infection have been significantly
associated with low socio-economic status and poor living conditions during childhood [12].
It has been demonstrated that prevalence of H. pylori infection in developing countries with
low socio-economic level and poor management of drinking water is much higher ( > 80%)
than that in developed countries ( < 40%) [13, 14].
Several studies have shown that acquisition of H. pylori infection occurs during early
childhood, and adults probably carry the same bacterial strain for sequential decades [15].
Studies from both developing and developed countries have also demonstrated that children
who have infected mothers (with low education) and infected older siblings are at highest risk
of infection [16, 17]. Furthermore, low rate (8.9%) of H. pylori infection in developed
countries [18] and high rates, 52% [19] and 98% [20], in developing ones refer to a strong
association between the prevalence of H. pylori infection and the socio economic status of a
population.
The annual rate of seroconversion in adult populations in developed countries appears to
be small, about 0.2-1.0 percent. Instead, adults under uncommon circumstances, such as
military and closed community, can seroconvert at higher rates than normal people up to
6.4% and 7.3% respectively [4].
In the USA a significant decrease in prevalence was recently observed only in the non-
Hispanic white population compared to the previous ten years [21].
A recent study found a prevalence of 58% in Caribbean territories [22]. In Australia the
H. pylori infection appears to be more prevalent in the elderly (> 70 years) than the youngest
people (< 40 years): 32% vs 5% respectively and in the born overseas as well as in the lowest
socio-economic areas [23].
In Europe the prevalence is still higher in the eastern than in the western countries. In
Denmark the infection is more prevalent in the older than 45 years compared to younger [24].
In Belgium the prevalence is also declining in the last 20 years: from 36.2 to 15.2 % and from
71.7 to 40% in the western European people and in north Africans patients, respectively with
a global prevalence of 37.7% [25].

There is now evidence that H. pylori infection is declining in both developed and
developing countries.
Queiroz et a.l followed a cohort of Brazilian children from low-income community and
they found that the prevalence of H. pylori infection increased during the observation period
(8 years) from 53.4% at baseline to 64.7%. Among them, 6.0% had lost the infection, while
17.3% became infected. Risk factors for H. pylori infection were a high number of children in
the household and male gender [26].
In a large cross-sectional survey of adults in the United Kingdom, male gender,
increasing age, shorter height, tobacco use, and lower socio-economic status were all
significantly associated with positive H. pylori serology [27].
While in a Czech survey study, a lack of formal education of the father, and
institutionalization of the child were all significantly associated with infection [28].
Instead no significant effects of gender, socio-economic status, number of children in the
household, parental education, or sharing a room or a bed with parents or siblings was
observed in a Greek study [29]. The same results were observed in a Taiwanese study unlike
of Pakistani children [8].
Transmission Routes
The route of transmission of H. pylori is not completely understood. The only known
reservoir is the human stomach [30] and since H. pylori appears to have a narrow host range,
new infections are thought to occur as a consequence of direct human-to-human transmission
or environmental contamination.
Person-to-person transmission can be subdivided in two main categories: vertical and
horizontal transmission.
The vertical mode is infection spread from ascendant to descendent within the same
family, (e.g. from parents to child), while horizontal transmission involves contact with
individuals outside the family or environmental contamination [31].
A lot of studies since before 2000 look at the relation between H. pylori infections and
familial exposure. Most of them [32, 33] provided support to the concept of intrafamilial
clustering of H. pylori infection. They suggest that person-to-person transmission occurred in
these families possibly because of close interpersonal contacts, that family members shared a
genetic predisposition to H. pylori infection, that family members were exposed to a common
source of infection or that spouses’ childhood socio-economic class was similar.
Instead in developed countries with low H. pylori prevalence, the infected mother is
likely to be the primary source for infant H. pylori infection [34].
In population with high H. pylori prevalence and poor socio-economic conditions,
infected mothers are less involved in the transmission inside the family, while transmission
among siblings as well as outside acquisition appears to play a major role in the transmission
pathway.
The person-to-person transmission may occur by three possible pathways: the gastro–
oral, the oral–oral and the faecal–oral routes, but no predominant mechanism of
transmission has been yet identified.

Gastro-Oral Transmission
H. pylori is acquired in early life and the vomiting of achlorhydric mucus may serve as a
vehicle for transmission. The transmission route could be by gastric juice, especially as a
result of epidemic vomiting in childhood [35].
Studies reported data about isolation percentage of H. pylori from gastric juice of
symptomatic patients; the microbe appears to survive outside the human body in unbuffered
gastric juice, being often present in high quantities in vomit with as many as 30,000 CFU/mL
of sample. These results are favourable to the gastro-oral transmission, especially during
childhood, in association with poor hygiene practices.
Oral-Oral Transmission
Saliva is another possible source of H. pylori transmission, since the gastric flora can
reach and colonize the mouth after regurgitation or vomiting. H. pylori has been cultured
directly from saliva and the DNA has been frequently amplified from saliva, subgingival
biofilm and dental plaque [36]. Based on these reports, the mouth might be a reservoir of H.
pylori [37]. The oral-oral transmission involves especially the mother-child transmission: the
oral secretions of the mother, which may be contaminated with H. pylori, can be transmitted
to the infant. Pre-masticating food before it is fed to children is a popular maternal practice
(specially in developing countries). Furthermore, the transmission may occur by the common
use of spoons, the licking of pacifiers or feeding bottles nipples or even by the chewing or
tasting of children’s food. Nevertheless other negative arguments against the oral–oral
transmission include the frequent absence of related strains infecting spouses [38, 39], but this
is controversial given reports that demonstrate the presence of common strains infecting
couples [40]. These data suggest that although saliva might work as a vehicle of transmission,
the oral–oral transmission is not the main mode of transmission of H. pylori, at least in adults.
Faecal-Oral Transmission
H. pylori DNA has been frequently detected in human faeces [41], but attempts to culture
H. pylori from faeces have had limited success because the bacterium exists there
predominantly in a non-culturable (coccoid) form. However transmission via faecal
contaminants is supported by some studies on animals and also by the occurrence of H. pylori
infections among institutionalized young people during outbreaks of gastroenteritis,
especially when the hygienic conditions are poor.
Transmission by Water
The exact way by which H. pylori gains access to the human stomach is unknown and
also environmental contamination should be considered. When hygienic conditions are poor,
household contamination of treated water cannot be ruled out.

Some authors hypothesize that water plays a role both as an environmental reservoir of
infection and as a medium in the fecal–oral transmission of H. pylori infection. They found
that children living in houses with an external water supply, or those consuming raw
vegetables, which are often irrigated with untreated sewage water, had a higher prevalence of
H. pylori infection [42, 43].
H. pylori is easily controlled by chlorination (the principal disinfection agent of water),
but recontamination of treated water is a widespread problem. Poor hygienic practices during
childhood, absence of a household bath, non-hygienic drinking water and absence of a
sewage disposal facility may lead to re-contamination of drinking water, that in this way may
serve as a vehicle of transmission.
The association of serum antibodies against H. pylori with serum antibodies against two
known waterborne pathogens (Hepatitis A virus) [44] and Giardia [45], suggests that the
infection may be waterborne or related to poor sanitary practices.
However, these associations are not always observed.
Indirect evidences of a possible waterborne transmission of H. pylori are the presence of
DNA and observation of coccoid forms in water samples, survival of H. pylori in artificially
contaminated water and growth of H. pylori from water samples.
Transmission by Food
As it happens with water, food products may also be contaminated while handling, under
poor hygienic conditions.
Several studies address the role of food in the transmission of H. pylori. Food products
analysed are mainly milk, meat and vegetables. Among these, milk products are the most
studied, probably because the infection is mainly acquired during childhood and milk is
mostly consumed during this period [46].
Recurrence of Infection
Recurrence of H. pylori is thought to occur via two distinct mechanisms, recrudescence
and reinfection.
Recrudescence reflects reappearance of the original strain of H. pylori following its
temporary suppression rather than successful eradication. Instead, true reinfection occurs
when, after successful eradication, a patient becomes infected with either the original strain or
a new strain of H. pylori [47].
Many investigators have found that recurrence rates during the first 3-12 months after
cure are due to late recrudescence. Demonstrated H. pylori negativity for 1 year post-
treatment is a reliable indicator of successful eradication without recrudescence which is
related to the efficacy of the regimen used. Studies showed that recurrence of H. pylori
infection more frequently occurred in patients treated with a low-efficacy therapy than in
those treated with a high-efficacy therapeutic regimen. It seems that low-efficacy therapy
does not actually cure H. pylori infection in the gastric mucosa, but only temporarily
suppresses it and does not completely eradicate it from the host [48-50].

H. pylori reinfection after successful eradication is an important problem in the

management of this disease. The reinfection rate of H. pylori varies considerably among
different studies.
Studies before 2000 reported considerably wide annual recurrence rates ranging between
0 and 100% in adults and between 2.0 and 13.5% in children. The studies carried out after
2000 also produced conflicting results, with relatively low recurrence rates.
Generally, low annual recurrence rates were observed in developed countries (up to 2%
for both adults and children), but high recurrence rates (>10%) were observed in developing
countries [51].
Recrudescence rather than reinfection is likely to be responsible for most recurrent cases
because the recurrences decrease with time and decline sharply after the first year, and
identified strains (before and after therapy) are usually genetically identical.
Reported “true” reinfection rates in adults generally varied from 0 to 23.4%. The annual
“true” reinfection rates were much lower than the reported annual recurrence rates within the
first years after eradication [47].
Intrafamilial transmission could be also involved in the reinfection of H. pylori; its
presence among asymptomatic family members may facilitate the transmission within
households [7].
The reinfection rate after eradication therapy for H. pylori is extremely low in developed
countries such as Europe and the USA. Here, the annual reinfection rates are reported to be
around 1%. In contrast to the low rates of H. pylori reinfection reported in western
populations, high recurrence rates have been reported in such developing countries as Peru,
Brazil, Chile, Vietnam and Bangladesh, all of which are countries with a high prevalence of
H. pylori infection [52-55]. Therefore the high prevalence of H. pylori infection may possibly
be associated with high recurrence of infection after eradication because of the high risk of re-
exposure to infection [56].
Genetic factors may also play a role in reinfection of H. pylori after successful
eradication although there has been no study that directly addresses this issue. Therefore,
susceptible individuals who have had H. pylori eradicated may be prone to reinfection with
the bacterium when they are exposed to H. pylori-positive persons [7].
The role of searching for and eradicating of H. pylori infection in general or in a risk
population to reduce the gastric cancer incidence, it is not well established, yet. Although
from several studies appears that this approach in population at high risk of develop gastric
cancer is a cost-effective strategy [57-60], re-testing and re-treating individuals shortly after
initial eradication therapy may not be cost-effective. In fact, Morgan and colleagues stated
that the recurrence at 1 year was significantly associated with study site, number of children
in the households, and non-adherence to therapy, but not with treatment used [61]. In their
study the risk of recurrent H. pylori infection following apparently successful eradication was
11.5%, regardless of the eradication therapy used. Meanwhile in a previous review by Gisbert
the overall annual recurrence risk varied from 3.4% to 8.7% in high-income and in lower-
income countries, respectively [62].

Conclusion
The prevalence of H. pylori is closely tied to socio-economic conditions and accordingly,
this infection is more common in developing than in developed countries such as the United
States. A mode of infection on which, perhaps, we do not put attention enough, but that
certainly plays a key role in the transmission of H. pylori infection, is the intrafamilial
transmission. The close and intimate contact among family members appears to be a key
route responsible for the transmission of H. pylori infection [63, 64].
Considering the substantial efforts that the H. pylori is forcing us every day, both to avoid
complications that can be very serious (e.g. peptic ulcer, gastric cancer) and the consequent
heavy financial burden, it's time to take more accurate actions toward this infection and adopt
a strategy to prevent it, also in western countries [10].
H. pylori infection still being sneaky, it is often overlooked in terms of prevention and we
are concerned only when a patient is symptomatic [65]. Therefore, probably we should better
control all family members of infected persons regardless of their symptoms. In this way,
perhaps, by reducing the undiagnosed patients, we could reduce the risk of development of
the H. pylori-related effects, decreasing the risk of reinfection within the family - although
rare - and then limit the spread of H. pylori infection [66].
H. pylori recurrence in the first year after eradication therapy is likely due to a mixture of
recrudescence of infection and reinfection. The reinfection predominates in subsequent years
after eradication while the risk of recurrence tends to decrease.
The recurrence 1 year after the treatment usually is associated with drugs non-adherence
and the regional zone of patients, demonstrating a possible marker of local antibiotic
resistance. Since the early age at acquisition of H. pylori infection may result in more intense
inflammation and the early development of atrophic gastritis and subsequent risk of gastric
ulcer, gastric cancer, or both, we need, perhaps, to implement health education programs
within the family (washing of hands and mouth, brushing teeth, no sharing of food plates or
drinking glasses, no sharing of spoons in feeding children) to minimize the spread of the
infections, including those of H. pylori.
Then, for having good results in eradication of H. pylori infection we should consider not
only the choice of antibiotics but also the geographic site, the demographic factors, and the
local infection recurrence rate.
References
[1] Linz B, Balloux F, Moodley Y, et al. (2007). An African origin for the intimate
association between humans and Helicobacter pylori. Nature., 445:915–8.
[2] Malfertheiner P, Megraud F, O’Morain CA, Atherton J, Axon ATR, Bazzoli F, Gensini
GF, Gisbert JP, Graham DY, Rokkas T, El-Omar EM, Kuipers EJ, The European
Helicobacter Study Group (EHSG), (2012). Management of Helicobacter pylori
infection – the Maastricht IV/ Florence Consensus Report. Gut., 61:646-664.
[3] IARC Working Group on the Evaluation of Carcinogenic Risks to Humans
Schistosomes, liver flukes and Helicobacter pylori 7-14 June 1994, Lyon IARC Monogr
Eval. Carcinog. Risks Hum., 61:1-241.

[4] Ghoshal U, Chaturvedi R, Correa P. The enigma of Helicobacter pylori and gastric
cancer. Indian J. Gastroenterology 2010,29: 95-100.
[5] Agha A, Graham D, (2005). Evidence-based examination of the African enigma in
relation to Helicobacter pylori infection. Scand. J. Gastroenterol. 40:523-529.
[6] Fock KM, Talley N, Moayyedi P, Hunt R, Azuma T, Sugano K, Xiao SD, Lam SK,
Goh KL, Chiba T, Uemura N, Kim JG, Kim N, Ang TL, Mahachai V, Mitchell H, Rani
AA, Liou JM, Vilaichone RK, Sollano J, (2008). Asia–Pacific consensus guidelines on
gastric cancer prevention. J. Gastroenterol. Hepatol, 23: 351-65.
[7] Ryu KH, Yi SY, Na YJ et al. (2010). Reinfection rate and endoscopic changes after
successful eradication of Helicobacter pylori. World. J. Gastroenterol., 16:251-255.
[8] Ford AC, Axon ATR, (2010). Epidemiology of Helicobacter pylori infection and
Public Health implications. Helicobacter, 15(suppl.1):1-6).
[9] Bruce MG, Maaroos HI, (2008). Epidemiology of Helicobacter pylori infection.
Helicobacter., 3 (suppl.1) :1-6.
[10] Salih BA, (2009). Helicobacter pylori infection in Developing Countries: the burden for
how long? Saudi J Gastroenterol, 15:201-207.
[11] Mitchel HM. Epidemiology of infection. In: Mobley HLT, Mendz GL, Hazell SL,
editors, Helicobacter pylori: Physiology and Genetics. Washington (DC): ASM Press;
2001. Chapter 2.
[12] Webb PM, Knight T, Greaves S et al. (1994). Relation between infection with
Helicobacter pylori and living conditions in childhood: evidence for person to person
transmission in early life. BMJ., 308:750–3.
[13] Pounder R, Ng D, (1995). The prevalence of Helicobacter pylori infection in different
countries. Aliment. Pharmacol. Ther., 9:33–9.
[14] Perez-Perez GI, Rothenbacher D, Brenner H, (2004). Epidemiology of Helicobacter
pylori infection. Helicobacter, 9:1–6.
[15] Cullen DJ, Collins BJ, Christiansen KJ et al. (1993). When is Helicobacter pylori
infection acquired? Gut., 34:1681–2.
[16] Rowland M, Daly L, Vaughan M et al. (2006). Age-specific incidence of Helicobacter
pylori. Gastroenterology., 130:65–72.
[17] Kivi M, Johansson AL, Reilly M, Tindberg Y, (2005). Helicobacter pylori status in
family members as risk factors for infection in children. Epidemiol. Infect., 133:645–52.
[18] Al-Shamahy HA, (2005). Seroprevalence of Helicobacter pylori among children in
Sana’a. Yemen. Ann. Saudi. Med., 25:299–303.
[19] Ndip RN, Malange AE, Akoachere JF, MacKay WG, Titanji VP, Weaver LT, (2004).
Helicobacter pylori antigens in the faeces of asymptomatic children in the Buea and
Limbe health districts of Cameroon: a pilot study. Trop. Med. Int. Health., 9:1036–40.
[20] Alborzi A, Soltani J, Pourabbas B et al. (2006). Prevalence of Helicobacter pylori
infection in children (south of Iran). Diagn. Microbiol. Infect. Dis., 54:259–61.
[21] Grad YH, Lipsitch M, Aiello AE, (2012). Secular trends in Helicobacter pylori
seroprevalence in adults in the United States: evidence for sustained race/ethnic
disparities. Am. J. Epidemiol., 175:54–9.
[22] Carter FP, Frankson T, Pintard J, Edgecombe B, (2011). Seroprevalence of
Helicobacter pylori infection in adults in the Bahamas. West. Indian. Med. J., 60:662–5.

[23] Pandeya N, Whiteman DC, (2011). Prevalence and determinants of Helicobacter pylori
sero-positivity in the Australian adult community. J. Gastroenterol. Hepatol., 26:
1283–9.
[24] Dahlerup S, Andersen RC, Nielsen BS, Schjodt I, Christensen LA, Gerdes LU, et al.
(2011). First-time urea breath tests performed at home by 36,629 patients: a study of
Helicobacter pylori prevalence in primary care. Helicobacter., 16:468–74.
[25] Queiroz DM, Carneiro JG, Braga-Neto MB, Fialho AB, Fialho AM, Goncalves MH, et.
al, (2012). Natural history of Helicobacter pylori infection in childhood: eight-year
follow-up cohort study in an urban community in north east of Brazil. Helicobacter.,
17:23–9.
[26] Jackson L, Britton J, Lewis SA, McKeever TM, Atherton J, Fullerton D, et al. (2009).
A population-based epidemiologic study of Helicobacter pylori infection and its
association with systemic inflammation. Helicobacter., 14:460–5
[27] Sykora J, Siala K, Varvarovska J, Pazdiora P, Pomahacova R, Huml M, (2009).
Epidemiology of Helicobacter pylori infection in asymptomatic children: a prospective
population-based study from the Czech Republic. Application of a monoclonal-based
antigen-in stool enzyme immunoassay. Helicobacter., 14:286–97
[28] Roma E, Panayiotou J, Pachoula J, Kafritsa Y, Constantinidou C, Mentis A, et al.
(2009). Intrafamilial spread of Helicobacter pylori infection in Greece. J. Clin.
Gastroenterol., 43:711–5.
[29] Megraud, F., Broutet, N., (2000). Review article: have we found the source of
Helicobacter pylori? Aliment. Pharmacol. Ther., 14 (Suppl 3), 7–12.
[30] Schwarz, S., Morelli, G., Kusecek, B., Manica, A., Balloux, F., Owen, R.J., Graham,
D.Y., van der, M.S., Achtman, M., Suerbaum, S., (2008). Horizontal versus familial
transmission of Helicobacter pylori. PLoS Pathogens 4, e1000180.
[31] Rothenbacher D, Bode G,Berg Get al. (1999). Helicobacter pylori among preschool
children and their parents: evidence of parent-child transmission. J. Infect. Dis.,
179:398-402.
[32] Parente F, Maconi G, Sangaletti O et al. (1996). Prevalence of Helicobacter pylori
infection and gastroduodenal lesions in spouses of Helicobacter pylori positive patients
with duodenal ulcere. Gut., 39:634-638.
[33] Weyermann M, Rothenbacher D, Brenner H., (2009). Acquisition of Helicobacter
pylori infection in early childhood: independent contributions of infected mothers,
fathers, and siblings. Am. J. Gastroenterol., 104, 182–189.
[34] Axon AT, (1995). Review article: is Helicobacter pylori transmitted by the gastro–oral
route? Aliment. Pharmacol .Ther., 9, 585–588.
[35] Burgers R, Schneider-Brachert W, Reischl U, Behr A, Hiller KA, Lehn N, Schmalz G,
Ruhl S, (2008). Helicobacter pylori in human oral cavity and stomach. Eur. J. Oral.
Sci., 116, 297–304.
[36] Gebara EC, Faria CM, Pannuti C, Chehter L, Mayer MP, Lima LA, (2006). Persistence
of Helicobacter pylori in the oral cavity after systemic eradication therapy. J. Clin.
Periodont., 33, 329–333.
[37] Gisbert JP, Arata IG, Boixeda D, Barba M, Canton R, Plaza AG, Pajares JM, (2002).
Role of partner's infection in reinfection after Helicobacter pylori eradication. Eur. J.
Gastroenterol. Hepatol., 14, 865–871.

[38] Vale FF, Vitor JM, (2007). Genomic methylation: a tool for typing Helicobacter pylori
isolates. Appl. Environ. Microbiol., 73, 4243–4249.
[39] Kivi M, Tindberg Y, Sorberg M, Casswall TH, Befrits R, Hellstrom PM, Bengtsson C,
Engstrand L, Granstrom M, (2003). Concordance of Helicobacter pylori strains within
families. J. Clin. Microbiol., 41, 5604–5608.
[40] Queralt N, Bartolome R, Araujo R, (2005). Detection of Helicobacter pylori DNA in
human faeces and water with different levels of faecal pollution in the north-east of
Spain. J. Appl. Microbiol., 98, 889–895.
[41] Klein PD, Graham DY, Gaillour A, Opekun AR, O’Brian Smith E, the Gastrointestinal
Physiology Working Group, (1991). Water source as risk factor for Helicobacter pylori
infection in Peruvian children. Lancet., 337:1503–6.
[42] Hopkins RJ, Vial PA, Ferreccio C, Ovalle J, Prado P, Sotomayor V, et al. (1993).
Seroprevalence of Helicobacter pylori in Chile: vegetables may serve as one route of
transmission. J. Infect Dis., 168:222–6.
[43] Bizri AR, Nuwayhid IA, Hamadeh GN, Steitieh SW, Choukair AM, Musharrafieh UM,
(2006). Association between hepatitis A virus and Helicobacter pylori in a developing
country: the saga continues. J. Gastroenterol. Hepatol., 21, 1615–1621.
[44] Moreira Jr ED, Nassri VB, Santos RS, Matos JF, de Carvalho WA, Silvani CS, Santana
S, (2005). Association of Helicobacter pylori infection and giardiasis: results from a
study of surrogate markers for fecal exposure among children. World. J. Gastroenterol.,
11, 2759–2763.
[45] Vale FF, Vitor JMB, (2010).Transmission pathway of Helicobacter pylori: Does food
play a role in rural and urban areas? Int. J. Food. Microbiol., 138: 1-12.
[46] Zhang YY, Xia HHX, Zhuan ZH, Zhong J, (2009). Review article: “True” re-infection
of Helicobacter pylori after successful eradication: worldwide annual rates, risk factors
and clinical implications. Aliment Pharmacol Ther., 29(2):145-60.
[47] Sachs G, Scott DR. Helicobacter pylori: eradication or preservation? F1000 Medicine
Reports 2012; 4:7.
[48] Adachi M, Mizuno M, Yokota K, Miyoshi M, Nagahara Y, Maga T, Ishiki K, Inaba T,
Okada H, Oguma K, Tsuji T, (2002). Reinfection rate following effective therapy
against Helicobacter pylori infection in Japan. J. Gastroenterol. Hepatol., 17: 27-31.
[49] Cameron EA, Bell GD, Baldwin L, Powell KU, Williams SG, (2006). Long-term study
of re-infection following successful eradication of Helicobacter pylori infection.
Aliment Pharmacol Ther., 23: 1355-1358.
[50] Zendehdel N, Nasseri-Moghaddam S, Malekzadeh R, Massarrat S, Sotoudeh M,
Siavoshi F, (2005). Helicobacter pylori reinfection rate 3 years after successful
eradication. J. Gastroenterol. Hepatol., 20: 401-404.
[51] Okimoto T, Murakami K, Sato R, Miyajima H, Nasu M, Kagawa J, Kodama M, Fujioka
T, (2003). Is the recurrence of Helicobacter pylori infection after eradication therapy
resultant from recrudescence or reinfection, in Japan. Helicobacter., 8: 186-191.
[52] Wheeldon TU, Hoang TT, Phung DC, Björkman A, Granström M, Sörberg M, (2005).
Long-term follow-up of Helicobacter pylori eradication therapy in Vietnam: reinfection
and clinical outcome. Aliment Pharmacol Ther., 21: 1047-1053.
[53] McMahon BJ, Bruce MG, Hennessy TW, Bruden DL, Sacco F, Peters H, Hurlburt DA,
Morris JM, Reasonover AL, Dailide G, Berg DE, Parkinson AJ, (2006). Reinfection

after successful eradication of Helicobacter pylori: a 2-year prospective study in Alaska

Natives. Aliment. Pharmacol. Ther., 23: 1215-1223.
[54] Soto G, Bautista CT, Roth DE, Gilman RH, Velapatiño B, Ogura M, Dailide G, Razuri
M, Meza R, Katz U, Monath TP, Berg DE, Taylor DN, (2003). Helicobacter pylori
reinfection is common in Peruvian adults after antibiotic eradication therapy. J. Infect
Dis., 188: 1263-1275.
[55] Seo M, Okada M, Shirotani T, Nishimura H, Maeda K, Aoyagi K, Sakisaka S, (2002).
Recurrence of Helicobacter pylori infection and the long-term outcome of peptic ulcer
after successful eradication in Japan. J. Clin. Gastroenterol., 34: 129-134.
[56] Parsonnet J, Harris RA, Hack HM, Owens DK, (1996). Modelling cost-effectiveness of
Helicobacter pylori screening to prevent gastric cancer: a mandate for clinical trials.
Lancet., 348:150–4.
[57] Wong BCY, Lam SK, Wong WM, Chen JS, Zheng TT, Feng RE, et al. (2004).
Helicobacter pylori eradication to prevent gastric cancer in a high-risk region of China:
a randomized controlled trial. JAMA., 291:187–94.
[58] You W-C, Brown LM, Zhang L, Li J-Y, Jin M-L, Chang Y-S, et al. (2006).
Randomized double-blind factorial trial of three treatments to reduce the prevalence of
precancerous gastric lesions. J. Natl. Cancer. Inst., 98:974–83.
[59] Ma JL, Zhang L. Brown LM et al. (2012). Fifteen-year effects of Helicobacter pylori,
garlic, and vitamin treatments on gastric cancer incidence and mortality. J. Natl.
Cancer. Inst., 104(6): 488-492.
[60] Morgan DR, Torres J, Sexton R, Herrero R, Salazar-Martinez E, Greenberg ER, Bravo
LE, Dominguez RL, Ferreccio C, Lazcano-Ponce EC, Meza-Montenegro MM, Pena
EM, Pena R, Correa P, Martinez ME, Chey WD, Valdivieso M, Anderson GL,
Goodman GE, Crowley JJ, Baker LH, (2013). Risk of recurrent Helicobacter pylori
infection 1 year after initial eradication therapy in 7 latin american communities.
JAMA., 309: 578-586.
[61] Gisbert JP, (2005). The recurrence of Helicobacter pylori infection: incidence and
variables influencing it: a critical review. Am. J. Gastroenterol., 100 (9): 2083-2099.
[62] Kivi M, Tindberg Y, (2006). Helicobacter pylori occurrence and transmission: a family
affair? Scand. J. Infect. Dis., 38(6-7):407-17.
[63] Goodman KJ, Correa P, (1995). The transmission of Helicobacter pylori. A critical
review of the evidence. Int. J. Epidemiol., 24:875-887.
[64] Nahar S, Kibria KMK, Hossain E et al. (2009). Evidence of intra-familial transmission
of Helicobacter pylori by PCR-based RAPD fingerprinting in Bangladesh. Eur. J. Clin.
Microbiol. Infect. Dis., 28:767–773.
[65] Niv Y, (2008). H. pylori recurrence after successful eradication. World. J.
Gastroenterol., 14:1477-1478.

Helicobacter pylori Infection
and Gastrointestinal
Diseases – Gastric Pathology

Chapter 7
Epithelial Dysfunction
in the Pathogenesis of
Tamia K. Lapointe1 and Andre G. Buret2

1
Department of Physiology and Pharmacology, 2Department of Biological Sciences,
Inflammation Research Network, Snyder Institute for Chronic Diseases,
University of Calgary, Calgary, Alberta, Canada
Abstract
The gastrointestinal epithelium serves as a mediator between several opposing
forces: proliferation and cell death, protective barrier and nutrient intake, primary defense
function and tissue damage. The key role of the epithelium in gastrointestinal
homeostasis is underlined by a growing body of evidence demonstrating epithelial
dysfunction in the context of a number of chronic inflammatory disorders, enteric
infections, and carcinogenesis. Helicobacter pylori has been shown to disrupt epithelial
functions by subverting signaling pathways involved in the regulation of cellular turnover
and epithelial barrier function, thus facilitating the development of epithelial-derived
cancers. While several H. pylori virulence factors have been associated with an increased
pathogenic potential (e.g. VacA and CagA), the host-microbial interactions influencing
the clinical outcome of H. pylori infection remain elusive. This chapter highlights our
current knowledge of the mechanisms by which H. pylori disrupts epithelial function to
promote the development of ulcerative diseases and cancer.
Keywords: Gastrointestinal epithelium, Apical junctional complex, Tight junction, Adherens

junction, Claudin, Occludin, Zonula occluden, Epithelial permeability, -catenin, E-
cadherin, Myosin light chain kinase, Rho-associated kinase, Apoptosis, Cell survival,
Interleukin-1, Matrix metalloproteinase, Cancer, Cytotoxin-Associated Gene (Cag) A,
Vacuolating toxin A (VacA), Epidermal growth factor receptor, Endocytosis, Calpain

116 Tamia K. Lapointe and Andre G. Buret
Introduction
Mucosal surfaces are privileged sites for host interactions with the external environment.
The gastrointestinal tract represents the largest mucosal surface of the human body, and is
continuously exposed to a plethora of antigens from food, resident bacteria, and pathogenic
microorganisms. The single layer of epithelial cells lining the gastrointestinal tract acts as a
selective physical barrier segregating the luminal environment and subepithelial tissues.
Apical junctional complexes (AJCs) (Figure 7.1), including tight junctions and immediately
subjacent adherens junctions, play a crucial role in maintaining mucosal homeostasis. By
selectively controlling paracellular exchanges and physically regulating cell polarity, AJCs
assume both a ‘gate’ and a ‘fence’ function in the gastrointestinal epithelium. Beyond cell-
cell adhesion, individual AJC components also act as signaling molecules and intermediary
scaffolds for intracellular signaling complexes, thus participating in the regulation of multiple
cellular functions including cell death, proliferation, differentiation, and migration (reviewed
in [1]). AJCs are dynamic structures and, under physiological conditions, several mechanisms
work together to acutely regulate their continuous remodeling and barrier properties. Cellular
turnover, governed by the balance between cell proliferation and apoptosis, also represent an
important factor in the maintenance of epithelial homoeostasis, especially with regards to
neoplastic transformation. In the last few decades, not only have we gained extensive
knowledge of the regulatory signals involved in epithelial integrity and barrier function, but
have also realized the undeniable involvement of epithelial dysfunction in disease initiation
and progression. Indeed, epithelial defects have been associated with the pathophysiology of a
broad range of disorders including Inflammatory Bowel Disease (IBD), food allergies, cancer,
and infection with enteric pathogens such Escherichia coli, Giardia sp. and, of particular
interest for this book, Helicobacter pylori (reviewed in [2-5])
H. pylori colonizes the gastric mucosa of over half of the human population, leading to
the development of asymptomatic gastritis. In a subset of H. pylori-infected individuals, this
chronic inflammation progresses into gastric ulcers, mucosal lymphomas, or gastric
adenocarcinomas, thus classifying H. pylori as a type I carcinogen. Certain H. pylori
virulence determinants have been associated with an increased pathogenic potential. Notably,
strains expressing the translocated Cytotoxin-Associated Gene (Cag) A protein and secreted
Vacuolating toxin A (VacA) are known to present a greater risk for the development of
ulcerative diseases and gastric cancers. Nevertheless, it is important to keep in mind that a
subset of patients infected with CagA+/VacA+ strains remains asymptomatic, indicating that
other microbial factors must influence disease outcome. While the specific host-microbial
interactions influencing disease severity remain elusive, it is now clear that epithelial
alterations represent an important component of H. pylori pathogenesis and a significant
determinant of disease progression.
This chapter elaborates on the importance of epithelial integrity in the maintenance of
gastrointestinal homeostasis and explores our current knowledge of the mechanisms by which
H. pylori disrupts this equilibrium to influence disease outcome.

Epithelial Dysfunction in the Pathogenesis of Helicobacter pylori Infection 117
The Gastrointestinal Mucosal Barrier

Tight Junctions
Epithelial tight junctions (Figure 7.1) are the most apical component of the AJCs. They
are formed by heterotypic interactions of over 50 distinct tight junction-related proteins
organized in broad classes. Zonula Occluden (ZO) -1, a large cytosolic, membrane-associated
protein, was the first component of the tight junctions to be identified [6]. While ZO proteins
(ZO-1, -2, and -3) are bound to F-actin through their C-terminal domain, they also form a
cytosolic anchoring platform for several transmembrane components of the tight junctions,
thus providing a structural and functional link between these transmembrane proteins and the
cytoskeleton [7-9].
Figure 7.1. Structure of the apical junctional complex. Tight junctions are composed of several
transmembrane proteins, including claudins, occludin, and junctional adhesion molecules (JAM). These
proteins are linked to cytosolic plaque proteins of the zonula occluden (ZO) family, which are themselves
anchored to the actinomyosin ring of epithelial cells. Adherens junctions are formed by the interaction of the
transmembrane protein E-cadherin with intracellular -catenin and catenin-p120, both of which link to the
actin cytoskeleton via -catenin.

Occludin was the first transmembrane protein of the tight junctions to be discovered [10].
Occludin interacts intracellularly with all three members of the ZO family and extracellularly
with equivalent loops on adjacent cells [8, 11-13]. Hyperphosphorylation of occludin is
required for its localization to the tight junctions [14]. The role of occludin in intestinal
epithelial barrier remains controversial. An important part of this debate originates from a
study revealing that occludin-deficient mice, although presenting a severely abnormal
phenotype and experiencing gastritis and gastric atrophy, do not show enhanced colonic
permeability [15]. Nevertheless, several studies published since have shown a functional role
for occludin in the gastrointestinal epithelium both in vitro and in vivo [13, 16-21]. Notably,
Balda et al. demonstrated that peptides mimicking the extracellular loop domain of occludin
induced tight junction disassembly and loss of epithelial barrier function [17]. These
observations suggest that while occludin may not be essential in the initial formation of
functional tight junction strands, it may be involved in their physiological and
pathophysiological remodeling.
The claudin family of tight junctional transmembrane proteins comprises at least 24
members [22]. Claudins are expressed in a tissue-specific manner and are considered to be the
main functional determinant of tight junctions [23]. Intracellularly, the C-terminal region of
claudin proteins binds with members of the ZO family [24, 25]. This interaction appears to be
essential for the formation of tight junctions, as the deletion of ZO-1 and ZO-2 has been
shown to prevent the targeting of claudins to the tight junctions and the establishment of
barrier function in vitro [26]. Strong evidence suggests that differential expression and homo-
versus hetero-typic interactions between specific claudins determine the barrier properties of
individual tight junction strands [27-29]. Furuse et al. have shown that claudin-1 and claudin-
4 enhance barrier function in vitro, whereas the expression of claudin-2 increases tight
junction permeability [28]. The essential role of claudins in barrier function has been
highlighted in several reports examining claudin-specific knockout mice: while claudin-15-
deficient animals exhibit decreased paracellular permeability to ions and develop
megaintestine, claudin-1 and claudin-5 -deficient mice show severe newborn lethal
phenotypes attributed to abnormal skin water loss and blood-brain barrier defects,
respectively [30-33].
Junctional Adhesion Molecule (JAMs), another transmembrane component of the tight
junctions, is a single membrane-spanning domain protein of the immunoglobulin gene
superfamily. JAM-A is expressed in both endothelial and epithelial cells and appears to be the
predominant isoform involved in tight junction structure and function [34, 35]. JAM-A
interacts intracellularly with ZO-1 and forms homotypic interaction between adjacent
epithelial cells [34-37].
Adherens Junctions
Adherens junctions (Figure 7.1) are required for the establishment of fully functional
tight junctions [38]. Whereas the main function of tight junctions is to seal the paracellular
space between neighboring epithelial cells, adherens junctions ensure cellular proximity,
polarization, and differentiation. Adherens junctions participate in cell-cell adhesion through
Ca2+-dependent homotypic binding of E-cadherin between adjacent epithelial cells [39]. E-
cadherin interacts directly with the cytosolic protein -catenin or the homologous p120-

catenin (p120), both of which are anchored to the actin cytoskeleton via the intermediary of
-catenin [40-43]. While membrane-bound -catenin serves as a structural platform for the
adherens junctions, cytosolic -catenin acts as a downstream component of the Wnt signaling
pathway. Activation of the Wnt pathway leads to nuclear accumulation of -catenin, where it
interacts with the transcription factor Lymphocyte Enhancer Factor/T Cell Factor (LEF/TCF)
to induce the expression of genes implicated in apoptosis, cell proliferation, and
carcinogenesis e.g. c-myc, matrix-metalloproteinase (MMP)-7 and cyclooxygenase (COX)-
2 (reviewed in [44]).
Physiological Regulation of Epithelial Barrier Function
It is important to understand that while epithelial AJCs form a protective barrier, these
complex structures are not completely impermeant and static. On the contrary, acute
regulation of AJCs allows the epithelium to modulate its level of permeability to allow the
exchange of nutrients, while at the same time restricting bacterial invasion. In order to fully
appreciate how disruption of epithelial function may contribute to pathogenesis, the next few
paragraphs will highlight the major signaling pathways involved in the physiological
regulation of epithelial barrier structure and function.
Myosin light chain kinase. The identification of Myosin Light Chain Kinase (MLCK) as
a critical physiological regulator of epithelial permeability in 1997 represented the first
significant advance in our understanding of epithelial barrier modulation. MLCK is a Ca2+-
dependent serine/threonine kinase that once activated, directly phosphorylates Myosin Light
Chains II (MLC) [45]. In the epithelium, MLC phosphorylation is known to stimulate the
contraction of perijunctional actin, thus inducing tight junction strand distension and a
consequent increase in paracellular permeability [46]. In contrast, protein kinase C is known
to inhibit MLCK, thus mediating the relaxation of the perijunctional actinomyosin ring of the
cell and a decrease in epithelial permeability [47]. MLCK has been extensively studied in the
context of both enteric infections and inflammatory disorders. Notably, Blair and colleagues
demonstrated an increase in MLCK expression in intestinal tissue from Crohn’s disease
patients, which correlated with the severity of colitis [48]. Furthermore, enteropathogenic E.
coli, H. pylori, and the protozoan pathogen Giardia lamblia are all known to mediate MLCK-
dependent disruption of epithelial barrier structure and function during infection [49-51].
Rho-associated kinase. The small Rho GTPase RhoA also plays an important role in the
regulation of epithelial barrier function; GTP-bound RhoA modulates epithelial permeability
by activating the serine/threonine Rho-associated Kinase (ROCK) , which modulates
cytoskeletal dynamic by 1) directly phosphorylating MLC, and 2) delaying MLC
dephosphorylation by inhibiting MLC phosphatase [52-60]. Several studies have
demonstrated that ROCK is essential for the formation of tight and adherens junctions in
intestinal epithelial cells [54, 58-61].
Because it plays a central role in cytoskeletal rearrangement, the Rho/ROCK signaling
axis also represents a key component of several other mucosal physiological processes. For
exemple, ROCK participates in the organized migration of epithelial cells; inhibition of
ROCK in intestinal epithelial cells induces the disorganization of the actin cytoskeleton,
leading to the formation of non-polarized protrusions at the spreading edge of epithelial

monolayers in vitro [61]. ROCK can also be constitutively activated by proteolytic cleavage
of its auto-inhibitory domain by caspase-3, the final executioner caspase involved in
programmed cell death (apoptosis) [62, 63]. This process is believed to play an important role
in regulating membrane blebbing during apoptosis [62, 63]. Consistent with the role of
ROCK in cell adhesion and migration, ROCK has been shown to promote tumor metastasis
and invasion [64, 65]; by modulating actin contraction, ROCK reduces cellular adhesion,
thereby promoting the infiltration of endothelial cells into tumors and the overall process of
angiogenesis [66]. Finally, ROCK has been shown to activate several members of the MMP
family of proteases (MMP-2, MMP-9, and MMP-13), known to promote cancer cell invasion
and metastasis by degrading components of the extracellular matrix [67, 68].
Endocytosis of AJC components. Endocytosis allows the internalization of extracellular
material and plasma membrane components via the formation of membrane transport
vesicles. Endocytic pathways can be divided into three distinct mechanisms: clathrin-,
caveolar-, and macropinocytosis- mediated endocytosis (reviewed in [69, 70]). Selective low-
grade endocytosis of AJC components is essential for the constant remodeling and repair of
the gastrointestinal epithelium. Once internalized, AJC proteins accumulate in cytosolic
early/common endosomes, from which they can either be recycled to the membrane, stored,
or targeted to late endosomes for degradation [71-74]. In contrast, the accelerated non-
selective internalization of AJC proteins observed during certain pathological conditions (e.g.
bacterial infections, inflammation, irradiation, etc.) promotes a rapid loss of intercellular
adhesion and a significant increase in epithelial permeability [12, 75-80]. In 2010, Utech and
colleagues proposed a hypothesis for cytokine-mediated internalization of individual tight
junction proteins wherein acute/transient inflammatory stimuli induce endocytosis of occludin
alone, while chronic/prolonged stimuli mediate internalization of occludin along with several
claudins [81].
Epithelial cell turnover and apoptosis. The epithelium of the gastrointestinal tract is in
constant remodeling. New cells generated at the bottom of gastric pits or intestinal crypt base
slowly migrate upwards, differentiate into either a secretory or absorptive phenotype, and
eventually engage in programmed cell death (apoptosis) before being shed from the epithelial
monolayers. In contrast to necrosis, which is associated with cellular damage and
inflammation, apoptosis is a self-limiting process; nuclear fragmentation and the formation of
membrane-bound vesicles (apoptotic bodies) prevent the release of cellular content into the
extracellular space and the initiation of inflammation and subsequent tissue damage. In
addition to its role in epithelial renewal, apoptosis also play an important anti-tumorigenic
function by eliminating DNA-damaged cells. In physiological conditions, the high apoptotic
rate of epithelial cell does not compromise epithelial barrier. Nevertheless, cellular
mechanisms must be in place to overcome the challenges of constant epithelial remodeling
and assure mucosal homeostasis. Elegant studies performed by Madara and colleagues in the
early 1990’s demonstrated that during epithelial cell apoptosis and shedding, neighbouring
cells extend processes beneath the cell about to shed [82]. As the cell is extruded from the
epithelium, these protrusions come together and form tight junction-like complexes in a
process called “zipper”, thereby preventing epithelial leaks at the shedding site [82]. While
this mechanism appears to be sufficient to prevent epithelial barrier defect in physiological
conditions, it cannot always sustain the heightened rates of apoptosis and cellular turnover
associated with certain pathological conditions such as enteric infections (reviewed in [3]).

Indeed, E. coli LPS and Giardia lambia have previously been shown to increase epithelial
permeability in vitro in a caspase-3-dependent manner [83, 84].
Epithelial Dysfunction during H. pylori Infection

The initial inflammatory response induced by H. pylori is largely driven by neutrophils
and macrophages. These innate immune cells are recruited to the site of infection by the
epithelial release of interleukin (IL)-8, a mechanism mediated essentially via toll-like receptor
(TLR)-2 and NFκB [85, 86]. Once within the gastric mucosa, macrophages and neutrophils
contribute to the establishment of chronic inflammation by secreting a wide array of
proinflammatory mediators including IL-1, IL-6, and tumor necrosis factor (TNF) .
Despite a growing body of evidence indicating that H. pylori can invade gastric epithelial
cells [87-89], H. pylori is primarily perceived as a non-invasive pathogen inducing
intracellular host signaling events from the outside via the intermediary of bacterial effectors
such as CagA and VacA. The cag pathogenicity island (cagPAI) is the most characterized of
H. pylori virulence factors. This 40kb DNA segment is conserved in 50-70% of all H. pylori
isolates and encodes several genes involved in pathogenesis [90]. While the cagPAI is not
essential for colonization, cag positive strains trigger a much more aggressive host
inflammatory response and are associated with an increased risk for the development of
ulcerative diseases and carcinomas [91, 92]. The cagPAI encodes a functional type 4
secretory system (T4SS) allowing the translocation of bacterial factors, including CagA, into
host cells [85, 93, 94]. Once inside epithelial cells, CagA is tyrosine-phosphorylated by
members of the Src family of kinases and then recruits and activates the eukaryotic tyrosine
phosphatase SHP2, which leads to rearrangement of the actin cytoskeleton and cell spreading
[95-97]. These changes are characteristic of H. pylori-infected cells and are referred to as the
“Hummingbird phenotype” [98, 99].
Expression of VacA by H. pylori has also been associated with an increased risk for
gastric cancer [100-102]. VacA interacts with epithelial cells either via direct contact of the
bacteria with host cells, or its release into the extracellular space. The contact of VacA with
epithelial cells induces the formation of anion-selective pores, allowing the leakage of
bicarbonate, chloride, and urea from host cells [103]. After its initial contact with the
epithelium, VacA is internalized into epithelial cells and induces the formation of larges
vacuoles, ultimately leading to epithelial cell death [104, 105]. While all strains of H. pylori
express VacA, the specific strains expressing active forms of the toxin ( 50%) are more
frequently associated with ulcerative diseases [106].
H. pylori is a highly manipulative pathogen; in addition to the virulence determinants
mentioned above, the bacterium has evolved several strategies in order to tailor the gastric
environment to its survival needs and establish a persistent infection. These include inhibition
of acid secretion, evasion of the host immune system, and the ability to move through the
mucus layer and firmly adhere to epithelial cells (reviewed in [107]). While these adaptations
are necessary for the establishment of the chronic state of mucosal inflammation
characteristic of H. pylori infection, the cellular and molecular mechanisms involved in these
processes extend beyond the scope of this chapter. The rest of this review will rather
emphasize on the ability of the bacterium to subvert normal cell signaling pathways involved

in the regulation of epithelial integrity and barrier function to influence disease progression
and outcome.
Direct Cleavage of AJC Components and Modulation of Actin

Dynamics by H. pylori
During the onset of carcinogenesis, fully differentiated epithelial cells transition into a
depolarized and migratory phenotype. This process, referred to as epithelial-to-mesenchymal
transition, critically relies on the remodeling of tight and adherens junctions. Indeed, aberrant
expression of AJC proteins is now recognized as a hallmark of cancer initiation and
progression. For example, downregulation of claudin-4 has been associated with increased
invasiveness and metastatic potential of pancreatic cancer, and poor gastric cancer prognosis
[108-110]. Similarly, loss of E-cadherin expression has been associated with invasiveness
potential, and germline E-cadherin mutations have been found in familiar gastric cancers
[111, 112]. Furthermore, an increase in claudin-1 expression and its redistribution from the
membrane to the cytosol and nucleus was observed in different cancer cell lines and human
primary colon carcinoma and metastasis [113]. H. pylori, by disrupting AJC structure
/function and promoting infiltration of luminal antigens, promotes the establishment of a
favorable inflammatory environment for the growth, migration, and invasion of tumor cells.
The following paragraphs will dissect this cascade of events hypothesized to represent an
early step in the progression from superficial gastritis to adenocarcinoma.
Tight junctions. While most H. pylori organisms present in infected individuals remain
within the mucous layer covering the gastric epithelium, a small percentage ( 20%) are
found adhering directly to epithelial cells, predominantly at the intercellular junction between
neighbouring cells [114, 115]. This direct contact of H. pylori with the gastric epithelium is
essential for insertion of the T4SS and subsequent transfer of CagA into epithelial cells. Once
in the intracellular space, CagA has been shown to associate with tight junctional complexes
and disrupts ZO-1 and JAM-A, consequently compromising epithelial barrier function and
cell morphology [116]. It was also reported that ectopic expression of CagA in mammalian
epithelial cells could disrupt epithelial polarity and tight junction structure and function, thus
suggesting a role for CagA in H. pylori-associated epithelial barrier defects [116].
Interestingly, several studies have also reported CagA-independent cytoskeletal
rearrangements during H. pylori infection. Experiments using isogenic mutants have shown
that H. pylori can disrupt claudin-4, claudin-5, and occludin in a CagA/VacA-independent
manner [49, 117]. It is important to mention that the mutant strains used in these studies
expressed a functional T4SS. This information is relevant in view of previous reports
demonstrating that some of the pathogenic events occurring during H. pylori infection,
including modulation of actinomyosin dynamic by Rho GTPase and NFB-dependent
expression of IL-8, were not actually dependent on the expression and translocation of CagA
into host cells, but rather the T4SS itself [118, 119]. Until recently, CagA was the only
identified virulence factor transported by the T4SS, and the translocated substrate inducing
NFB-mediated gene expression remained unclear. We now know that H. pylori
peptidoglycan is also transported by the T4SS into host cells, where it is recognized by
intracellular Nucleotide Oligomerization binding Domain (Nod) 1, a pattern recognition
receptor upstream of NFB [120].
CagA-independent disruption of epithelial tight junctions could also represent an early

event in H. pylori-induced epithelial barrier defects. Indeed, it was shown that injection of
CagA into host cell requires the interaction of the T4SS with basolaterally-expressed
epithelial 1 integrin [121]. This suggests that CagA-independent opening of the paracellular
pathway could precede CagA translocation into epithelial cells and its direct effect on tight
junctions. It is worth mentioning at this point that, regardless of the involvement of CagA in
this mechanism, it is hypothesized that this loss of epithelial barrier function is not used by
the bacteria to colonize subepithelial compartments, but rather to increase nutrient availability
and bacterial survival. In addition, this process may also contribute to the establishment of
chronic inflammation by increasing the antigenic load in the mucosal tissue.
The exact mechanism by which H. pylori disrupts epithelial barrier structure and function
remains a current topic of investigation (reviewed in Figure 7.2). H. pylori has been show to
disrupt claudin-4, claudin-5 and occludin in a MLCK-dependent manner [19, 49]. While this
effect appears to be independent of CagA, VacA, or a functional cagPAI, H. pylori-secreted
urease has been suggested as a potential player in this mechanism [19]. Urease is required for
H. pylori colonization and is known to contribute to tissue inflammation and injury [122,
123]. Using isogenic mutants, Wroblewski et al. have shown that H. pylori urease could
induce MLC phosphorylation via both MLCK and ROCK, and thus disrupt epithelial barrier
structure and function [19].
Proinflammatory mediators released by host cells during H. pylori infection are also
believed to play an important role in epithelial barrier defects. IL-1 has been extensively
studied in the context of H. pylori pathogenesis, most notably for its ability to modulate acid
secretion and consequent involvement in the development of gastric cancer [94, 124].
Importantly, it was demonstrated that IL-1 gene cluster polymorphisms associated with
increased IL-1 production pose a greater risk for the development of H. pylori-associated
gastric cancer [125, 126]. Furthermore, IL-1 was recently shown to activate both MLCK and
ROCK, and alter the expression and cellular distribution of tight junctional occludin and
claudin-4 [117, 127, 128]. In human gastric epithelial cells, H. pylori has been shown to
transactivate the IL-1 receptor I (IL-1RI) to induce ROCK activation, and consequently
mediate claudin-4 disruption [117]. The small GTPase RhoA is believed to act as a signaling
intermediate linking IL-1RI phosphorylation and ROCK activation in this process. Indeed,
upon IL-1 stimulation, IL-1RI has been shown to recruit and activate RhoA, independently
of the NFB pathway [129]. Although pharmacological inhibitors were not used in these
studies, an increase in RhoA activation was observed under H. pylori challenge in vitro [117].
TNF also represents an important proinflammatory mediator involved in the
pathogenesis of H. pylori. Similarly tp IL-1, a gene polymorphisms associated with elevated
expression of TNF has been linked to an increased risk of H. pylori-induced gastric cancer,
potentially via its ability to modulate gastric secretion [130]. TNF has also been extensively
studied for its detrimental effect on epithelial barrier structure and function; in vitro
experiments conducted on intestinal epithelial cells have demonstrated that TNF can
decrease the number of tight junction strands and induce the redistribution of occludin,
claudin-1, and ZO-1 away from the tight junctions in an MLCK-dependent manner [131-135].
TNF also synergizes with interferon  to increase paracellular permeability in vitro,
independently of the pro-apoptotic effect of both cytokines [133]. This was associated with
the cellular redistribution of JAM-A, occludin, claudin-1, and claudin-4, and involved the

activation of both ROCK and MLCK [75, 133, 136-139]. Although the effect of TNF on the
epithelium during H. pylori infection has not been investigated directly, its role in H. pylori-
mediated tight junctional alterations would be an interesting avenue for further investigation.
Figure 7.2. Effect of H. pylori on apical junctional complexes. H. pylori modulates tight junction structure
and function via direct binding of CagA to junctional proteins, activation of MLCK and ROCK, and
induction of proinflammatory cytokines release by macrophages. H. pylori also affect adherens junctions by
engaging the PI3K/AKT signaling pathway, mediating the cleavage of E-cadherin, and inducing the cellular
redistribution of -catenin and p120, thus promoting hyperproliferation, differentiation defects, and
metastasis. Abbreviations: ADAM: a disintegrin and matrix metalloproteinase, GSK3: Glycogen Synthase
Kinase 3, IL-1: Interleukin-1, MLCK: myosin light chain kinase, MMP: Matrix metalloproteinase, PI3K:
Phosphoinositide-3-kinase, ROCK: Rho-associated kinase, T4SS: Type 4 secretion system, TLR-2: Toll-like
receptor-2, TNF: Tumor necrosis factor ..
While internalization of transmembrane proteins is known to be involved in the

physiological remodeling of AJCs, endocytosis of tight junctional components has also been
observed in various pathological conditions including enteric infections [12, 75, 76]. For
example, cytotoxic necrotizing factor-1 has been shown to induce caveolar-mediated
endocytosis of ZO-1 and JAM-A in the context of E. coli infection [76]. Moreover, recent
observations suggest that H. pylori could mediate clathrin-coated pit-dependent endocytosis
of claudin-4 (unpublished data, Lapointe T. K.). Although the presence of the bacterium in
claudin-4-associated vesicle-like structures was not assessed in this study, it could perhaps
explain, at least in part, how H. pylori gains access to the intracellular space. Indeed, a
growing body of evidence indicates that H. pylori can invade gastric epithelial cells [87-89].
It has been suggested that VacA-dependent internalization of H. pylori in epithelial cells
could contribute to its persistence in the stomach despite host immune responses [87]. This
could explain, at least in part, why H. pylori induces a T-helper type I adaptive response,
normally characteristic of intracellular pathogens [140].

Adherens junctions. Adherens junctions play an important role in the maintenance of

tissue architecture and cellular polarity in the gastrointestinal mucosa. The tumor-suppressive
properties of intact adherens junctions have been highlighted in several studies demonstrating
an association between adherens junction disruption and carcinogenesis. Notably, hepatocyte
growth factor (HGF) has been shown to mediate MMP-7-dependent cleavage of E-cadherin
extracellular domain, thus producing a fragment capable of promoting cellular invasion by
inhibiting cadherin-based cell contacts in a paracrine fashion [141-144]. In agreement with
this observation, serum level of soluble E-cadherin has been validated as a prognostic marker
for gastric adenocarcinoma [145]. H. pylori has been shown to induce the shedding of E-
cadherin ectodomain both in vitro and in human samples [146-150]. Host-derived proteases
MMP-7 and a disintegrin and matrix metalloproteinase (ADAM)-10 have been hypothesized
to play a role in this process [148-151]. Alternatively, proteases secreted by H. pylori itself
could also be responsible for the extracellular cleavage of E-cadherin, a process previously
documented in the context of Bacterioides fragilis infections [152, 153].
Aside from promoting E-cadherin’s intrinsic carcinogenic potential, several studies have
also demonstrated the ability of H. pylori to induce E-cadherin gene promoter
hypermethylation, thus leading to a decrease in E-cadherin expression and an important
increase in free cytosolic -catenin [154-159]. In physiological conditions, Glycogen
Synthase Kinase (GSK)-3-mediated phosphorylation of cytosolic -catenin targets the
protein for ubiquitination and degradation, thus preventing -catenin-dependent expression of
target genes involved in carcinogenesis [160]. H. pylori has been show to subvert this
regulatory mechanism by transactivating the Epidermal Growth Factor Receptor (EGFR) both
in vitro and in vivo, thus leading to the activation of the Phosphoinositide-3-kinase
(PI3K)/AKT signaling cascade and inhibition of GSK-3 [161-165]. While the involvement
of CagA in this process remains controversial, VacA and the outer membrane protein OipA
have been suggested as potential bacterial factors responsible for these effects [161-163].
Using a mouse model of H. felis infection, a recent study demonstrated that macrophage-
derived TNF also phosphorylate AKT and inhibit GSK-3, thus promoting nuclear
accumulation of -catenin and the development of gastric dysplasia [166]. Moreover, CagA
has been shown to directly bind and destabilize -catenin/E-cadherin complexes, thus
promoting -catenin-mediated mitogenic signaling [167-169]. Notably, increased levels of
nuclear -catenin have been observed in gerbils infected with a carcinogenic strain of H.
pylori [169]. Significantly elevated levels of nuclear -catenin were also reported in gastric
biopsies from patients infected with CagA positive strains of H. pylori, as compared to
individual colonized with CagA-deficient strains [169].
H. pylori can also affect adherens junctions independently of the expression of E-
cadherin or -catenin. For example, H. pylori has recently been shown to disrupt adherens
junctions by activating the cell surface receptor TLR-2 and host-derived protease calpain in a
CagA/VacA-independent manner [147]. Calpain enzymes ( and m) are Ca2+-dependent
proteases involved in several cellular processes including cell motility, survival, apoptosis,
and inflammation [170-172]. Both calpain isoforms are known to cleave the cytoplasmic
domain of E-cadherin between residues 782 and 787, thus producing a truncated form of the
protein lacking the -catenin binding domain [173]. Calpain is also known to directly cleave
membrane-bound -catenin, leading to its nuclear transport and expression of several
oncogenes [174]. Taken together, these observations suggest that activation of -catenin

signaling may represent a pivotal determinant in the establishment of pre-malignant lesions in

response to H. pylori infection.
Although it has not gained as much attention as its homologue -catenin, p120-catenin
also appears to be modulated during H. pylori infection. In physiological conditions, p120
mainly remains membrane-bound at the level of adherens junctions. However, loss of E-
cadherin expression or overexpression of p120 has been shown to result in the cytosolic
redistribution of p120 and its association with Rho GTPases, thus promoting cellular motility
and metastasis [175-178]. Nuclear accumulation of p120 has been documented in ovarian and
gastric carcinomas [179, 180], and in H. pylori-infected human and murine gastric epithelial
cells [181, 182]. In the nucleus, p120 is believed to release the transcriptional repression of
mmp-7, thus promoting mitogenic signaling cascades culminating in the loss of cell contact
and attachment, an important step in epithelial-to-mesenchymal transition and metastasis
[182].
Modulation of Cellular Turnover and Apoptosis by H. pylori
Constant self-renewal of the gastrointestinal epithelium is ensured by the high

proliferative rate of progenitor cells and rapid apoptosis/shedding of differentiated epithelial
cells into the lumen. In physiological conditions, this tightly regulated process is essential to
eliminate damaged or infected cells. Nevertheless, it also puts the gastrointestinal tract in a
precarious state for the development of epithelial-derived cancers, should this fine balance be
disturbed by external factors. It has been suggested that infection with H. pylori increases the
risk of carcinogenesis by modulating the rate of epithelial turnover. Indeed, several studies
have demonstrated the pro-apoptotic effect of H. pylori in vitro, in vivo, and in human
biopsies (Figure 7.3) [105, 183-187]. VacA has been reported to play a pro-apoptotic role
during H. pylori infection. Indeed, it was demonstrated that strains of H. pylori expressing
active forms of VacA can induce gastric epithelial cell apoptosis, and are associated with
enhanced gastric epithelial injury [105, 188-190]. This process appears to require acid-
mediated disassembly and internalization of VacA into host cells [191, 192]. Recently, VacA
was also shown to induce apoptosis by inducing mitochondrial fission via the recruitment and
activation of dynamin-related protein 1 (DRP1), thus leading to the activation of the pro-
apoptotic factor BCL-2-associated protein X (BAX), release of mitochondrial cytochrome c,
and caspase-3-mediated apoptosis [193-196]. H. pylori has also been shown to induce
epithelial cell death via binding of urease to class II Major Histocompatibility Complex
(MHC) molecules, induction of the cell surface receptor Fas and Fas ligand expression, and
activation and nuclear translocation of NFB [185, 197-200].
Nevertheless, numerous studies also demonstrated the pro-survival potential of H. pylori.
For exemple, CagA has been shown to induce epithelial cell hyperproliferation and prevent
cell death, a mechanism regulated, at least in part, by the transactivation of the EGFR [201-
204]. Furthermore, H. pylori-induced epithelial hyperplasia and gastric atrophy has been
shown to correlate with the dysregulation of EGFR activation and its ligands, including
membrane-bound Heparin-Binding EGF-like Growth Factor (HB-EGF) [201, 202]. Indeed,
H. pylori has been shown to induce the release of HB-EGF by activating the proteolytic
enzyme ADAM-17 [201-203]. This in turns leads to the activation of the PI3K/AKT
signaling pathway, known to induce epithelial cell migration, disrupt cellular polarity, and
prevent epithelial apoptosis via the inhibition BAX [201-203]. MMP-7 is another proteolytic
enzyme hypothesized to be involved in H. pylori-mediated HB-EGF-dependent
transactivation of EGFR; while CagA+ strain of H. pylori have been shown to induce MMP-7
expression, genetic polymorphisms resulting in increased MMP-7 expression have been
associated with ulcerative disease and poor gastric cancer prognostic [205-207]. Of note,
EGFR-mediated cell survival also appears to be further amplified by CagA-mediated
inhibition of EGFR endocytosis and degradation [204]. COX-2 metabolites have also been
implicated in the pro-survival effect of H. pylori. Indeed, both PGE2 and 15-deoxy12,14-J2
(15d-PGJ2) are known to inhibit epithelial cell apoptosis, the latter via the activation of the
nuclear hormone receptor Peroxisome Proliferator-Activated Receptor γ (PPARγ) [208-210].
An imbalance between apoptosis and survival of DNA-damaged epithelial cells is believe
to play an important role in H. pylori-associated carcinogenesis. CagA has been shown to
physically interact with the tumor suppressor Apoptosis-Stimulating Protein of p53-2
(ASPP2) [211]. Upon DNA damage or oncogenic stimulation, ASPP2 binds and activates the
tumor suppressor p53, thus leading to apoptosis [212]. However, in the context of H. pylori
infection, the association of CagA to ASPP2 and subsequently p53 appears to target the
complex for proteosomal degradation, therefore inhibiting the apoptotic machinery of the cell
[211]. A recent study by Chaturvedi et al. reported that CagA could induce apoptosis both in
vitro and in vivo by promoting the expression of spermine oxidase, a catabolic enzyme
involved in DNA damage and apoptosis through the production of reactive oxygen species
[213]. Interestingly, these experiments also revealed that a subpopulation of gastric epithelial
cells expressing spermine oxidase and showing oxidative DNA damage were resistant to
apoptosis, thus presenting a higher risk for neoplastic transformation [213]. These
observations are consistent with a previous study indicating that H. pylori could prevent
apoptosis of gastric epithelial pit cells induced by the chemotherapeutic agent Etoposide
[214]. This process appears to be mediated by CagA-dependent activation of mitogen-
activated protein kinases MEK and ERK and Serum Response Element (SRE)/Serum
Response Factor (SRF)-driven transcription of the survival protein Myeloid Cell Leukemia
Sequence-1 (MCL1), known to promote cell survival [214].
Rather than being considered as experimental disparity, the pro- versus anti-apoptotic
effects of H. pylori should be regarded as a balancing act. Therefore, we suggest a
consolidating model in which, during the onset of infection, H. pylori-mediated pro-apoptotic
pathways could be involved in the initial weakening of the epithelial barrier, thus facilitating
access to nutrients and promoting low-grade chronic inflammation. The pro-survival effect of
the bacteria on progenitor epithelial cells would then come into play to promote the persistent
colonization of the stomach, and eventually malignant transformation.

Figure 7.3. Effect of H. pylori on cellular turnover. H. pylori is know to induce both pro- and anti-apoptotic
signaling cascades in gastric epithelial cells. The pro-apoptotic effect of H. pylori is driven by expression of
Fas and Fas ligand, mitochondrial release of cytochrome c, induction of ROS production, and activation of
NFB. In contrast, the pro-survival effect of the bacterium is mediated primarily by transactivation of the
EGFR, activation of the PI3K/AKT and MEK/ERK signaling pathways, inhibition of the pro-apoptotic effect
of p53, and PPAR. Abbreviations: ADAM: a disintegrin and matrix metalloproteinase, ASPP2: Apoptosis-
Stimulating Protein of p53-2, BAX: BCL-2-associated protein X, BCL-2: B-cell lymphoma 2, COX-2:
Cyclooxygenase-2, Cyt c: Cytochrome c, DRP1: dynamin-related protein 1, EGFR: Epidermal growth factor
receptor, FAS-L: Fas ligand , HB-EGF: Heparin-binding EGF-like growth factor, MHC-II: Major
Histocompatibility Complex class II, MMP: Matrix metalloproteinase, NFB: Nuclear factor B, PGE2:
prostaglandin E2, PI3K: Phosphoinositide-3-kinase, PPAR: Peroxisome proliferator-activated receptor γ,
ROS: Reactive oxygen species, T4SS: Type 4 secretion system, 15d-PGJ2: 15-deoxy12,14-J2.

Conclusion
The gastrointestinal epithelium serves as mediator reconciling opposing forces;
proliferation versus cell death, protective barrier versus nutrient intake, primary defense
function versus tissue damage. This equilibrium is orchestrated by several redundant
regulatory pathways that assure its absorptive, protective, and remodeling functions. The
crucial role of the epithelium in gastrointestinal homeostasis is underlined by numerous
studies describing epithelial dysfunction as a key pathogenic process in various disorders
including chronic inflammatory diseases, enteric infections, and carcinogenesis. Gastric
adenocarcinoma is the second leading cause of cancer-related death worldwide and is strongly
associated with H. pylori infection. While much remains to be elucidated, the last twenty
years of research have shed light on some important pathways involved in H. pylori-induced
carcinogenesis including the modulation of cellular turnover, disruption of tight and adherens
junctions, and promotion of cellular invasion. This knowledge grants us valuable tools to
identify subpopulations at high risk of developing H. pylori-associated gastric cancer, as well
as new targets for the development of prophylactic approaches to prevent eventual
complications associated with H. pylori infections.
References
[1] Matter, K., and Balda, M.S. (2003). Signalling to and from tight junctions. Nat Rev Mol
Cell Biol 4, 225-236.
[2] O'Hara, J.R., and Buret, A.G. (2008). Mechanisms of intestinal tight junctional
disruption during infection. Front Biosci 13, 7008-7021.
[3] Buret, A.G., and Bhargava, A. (2013). Modulatory mechanisms of enterocyte apoptosis
by viral, bacterial and parasitic pathogens. Crit Rev Microbiol.
[4] Macdonald, T.T. and Monteleone, G. (2005). Immunity, inflammation, and allergy in
the gut. Science 307, 1920-1925.
[5] Marchiando, A.M., Graham, W.V., and Turner, J.R. (2010). Epithelial barriers in
homeostasis and disease. Annu Rev Pathol 5, 119-144.
[6] Stevenson, B.R., Siliciano, J.D., Mooseker, M.S., and Goodenough, D.A. (1986).
Identification of ZO-1: a high molecular weight polypeptide associated with the tight
junction (zonula occludens) in a variety of epithelia. J Cell Biol 103, 755-766.
[7] Fanning, A.S., Jameson, B.J., Jesaitis, L.A., and Anderson, J.M. (1998). The tight
junction protein ZO-1 establishes a link between the transmembrane protein occludin
and the actin cytoskeleton. J Biol Chem 273, 29745-29753.
[8] Furuse, M., Itoh, M., Hirase, T., Nagafuchi, A., Yonemura, S., and Tsukita, S. (1994).
Direct association of occludin with ZO-1 and its possible involvement in the
localization of occludin at tight junctions. J Cell Biol 127, 1617-1626.
[9] Wittchen, E.S., Haskins, J., and Stevenson, B.R. (1999). Protein interactions at the tight
junction. Actin has multiple binding partners, and ZO-1 forms independent complexes
with ZO-2 and ZO-3. J Biol Chem 274, 35179-35185.

[10] Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., and Tsukita, S. (1993).
Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol
123, 1777-1788.
[11] Haskins, J., Gu, L., Wittchen, E.S., Hibbard, J., and Stevenson, B.R. (1998). ZO-3, a
novel member of the MAGUK protein family found at the tight junction, interacts with
ZO-1 and occludin. J Cell Biol 141, 199-208.
[12] Nusrat, A., von Eichel-Streiber, C., Turner, J.R., Verkade, P., Madara, J.L., and Parkos,
C.A. (2001). Clostridium difficile toxins disrupt epithelial barrier function by altering
membrane microdomain localization of tight junction proteins. Infect Immun 69, 1329-
1336.
[13] Wong, V., and Gumbiner, B.M. (1997). A synthetic peptide corresponding to the
extracellular domain of occludin perturbs the tight junction permeability barrier. J Cell
Biol 136, 399-409.
[14] Sakakibara, A., Furuse, M., Saitou, M., Ando-Akatsuka, Y., and Tsukita, S. (1997).
Possible involvement of phosphorylation of occludin in tight junction formation. J Cell
Biol 137, 1393-1401.
[15] Saitou, M., Furuse, M., Sasaki, H., Schulzke, J.D., Fromm, M., Takano, H., Noda, T.,
and Tsukita, S. (2000). Complex phenotype of mice lacking occludin, a component of
tight junction strands. Mol Biol Cell 11, 4131-4142.
[16] McCarthy, K.M., Skare, I.B., Stankewich, M.C., Furuse, M., Tsukita, S., Rogers, R.A.,
Lynch, R.D., and Schneeberger, E.E. (1996). Occludin is a functional component of the
tight junction. J Cell Sci 109 ( Pt 9), 2287-2298.
[17] Balda, M.S., Whitney, J.A., Flores, C., Gonzalez, S., Cereijido, M., and Matter, K.
(1996). Functional dissociation of paracellular permeability and transepithelial
electrical resistance and disruption of the apical-basolateral intramembrane diffusion
barrier by expression of a mutant tight junction membrane protein. J Cell Biol 134,
1031-1049.
[18] Yu, A.S., McCarthy, K.M., Francis, S.A., McCormack, J.M., Lai, J., Rogers, R.A.,
Lynch, R.D., and Schneeberger, E.E. (2005). Knockdown of occludin expression leads
to diverse phenotypic alterations in epithelial cells. Am J Physiol Cell Physiol 288,
C1231-1241.
[19] Wroblewski, L.E., Shen, L., Ogden, S., Romero-Gallo, J., Lapierre, L.A., Israel, D.A.,
Turner, J.R., and Peek, R.M., Jr. (2009). Helicobacter pylori dysregulation of gastric
epithelial tight junctions by urease-mediated myosin II activation. Gastroenterology
136, 236-246.
[20] Nusrat, A., Brown, G.T., Tom, J., Drake, A., Bui, T.T., Quan, C., and Mrsny, R.J.
(2005). Multiple protein interactions involving proposed extracellular loop domains of
the tight junction protein occludin. Mol Biol Cell 16, 1725-1734.
[21] Lapointe, T.K., and Buret, A.G. (2012). Interleukin-18 facilitates neutrophil
transmigration via myosin light chain kinase-dependent disruption of occludin, without
altering epithelial permeability. Am J Physiol Gastrointest Liver Physiol 302, G343-
351.
[22] Tsukita, S., Furuse, M., and Itoh, M. (2001). Multifunctional strands in tight junctions.
Nat Rev Mol Cell Biol 2, 285-293.

[23] Morita, K., Furuse, M., Fujimoto, K., and Tsukita, S. (1999). Claudin multigene family
encoding four-transmembrane domain protein components of tight junction strands.
Proc Natl Acad Sci U S A 96, 511-516.
[24] Furuse, M., Fujita, K., Hiiragi, T., Fujimoto, K., and Tsukita, S. (1998). Claudin-1 and -
2: novel integral membrane proteins localizing at tight junctions with no sequence
similarity to occludin. J Cell Biol 141, 1539-1550.
[25] Itoh, M., Furuse, M., Morita, K., Kubota, K., Saitou, M., and Tsukita, S. (1999). Direct
binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the
COOH termini of claudins. J Cell Biol 147, 1351-1363.
[26] Umeda, K., Ikenouchi, J., Katahira-Tayama, S., Furuse, K., Sasaki, H., Nakayama, M.,
Matsui, T., Tsukita, S., and Furuse, M. (2006). ZO-1 and ZO-2 independently
determine where claudins are polymerized in tight-junction strand formation. Cell 126,
741-754.
[27] Daugherty, B.L., Ward, C., Smith, T., Ritzenthaler, J.D., and Koval, M. (2007).
Regulation of heterotypic claudin compatibility. J Biol Chem 282, 30005-30013.
[28] Furuse, M., Furuse, K., Sasaki, H., and Tsukita, S. (2001). Conversion of zonulae
occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby
canine kidney I cells. J Cell Biol 153, 263-272.
[29] Mrsny, R.J., Brown, G.T., Gerner-Smidt, K., Buret, A.G., Meddings, J.B., Quan, C.,
Koval, M., and Nusrat, A. (2008). A key claudin extracellular loop domain is critical
for epithelial barrier integrity. Am J Pathol 172, 905-915.
[30] Furuse, M., Hata, M., Furuse, K., Yoshida, Y., Haratake, A., Sugitani, Y., Noda, T.,
Kubo, A., and Tsukita, S. (2002). Claudin-based tight junctions are crucial for the
mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 156,
1099-1111.
[31] Morita, K., Sasaki, H., Furuse, M., and Tsukita, S. (1999). Endothelial claudin: claudin-
5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol 147, 185-
194.
[32] Nitta, T., Hata, M., Gotoh, S., Seo, Y., Sasaki, H., Hashimoto, N., Furuse, M., and
Tsukita, S. (2003). Size-selective loosening of the blood-brain barrier in claudin-5-
deficient mice. J Cell Biol 161, 653-660.
[33] Tamura, A., Kitano, Y., Hata, M., Katsuno, T., Moriwaki, K., Sasaki, H., Hayashi, H.,
Suzuki, Y., Noda, T., Furuse, M., et al. (2008). Megaintestine in claudin-15-deficient
mice. Gastroenterology 134, 523-534.
[34] Bazzoni, G., Martinez-Estrada, O.M., Orsenigo, F., Cordenonsi, M., Citi, S., and
Dejana, E. (2000). Interaction of junctional adhesion molecule with the tight junction
components ZO-1, cingulin, and occludin. J Biol Chem 275, 20520-20526.
[35] Ebnet, K., Schulz, C.U., Meyer Zu Brickwedde, M.K., Pendl, G.G., and Vestweber, D.
(2000). Junctional adhesion molecule interacts with the PDZ domain-containing
proteins AF-6 and ZO-1. J Biol Chem 275, 27979-27988.
[36] Bazzoni, G., Martinez-Estrada, O.M., Mueller, F., Nelboeck, P., Schmid, G., Bartfai,
T., Dejana, E., and Brockhaus, M. (2000). Homophilic interaction of junctional
adhesion molecule. J Biol Chem 275, 30970-30976.
[37] Kostrewa, D., Brockhaus, M., D'Arcy, A., Dale, G.E., Nelboeck, P., Schmid, G.,
Mueller, F., Bazzoni, G., Dejana, E., Bartfai, T., et al. (2001). X-ray structure of

junctional adhesion molecule: structural basis for homophilic adhesion via a novel
dimerization motif. Embo J 20, 4391-4398.
[38] Turner, J.R. (2009). Intestinal mucosal barrier function in health and disease. Nat Rev
Immunol 9, 799-809.
[39] Nose, A., Nagafuchi, A., and Takeichi, M. (1988). Expressed recombinant cadherins
mediate cell sorting in model systems. Cell 54, 993-1001.
[40] Aberle, H., Butz, S., Stappert, J., Weissig, H., Kemler, R., and Hoschuetzky, H. (1994).
Assembly of the cadherin-catenin complex in vitro with recombinant proteins. J Cell
Sci 107 ( Pt 12), 3655-3663.
[41] Drees, F., Pokutta, S., Yamada, S., Nelson, W.J., and Weis, W.I. (2005). Alpha-catenin
is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament
assembly. Cell 123, 903-915.
[42] Nagafuchi, A. (2001). Molecular architecture of adherens junctions. Curr Opin Cell
Biol 13, 600-603.
[43] Rimm, D.L., Koslov, E.R., Kebriaei, P., Cianci, C.D., and Morrow, J.S. (1995). Alpha
1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-
actin to the membrane adhesion complex. Proc Natl Acad Sci U S A 92, 8813-8817.
[44] White, B.D., Chien, A.J., and Dawson, D.W. (2012). Dysregulation of Wnt/beta-catenin
signaling in gastrointestinal cancers. Gastroenterology 142, 219-232.
[45] Kamm, K.E., and Stull, J.T. (2001). Dedicated myosin light chain kinases with diverse
cellular functions. J Biol Chem 276, 4527-4530.
[46] Turner, J.R., Rill, B.K., Carlson, S.L., Carnes, D., Kerner, R., Mrsny, R.J., and Madara,
J.L. (1997). Physiological regulation of epithelial tight junctions is associated with
myosin light-chain phosphorylation. Am J Physiol 273, C1378-1385.
[47] Turner, J.R., Angle, J.M., Black, E.D., Joyal, J.L., Sacks, D.B., and Madara, J.L.
(1999). PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC
kinase. Am J Physiol 277, C554-562.
[48] Blair, S.A., Kane, S.V., Clayburgh, D.R., and Turner, J.R. (2006). Epithelial myosin
light chain kinase expression and activity are upregulated in inflammatory bowel
disease. Lab Invest 86, 191-201.
[49] Fedwick, J.P., Lapointe, T.K., Meddings, J.B., Sherman, P.M., and Buret, A.G. (2005).
Helicobacter pylori activates myosin light-chain kinase to disrupt claudin-4 and
claudin-5 and increase epithelial permeability. Infect Immun 73, 7844-7852.
[50] Scott, K.G., Meddings, J.B., Kirk, D.R., Lees-Miller, S.P., and Buret, A.G. (2002).
Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin
light chain kinase-dependent fashion. Gastroenterology 123, 1179-1190.
[51] Yuhan, R., Koutsouris, A., Savkovic, S.D., and Hecht, G. (1997). Enteropathogenic
Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial
permeability. Gastroenterology 113, 1873-1882.
[52] Hall, A. (1994). Small GTP-binding proteins and the regulation of the actin
cytoskeleton. Annu Rev Cell Biol 10, 31-54.
[53] Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatsu, A., Fujita, A.,
Watanabe, N., Saito, Y., Kakizuka, A., Morii, N., et al. (1996). The small GTP-binding
protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to
myotonic dystrophy kinase. Embo J 15, 1885-1893.

[54] Leung, T., Chen, X.Q., Manser, E., and Lim, L. (1996). The p160 RhoA-binding kinase
ROK alpha is a member of a kinase family and is involved in the reorganization of the
cytoskeleton. Mol Cell Biol 16, 5313-5327.
[55] Nakagawa, O., Fujisawa, K., Ishizaki, T., Saito, Y., Nakao, K., and Narumiya, S.
(1996). ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming
protein serine/threonine kinase in mice. FEBS Lett 392, 189-193.
[56] Leung, T., Manser, E., Tan, L., and Lim, L. (1995). A novel serine/threonine kinase
binding the Ras-related RhoA GTPase which translocates the kinase to peripheral
membranes. J Biol Chem 270, 29051-29054.
[57] Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., Nakano, T.,
Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996). Rho-associated kinase, a novel
serine/threonine kinase, as a putative target for small GTP binding protein Rho. Embo J
15, 2208-2216.
[58] Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y.,
and Kaibuchi, K. (1996). Phosphorylation and activation of myosin by Rho-associated
kinase (Rho-kinase). J Biol Chem 271, 20246-20249.
[59] Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B.,
Feng, J., Nakano, T., Okawa, K., et al. (1996). Regulation of myosin phosphatase by
Rho and Rho-associated kinase (Rho-kinase). Science 273, 245-248.
[60] Walsh, S.V., Hopkins, A.M., Chen, J., Narumiya, S., Parkos, C.A., and Nusrat, A.
(2001). Rho kinase regulates tight junction function and is necessary for tight junction
assembly in polarized intestinal epithelia. Gastroenterology 121, 566-579.
[61] Hopkins, A.M., Pineda, A.A., Winfree, L.M., Brown, G.T., Laukoetter, M.G., and
Nusrat, A. (2007). Organized migration of epithelial cells requires control of adhesion
and protrusion through Rho kinase effectors. Am J Physiol Gastrointest Liver Physiol
292, G806-817.
[62] Coleman, M.L., Sahai, E.A., Yeo, M., Bosch, M., Dewar, A., and Olson, M.F. (2001).
Membrane blebbing during apoptosis results from caspase-mediated activation of
ROCK I. Nat Cell Biol 3, 339-345.
[63] Sebbagh, M., Renvoize, C., Hamelin, J., Riche, N., Bertoglio, J., and Breard, J. (2001).
Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic
membrane blebbing. Nat Cell Biol 3, 346-352.
[64] Horiuchi, A., Imai, T., Wang, C., Ohira, S., Feng, Y., Nikaido, T., and Konishi, I.
(2003). Up-regulation of small GTPases, RhoA and RhoC, is associated with tumor
progression in ovarian carcinoma. Lab Invest 83, 861-870.
[65] Kamai, T., Tsujii, T., Arai, K., Takagi, K., Asami, H., Ito, Y., and Oshima, H. (2003).
Significant association of Rho/ROCK pathway with invasion and metastasis of bladder
cancer. Clin Cancer Res 9, 2632-2641.
[66] Croft, D.R., Sahai, E., Mavria, G., Li, S., Tsai, J., Lee, W.M., Marshall, C.J., and Olson,
M.F. (2004). Conditional ROCK activation in vivo induces tumor cell dissemination
and angiogenesis. Cancer Res 64, 8994-9001.
[67] Liotta, L.A., Tryggvason, K., Garbisa, S., Hart, I., Foltz, C.M., and Shafie, S. (1980).
Metastatic potential correlates with enzymatic degradation of basement membrane
collagen. Nature 284, 67-68.
[68] Vishnubhotla, R., Sun, S., Huq, J., Bulic, M., Ramesh, A., Guzman, G., Cho, M., and
Glover, S.C. (2007). ROCK-II mediates colon cancer invasion via regulation of MMP-2

and MMP-13 at the site of invadopodia as revealed by multiphoton imaging. Lab Invest
87, 1149-1158.
[69] Bonifacino, J.S., and Glick, B.S. (2004). The mechanisms of vesicle budding and
fusion. Cell 116, 153-166.
[70] Mukherjee, S., Ghosh, R.N., and Maxfield, F.R. (1997). Endocytosis. Physiol Rev 77,
759-803.
[71] Ivanov, A.I., Nusrat, A., and Parkos, C.A. (2004). Endocytosis of epithelial apical
junctional proteins by a clathrin-mediated pathway into a unique storage compartment.
Mol Biol Cell 15, 176-188.
[72] Le, T.L., Yap, A.S., and Stow, J.L. (1999). Recycling of E-cadherin: a potential
mechanism for regulating cadherin dynamics. J Cell Biol 146, 219-232.
[73] Low, S.H., Miura, M., Roche, P.A., Valdez, A.C., Mostov, K.E., and Weimbs, T.
(2000). Intracellular redirection of plasma membrane trafficking after loss of epithelial
cell polarity. Mol Biol Cell 11, 3045-3060.
[74] Matsuda, M., Kubo, A., Furuse, M., and Tsukita, S. (2004). A peculiar internalization
of claudins, tight junction-specific adhesion molecules, during the intercellular
movement of epithelial cells. J Cell Sci 117, 1247-1257.
[75] Bruewer, M., Luegering, A., Kucharzik, T., Parkos, C.A., Madara, J.L., Hopkins, A.M.,
and Nusrat, A. (2003). Proinflammatory cytokines disrupt epithelial barrier function by
apoptosis-independent mechanisms. J Immunol 171, 6164-6172.
[76] Hopkins, A.M., Walsh, S.V., Verkade, P., Boquet, P., and Nusrat, A. (2003).
Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and
epithelial barrier function. J Cell Sci 116, 725-742.
[77] McNamara, B.P., Koutsouris, A., O'Connell, C.B., Nougayrede, J.P., Donnenberg,
M.S., and Hecht, G. (2001). Translocated EspF protein from enteropathogenic
Escherichia coli disrupts host intestinal barrier function. J Clin Invest 107, 621-629.
[78] Porvaznik, M. (1979). Tight junction disruption and recovery after sublethal gamma
irradiation. Radiat Res 78, 233-250.
[79] Lerch, M.M., Lutz, M.P., Weidenbach, H., Muller-Pillasch, F., Gress, T.M., Leser, J.,
and Adler, G. (1997). Dissociation and reassembly of adherens junctions during
experimental acute pancreatitis. Gastroenterology 113, 1355-1366.
[80] Bruewer, M., Utech, M., Ivanov, A.I., Hopkins, A.M., Parkos, C.A., and Nusrat, A.
(2005). Interferon-gamma induces internalization of epithelial tight junction proteins
via a macropinocytosis-like process. Faseb J 19, 923-933.
[81] Utech, M., Mennigen, R., and Bruewer, M. (2010). Endocytosis and recycling of tight
junction proteins in inflammation. J Biomed Biotechnol 2010, 484987.
[82] Madara, J.L. (1990). Maintenance of the macromolecular barrier at cell extrusion sites
in intestinal epithelium: physiological rearrangement of tight junctions. J Membr Biol
116, 177-184.
[83] Chin, A.C., Flynn, A.N., Fedwick, J.P., and Buret, A.G. (2006). The role of caspase-3
in lipopolysaccharide-mediated disruption of intestinal epithelial tight junctions. Can J
Physiol Pharmacol 84, 1043-1050.
[84] Chin, A.C., Teoh, D.A., Scott, K.G., Meddings, J.B., Macnaughton, W.K., and Buret,
A.G. (2002). Strain-dependent induction of enterocyte apoptosis by Giardia lamblia
disrupts epithelial barrier function in a caspase-3-dependent manner. Infect Immun 70,
3673-3680.

[85] Fischer, W., Puls, J., Buhrdorf, R., Gebert, B., Odenbreit, S., and Haas, R. (2001).
Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential
genes for CagA translocation in host cells and induction of interleukin-8. Mol Microbiol
42, 1337-1348.
[86] Mandell, L., Moran, A.P., Cocchiarella, A., Houghton, J., Taylor, N., Fox, J.G., Wang,
T.C., and Kurt-Jones, E.A. (2004). Intact gram-negative Helicobacter pylori,
Helicobacter felis, and Helicobacter hepaticus bacteria activate innate immunity via
toll-like receptor 2 but not toll-like receptor 4. Infect Immun 72, 6446-6454.
[87] Terebiznik, M.R., Vazquez, C.L., Torbicki, K., Banks, D., Wang, T., Hong, W., Blanke,
S.R., Colombo, M.I., and Jones, N.L. (2006). Helicobacter pylori VacA toxin promotes
bacterial intracellular survival in gastric epithelial cells. Infect Immun 74, 6599-6614.
[88] Dubois, A. (1995). Spiral bacteria in the human stomach: the gastric helicobacters.
Emerg Infect Dis 1, 79-85.
[89] Wyle, F.A., Tarnawski, A., Schulman, D., and Dabros, W. (1990). Evidence for gastric
mucosal cell invasion by C. pylori: an ultrastructural study. J Clin Gastroenterol 12
Suppl 1, S92-98.
[90] Backert, S., and Selbach, M. (2008). Role of type IV secretion in Helicobacter pylori
pathogenesis. Cell Microbiol 10, 1573-1581.
[91] Blaser, M.J., Perez-Perez, G.I., Kleanthous, H., Cover, T.L., Peek, R.M., Chyou, P.H.,
Stemmermann, G.N., and Nomura, A. (1995). Infection with Helicobacter pylori strains
possessing cagA is associated with an increased risk of developing adenocarcinoma of
the stomach. Cancer Res 55, 2111-2115.
[92] Tham, K.T., Peek, R.M., Jr., Atherton, J.C., Cover, T.L., Perez-Perez, G.I., Shyr, Y.,
and Blaser, M.J. (2001). Helicobacter pylori genotypes, host factors, and gastric
mucosal histopathology in peptic ulcer disease. Hum Pathol 32, 264-273.
[93] Censini, S., Lange, C., Xiang, Z., Crabtree, J.E., Ghiara, P., Borodovsky, M., Rappuoli,
R., and Covacci, A. (1996). cag, a pathogenicity island of Helicobacter pylori, encodes
type I-specific and disease-associated virulence factors. Proc Natl Acad Sci U S A 93,
14648-14653.
[94] Peek, R.M., Jr., Miller, G.G., Tham, K.T., Perez-Perez, G.I., Zhao, X., Atherton, J.C.,
and Blaser, M.J. (1995). Heightened inflammatory response and cytokine expression in
vivo to cagA+ Helicobacter pylori strains. Lab Invest 73, 760-770.
[95] Odenbreit, S., Puls, J., Sedlmaier, B., Gerland, E., Fischer, W., and Haas, R. (2000).
Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV
secretion. Science 287, 1497-1500.
[96] Selbach, M., Moese, S., Hurwitz, R., Hauck, C.R., Meyer, T.F., and Backert, S. (2003).
The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin
rearrangement by c-Src inactivation. Embo J 22, 515-528.
[97] Yamazaki, S., Yamakawa, A., Ito, Y., Ohtani, M., Higashi, H., Hatakeyama, M., and
Azuma, T. (2003). The CagA protein of Helicobacter pylori is translocated into
epithelial cells and binds to SHP-2 in human gastric mucosa. J Infect Dis 187, 334-337.
[98] Backert, S., Moese, S., Selbach, M., Brinkmann, V., and Meyer, T.F. (2001).
Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for
induction of a scattering phenotype in gastric epithelial cells. Mol Microbiol 42, 631-
644.

[99] Segal, E.D., Cha, J., Lo, J., Falkow, S., and Tompkins, L.S. (1999). Altered states:
involvement of phosphorylated CagA in the induction of host cellular growth changes
by Helicobacter pylori. Proc Natl Acad Sci U S A 96, 14559-14564.
[100] Gerhard, M., Lehn, N., Neumayer, N., Boren, T., Rad, R., Schepp, W., Miehlke, S.,
Classen, M., and Prinz, C. (1999). Clinical relevance of the Helicobacter pylori gene for
blood-group antigen-binding adhesin. Proc Natl Acad Sci U S A 96, 12778-12783.
[101] Miehlke, S., Kirsch, C., Agha-Amiri, K., Gunther, T., Lehn, N., Malfertheiner, P.,
Stolte, M., Ehninger, G., and Bayerdorffer, E. (2000). The Helicobacter pylori vacA s1,
m1 genotype and cagA is associated with gastric carcinoma in Germany. Int J Cancer
87, 322-327.
[102] Louw, J.A., Kidd, M.S., Kummer, A.F., Taylor, K., Kotze, U., and Hanslo, D. (2001).
The relationship between Helicobacter pylori infection, the virulence genotypes of the
infecting strain and gastric cancer in the African setting. Helicobacter 6, 268-273.
[103] Tombola, F., Morbiato, L., Del Giudice, G., Rappuoli, R., Zoratti, M., and Papini, E.
(2001). The Helicobacter pylori VacA toxin is a urea permease that promotes urea
diffusion across epithelia. J Clin Invest 108, 929-937.
[104] Cover, T.L., and Blaser, M.J. (1992). Purification and characterization of the
vacuolating toxin from Helicobacter pylori. J Biol Chem 267, 10570-10575.
[105] Cover, T.L., Krishna, U.S., Israel, D.A., and Peek, R.M., Jr. (2003). Induction of gastric
epithelial cell apoptosis by Helicobacter pylori vacuolating cytotoxin. Cancer Res 63,
951-957.
[106] Atherton, J.C., Cao, P., Peek, R.M., Jr., Tummuru, M.K., Blaser, M.J., and Cover, T.L.
(1995). Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association
of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem
270, 17771-17777.
[107] Wroblewski, L.E., Peek, R.M., Jr., and Wilson, K.T. (2010). Helicobacter pylori and
gastric cancer: factors that modulate disease risk. Clin Microbiol Rev 23, 713-739.
[108] Michl, P., Barth, C., Buchholz, M., Lerch, M.M., Rolke, M., Holzmann, K.H., Menke,
A., Fensterer, H., Giehl, K., Lohr, M., et al. (2003). Claudin-4 expression decreases
invasiveness and metastatic potential of pancreatic cancer. Cancer Res 63, 6265-6271.
[109] Ohtani, S., Terashima, M., Satoh, J., Soeta, N., Saze, Z., Kashimura, S., Ohsuka, F.,
Hoshino, Y., Kogure, M., and Gotoh, M. (2009). Expression of tight-junction-
associated proteins in human gastric cancer: downregulation of claudin-4 correlates
with tumor aggressiveness and survival. Gastric Cancer 12, 43-51.
[110] Lee, S.K., Moon, J., Park, S.W., Song, S.Y., Chung, J.B., and Kang, J.K. (2005). Loss
of the tight junction protein claudin 4 correlates with histological growth-pattern and
differentiation in advanced gastric adenocarcinoma. Oncol Rep 13, 193-199.
[111] Guilford, P., Hopkins, J., Harraway, J., McLeod, M., McLeod, N., Harawira, P., Taite,
H., Scoular, R., Miller, A., and Reeve, A.E. (1998). E-cadherin germline mutations in
familial gastric cancer. Nature 392, 402-405.
[112] Huber, M.A., Kraut, N., and Beug, H. (2005). Molecular requirements for epithelial-
mesenchymal transition during tumor progression. Curr Opin Cell Biol 17, 548-558.
[113] Dhawan, P., Singh, A.B., Deane, N.G., No, Y., Shiou, S.R., Schmidt, C., Neff, J.,
Washington, M.K., and Beauchamp, R.D. (2005). Claudin-1 regulates cellular
transformation and metastatic behavior in colon cancer. J Clin Invest 115, 1765-1776.

[114] Falk, P., Roth, K.A., Boren, T., Westblom, T.U., Gordon, J.I., and Normark, S. (1993).
An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-
specific tropism in the human gastric epithelium. Proc Natl Acad Sci U S A 90, 2035-
2039.
[115] Thomsen, L.L., Gavin, J.B., and Tasman-Jones, C. (1990). Relation of Helicobacter
pylori to the human gastric mucosa in chronic gastritis of the antrum. Gut 31, 1230-
1236.
[116] Amieva, M.R., Vogelmann, R., Covacci, A., Tompkins, L.S., Nelson, W.J., and
Falkow, S. (2003). Disruption of the epithelial apical-junctional complex by
Helicobacter pylori CagA. Science 300, 1430-1434.
[117] Lapointe, T.K., O'Connor, P.M., Jones, N.L., Menard, D., and Buret, A.G. (2010).
Interleukin-1 receptor phosphorylation activates Rho kinase to disrupt human gastric
tight junctional claudin-4 during Helicobacter pylori infection. Cell Microbiol 12, 692-
703.
[118] Sharma, S.A., Tummuru, M.K., Blaser, M.J., and Kerr, L.D. (1998). Activation of IL-8
gene expression by Helicobacter pylori is regulated by transcription factor nuclear
factor-kappa B in gastric epithelial cells. J Immunol 160, 2401-2407.
[119] Tummuru, M.K., Sharma, S.A., and Blaser, M.J. (1995). Helicobacter pylori picB, a
homologue of the Bordetella pertussis toxin secretion protein, is required for induction
of IL-8 in gastric epithelial cells. Mol Microbiol 18, 867-876.
[120] Viala, J., Chaput, C., Boneca, I.G., Cardona, A., Girardin, S.E., Moran, A.P., Athman,
R., Memet, S., Huerre, M.R., Coyle, A.J., et al. (2004). Nod1 responds to peptidoglycan
delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol 5, 1166-
1174.
[121] Kwok, T., Zabler, D., Urman, S., Rohde, M., Hartig, R., Wessler, S., Misselwitz, R.,
Berger, J., Sewald, N., Konig, W., et al. (2007). Helicobacter exploits integrin for type
IV secretion and kinase activation. Nature 449, 862-866.
[122] Eaton, K.A., and Krakowka, S. (1994). Effect of gastric pH on urease-dependent
colonization of gnotobiotic piglets by Helicobacter pylori. Infect Immun 62, 3604-3607.
[123] Gobert, A.P., Mersey, B.D., Cheng, Y., Blumberg, D.R., Newton, J.C., and Wilson,
K.T. (2002). Cutting edge: urease release by Helicobacter pylori stimulates macrophage
inducible nitric oxide synthase. J Immunol 168, 6002-6006.
[124] Yamaoka, Y., Kita, M., Kodama, T., Sawai, N., and Imanishi, J. (1996). Helicobacter
pylori cagA gene and expression of cytokine messenger RNA in gastric mucosa.
Gastroenterology 110, 1744-1752.
[125] El-Omar, E.M. (2001). The importance of interleukin 1beta in Helicobacter pylori
associated disease. Gut 48, 743-747.
[126] El-Omar, E.M., Carrington, M., Chow, W.H., McColl, K.E., Bream, J.H., Young, H.A.,
Herrera, J., Lissowska, J., Yuan, C.C., Rothman, N., et al. (2000). Interleukin-1
polymorphisms associated with increased risk of gastric cancer. Nature 404, 398-402.
[127] Al-Sadi, R., Ye, D., Dokladny, K., and Ma, T.Y. (2008). Mechanism of IL-1beta-
induced increase in intestinal epithelial tight junction permeability. J Immunol 180,
5653-5661.
[128] Al-Sadi, R.M., and Ma, T.Y. (2007). IL-1beta causes an increase in intestinal epithelial
tight junction permeability. J Immunol 178, 4641-4649.

[129] Singh, R., Wang, B., Shirvaikar, A., Khan, S., Kamat, S., Schelling, J.R.,
Konieczkowski, M., and Sedor, J.R. (1999). The IL-1 receptor and Rho directly
associate to drive cell activation in inflammation. J Clin Invest 103, 1561-1570.
[130] El-Omar, E.M., Rabkin, C.S., Gammon, M.D., Vaughan, T.L., Risch, H.A.,
Schoenberg, J.B., Stanford, J.L., Mayne, S.T., Goedert, J., Blot, W.J., et al. (2003).
Increased risk of noncardia gastric cancer associated with proinflammatory cytokine
gene polymorphisms. Gastroenterology 124, 1193-1201.
[131] Graham, W.V., Wang, F., Clayburgh, D.R., Cheng, J.X., Yoon, B., Wang, Y., Lin, A.,
and Turner, J.R. (2006). Tumor necrosis factor-induced long myosin light chain kinase
transcription is regulated by differentiation-dependent signaling events.
Characterization of the human long myosin light chain kinase promoter. J Biol Chem
281, 26205-26215.
[132] Ma, T.Y., Boivin, M.A., Ye, D., Pedram, A., and Said, H.M. (2005). Mechanism of
TNF-{alpha} modulation of Caco-2 intestinal epithelial tight junction barrier: role of
myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol
288, G422-430.
[133] Wang, F., Graham, W.V., Wang, Y., Witkowski, E.D., Schwarz, B.T., and Turner, J.R.
(2005). Interferon-gamma and tumor necrosis factor-alpha synergize to induce
intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase
expression. Am J Pathol 166, 409-419.
[134] Clayburgh, D.R., Barrett, T.A., Tang, Y., Meddings, J.B., Van Eldik, L.J., Watterson,
D.M., Clarke, L.L., Mrsny, R.J., and Turner, J.R. (2005). Epithelial myosin light chain
kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in
vivo. J Clin Invest 115, 2702-2715.
[135] Zolotarevsky, Y., Hecht, G., Koutsouris, A., Gonzalez, D.E., Quan, C., Tom, J., Mrsny,
R.J., and Turner, J.R. (2002). A membrane-permeant peptide that inhibits MLC kinase
restores barrier function in in vitro models of intestinal disease. Gastroenterology 123,
163-172.
[136] Bruewer, M., Utech, M., Ivanov, A., Hopkins, A., Parkos, C., and Nusrat, A. (2005).
Interferon-gamma induces internalization of epithelial tight junction proteins via a
macropinocytosis-like process. Faseb J 19, 923-933.
[137] Segain, J.P., Raingeard de la Bletiere, D., Sauzeau, V., Bourreille, A., Hilaret, G.,
Cario-Toumaniantz, C., Pacaud, P., Galmiche, J.P., and Loirand, G. (2003). Rho kinase
blockade prevents inflammation via nuclear factor kappa B inhibition: evidence in
Crohn's disease and experimental colitis. Gastroenterology 124, 1180-1187.
[138] Utech, M., Ivanov, A.I., Samarin, S.N., Bruewer, M., Turner, J.R., Mrsny, R.J., Parkos,
C.A., and Nusrat, A. (2005). Mechanism of IFN-gamma-induced endocytosis of tight
junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane.
Mol Biol Cell 16, 5040-5052.
[139] Wang, F., Schwarz, B.T., Graham, W.V., Wang, Y., Su, L., Clayburgh, D.R., Abraham,
C., and Turner, J.R. (2006). IFN-gamma-induced TNFR2 expression is required for
TNF-dependent intestinal epithelial barrier dysfunction. Gastroenterology 131, 1153-
1163.
[140] Bamford, K.B., Fan, X., Crowe, S.E., Leary, J.F., Gourley, W.K., Luthra, G.K., Brooks,
E.G., Graham, D.Y., Reyes, V.E., and Ernst, P.B. (1998). Lymphocytes in the human

gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype.

[141] Lee, K.H., Choi, E.Y., Hyun, M.S., Jang, B.I., Kim, T.N., Kim, S.W., Song, S.K., Kim,
J.H., and Kim, J.R. (2007). Association of extracellular cleavage of E-cadherin
mediated by MMP-7 with HGF-induced in vitro invasion in human stomach cancer
cells. Eur Surg Res 39, 208-215.
[142] Wang, F., Sloss, C., Zhang, X., Lee, S.W., and Cusack, J.C. (2007). Membrane-bound
heparin-binding epidermal growth factor like growth factor regulates E-cadherin
expression in pancreatic carcinoma cells. Cancer Res 67, 8486-8493.
[143] Noe, V., Fingleton, B., Jacobs, K., Crawford, H.C., Vermeulen, S., Steelant, W.,
Bruyneel, E., Matrisian, L.M., and Mareel, M. (2001). Release of an invasion promoter
E-cadherin fragment by matrilysin and stromelysin-1. J Cell Sci 114, 111-118.
[144] Davies, G., Jiang, W.G., and Mason, M.D. (2001). Matrilysin mediates extracellular
cleavage of E-cadherin from prostate cancer cells: a key mechanism in hepatocyte
growth factor/scatter factor-induced cell-cell dissociation and in vitro invasion. Clin
Cancer Res 7, 3289-3297.
[145] Chan, A.O., Lam, S.K., Chu, K.M., Lam, C.M., Kwok, E., Leung, S.Y., Yuen, S.T.,
Law, S.Y., Hui, W.M., Lai, K.C., et al. (2001). Soluble E-cadherin is a valid prognostic
marker in gastric carcinoma. Gut 48, 808-811.
[146] Weydig, C., Starzinski-Powitz, A., Carra, G., Lower, J., and Wessler, S. (2007). CagA-
independent disruption of adherence junction complexes involves E-cadherin shedding
and implies multiple steps in Helicobacter pylori pathogenicity. Exp Cell Res 313,
3459-3471.
[147] O'Connor, P.M., Lapointe, T.K., Jackson, S., Beck, P.L., Jones, N.L., and Buret, A.G.
(2011). Helicobacter pylori activates calpain via toll-like receptor 2 to disrupt adherens
junctions in human gastric epithelial cells. Infect Immun 79, 3887-3894.
[148] McCaig, C., Duval, C., Hemers, E., Steele, I., Pritchard, D.M., Przemeck, S., Dimaline,
R., Ahmed, S., Bodger, K., Kerrigan, D.D., et al. (2006). The role of matrix
metalloproteinase-7 in redefining the gastric microenvironment in response to
Helicobacter pylori. Gastroenterology 130, 1754-1763.
[149] Wroblewski, L.E., Noble, P.J., Pagliocca, A., Pritchard, D.M., Hart, C.A., Campbell, F.,
Dodson, A.R., Dockray, G.J., and Varro, A. (2003). Stimulation of MMP-7 (matrilysin)
by Helicobacter pylori in human gastric epithelial cells: role in epithelial cell migration.
J Cell Sci 116, 3017-3026.
[150] Schirrmeister, W., Gnad, T., Wex, T., Higashiyama, S., Wolke, C., Naumann, M., and
Lendeckel, U. (2009). Ectodomain shedding of E-cadherin and c-Met is induced by
Helicobacter pylori infection. Exp Cell Res 315, 3500-3508.
[151] Maretzky, T., Reiss, K., Ludwig, A., Buchholz, J., Scholz, F., Proksch, E., de Strooper,
B., Hartmann, D., and Saftig, P. (2005). ADAM10 mediates E-cadherin shedding and
regulates epithelial cell-cell adhesion, migration, and beta-catenin translocation. Proc
Natl Acad Sci U S A 102, 9182-9187.
[152] Wu, S., Rhee, K.J., Zhang, M., Franco, A., and Sears, C.L. (2007). Bacteroides fragilis
toxin stimulates intestinal epithelial cell shedding and gamma-secretase-dependent E-
cadherin cleavage. J Cell Sci 120, 1944-1952.

[153] Wu, S., Lim, K.C., Huang, J., Saidi, R.F., and Sears, C.L. (1998). Bacteroides fragilis
enterotoxin cleaves the zonula adherens protein, E-cadherin. Proc Natl Acad Sci U S A
95, 14979-14984.
[154] Perri, F., Cotugno, R., Piepoli, A., Merla, A., Quitadamo, M., Gentile, A., Pilotto, A.,
Annese, V., and Andriulli, A. (2007). Aberrant DNA methylation in non-neoplastic
gastric mucosa of H. Pylori infected patients and effect of eradication. Am J
Gastroenterol 102, 1361-1371.
[155] Leung, W.K., Man, E.P., Yu, J., Go, M.Y., To, K.F., Yamaoka, Y., Cheng, V.Y., Ng,
E.K., and Sung, J.J. (2006). Effects of Helicobacter pylori eradication on methylation
status of E-cadherin gene in noncancerous stomach. Clin Cancer Res 12, 3216-3221.
[156] Huang, F.Y., Chan, A.O., Rashid, A., Wong, D.K., Cho, C.H., and Yuen, M.F. (2012).
Helicobacter pylori induces promoter methylation of E-cadherin via interleukin-1beta
activation of nitric oxide production in gastric cancer cells. Cancer 118, 4969-4980.
[157] Qian, X., Huang, C., Cho, C.H., Hui, W.M., Rashid, A., and Chan, A.O. (2008). E-
cadherin promoter hypermethylation induced by interleukin-1beta treatment or H.
pylori infection in human gastric cancer cell lines. Cancer Lett 263, 107-113.
[158] Chan, A.O., Lam, S.K., Wong, B.C., Wong, W.M., Yuen, M.F., Yeung, Y.H., Hui,
W.M., Rashid, A., and Kwong, Y.L. (2003). Promoter methylation of E-cadherin gene
in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer.
Gut 52, 502-506.
[159] Chan, A.O., Peng, J.Z., Lam, S.K., Lai, K.C., Yuen, M.F., Cheung, H.K., Kwong, Y.L.,
Rashid, A., Chan, C.K., and Wong, B.C. (2006). Eradication of Helicobacter pylori
infection reverses E-cadherin promoter hypermethylation. Gut 55, 463-468.
[160] Aberle, H., Bauer, A., Stappert, J., Kispert, A., and Kemler, R. (1997). beta-catenin is a
target for the ubiquitin-proteasome pathway. Embo J 16, 3797-3804.
[161] Franco, A.T., Johnston, E., Krishna, U., Yamaoka, Y., Israel, D.A., Nagy, T.A.,
Wroblewski, L.E., Piazuelo, M.B., Correa, P., and Peek, R.M., Jr. (2008). Regulation of
gastric carcinogenesis by Helicobacter pylori virulence factors. Cancer Res 68, 379-
387.
[162] Nakayama, M., Hisatsune, J., Yamasaki, E., Isomoto, H., Kurazono, H., Hatakeyama,
M., Azuma, T., Yamaoka, Y., Yahiro, K., Moss, J., et al. (2009). Helicobacter pylori
VacA-induced inhibition of GSK3 through the PI3K/Akt signaling pathway. J Biol
Chem 284, 1612-1619.
[163] Sokolova, O., Bozko, P.M., and Naumann, M. (2008). Helicobacter pylori suppresses
glycogen synthase kinase 3beta to promote beta-catenin activity. J Biol Chem 283,
29367-29374.
[164] Suzuki, M., Mimuro, H., Kiga, K., Fukumatsu, M., Ishijima, N., Morikawa, H., Nagai,
S., Koyasu, S., Gilman, R.H., Kersulyte, D., et al. (2009). Helicobacter pylori CagA
phosphorylation-independent function in epithelial proliferation and inflammation. Cell
Host Microbe 5, 23-34.
[165] Tabassam, F.H., Graham, D.Y., and Yamaoka, Y. (2009). Helicobacter pylori activate
epidermal growth factor receptor- and phosphatidylinositol 3-OH kinase-dependent Akt
and glycogen synthase kinase 3beta phosphorylation. Cell Microbiol 11, 70-82.
[166] Oguma, K., Oshima, H., Aoki, M., Uchio, R., Naka, K., Nakamura, S., Hirao, A., Saya,
H., Taketo, M.M., and Oshima, M. (2008). Activated macrophages promote Wnt

signalling through tumour necrosis factor-alpha in gastric tumour cells. Embo J 27,
1671-1681.
[167] Kurashima, Y., Murata-Kamiya, N., Kikuchi, K., Higashi, H., Azuma, T., Kondo, S.,
and Hatakeyama, M. (2008). Deregulation of beta-catenin signal by Helicobacter pylori
CagA requires the CagA-multimerization sequence. Int J Cancer 122, 823-831.
[168] Murata-Kamiya, N., Kurashima, Y., Teishikata, Y., Yamahashi, Y., Saito, Y., Higashi,
H., Aburatani, H., Akiyama, T., Peek, R.M., Jr., Azuma, T., et al. (2007). Helicobacter
pylori CagA interacts with E-cadherin and deregulates the beta-catenin signal that
promotes intestinal transdifferentiation in gastric epithelial cells. Oncogene 26, 4617-
4626.
[169] Franco, A.T., Israel, D.A., Washington, M.K., Krishna, U., Fox, J.G., Rogers, A.B.,
Neish, A.S., Collier-Hyams, L., Perez-Perez, G.I., Hatakeyama, M., et al. (2005).
Activation of beta-catenin by carcinogenic Helicobacter pylori. Proc Natl Acad Sci U S
A 102, 10646-10651.
[170] Tan, Y., Wu, C., De Veyra, T., and Greer, P.A. (2006). Ubiquitous calpains promote
both apoptosis and survival signals in response to different cell death stimuli. J Biol
Chem 281, 17689-17698.
[171] Liu, L., Xing, D., and Chen, W.R. (2009). Micro-calpain regulates caspase-dependent
and apoptosis inducing factor-mediated caspase-independent apoptotic pathways in
cisplatin-induced apoptosis. Int J Cancer 125, 2757-2766.
[172] Wiemer, A.J., Lokuta, M.A., Surfus, J.C., Wernimont, S.A., and Huttenlocher, A.
(2009). Calpain inhibition impairs TNF-alpha-mediated neutrophil adhesion, arrest and
oxidative burst. Mol Immunol 47, 894-902.
[173] Rios-Doria, J., Day, K.C., Kuefer, R., Rashid, M.G., Chinnaiyan, A.M., Rubin, M.A.,
and Day, M.L. (2003). The role of calpain in the proteolytic cleavage of E-cadherin in
prostate and mammary epithelial cells. J Biol Chem 278, 1372-1379.
[174] Rios-Doria, J., Kuefer, R., Ethier, S.P., and Day, M.L. (2004). Cleavage of beta-catenin
by calpain in prostate and mammary tumor cells. Cancer Res 64, 7237-7240.
[175] Anastasiadis, P.Z., and Reynolds, A.B. (2001). Regulation of Rho GTPases by p120-
catenin. Curr Opin Cell Biol 13, 604-610.
[176] Grosheva, I., Shtutman, M., Elbaum, M., and Bershadsky, A.D. (2001). p120 catenin
affects cell motility via modulation of activity of Rho-family GTPases: a link between
cell-cell contact formation and regulation of cell locomotion. J Cell Sci 114, 695-707.
[177] Noren, N.K., Liu, B.P., Burridge, K., and Kreft, B. (2000). p120 catenin regulates the
actin cytoskeleton via Rho family GTPases. J Cell Biol 150, 567-580.
[178] Reynolds, A.B., and Carnahan, R.H. (2004). Regulation of cadherin stability and
turnover by p120ctn: implications in disease and cancer. Semin Cell Dev Biol 15, 657-
663.
[179] Wijnhoven, B.P., Pignatelli, M., Dinjens, W.N., and Tilanus, H.W. (2005). Reduced
p120ctn expression correlates with poor survival in patients with adenocarcinoma of the
gastroesophageal junction. J Surg Oncol 92, 116-123.
[180] Sarrio, D., Moreno-Bueno, G., Sanchez-Estevez, C., Banon-Rodriguez, I., Hernandez-
Cortes, G., Hardisson, D., and Palacios, J. (2006). Expression of cadherins and catenins
correlates with distinct histologic types of ovarian carcinomas. Hum Pathol 37, 1042-
1049.

[181] Krueger, S., Hundertmark, T., Kuester, D., Kalinski, T., Peitz, U., and Roessner, A.
(2007). Helicobacter pylori alters the distribution of ZO-1 and p120ctn in primary
human gastric epithelial cells. Pathol Res Pract 203, 433-444.
[182] Ogden, S.R., Wroblewski, L.E., Weydig, C., Romero-Gallo, J., O'Brien, D.P., Israel,
D.A., Krishna, U.S., Fingleton, B., Reynolds, A.B., Wessler, S., et al. (2008). p120 and
Kaiso regulate Helicobacter pylori-induced expression of matrix metalloproteinase-7.
Mol Biol Cell 19, 4110-4121.
[183] Jones, N.L., Shannon, P.T., Cutz, E., Yeger, H., and Sherman, P.M. (1997). Increase in
proliferation and apoptosis of gastric epithelial cells early in the natural history of
Helicobacter pylori infection. Am J Pathol 151, 1695-1703.
[184] Moss, S.F., Calam, J., Agarwal, B., Wang, S., and Holt, P.R. (1996). Induction of
gastric epithelial apoptosis by Helicobacter pylori. Gut 38, 498-501.
[185] Rudi, J., Kuck, D., Strand, S., von Herbay, A., Mariani, S.M., Krammer, P.H., Galle,
P.R., and Stremmel, W. (1998). Involvement of the CD95 (APO-1/Fas) receptor and
ligand system in Helicobacter pylori-induced gastric epithelial apoptosis. J Clin Invest
102, 1506-1514.
[186] Peek, R.M., Jr., Wirth, H.P., Moss, S.F., Yang, M., Abdalla, A.M., Tham, K.T., Zhang,
T., Tang, L.H., Modlin, I.M., and Blaser, M.J. (2000). Helicobacter pylori alters gastric
epithelial cell cycle events and gastrin secretion in Mongolian gerbils. Gastroenterology
118, 48-59.
[187] Jones, N.L., Day, A.S., Jennings, H., Shannon, P.T., Galindo-Mata, E., and Sherman,
P.M. (2002). Enhanced disease severity in Helicobacter pylori-infected mice deficient
in Fas signaling. Infect Immun 70, 2591-2597.
[188] Atherton, J.C., Peek, R.M., Jr., Tham, K.T., Cover, T.L., and Blaser, M.J. (1997).
Clinical and pathological importance of heterogeneity in vacA, the vacuolating
cytotoxin gene of Helicobacter pylori. Gastroenterology 112, 92-99.
[189] Cover, T.L., Reddy, L.Y., and Blaser, M.J. (1993). Effects of ATPase inhibitors on the
response of HeLa cells to Helicobacter pylori vacuolating toxin. Infect Immun 61, 1427-
1431.
[190] Ghiara, P., Marchetti, M., Blaser, M.J., Tummuru, M.K., Cover, T.L., Segal, E.D.,
Tompkins, L.S., and Rappuoli, R. (1995). Role of the Helicobacter pylori virulence
factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease. Infect
Immun 63, 4154-4160.
[191] McClain, M.S., Schraw, W., Ricci, V., Boquet, P., and Cover, T.L. (2000). Acid
activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin
internalization by eukaryotic cells. Mol Microbiol 37, 433-442.
[192] Cover, T.L., Hanson, P.I., and Heuser, J.E. (1997). Acid-induced dissociation of VacA,
the Helicobacter pylori vacuolating cytotoxin, reveals its pattern of assembly. J Cell
Biol 138, 759-769.
[193] Jain, P., Luo, Z.Q., and Blanke, S.R. (2011). Helicobacter pylori vacuolating cytotoxin
A (VacA) engages the mitochondrial fission machinery to induce host cell death. Proc
Natl Acad Sci U S A 108, 16032-16037.
[194] Lan, C.H., Sheng, J.Q., Fang, D.C., Meng, Q.Z., Fan, L.L., and Huang, Z.R. (2010).
Involvement of VDAC1 and Bcl-2 family of proteins in VacA-induced cytochrome c
release and apoptosis of gastric epithelial carcinoma cells. J Dig Dis 11, 43-49.

[195] Yamasaki, E., Wada, A., Kumatori, A., Nakagawa, I., Funao, J., Nakayama, M.,
Hisatsune, J., Kimura, M., Moss, J., and Hirayama, T. (2006). Helicobacter pylori
vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak,
leading to cytochrome c release and cell death, independent of vacuolation. J Biol Chem
281, 11250-11259.
[196] Galmiche, A., Rassow, J., Doye, A., Cagnol, S., Chambard, J.C., Contamin, S., de
Thillot, V., Just, I., Ricci, V., Solcia, E., et al. (2000). The N-terminal 34 kDa fragment
of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces
cytochrome c release. Embo J 19, 6361-6370.
[197] Fan, X., Gunasena, H., Cheng, Z., Espejo, R., Crowe, S.E., Ernst, P.B., and Reyes, V.E.
(2000). Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and
induces their apoptosis. J Immunol 165, 1918-1924.
[198] Houghton, J., Korah, R.M., Condon, M.R., and Kim, K.H. (1999). Apoptosis in
Helicobacter pylori-associated gastric and duodenal ulcer disease is mediated via the
Fas antigen pathway. Dig Dis Sci 44, 465-478.
[199] Jones, N.L., Day, A.S., Jennings, H.A., and Sherman, P.M. (1999). Helicobacter pylori
induces gastric epithelial cell apoptosis in association with increased Fas receptor
expression. Infect Immun 67, 4237-4242.
[200] Gupta, R.A., Polk, D.B., Krishna, U., Israel, D.A., Yan, F., DuBois, R.N., and Peek,
R.M., Jr. (2001). Activation of peroxisome proliferator-activated receptor gamma
suppresses nuclear factor kappa B-mediated apoptosis induced by Helicobacter pylori
in gastric epithelial cells. J Biol Chem 276, 31059-31066.
[201] Wallasch, C., Crabtree, J.E., Bevec, D., Robinson, P.A., Wagner, H., and Ullrich, A.
(2002). Helicobacter pylori-stimulated EGF receptor transactivation requires
metalloprotease cleavage of HB-EGF. Biochem Biophys Res Commun 295, 695-701.
[202] Keates, S., Sougioultzis, S., Keates, A.C., Zhao, D., Peek, R.M., Jr., Shaw, L.M., and
Kelly, C.P. (2001). cag+ Helicobacter pylori induce transactivation of the epidermal
growth factor receptor in AGS gastric epithelial cells. J Biol Chem 276, 48127-48134.
[203] Yan, F., Cao, H., Chaturvedi, R., Krishna, U., Hobbs, S.S., Dempsey, P.J., Peek, R.M.,
Jr., Cover, T.L., Washington, M.K., Wilson, K.T., et al. (2009). Epidermal growth
factor receptor activation protects gastric epithelial cells from Helicobacter pylori-
induced apoptosis. Gastroenterology 136, 1297-1307, e1291-1293.
[204] Bauer, B., Bartfeld, S., and Meyer, T.F. (2009). H. pylori selectively blocks EGFR
endocytosis via the non-receptor kinase c-Abl and CagA. Cell Microbiol 11, 156-169.
[205] Kubben, F.J., Sier, C.F., Meijer, M.J., van den Berg, M., van der Reijden, J.J.,
Griffioen, G., van de Velde, C.J., Lamers, C.B., and Verspaget, H.W. (2006). Clinical
impact of MMP and TIMP gene polymorphisms in gastric cancer. Br J Cancer 95, 744-
751.
[206] Hellmig, S., Ott, S., Rosenstiel, P., Robert Folsch, U., Hampe, J., and Schreiber, S.
(2006). Genetic variants in matrix metalloproteinase genes are associated with
development of gastric ulcer in H. Pylori infection. Am J Gastroenterol 101, 29-35.
[207] Crawford, H.C., Krishna, U.S., Israel, D.A., Matrisian, L.M., Washington, M.K., and
Peek, R.M., Jr. (2003). Helicobacter pylori strain-selective induction of matrix
metalloproteinase-7 in vitro and within gastric mucosa. Gastroenterology 125, 1125-
1136.

[208] Forman, B.M., Tontonoz, P., Chen, J., Brun, R.P., Spiegelman, B.M., and Evans, R.M.
(1995). 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte
determination factor PPAR gamma. Cell 83, 803-812.
[209] Nakajima, A., Wada, K., Miki, H., Kubota, N., Nakajima, N., Terauchi, Y., Ohnishi, S.,
Saubermann, L.J., Kadowaki, T., Blumberg, R.S., et al. (2001). Endogenous PPAR
gamma mediates anti-inflammatory activity in murine ischemia-reperfusion injury.
[210] Su, C.G., Wen, X., Bailey, S.T., Jiang, W., Rangwala, S.M., Keilbaugh, S.A., Flanigan,
A., Murthy, S., Lazar, M.A., and Wu, G.D. (1999). A novel therapy for colitis utilizing
PPAR-gamma ligands to inhibit the epithelial inflammatory response. J Clin Invest 104,
383-389.
[211] Buti, L., Spooner, E., Van der Veen, A.G., Rappuoli, R., Covacci, A., and Ploegh, H.L.
(2011). Helicobacter pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-
stimulating protein of p53 (ASPP2) tumor suppressor pathway of the host. Proc Natl
Acad Sci U S A 108, 9238-9243.
[212] Samuels-Lev, Y., O'Connor, D.J., Bergamaschi, D., Trigiante, G., Hsieh, J.K., Zhong,
S., Campargue, I., Naumovski, L., Crook, T., and Lu, X. (2001). ASPP proteins
specifically stimulate the apoptotic function of p53. Mol Cell 8, 781-794.
[213] Chaturvedi, R., de Sablet, T., Peek, R.M., and Wilson, K.T. (2012). Spermine oxidase,
a polyamine catabolic enzyme that links Helicobacter pylori CagA and gastric cancer
risk. Gut Microbes 3, 48-56.
[214] Mimuro, H., Suzuki, T., Nagai, S., Rieder, G., Suzuki, M., Nagai, T., Fujita, Y.,
Nagamatsu, K., Ishijima, N., Koyasu, S., et al. (2007). Helicobacter pylori dampens gut
epithelial self-renewal by inhibiting apoptosis, a bacterial strategy to enhance
colonization of the stomach. Cell Host Microbe 2, 250-263.

Chapter 8
Clinical Aspects and Complications

of Helicobacter pylori Infection
Marino Venerito and Peter Malfertheiner*

Department of Gastroenterology, Hepatology and Infectious Diseases,
Otto-von-Guericke University Hospital, Magdeburg, Germany
Abstract
Helicobacter pylori (H. pylori) infection is still one of the world’s most frequent
infections. Infected individuals have a chronic active gastritis, which is generally
asymptomatic. About 20% of subjects infected with the bacterium will develop clinical
manifestations of the infection including peptic ulcer disease, adenocarcinoma of the
stomach and gastric MALT lymphoma. H. pylori infection is also one of the most likely
causes of functional dyspepsia. The clinical outcome of the infection depends on many
variables, including H. pylori genotype, innate host immune response, genetic
predisposition and environmental factors. H. pylori eradication decreases the incidence of
peptic ulcer disease and prevents its recurrence. H. pylori eradication improves symptoms
in a subset of patients with functional dyspepsia. Cure of H. pylori infection decreases
significantly the incidence of gastric cancer in infected subjects without premalignant
conditions of gastric mucosa. At early stages, gastric MALT lymphoma can be cured
completely by eradication of H. pylori.
Keywords: Helicobacter pylori, chronic gastritis, functional dyspepsia, peptic ulcer disease,
gastric cancer, MALT lymphoma
*
Corresponding Author address: Prof. Dr. med. habil. Dr. h.c. Peter Malfertheiner, Head of the Department of
Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke University Hospital, Leipziger str.
44, 39120, Magdeburg, Germany. Email: peter.malfertheiner@med.ovgu.de.

146 Marino Venerito and Peter Malfertheiner
Introduction
Helicobacter pylori (H. pylori) infection is etiologically linked to histologic chronic
active gastritis, peptic ulcer disease, adenocarcinoma of the stomach and primary B-cell
gastric lymphoma (gastric MALT lymphoma). Most infected individuals have a chronic
active, superficial gastritis, which is generally asymptomatic. About 20% of subjects infected
with the bacterium will develop clinical manifestations of the infection.
Factors determining whether the infection will produce disease are abnormalities in
gastric acid regulation and the infiltration of immune cells in the gastric mucosa with cytokine
release leading to gastric damage [1]. H. pylori impairs the homeostasis of gastric acid
secretion via different mechanisms depending on the severity and distribution of the H. pylori
induced gastritis [2]. The infection is associated with 3 potential pathophysiological and
clinical outcomes, and the figure 1 illustrates the histological, physiologic, and clinical
characteristics of each. Figure 2 shows a proposed natural history of H. pylori infection in
humans [3].
Figure 1. Pathophysiologic and clinical outcomes of chronic Helicobacter pylori infection. The
infection is associated with 3 potential outcomes, and the Figure illustrates the histologic, physiologic,
and clinical characteristics of each. Modified from Amieva MR and El Omar EM. [2].
Most infected subjects present a gastritis phenotype characterized by mild pangastritis

and little impairment of gastric acid secretion [2]. In up to 15% of infected individuals, H.
pylori induces an antral-predominant pattern of gastritis, which is associated with duodenal
ulcer (“duodenal ulcer phenotype”) [4]. In patients with duodenal ulcer, the severity of H.
pylori induced inflammation is greatest in the distal antral region by sparing the acid-
secreting corpus mucosa [5]. These modifications are associated with an impaired negative
feedback regulation of gastrin release (which results in high gastrin levels) and an increased

Clinical Aspects and Complications of Helicobacter pylori Infection 147
acid response to gastrin due to an increased number of acid-secreting parietal cells as a

consequence of the trophic effect of hypergastrinaemia [5]. About 1% of infected individuals
present a gastritis pattern in which the severity of inflammation is higher in the gastric body
and fundus of the stomach. The chronic inflammation leads on the long run to the loss of
acid- and mucus-secreting cells (atrophy, hypo- or achlorhydria), leaving the stomach
vulnerable for the development of ulcers in the gastric body and fundus [6]. Atrophy of the
gastric mucosa is a risk factor for dysplasia and gastric cancer, usually the intestinal type.
This gastritis phenotype is particularly prevalent in certain population groups including
African Americans, Asians, Scandinavians, Hispanics and immigrants from Japan, China and
South America, where also gastric cancer is common [7].
Environmental
H. pylori factors
Gastric
cancer
Multifocal
Acute Gastric ulcer
atrophic
gastritis
gastritis
Lymphoma
Chronic active gastritis
Antral- duodenal ulcer

predominant
gastritis Lymphoma
Figure 2. Proposed natural history of Helicobacter pylori infection in humans. Modified from Graham
DY and Sung JJY [3].
Functional Dyspepsia
Definition
The term functional dyspepsia describes the presence of symptoms originating from the
stomach and duodenum in the absence of organic disease (i.e. peptic ulcer disease) that is
likely to explain the symptoms. Symptoms originating from the esophagus like heartburn or
regurgitation are not included in the current definition. Dyspeptic symptoms include
postprandial fullness, early satiation, epigastric pain and epigastric burning. For diagnosis of
functional dyspepsia the presence of one or more dyspeptic symptoms for the last three
months with symptoms onset at least 6 months before diagnosis is required [8].

Epidemiology
Functional dyspepsia is a highly prevalent disorder, affecting 5-12% of the general

population [9, 10] and accounting for 2-5% of medical consultations [11].
Prevalence of dyspeptic symptoms is slightly higher in women than in men and appears
to decline with age.
The incidence of dyspepsia (number of new cases in a population at risk) is poorly
documented. In a Scandinavian study conducted on a period of 3 months, the incidence of
dyspepsia was lower than 1% [12]. Longitudinal studies suggest that symptoms improve or
disappear over the time in less than half of the patients [13]. The probability of remission is
lower in patients with a longer history of dyspeptic symptoms, lower educational level or
psychosocial stress.
Pathophysiology
A number of theories have been proposed to describe the pathophysiology of functional

dyspeptic symptoms, including visceral hypersensitivity and alterations of motility in the
gastroduodenal tract [14]. In some cases, these symptoms might have a post-infectious cause
[15]. A variety of bacterial infections have been implicated in the pathogenesis of functional
dyspepsia. H. pylori infection represents one of the most likely causes, as it is common
worldwide and involves primarily the gastric mucosa. Pathophysiologic mechanisms thought
to induce functional dyspeptic symptoms in patients infected with H. pylori are enhanced acid
secretion and decreased secretion of ghrelin (a hormone produced by the gastric
enteroendocrine cell compartment involved in hunger sensation, acid secretion and
gastrointestinal motility) [16]. Increased acid secretion is a condition associated with the
antral-predominant pattern of gastritis whereas significantly lower plasma levels of total and
active ghrelin have been observed in patients with atrophy of gastric mucosa. However, the
relationship between gastritis pattern and functional dyspeptic symptoms has not been
investigated.
H. pylori Eradication in Functional Dyspepsia
In a Cochrane meta-analysis of 21 trials involving 4331 patients with functional

dyspepsia eradication therapy for H. pylori infection compared with controls induced at 12
months a small but statistically significant reduction in the frequency of dyspeptic symptoms
[17] The clinical significance of this findings is unclear because the effect occurs only late
and is relatively small, with a number needed to treat of 14 (95% CI: 10-20) H. pylori-
positive patients to achieve one cure.
Thus, in a small subset of H. pylori-positive patients with functional dyspepsia
eradication therapy improves symptoms and the effect is long-term. Guidelines strongly
recommend H. pylori eradication despite the small benefit as no therapy has been shown to be
particularly effective in functional dyspepsia. Moreover, H. pylori eradication in patients with
functional dyspepsia has other benefits such as the prevention of peptic ulcer disease and the
possible reduction in the risk of gastric malignancies.

Peptic Ulcer Disease (See Also Chapter 3)

Definition
Peptic ulcer disease, which embraces both gastric and duodenal ulcers, is defined
pathologically as a defect in the gastric or duodenal wall that extends through the muscularis
mucosae into the deeper layers of the wall (submucosa or the muscularis propria); a mucosal
break without penetration of the muscularis mucosae is called erosion [18]. Complications of
peptic ulcer disease include bleeding, penetration, perforation and obstruction.
Risk Factors
H. pylori and the intake of non-steroidal anti-inflammatory drugs (NSAIDs) including

low-dose aspirin are the most important risk factors in the pathogenesis of peptic ulcers [19].
H. pylori infection and NSAID use are independent risk factors for the development of an
ulcer. A meta-analysis of observational studies showed that the presence of both risk factors
increases synergistically the risk for developing a gastroduodenal ulcer, whereas the odds
ratio for developing gastroduodenal ulcer bleeding was almost the sum of the two individual
odds ratios estimated for H. pylori infection and NSAID use alone [20].
Epidemiology
Peptic ulcer disease has been a major threat to the world’s population over the past two
centuries, with a high morbidity and substantial mortality. Epidemiological data for this
disease and its complications have shown striking geographical variations in incidence and
prevalence. Development of ulcer disease and death from it has been associated with the birth
of urbanisation and was interpreted as a birth-cohort event with the peak of disease in those
born during the late 19th century [21, 22].
Data on current prevalence of uncomplicated peptic ulcer are very scarce, as according to
current recommendations endoscopic diagnosis is not necessary for treatment of
uncomplicated peptic ulcer disease suspected in the presence of dyspeptic symptoms.
The most frequent and severe complication of peptic ulcers is bleeding, which is reported
in 50–170 per 100.000, with the highest risk in people aged older than 60 years [23, 24, 25].
Perforation is less frequent than bleeding, with an incidence of around seven to ten per
100.000 [14, 15]. Penetration of retroperitoneal organs is characterised by constant severe
pain but fortunately is rare [15]. Gastric outlet obstruction due to ulcer-induced fibrosis is also
rare, and should raise suspicion of underlying malignant disease [26].
Clinical Features
The predominant symptom of uncomplicated peptic ulcer is a burning pain in the

epigastrium, which can be accompanied by other dyspeptic symptoms such as fullness,

bloating, early satiety, and nausea. In patients with gastric ulcer symptoms may occur after a
meal and in some patients, eating may precipitate pain immediately. As a consequence,
anorexia and weight loss may occur in up to 50% of patients. In patients with duodenal ulcer,
epigastric pain occurs typically during the fasting state or even during the night and is usually
relieved by food intake or acid-neutralising agents. Roughly a third of these patients also have
heartburn, mostly without erosive oesophagitis [27]. Chronic ulcers can be asymptomatic
[28].
In particular, this absence of symptoms is seen in NSAID-induced ulcers, for which
upper gastrointestinal bleeding or perforation might be the first clinical manifestation of
disease.
Diagnosis
A peptic ulcer is diagnosed at endoscopy when there is a mucosal break of diameter 5

mm or larger, covered with fibrin; a mucosal break smaller than 5 mm is called an erosion.
The 5 mm criterion is arbitrary, but is used in clinical trials. The extent to which this criterion
relates to the pathological criterion of penetration of the muscularis mucosa is unclear. Peptic
ulcers can be single or many. The typical location of the duodenal ulcer is in the bulb, where
gastric contents enter the small intestine. The site of predilection for gastric ulcers is the
angulus of the lesser curvature; however, they can occur at any location from the pylorus to
the cardia. Occasionally, kissing ulcers are seen located face to face on the anterior and
posterior walls of the duodenal bulb. If ulceration is seen in the more distal duodenum, then
causes other than H. pylori infection (i.e. underlying Crohn’s disease, ischaemia, or the rare
Zollinger-Ellison syndrome) should be suspected. On endoscopic diagnosis of peptic ulcer,
biopsy samples of the antral and body or fundus mucosa should be taken for detection of H.
pylori infection by rapid urease and histological tests.
In many developed countries, ulcer-like symptoms in patients aged up to 55 years are
generally not investigated by endoscopic examination but by testing non-invasively for H.
pylori (13C-urea breath test [UBT], stool antigen test) and treated with H. pylori eradication if
positive [29, 30].
The rationale for this test-and-treat strategy is that symptoms in a proportion of patients
will be due to underlying ulcer disease that will be cured by H. pylori treatment. Moreover,
malignant disease is rare in young people and in the absence of alarm symptoms such as loss
of appetite, weight loss, anaemia, and vomiting.
Treatment
The discovery of H. pylori in 1982 by Warren and Marshall changed greatly our
understanding of peptic ulcer disease and its treatment, switching the notion from an acid-
driven disease to an infectious disease [31, 32]. Maintenance acid suppressive therapy for
duodenal ulcer, which followed decades of dominance of surgical interventions (subtotal
gastric resections, several forms of vagotomy), was replaced with a short-term antibiotic
regimen targeting eradication of H. pylori infection [33, 34].

Adenocarcinoma of the Stomach

Definition and Classification
Adenocarcinoma of the stomach (gastric cancer) is a malignant disease in which cancer

cells grow in the stomach. Gastric cancer can develop in any part of the stomach and spread
to other organs through tumor growth, the bloodstream, or the lymphatic system.
Approximately 97% of stomach tumors are adenocarcinomas.
Gastric adenocarcinomas are classified anatomically as proximal (cardia) and distal (non-
cardia). According to the Lauren classification gastric cancer can be subdivided in two
distinct pathological entities: the intestinal type and the diffuse type [35]. These two entities
have different morphologic appearance, epidemiology, pathogenesis and prognostic features.
In the intestinal form of gastric cancer tumor cells are cohesively arranged, forming irregular
tubular structures that mimick intestinal glands. In the diffuse form of gastric cancer tumor
cells show lack of cohesiveness and infiltrate diffusely the neighbouring structures. In this
subtype, also called the signet-ring adenocarcinoma, the nuclei are pushed to the periphery
due to the abundant mucinous cytoplasmic content. Non-cardia adenocarcinomas of both
intestinal and diffuse type are commonly associated with H. pylori infection, whereas the
association of this infection with cardia adenocarcinomas is less well defined.
Finally, gastric cancer can be sporadic or associated with family cancer syndromes. Non-
cardia gastric cancer of the sporadic type has three major contributing factors: Helicobacter
pylori infection, specific host immune response, and environmental influences. Completely
distinct is the development of hereditary diffuse gastric cancer (HDGC) which is a familial
cancer syndrome caused, in 30-40% of cases, by germ-line mutations of the E-
cadherin/CDH1 gene.
Other family cancer syndromes associated with an increased risk for developing gastric
cancer are the familial adenomatous polyposis (FAP), the Lynch syndrome (hereditary
nonpolyposis colorectal cancer, HNPCC), and juvenile polyposis syndrome.
In the present chapter we give a special emphasis to non-cardia gastric cancer for its close
association to H. pylori infection.
Epidemiology
About one million new cases of gastric cancer were estimated to have occurred in 2008
(988.000 cases, 7.8% of the total), making it currently the fourth most common malignancy in
the world, behind cancers of the lung, breast and colo-rectum. More than 70% of cases occur
in developing countries, and half the world total occurs in Eastern Asia (mainly in China).
Age-standardised incidence rates are about twice as high in men as in women, ranging from
3.9 in Northern Africa to 42.4 in Eastern Asia for men, and from 2.2 in Southern Africa to
18.3 in Eastern Asia for women [36].
Gastric cancer is still the second leading cause of cancer death in both sexes worldwide
(736 000 deaths, 9.7% of the total). The highest mortality rates are estimated in Eastern Asia
(28.1 per 100,000 in men, 13.0 per 100,000 in women), the lowest in Northern America (2.8

and 1.5 respectively). High mortality rates are also present in both sexes in Central and
Eastern Europe, and in Central and South America (Table 1) [36].
The intestinal form of gastric cancer is more frequently observed in older patients with
gastric atrophy, is more closely linked to environmental and dietary risk factors, represents
the predominant form in regions with a high incidence of gastric cancer, and is the form of
cancer that is now declining worldwide [37]. Non-cardia gastric cancer has a male-to-female
ratio of approximately 2:1 [38]. Incidence rates are significantly higher among non-white
subjects, lower socio-economic groups, and in developing countries [37]. Incidence rises
progressively with age, with a peak incidence between 50 and 70 years.
The frequency of the diffuse form of gastric cancer instead is the same throughout the
world, occurs at a lower age, is more virulent in women, and is associated with a worse
prognosis than the intestinal form.
Table 1.
Worldwide Incidence and Mortality Rates per 100,000 Cases (Age adjusted) of Gastric Cancer
for 2008 (selected countries). Source: Globocan 2008, IARC
Incidence Mortality
Country/Area Male Female Male Female
Republic of Korea 62.2 24.6 22.8 8.6
Mongolia 48.2 22.3 36.9 17.6
Japan 46.8 18.2 20.5 8
China 41.3 18.5 30.1 14.6
Belarus 34.2 15 30.1 11.8
Costa Rica 28.5 15.6 23.6 10.6
Ecuador 28 19.8 24.1 17.5
Chile 27.3 10.3 23.1 8.6
Russian Federation 26.9 11.7 24 9.9
Romania 15.9 5.8 15.2 5.4
Italy 14.8 7.7 9 4.6
Poland 13.6 5.1 12.7 4.6
Spain 12.1 5.3 8.7 3.8
Germany 10.4 5.5 6.8 3.7
Australia 7.3 3.2 4 2
Canada 6.7 3.1 4.1 2.1
USA 5.7 2.8 2.7 1.4
India 4.7 2.9 4.6 2.7
Nigeria 3.2 1.4 2.6 1.7
Botswana 0.6 0.2 0.6 0.2
Clinical Features
In more than two thirds of patients, gastric cancer is advanced at initial presentation. The
clinical history of tumor-related symptoms is usually of recent date and symptoms are often
unspecific. Dyspeptic symptoms like a vague discomfort in the epigastrium, abdominal

fullness or pain and nausea can be present, although about 40% of patients never report
dyspeptic symptoms within their recent or remote medical history [39]. Alarm features, which
should promptly raise the suspicion of advanced gastric cancer, are weight loss due to
dyspeptic symptoms, recurrent or persisting vomiting and signs of bleeding like anemia,
hemathemesis or melena. Dysphagia is common in patients with cancer arising in the
proximal stomach.
H. pylori Infection and Gastric Cancer (See Also Chapter 3)
Infection with H. pylori is the major risk factor for the development of sporadic non-
cardia gastric cancer of both intestinal and diffuse type. H. pylori was classified in 1994 as
class I carcinogen (definite carcinogen) by the International Agency for Research on Cancer
(IARC), a subordinate organization of the World Health Organization (WHO) [40]. The
IARC concluded that H. pylori has certainly a role in the pathogenesis of gastric
adenocarcinoma based mainly on epidemiological evidence. The more recent WHO
contribution by the IARC published in 2010 confirmed that chronic infection with H. pylori is
carcinogenic to humans. This confirmation is based on multiple lines of evidence pointing to
a central role for the chronic gastric inflammatory response and resulting oxidative stress in
H. pylori-associated gastric carcinogenesis [41].
Two coexisting mechanisms explain the association between H. pylori and gastric cancer:
the promotion of carcinogenesis by H. pylori itself and the establishment of a carcinogenic
environment in the stomach due to long-term infection with H. pylori. Both mechanisms are
responsible for the development of intestinal type gastric cancer, whereas only the first
mechanism may be responsible for the development of diffuse type gastric cancer.
The development of non-cardia intestinal type gastric cancer is a multistep process
triggered by H. pylori infection (Figure 3). This multistep model, which was in large part
developed by Correa and colleagues, is based on a temporal sequence of precancerous
changes that eventually leads to the development of gastric cancer [42]. H. pylori-induced
gastric inflammation represents a common feature of the initiation and progression to
intestinal type gastric cancer. In a subset of patients, the chronic inflammatory process leads
to the loss of gastric glandular tissue (atrophy) and replacement of the normal gastric lineages
with metaplastic cells (intestinal metaplasia). Dysplasia, early gastric cancer and advanced
gastric cancer may follow.
Currently it remains uncertain whether the diffuse type of gastric cancer follows a
histological progression similar to the proposed pathway for the intestinal type of cancer.
First substantial evidence for the key role of H. pylori infection in the development of
gastric cancer was obtained from studies on Mongolian gerbils, where H. pylori infection
over a period of 62 weeks induced intestinal type adenocarcinomas in 37% of the infected
gerbils without the influence of any cocarcinogens [43].
There were also hints for the multifactorial aetiology of gastric cancer, as
supplementation of the animals with nitrosamines increased cancer incidence and lead to a
more rapid carcinogenesis [44, 45].
Numerous studies attempted to assess the attributable risk of H. pylori infection for
gastric carcinogenesis in humans. In 2003, the Helicobacter and Cancer Collaborative Group
combined data from all available case–control studies nested with prospective cohorts to

assess more reliably the relative risk for gastric cancer. In this analysis, 1.228 patients were
included and a clear association of H. pylori infection with non-cardia gastric cancer (OR 3.0;
95% CI 2.3–3.8) was reported. This association was even stronger when blood samples for H.
pylori serology were obtained 10 years or longer before cancer diagnosis (OR 5.9; 95% CI
3.4–10.3) [46].
The loss of H. pylori colonisation in the presence of extensive gastric atrophy best
explains this phenomenon. Indeed, H. pylori-induced extensive gastric atrophy, that is present
in patients with intestinal type gastric cancer, does not allow a further colonization of the
bacterium. As a consequence, patients with gastric cancer have often lower levels of anti-H.
pylori antibodies in serum at the time of disease manifestation. These results were confirmed
by a meta-analysis of 19 studies including 2491 patients with gastric cancer and 3959
controls, where H. pylori infection was associated with a 2-fold increased risk of developing
non-cardia gastric cancer (OR=1.92; 95% CI:1.32–2.78) [47].
decades
GC GC
intestinal type diffuse type
Dysplasia
years
Intestinal metaplasia
gastric atrophy
months
chronic-active gastritis
H. pylori
Non-infected gastric mucosa

Figure 8.3
Figure 3. Schematic image of the Correa-cascade. Multistep model based on a temporal sequence of
precancerous changes that eventually leads to the development of gastric cancer on the basis of
Helicobacter pylori-driven chronic active gastritis. Modified from Bornschein et al. [69].
For a more meticulous assessment of the H. pylori attributable risk for gastric
carcinogenesis, bacterial virulence factors have been taken into consideration for risk
analysis. Several studies have shown that the risk of gastric cancer is influenced by the
presence of cytotoxin-associated antigen (CagA)-positive H. pylori strains. Furthermore,
antibodies against CagA persist longer in the serum and are useful to identify patients with
past H. pylori infection [48]. Ekström et al. reported an increase of the H. pylori-attributable
OR for non-cardia cancer from 2.2 to 21.0 if the CagA status was co-evaluated by
immunoblot analysis [49]. When both H. pylori IgG antibodies and CagA were analysed, the
seroprevalence of H. pylori infection in patients with non-cardia gastric cancer was 97% for
the intestinal type and 96% for the diffuse type, respectively. Further relevance is given by
the time of serum sampling. The associated OR for gastric cancer development in case of H.
pylori infection increases significantly if serum samples are taken within 90 days after tumor

gastrectomy [50]. Finally, H. pylori infection increases the risk of developing gastric cancer
of both intestinal and diffuse type at the same extent [51].
Eradication of H. pylori for Gastric Cancer Prevention
Growing evidence, mostly deriving from studies in high-risk populations in Asia, provide
support of a benefit of H. pylori eradication on gastric cancer development. In a long-term
prospective study by Uemura and colleagues, 1.526 infected and uninfected Japanese patients
with dyspeptic symptoms underwent endoscopy, after which some of the patients received H.
pylori eradication therapy. Although the patients were not randomly assigned to treatment and
the follow-up periods were inconsistent, gastric cancer developed in 36 (2.9%) of the H.
pylori-positive patients who did not receive eradication therapy but in none (0%) of the
patients that were successfully eradicated [52].
In another prospective interventional study from Japan on 1.342 patients with H. pylori-
induced peptic ulcer disease followed up for a median of 3.4 years gastric cancer occurred in
0.8% of the successfully eradicated patients in contrast to 2.3% of patients with eradication
failure [128].
A meta-analysis including data of 6.695 patients from 6 randomized trials has shown that
eradication of H. pylori reduces gastric cancer risk (relative risk 0.65, 95% CI 0.43–0.98)
[53]. Overall, over a follow-up period ranging from 4 to10 years gastric cancer developed in
56 of 3.307 (1.7%) untreated (control) participants compared with 37 of 3.388 (1.1%) treated
participants. All studies but 1 included in this meta-analysis were performed in Asia. At
histopathology baseline most of the subjects presented a diagnosis of gastric atrophy,
intestinal metaplasia or dysplasia. Thus, H. pylori eradication treatment modestly reduces
gastric cancer risk even in subjects with already established pre-neoplastic lesions, although
the risk is not abolished.
In a prospective, randomized, placebo-controlled, population-based primary prevention
study performed in a high-risk population in China, 1.630 healthy individuals infected with
H. pylori were recruited for randomization on either H. pylori eradication or placebo
treatment [54]. Although no benefit overall was observed in participants who received
eradication therapy compared to those who did not, there was a clear reduction in gastric
cancer incidence in the subgroup of participants with no precancerous changes (gastric
atrophy, intestinal metaplasia or dysplasia) at study initiation.
In another randomized intervention trial from China, the odds of developing gastric
cancer were reduced by 39 per cent in patients who received treatment for H. pylori compared
with the placebo group after 14 years of follow-up (absolute risk 3.0% versus 4.6%; odds
ratio 0.61, 95% CI 0.38-0.96; p = 0.03) [55].
In summary, eradication treatment reduces the incidence of gastric cancer, the earlier the
treatment, the higher the benefit. Subjects with pre-neoplastic changes may still have a
modest benefit from eradication treatment, but endoscopic surveillance in these patients
remains mandatory, as H. pylori infection is not necessary for gastric atrophy and intestinal
metaplasia to further progress towards gastric cancer [56].

Gastric Marginal Zone B Cell Lymphoma

of Malt Type (Gastric Malt Lymphoma)
(See Also Chapter 3 and 14)
Definition
Extranodal marginal zone B cell lymphoma of MALT type (MALT lymphoma) results
from the malignant transformation of B cells from the marginal zone of the mucosa-
associated lymphoid tissue (MALT). These lymphomas account for approximately 7–8% of
all non-Hodgkin’s lymphomas, and can arise at any extranodal site [57]. The gastrointestinal
tract is the most common site of the disease and at least one third of MALT lymphomas
present as a primary gastric lymphoma. MALT lymphomas may arise from MALT that
already exist (e.s. Peyer´s patches of the gut) or from MALT that has been acquired in sites of
inflammation in response to infection or autoimmune processes. Healthy stomach does not
contain MALT, but may acquire it in response to chronic H. pylori infection. Malignant
transformation occurs only in a small percentage of patients with acquired gastric MALT.
Epidemiology
The incidence of MALT lymphoma among H. pylori infected subjects ranges from
1:30.000 to 1:80.000. The median age at diagnosis is approximately 60 years, with a wide age
range. The male-to-female ratio is equal.
Clinical Features
The clinical manifestations of gastric MALT lymphoma are similar to those of gastric
adenocarcinoma. MALT lymphoma is often indolent in the early stages. Patients with
advanced disease may present with nonspecific symptoms including epigastric or abdominal
pain or dyspepsia [58]. Nausea, vomiting, signs of gastric bleeding and B symptoms (weight
loss, fever, night sweats) are unusual. Serum levels of LDH and β2-microglobulin are usually
normal [59].
H. pylori Infection and MALT Lymphoma
Malignant transformation of acquired gastric MALT is strongly driven by chronic H.

pylori infection. Several lines of evidence support the key role of H. pylori in the
development of gastric MALT lymphoma. First, infection by H. pylori is detected in 78-90%
of cases of gastric MALT lymphoma examined histologically and in 98% of cases studied by
serology [60, 61]. Second, gastric MALT lymphoma tissue contains T cells that are
specifically reactive to H. pylori. These T cells support the proliferation of neoplastic B cells
in vitro [62]. Third, low grade gastric MALT lymphoma completely regresses within 12
months in 62-75% of the patients after eradication of H. pylori [63, 64]. Thus, according to

current guidelines, eradication of H. pylori should be employed as the sole initial treatment of
localized (i.e. confined to the stomach) H. pylori-positive gastric MALT lymphoma [65, 66].
The presence of the t(11,18) translocation, however, strongly predicts failure to respond to
eradication therapy. These patients need adjunctive and alternative treatments [67]. After H.
pylori treatment all patients deserve a close follow up and eventually alternative treatments
(chemotherapy or radiotherapy) if the lymphoma fails to respond or progresses [68].
Conclusion
H. pylori eradication cures peptic ulcer disease and prevents its relapse, induces complete
regression in the majority of patients with low-grade gastric MALT lymphoma and improves
symptoms in patients with functional dyspepsia. Moreover, H. pylori eradication has proven
to be effective in the primary prevention of gastric cancer and to be even more effective if
provided before the development of atrophic changes of the gastric mucosa. A vaccine is
likely to represent the best strategy to protect from H. pylori-related diseases. From the socio-
economic point of view, the use of a prophylactic vaccine would be cost-effective [70]. An
effective vaccine against the bacterium would be invaluable, especially considering
decreasing eradication rates using antibiotic regimens [71]. Studies in animal models as well
as data from first trials on healthy human volunteers revealed promising results that deserve a
committed follow-up [72, 73].
References
[1] Tummala S, Keates S, Kelly CP (2004). Update on the immunologic basis of
Helicobacter pylori gastritis. Curr. Opin. Gastroenterol., 20, 592-7.
[2] Amieva MR, El Omar EM (2008). Host-Bacterial Interactions in Helicobacter pylori
Infection. Gastroenterology, 134, 306–323.
[3] Graham DY, Sung JJY (2009). In: Feldman M, Friedman LS, Brandt LJ. Sleisenger &
Fordtran's Gastrointestinal and Liver Disease. 8th ed. Philadelphia: Saunders Elsevier,
121-142.
[4] El-Omar EM, Penman ID, Ardill JE Chittajallu RS, Howie C, McColl KE (1995).
Helicobacter pylori infection and abnormalities of acid secretion in patients with
duodenal ulcer disease. Gastroenterol, 109, 681–91.
[5] Malfertheiner P, Chan FKL, McColl KEL (2009). Peptic ulcer disease. Lancet, 374,
1449–61.
[6] El-Omar EM, Oien K, El Nujumi A, Gillen D, Wirz A, Dahill S, Williams C, Ardill JE,
McColl KE (1997). Helicobacter pylori infection and chronic gastric acid
hyposecretion. Gastroenterol, 113, 15–24.
[7] Naylor GM, Gotoda T, Dixon M, Shimoda T, Gatta L, Owen R, Tompkins D, Axon A.
(2006). Why does Japan have a high incidence of gastric cancer? Comparison of
gastritis between UK and Japanese patients. Gut, 55, 1545–1552.
[8] Tack J, Talley NJ, Camilleri M, et al. (2006). Functional gastroduodenal disorders.
Gastroenterology, 130, 1466-1479.

[9] El-Serag HB, Talley NJ (2004). Systemic review: the prevalence and clinical course of
functional dyspepsia. Aliment. Pharmacol. Ther., 19, 643-654.
[10] Zagari RM, Law GR, Fuccio L, et al. (2010). Epidemiology of functional dyspepsia and
subgroups in the Italian general population: an endoscopic study. Gastroenterology,
138, 1302-1311.
[11] Koloski NA, Talley NJ, Huskic SS, Boyce PM (2003). Predictors of conventional and
alternative health care seeking for irritable bowel syndrome and functional dyspepsia.
Aliment Pharmacol. Ther., 17, 841-851.
[12] Agréus L, Svärdsudd K, Nyrén O, Tibblin G (1995). Irritable bowel syndrome and
dyspepsia in the general population: overlap and lack of stability over time.
[13] McQuaid KR (2009). Dyspepsia. In: Feldman M, Friedman LS, Brandt LJ. Sleisenger
& Fordtran's Gastrointestinal and Liver Disease. 8th ed. Philadelphia: Saunders
Elsevier, 121-142.
[14] Venerito M, Kandulski A, Malfertheiner P (2010). Functional (Nonulcer) Dyspepsia. In
Marko Duvnjak, “Dyspepsia in Clinical Practice” Springer ISBN, 1441917292.
[15] Porter CK, Gormley R, Tribble DR, Cash BD, Riddle MS (2011). The Incidence and
gastrointestinal infectious risk of functional gastrointestinal disorders in a healthy US
adult population. Am. J. Gastroenterol., 106, 130-8.
[16] Mori M, Suzuki H, Masaoka T, Imaeda H, Nomoto Y, Hosoda H, Nishizawa T,
Kangawa K, Hibi T (2006). Intravenous ghrelin administration enhances gastric acid
secretion—evaluation using wireless pH capsule. Aliment. Pharmacol. Ther., 24, 96–
103.
[17] Moayyedi P (2011). Helicobacter pylori eradication for functional dyspepsia: what are
we treating?: comment on “Helicobacter pylori eradication in functional dyspepsia”.
Arch. Intern. Med., 171, 1936–1937.
[18] Spechler SJ (2002). Peptic ulcer disease and its complications. In: Feldman M,
Friedman LS, Sleisenger MH, eds. Sleisenger & Fordtran’s Gastrointestinal and Liver
Disease, 7th edn. Philadelphia, PA: W.B. Saunders Company.
[19] Huang, J.Q.; Sridhar, S.; Hunt, R.H (2002). Role of Helicobacter pylori infection and
non-steroidal anti-inflammatory drugs in peptic-ulcer disease, a meta-analysis. Lancet,
359, 14-22.
[20] Huang, J.Q.; Sridhar, S.; Hunt, R.H (2002). Role of Helicobacter pylori infection and
non-steroidal anti-inflammatory drugs in peptic-ulcer disease, a meta-analysis. Lancet,
359, 14-22.
[21] Susser M, Stein Z (1962). Civilisation and peptic ulcer. Lancet, 279: 116–19.
[22] Sonnenberg A (2006). Causes underlying the birth-cohort phenomenon of peptic ulcer:
analysis of mortality data 1911–2000, England and Wales. Int. J. Epidemiol., 35, 1090–
97.
[23] Blatchford O, Davidson LA, Murray WR, Blatchford M, Pell J (1997). Acute upper
gastrointestinal haemorrhage in west of Scotland: case ascertainment study. BMJ, 315,
510–14.
[24] Rockall TA, Logan RF, Devlin HB, Northfield TC (1995). Incidence of and mortality
from acute upper gastrointestinal haemorrhage in the United Kingdom. Steering
Committee and members of the National Audit of Acute Upper Gastrointestinal
Haemorrhage. BMJ, 311, 222–26.

[25] Longstreth GF (1995). Epidemiology of hospitalization for acute upper gastrointestinal

hemorrhage: a population-based study. Am. J. Gastroenterol., 90, 206–10.
[26] Kumar D, Graham DY (1992). The stomach. Edinburgh, London, Madrid, Melbourne,
New York, Tokyo: Churchill Livingstone, 266–77.
[27] Malfertheiner P, Dent J, Zeijlon L, et al. (2002). Impact of Helicobacter pylori
eradication on heartburn in patients with gastric or duodenal ulcer disease—results from
a randomized trial programme. Aliment Pharmacol. Ther., 16, 1431–42.
[28] Dew MJ (1987). Asymptomatic peptic ulcer disease. Br Med J (Clin Res Ed). 15, 401.
[29] McColl KEL (2000). Should non-invasive Helicobacter pylori testing replace
endoscopy in investigation of dyspepsia? Helicobacter, 5, S11–15.
[30] McColl K E L, Murray L S, Gillen D, et al. (2002). Randomised trial of endoscopy with
testing for Helicobacter pylori compared with noninvasive H. pylori testing alone in the
management for dyspepsia. BMJ, 324, 999–1002.
[31] Warren JR, Marshall B (1983). Unidentified curved bacilli on gastric epithelium in
active chronic gastritis. Lancet, 321, 1273–75.
[32] Marshall BJ, Warren JR (1984). Unidentified curved bacilli in the stomach of patients
with gastritis and peptic ulceration. Lancet, 323, 1311–15.
[33] NIH Consensus Conference (1994). Helicobacter pylori in peptic ulcer disease. NIH
Consensus Development Panel on Helicobacter pylori in peptic ulcer disease. JAMA,
272, 65–69.
[34] Malfertheiner P, Megraud F, O’Morain C, et al. (2007). Current concepts in the
management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut.
56, 772–81.
[35] Lauren P (1965). The two histological main types of gastric carcinoma: diffuse and so-
called intestinal-type carcinoma. An attempt at a histoclinical classification. Acta.
Pathol. Microbiol. Scand., 64, 31-49.
[36] http://globocan.iarc.fr/factsheets/cancers/stomach.asp, 10/03/2013
[37] Houghton J and Wang TC (2009). Tumors of the stomach. In: Feldman M, Friedman
LS, Sleisenger MH, eds. Sleisenger & Fordtran’s Gastrointestinal and Liver Disease,
8th edn. Philadelphia, PA: W.B. Saunders Company.
[38] Parkin DM, Whelan SL, Ferlay J (1997). Cancer Incidence in Five Continents, vol VII.
Lyon, France: International Agency for Research on Cancer, 822-823.
[39] Coleman MP, Gatta G, Verdecchia A, Esteve J, Sant M, Storm H, Allemani C,
Ciccolallo L, Santaquilani M, Berrino F (2003) EUROCARE-3 summary: cancer
survival in Europe at the end of the 20th century. Ann. Oncol., 14, 128–149.
[40] Schistosomes, liver flukes and Helicobacter pylori (1994). IARC Working Group on the
Evaluation of Carcinogenic Risks to Humans. IARC Monogr Eval Carcinog Risks
Hum., 61, 1–241.
[41] http://monographs.iarc.fr/ENG/Monographs/vol100B/mono100B-15.pdf.
[42] Correa P, Haenszel W, Cuello C, Tannenbaum S, Archer M (1975). A model for gastric
cancer epidemiology. Lancet, 2, 58-60.
[43] Watanabe T, Tada M, Nagai H, Sasaki S, Nakao M (1998) Helicobacter pylori infection
induces gastric cancer in Mongolian gerbils. Gastroenterology, 115, 642–648.
[44] Shimizu N, Inada K, Nakanishi H, Tsukamoto T, Ikehara Y, Kaminishi M, Kuramoto S,
Sugiyama A, Katsuyama T, Tatematsu M (1999) Helicobacter pylori infection enhances

glandular stomach carcinogenesis in Mongolian gerbils treated with chemical

carcinogens. Carcinogenesis, 20, 669–676.
[45] Maruta F, Sugiyama A, Ishida K, Ikeno T, Murakami M, Kawasaki S, Ota H,
Tatematsu M, Katsuyama T (2000) Timing of N-methyl-N-nitrosourea administration
affects gastric carcinogenesis in Mongolian gerbils infected with Helicobacter pylori.
Cancer Lett., 160, 99–105.
[46] Helicobacter and Cancer Collaborative Group (2001) Gastric cancer and Helicobacter
pylori: a combined analysis of 12 case control studies nested within prospective
cohorts. Gut, 49, 347– 353.
[47] Huang JQ, Sridhar S, Chen Y, Hunt RH (1998) Meta-analysis of the relationship
between Helicobacter pylori seropositivity and gastric cancer. Gastroenterology, 114,
1169–1179.
[48] Sörberg M, Engstrand L, Ström M, Jönsson KA, Jörbeck H, Granström M (1997). The
diagnostic value of enzyme immunoassay and immunoblot in monitoring eradication of
Helicobacter pylori. Scand. J. Infect. Dis., 29, 147-51.
[49] Ekström AM, Held M, Hansson LE, Engstrand L, Nyrén O (2001). Helicobacter pylori
in gastric cancer established by CagA immunoblot as a marker of past infection.
[50] Brenner H, Arndt V, Stegmaier C, Ziegler H, Rothenbacher D (2004). Is Helicobacter
pylori infection a necessary condition for noncardia gastric cancer? Am. J. Epidemiol.,
159, 252-8.
[51] Bornschein J, Selgrad M, Warnecke M, Kuester D, Wex T, Malfertheiner P (2010). H.
pylori infection is a key risk factor for proximal gastric cancer. Dig. Dis. Sci., 55, 3124-
31.
[52] Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M,
Taniyama K, Sasaki N, Schlemper RJ (2001). Helicobacter pylori infection and the
development of gastric cancer. N. Engl. J. Med., 345, 784-9.
[53] Fuccio L, Zagari RM, Eusebi LH, Laterza L, Cennamo V, Ceroni L, Grilli D, Bazzoli F
(2009). Meta-analysis: can Helicobacter pylori eradication treatment reduce the risk for
gastric cancer? Ann. Intern. Med., 151, 121-8.
[54] Wong BC, Lam SK, Wong WM, Chen JS, Zheng TT, Feng RE, Lai KC, Hu WH, Yuen
ST, Leung SY, Fong DY, Ho J, Ching CK, Chen JS; China Gastric Cancer Study Group
(2004). Helicobacter pylori eradication to prevent gastric cancer in a high-risk region of
China: a randomized controlled trial. JAMA, 291, 187-94.
[55] Ma JL, Zhang L, Brown LM, Li JY, Shen L, Pan KF et al. (2012). Fifteen-year effects
of Helicobacter pylori, garlic, and vitamin treatments on gastric cancer incidence and
mortality. J. Natl. Cancer Inst., 104, 488–492.
[56] Dinis-Ribeiro M, Areia M, de Vries AC, Marcos-Pinto R, Monteiro-Soares M,
O'Connor A, Pereira C, Pimentel-Nunes P, Correia R, Ensari A, Dumonceau JM,
Machado JC, Macedo G, Malfertheiner P, Matysiak-Budnik T, Megraud F, Miki K,
O'Morain C, Peek RM, Ponchon T, Ristimaki A, Rembacken B, Carneiro F, Kuipers
EJ; MAPS Participants; European Society of Gastrointestinal Endoscopy; European
Helicobacter Study Group; European Society of Pathology; Sociedade Portuguesa de
Endoscopia Digestiva (2012). Management of precancerous conditions and lesions in
the stomach (MAPS): guideline from the European Society of Gastrointestinal
Endoscopy (ESGE), European Helicobacter Study Group (EHSG), European Society of

Pathology (ESP), and the Sociedade Portuguesa de Endoscopia Digestiva (SPED).

Virchows Arch., 460, 19-46.
[57] Fischbach W, Kestel W, Kirchner T, Mossner J, Wilms K (1992). Malignant
lymphomas of the upper gastrointestinal tract. Results of a prospective study in 103
patients. Cancer,70, 1075–80.
[58] Pinotti G, Zucca E, Roggero E, Pascarella A, Bertoni F, Savio A, Savio E, Capella C,
Pedrinis E, Saletti P, Morandi E, Santandrea G, Cavalli F (1997). Clinical features,
treatment and outcome in a series of 93 patients with low-grade gastric MALT
lymphoma. Leuk. Lymphoma., 26, 527-37.
[59] Collins RH Jr (2009). In: Feldman M, Friedman LS, Brandt LJ. Sleisenger & Fordtran's
Gastrointestinal and Liver Disease. 8th ed. Philadelphia: Saunders Elsevier, 121-142.
[60] Zucca E, Bertoni F, Roggero E, Bosshard G, Cazzaniga G, Pedrinis E, Biondi A,
Cavalli F (1998). Molecular analysis of the progression from Helicobacter pylori-
associated chronic gastritis to mucosa-associated lymphoid-tissue lymphoma of the
stomach. N. Engl. J. Med., 338, 804-10.
[61] Eck M, Greiner A, Schmausser B, Eck H, Kolve M, Fischbach W, Strecker P, Müller-
Hermelink HK (1999). Evaluation of Helicobacter pylori in gastric MALT-type
lymphoma: differences between histologic and serologic diagnosis. Mod. Pathol., 12,
1148-51.
[62] Hussell T, Isaacson PG, Crabtree JE, Spencer J (1993). The response of cells from low-
grade B-cell gastric lymphomas of mucosa-associated lymphoid tissue to Helicobacter
pylori. Lancet, 342, 571-4.
[63] Fischbach W, Goebeler-Kolve ME, Dragosics B, Greiner A, Stolte M (2004). Long
term outcome of patients with gastric marginal zone B cell lymphoma of mucosa
associated lymphoid tissue (MALT) following exclusive Helicobacter pylori
eradication therapy: experience from a large prospective series. Gut, 53, 34–7.
[64] Zullo A, Hassan C, Cristofari F, Andriani A, De Francesco V, Ierardi E, Tomao S,
Stolte M, Morini S, Vaira D (2010). Effects of Helicobacter pylori eradication on early
stage gastric mucosa-associated lymphoid tissue lymphoma. Clin. Gastroenterol.
Hepatol., 8, 105-10.
[65] Zucca E, Dreyling M; ESMO Guidelines Working Group (2009). Gastric marginal zone
lymphoma of MALT type: ESMO clinical recommendations for diagnosis, treatment
and follow-up. Ann. Oncol., Suppl 4, 113-4.
[66] Malfertheiner P, Megraud F, O'Morain CA, Atherton J, Axon AT, Bazzoli F, Gensini
GF, Gisbert JP, Graham DY, Rokkas T, El-Omar EM, Kuipers EJ; European
Helicobacter Study Group (2012). Management of Helicobacter pylori infection--the
Maastricht IV/ Florence Consensus Report. Gut, 61, 646-64.
[67] Wundisch T, Thiede C, Morgner A, et al. (2005). Long-term follow-up of gastric
MALTlymphoma after Helicobacter pylori eradication. J. Clin. Oncol., 23, 8018–24.
[68] Ruskone-Fourmestraux A, Fischbach W, Aleman BM, et al. (2011). EGILS consensus
report. Gastric extranodal marginal zone B-cell lymphoma of MALT. Gut, 60, 747–58.
[69] Bornschein J, Malfertheiner P (2011). Gastric carcinogenesis. Langenbecks Arch. Surg.,
396, 729-42.
[70] Rupnow MF, Chang AH, Shachter RD, Owens DK, Parsonnet J (2009) Cost-
effectiveness of a potential prophylactic Helicobacter pylori vaccine in the United
States. J. Infect. Dis., 200, 1311–1317.

[71] Del Guidice G, Malfertheiner P, Rappuoli R (2009) Development of vaccines against

Helicobacter pylori. Expert. Rev. Vaccin., 8, 1037–1049.
[72] Malfertheiner P, Schultze V, Rosenkranz B, Kaufmann SH, Ulrichs T, Novicki D,
Norelli F, Contorni M, Peppoloni S, Berti D, Tornese D, Ganju J, Palla E, Rappuoli R,
Scharschmidt BF, Del Guidice G (2008). Safety and immunogenicity of an
intramuscular Helicobacter pylori vaccine in noninfected volunteers: a phase I study.
Gastroenterology, 135, 787–795.
[73] Czinn SJ, Blanchard T (2011). Vaccinating against Helicobacter pylori infection. Nat.
Rev. Gastroenterol. Hepatol., 8, 133-40.

Helicobacter pylori Infection and
Associated Extra-Gastric Diseases

Chapter 9
Esophageal and Colon Diseases
Sergei F. Tatishchev and Hanlin L. Wang

Department of Pathology and Laboratory Medicine
David Geffen School of Medicine at University of California
Los Angeles, California, US
Abstract
While the proinflammatory and carcinogenic properties of Helicobacter pylori (H.
pylori) are well-established in the stomach, the effects of H. pylori infection on other
parts of the gastrointestinal tract are much less well-investigated. In this chapter, the
relationship between H. pylori infection and selected esophageal and colorectal diseases
is discussed. Studies have shown that H. pylori infection is inversely associated with the
occurrence of eosinophilic esophagitis, Barrett esophagus, esophageal adenocarcinoma
and inflammatory bowel disease.
In contrast, H. pylori infection, particularly by more virulent strains, appears to be
associated with an increased risk for the development of colorectal adenocarcinoma. It
remains to be investigated whether there is indeed a causal association between H. pylori
infection and these extragastric diseases. Currently available evidence suggests that the
modulating effects of H. pylori infection on these diseases may involve multiple
mechanisms such as hypergastrinemia, enhanced cytokine production, immunomodu-
lation and changes in intestinal microflora. It appears unlikely that H. pylori organisms
directly contribute to the pathogenesis of these extragastric diseases by physical
colonization as they do in the stomach.
Keywords: H. pylori, eosinophilic esophagitis, Barrett esophagus, esophageal

adenocarcinoma, ulcerative colitis, Crohn disease, colorectal cancer

Corresponding Author: Sergei F. Tatishchev, MD, UCLA Department of Pathology & Laboratory Medicine,
10833 Le Conte Avenue, Los Angeles, CA 90095, USA. Email: STatishchev@mednet.ucla.edu.

166 Sergei F. Tatishchev and Hanlin L. Wang
Introduction
In the last several decades, certain benign and malignant gastrointestinal diseases have
seen noticeable changes in epidemiology. For instance, the incidence of eosinophilic
esophagitis and inflammatory bowel diseases is on the rise, especially in developed countries.
The same is true for Barrett’s esophagus, esophageal adenocarcinoma and colorectal
adenocarcinoma. Helicobacter pylori (H. pylori) microorganisms, particularly the more
virulent strains, are slowly disappearing from the human population after almost twenty years
of aggressive antibiotic treatment. A number of studies have suggested that declining H.
pylori infection may play a role in the changing epidemiology of some digestive disorders.
This chapter examines current literature and focuses on the effects of H. pylori infection on
selected esophageal and colorectal diseases.
H. pylori Infection and Eosinophilic Esophagitis
Eosinophilic esophagitis (EE) is an allergic inflammatory condition of the esophagus

characterized by infiltration of the esophageal squamous epithelium by eosinophils. It has
been recognized as one of the major causes of dysphagia and has shown an increasing
incidence over the last several decades [1, 2]. The etiopathogenesis of EE remains largely
unknown, but is believed to be driven by an allergic-type T-cell-mediated immune process [3,
4]. Environmental factors are thought to play a role in the pathogenesis of EE.
Current scientific evidence points to an inverse association between H. pylori infection
and various allergic conditions, such as bronchial asthma, allergic rhinitis and atopic
dermatitis [5, 6]. In view of this inverse association, it has been suggested that H. pylori
organisms may be responsible for decreasing esophageal eosinophilia. To date, the largest and
most comprehensive study to evaluate the relationship between H. pylori infection and EE
was conducted by Dellon et al. [7]. A cross-sectional analysis of 165,017 US patients who
underwent both esophageal and gastric biopsies from 2008 through 2010 showed strong
evidence that H. pylori infection is inversely associated with esophageal eosinophilia (odds
ratio = 0.77; 95% confidence interval, 0.69 – 0.87). The authors indicated that increasing
clinical suspicion for EE was associated with decreasing odds of H. pylori infection. An
earlier and much smaller population-based study from Sweden also reported a negative
association between H. pylori infection and esophageal intraepithelial eosinophils (odds ratio
= 0.41; 95% confidence interval 0.19 – 0.92) [8].
A French group conducted analysis of biopsy samples from a pediatric population that
underwent esophagogastroduodenoscopy. In contrast to the observations reported in the
above mentioned studies, Kalach et al. found that children with H. pylori infection had higher
eosinophil counts in the esophagus than H. pylori-negative children [9]. This discrepant
observation might be explained by a relatively small sample size of the study (only 7 children
had met both clinical and histologic criteria for the diagnosis of EE) and differences between
adults with competent immune systems and pediatric patients with immature immune systems
(median age of children in this study was 9 months). Interestingly, children with H. pylori
infection also had higher eosinophil counts in their gastric antrum when compared to children
without H. pylori. This finding may further suggest a unique eosinophilic response to H.

Esophageal and Colon Diseases 167
pylori infection and a unique distribution of eosinophils within the gastrointestinal tract in the
pediatric population.
The inverse association between H. pylori infection and esophageal eosinophilia
observed in the adult population might be explained by the fact that H. pylori organisms
preferentially elicit T-helper 1 (Th1) mucosal immune response, which in turn drives
inhibition of the T-helper 2 (Th2) allergic response [6]. In the late 1980s, it was suggested
that allergic conditions could be prevented by a variety of infections in early childhood [10].
Around the same time, the paradigm of Th1/Th2 interplay within the adaptive immune system
began to emerge [11, 12]. Proponents of the Th1/Th2 hypothesis advocate that the differential
expression of proinflammatory cytokines by T-helper cells determines the Th1/Th2 balance.
Colonization with H. pylori organisms and exposure to H. pylori neutrophil-activating protein
(HP-NAP) is associated with the production of IL-12, IL-23, TNF-α and IFN-γ cytokines that
shift the balance towards Th1 immune response [13-15]. Thus, it is not unreasonable to
suggest that infection with H. pylori organisms early in life drives the differentiation of
antigen-stimulated T-cells towards a polarized Th1 phenotype and provides some degree of
protection against an allergic condition such as EE. D’Elios et al. demonstrated that not all H.
pylori strains are equal in terms of eliciting a polarized Th1 response [13]. There is evidence
that the type of T-helper cell response to H. pylori infection varies according to the H. pylori
antigen involved. The findings by these authors suggest that the CagA-positive H. pylori
strains are more likely to induce Th1 immune response, whereas CagA-negative strains
generate T-cell clones that produce both Th1 and Th2 cytokines. Furthermore, T cell
responses to certain soluble H. pylori antigens, such as VacA and urease, are also
characterized by the release of both Th1 and Th2 cytokines.
It should be mentioned that to date all studies that have looked into the relationship
between H. pylori and EE have only been able to establish associations and were unable to
provide definitive conclusions about causality or mechanisms. Uncovering the
etiopathogenesis of EE and better understanding of the effect of different H. pylori strains on
Th1/Th2 balance will likely provide additional clues to the nature of the observed inverse
association between H. pylori infection and EE in the adult population.
H. pylori and Barrett Esophagus / Esophageal Adenocarcinoma
Barrett esophagus (BE) is a well-recognized complication of chronic gastroesophageal

reflux disease (GERD). It is characterized by replacement of the squamous cell lining of the
distal esophagus by specialized columnar epithelium containing goblet cells. It is widely
accepted as the precursor lesion for the development of dysplasia and subsequent
adenocarcinoma of the esophagus. In fact, it is the only clearly recognized risk factor for
esophageal adenocarcinoma [16, 17]. Although there is some uncertainty about the absolute
risk of progression of BE to esophageal adenocarcinoma, a recent study out of Denmark
reports a much lower annual risk rate (0.12%; 95% CI, 0.09 – 0.15) than previously estimated
[18]. The reasons why only a relatively small number of patients with BE progress to
esophageal adenocarcinoma are not entirely clear. Given that the incidence of esophageal
adenocarcinoma continues to rise, in particular in developed countries, numerous
epidemiologic studies have examined other potential risk factors, as well as protective factors.

Multiple meta-analysis studies have shown that H. pylori reduces the risk of BE and/or
adenocarcinoma of the esophagus [19-23]. The most recent meta-analysis examining the
connection between H. pylori infection and BE has confirmed the inverse relationship
between these two conditions [19]. In this study, Fischbach et al. analyzed 49 studies from
different regions around the world that had examined the effect of H. pylori infection on BE.
The authors identified selection and/or information biases as the major sources of conflicting
observations. Only 4 of the 49 studies had no obvious biases and all 4 studies consistently
revealed a strong protective effect of H. pylori infection on BE with the relative risk of 0.46
(95% CI, 0.35 – 0.65) [24-27]. A subgroup of 7 studies included in this meta-analysis showed
a pooled estimated relative risk of 0.38 (95% CI, 0.19 – 0.78) for BE in patients infected with
CagA-positive strains of H. pylori [24, 25, 28-32]. Interestingly, one of these 7 studies
identified CagA-positive H. pylori as a risk factor for BE with a relative risk of 1.5 (95% CI,
0.93 – 2.46) [32]. However, this particular case-control study had a strong selection bias by
choosing young healthy blood donors as the control group instead of using the base
population from which the cases arose. In fact, one of the earlier meta-analyses performed by
Wang et al. concluded that the major impact on the prevalence of H. pylori infection in BE
patients was the selection of the control group [20].
Whenever healthy blood donors were chosen as normal controls, H. pylori infection was
either more prevalent in patients with BE or independent of BE [32-34]. Ethnic and
geographic differences may also contribute to the heterogeneity among studies. Several
observational studies identified noticeable differences in the incidence of BE among various
racial groups suggesting that H. pylori infection may have unequal effect on BE in patients
with different ethnic backgrounds [35, 36]. Finally, all meta-analyses recognized that the
observed relationship between H. pylori infection and BE depends on the definition of BE,
which was inconsistent among various studies.
To date, there are 3 meta-analysis studies that have examined the relationship between H.
pylori infection and esophageal adenocarcinoma [21-23]. All 3 studies have demonstrated an
inverse association between these two conditions with odds ratios ranging from 0.50 to 0.58.
Compared with CagA-negative H. pylori stains, CagA-positive H. pylori stains are more
significantly associated with markedly decreased esophageal cancer risk. In the study by
Islami and Kamangar [23], the inverse association was not observed with CagA-negative
strains. It has been speculated that the increasing incidence of esophageal adenocarcinoma
might be partially attributed to the declining rate of H. pylori infection in humans. In fact,
some regions of the world with still relatively high rates of H. pylori infection have not seen
an increase in the incidence of esophageal adenocarcinoma [37, 38]. Interestingly, no
statistically significant relationship between H. pylori infection and squamous cell carcinoma
of the esophagus was demonstrated [21-23].
The exact mechanisms for the observed inverse association between H. pylori infection
and BE and esophageal adenocarcinoma are unclear. It has been suggested that the decreased
gastric acid output associated with atrophic changes in the stomach in the setting of H. pylori
gastritis lowers exposure of the esophageal mucosa to the harmful effects of acidic gastric
juices. If this hypothesis holds true, declining rates of H. pylori infection in the developed
nations, particularly with CagA-positive H. pylori strains [39], may change the epidemiology
of BE and esophageal adenocarcinoma.

H. pylori Infection and Inflammatory Bowel Disease
Idiopathic inflammatory bowel disease (IBD) encompasses two nosological entities:

ulcerative colitis (UC) and Crohn disease (CD). Both conditions share a number of clinical
and histopathologic similarities, but each disease has its own unique features. The etiology
and pathogenesis of these conditions remain largely unknown. Since the 1950s, the incidence
of IBD has risen dramatically, particularly in developed countries. In the last two decades, the
incidence of both UC and CD has also increased in the rest of the world, especially the Asia-
Pacific countries, Eastern Europe and some parts of South America [40-42]. To uncover
potential mechanisms of UC and CD development, a significant number of epidemiologic
studies have been devoted to exploring associations between these conditions and a variety of
environmental factors.
In the last 20 years, numerous observational studies indicated that patients with IBD have
a low prevalence of H. pylori infection. In 2010, Luther et al conducted a meta-analysis with
a systematic review of the literature to estimate the relative risk of H. pylori infection in
patients with and without IBD [43]. These authors used relative risk to describe the ratio of
the probability of H. pylori infection occurring in patients with IBD over the control group.
Following a detailed analysis of relevant articles retrieved from MEDLINE, EMBASE and
other databases from inception to March 2009, the authors selected 23 studies with 5903
patients. Inclusion criteria for evidence of H. pylori infection in selected articles comprised of
any of the following: serologic (IgG antibody) diagnosis, urea breath test, rapid urease test,
fecal antigen test and/or histological diagnosis. The analysis showed evidence of H. pylori
infection in 27% of patients with IBD and in 41% of the control population. The relative risk
of H. pylori infection was 0.60 (95% confidence interval: 0.49 – 0.72) in CD patients and
0.75 (95% confidence interval: 0.62 – 0.90) in UC patients. Overall, the relative risk of H.
pylori infection in patients with IBD compared to controls was 0.64 (95% confidence interval:
0.54 – 0.75).
The studies used in this meta-analysis have certain limitations. First, the use of IgG
antibodies as evidence of H. pylori infection may show a higher than expected rate of false
positivity due to the high sensitivity and low specificity of serologic testing. Identification of
anti-H. pylori antibodies merely indicates that the individual had an exposure to H. pylori
organisms at some time in his/her life and does not necessarily indicate current disease.
Although IgG titers drop with time [44-46], the anti-H. pylori antibodies usually persist for
years and can be detected even after a patient is successfully treated [47]. Thus, it is difficult
to establish the temporal relationship with the development of IBD in these patients. Second,
in several studies included in the meta-analysis, the history of prior eradication treatment for
H. pylori infection or antibiotic use was not known for many patients. As a result, H. pylori
infection rate in IBD patients might have been misleadingly low. Finally, the criteria for IBD
diagnosis were not clearly defined in some studies. In others, the diagnosis of H. pylori
infection was not confirmed by histologic examination. Despite these flaws, the results of this
meta-analysis suggest a protective benefit of H. pylori infection against the occurrence of CD
and UC.
To date, the most comprehensive study on the relationship between H. pylori infection
and IBD was conducted by Sonnenberg and Genta [48]. To overcome deficiencies of previous
studies, the authors analyzed a large US-based patient population who underwent concurrent
upper gastrointestinal endoscopy and colonoscopy with biopsies. The diagnoses of H. pylori

infection and IBD were confirmed histologically. Of 65,515 subjects included in the study,
1064 patients were diagnosed with IBD (560 patients with UC, 371 patients with CD, and 133
with indeterminate colitis). The remaining 64,451 patients served as controls without IBD.
The statistical analyses showed strong evidence that H. pylori infection was inversely
associated with IBD. The adjusted odds ratios were 0.59 (95% confidence interval, 0.39 –
0.84) and 0.48 (95% confidence interval, 0.27 – 0.79) for UC and CD, respectively. Overall,
the authors concluded that among patients with H. pylori infection the prevalence of IBD was
roughly reduced by half.
Thus far, all the studies looking into the relationship between H. pylori infection and IBD
were only able to draw conclusions about associations, leaving causality and mechanisms
subject to open interpretation. In this regard, a research group from Germany showed that H.
pylori organisms induce a strong FoxP3+ T-cell response [49]. In healthy individuals,
regulatory FoxP3+ T-cells control intestinal mucosa homeostasis and suppress pathogenic T-
cell responses via IL-10 and TGF-β action [50, 51]. It has been proposed that failure of
regulatory immune networks to control excessive T-cell responses breaks homeostatic
mucosal T-cell equilibrium and leads to chronic inflammation [52]. For instance, IL-10
knock-out mice invariably develop intestinal inflammation and exogenous IL-10
administration prevents or at least ameliorates the development of colitis in certain IBD
models [53]. Higgins et al demonstrated that H. pylori infection in the stomach induces IL-10
expression in the mesenteric lymph nodes, suppresses the Th17 cytokine response to
Salmonella typhimurium in the cecum, and reduces S. typhimurium-induced colitis in a
mouse model [54]. Experimentally induced colitis in immunodeficient IBD mice has also
been shown to be abrogated by a transfer of a subset of regulatory CD4+ T-cells (Tregs) [55],
proving further support for the importance of Tregs (such as CD4+/FoxP3+ T-cell) in
regulating intestinal inflammation. These studies thus suggest that gastric H. pylori infection
may incite extragastric immunomodulatory effects, which may help explain the observed
negative association between H. pylori and IBD.
A number of studies have reported that the lower prevalence of H. pylori infection in IBD
patients could be the result of concurrent or previous use of sulfasalazine [56-58], an
antinflammatory drug that is commonly used to treat IBD. Whether or not sulfasalazine
contributes to the observed inverse association is unclear because the available data are
conflicting and controversial [59-61].
Disruption of the normal intestinal microflora has been proposed by many as a likely
culprit in the development of IBD. Whether or not H. pylori infection has any effect on
eubiotic intestinal microflora is unclear. Decreased gastric acidity secondary to long-standing
H. pylori infection and the disruption of the physiologic barrier function of the stomach likely
permit passage and colonization of the intestinal tract by microorganisms that would
otherwise be digested by gastric juices. How this fits in with the inverse relationship between
H. pylori infection and IBD is not understood.
Recently, it has been demonstrated that H. pylori DNA is capable of down-regulating
proinflammatory responses from dendritic cells and ameliorating dextran sodium sulphate
(DSS)-induced colitis [62]. These additional findings provide another potential explanation
for the inverse association between H. pylori infection and IBD.

H. pylori Infection and Colorectal Cancer
Colorectal cancer (CRC) is a heterogeneous disease involving diverse etiopathogenetic

mechanisms. The majority of CRCs are sporadic, with a variety of epidemiologic
associations. Ever since the oncogenic properties of H. pylori were firmly established in the
stomach, researches have been attempted to expand its oncogenicity to other parts of the
gastrointestinal tract, the colorectum in particular [63, 64]. The connection between H. pylori
infection and CRC to this day remains controversial, with some reports showing a strong
association [64-69] while others none [70-74]. The same inconsistency is true in the
association between H. pylori infection and colonic adenoma [75, 76]. The results of two
meta-analyses published in 2006 and 2008 both suggested a possible small increased risk of
CRC in association with H. pylori infection [77, 78].
Several hypotheses have attempted to explain the possible link between H. pylori
infection and CRC. At least four mechanisms by which H. pylori infection might affect CRC
development have been suggested: hypergastrinemia, mutagenic toxin production, change in
colorectal microflora, and H. pylori-mediated chronic inflammation in the colon by direct
colonization.
Hypergastrinemia
In the 1980s and 1990s, gastrin and gastrin-like peptides have received considerable
attention as having growth-promoting properties on normal and neoplastic colon epithelial
cells. Early in vitro studies demonstrated that gastrins have a direct mitogenic effect on
cultured colon tumor cells [79-82]. Later research using animal models has confirmed that
induced hypergastrinemia resulted in hyperproliferation of colonic mucosa in transgenic mice
[83-85]. In addition, gastrin gene knock-out mice showed decreased proliferation of large
intestinal mucosa [86]. Around the same time, several case-control studies reported elevated
serum gastrin levels in patients with adenomatous polyps and/or colorectal adenocarcinoma
[87-89]. Based on these observations, it has been proposed that hypergastrinemia seen in the
setting of H. pylori-associated atrophic gastritis is the promoter of colorectal carcinogenesis.
From the beginning, the link between hypergastrinemia and CRC has been in question.
Under normal physiologic conditions there are two major circulating forms of gastrins:
amidated and nonamidated. Transgenic mouse experiments have showed that amidated form
of gastrins, which are the major gastrins responsible for stimulating gastric acid secretion,
have no proliferative effect on colonic mucosa [85]. Animal studies on drug-induced
hypergastrinemia in the absence of carcinogens also showed no effect on CRC development
[90, 91]. More importantly, a number of studies have demonstrated that CRC cells express
gastrins that function as autocrine growth factors [92-96]. Therefore, autocrine gastrin
secretion by tumor cells may have been the source of hypergastrinemia observed in some
patients with CRC. In fact, some studies have demonstrated that following surgical resection
of CRC fasting serum gastrin levels drop in those patients [97, 98]. One of the strongest
arguments against the link between hypergastrinemia and CRC comes from patients with
Zollinger-Ellison syndrome. Orbuch et al. demonstrated that prolonged marked plasma
elevation of all forms of gastrin associated with the syndrome does not result in an increased
risk of developing CRC in this group of patients [99]. Furthermore, therapy-induced chronic
hypergastrinemia by proton pump inhibitors, H2-receptor antagonists or gastric vagotomy has
not been shown to increase the risk CRC development [90, 100-104]. Finally, several case-

control studies demonstrated that endogenous gastrin is unlikely to play any significant role in
colorectal carcinogenesis [105-108].
Mutagenic Toxin Production

It is known that patients infected with CagA-positive H. pylori organisms are more likely
to develop gastric cancer than those infected with CagA-negative strains [109]. Shmuely et al
examined serum IgG antibodies against H. pylori and CagA protein in patients with various
malignancies, which demonstrated that among the patients who had serologic evidence of H.
pylori infection, a significantly higher frequency of CagA positivity was observed in patients
with gastric and colon cancers when compared with others. The authors concluded that
infection with CagA-positive H. pylori is associated with an increased risk for CRC when
compared to infection with CagA-negative strains [110]. These observations should be
interpreted with caution, however. As the authors pointed out, testing for H. pylori was
performed at the same time as the cancer diagnosis, which raises questions about the temporal
relationship between these two conditions. The conclusions of the study were made under the
assumption that H. pylori infection occurred before CRC, as for gastric adenocarcinoma. A
seemingly apparent association between a more virulent CagA-positive H. pylori strain and
CRC might simply be the reflection of the fact that patients with CRC have altered immune
status. In the setting of impaired immune response, H. pylori organisms, especially the more
virulent strains, have a greater chance of successfully establishing infection in the host. For
instance, enhanced production of IL-4 has been associated with the progression or persistence
of H. pylori infection [111]. At the same time, it has been shown that IL-4 producing T2-
helper lymphocytes are significantly increased in patients with CRC [112].
If H. pylori infection does in fact precede and contribute to the development of CRC, it is
possible that CagA/VacA-positive strains are more capable of influencing carcinogenesis by
altering chemokines. It has been shown that CagA toxin released from H. pylori shed in the
stool may have a local proliferative effect on colonic mucosa through enhanced production of
such chemokines as IL-8 [110]. The VacA toxin also upregulates expression of IL-8 and other
inflammatory cytokines such as GRO-alpha (growth-related oncogene alpha), MCP-1
(monocyte chemotactic protein 1) and RANTES (regulated on activation, normal, T-cell
expressed and secreted), in immune cells [113].
Dramatic shifts in intestinal microflora could also potentially explain the increased risk
for CRC in patients infected with CagA-positive H. pylori. It has been shown that H. pylori
strains carrying cagA and vacA genes induce more severe gastric injury and subsequent
atrophy compared to less virulent strains [114]. Therefore, patients infected with CagA-
positive and/or VacA-positive H. pylori strains would be expected to sustain an even more
drastic disruption of the gastric acid barrier function. This, in turn, would allow for an
abnormal colonization of the intestinal tract with bacterial species linked to CRC.
Change in Colorectal Microflora

High gastric acidity is important for digestive function and also serves as the first line of
defense against microorganisms consumed with food and water. The gastric acid barrier is an
important regulator of the intestinal microflora composition [115-118]. As discusses earlier,
atrophic gastritis in the setting of H. pylori infection is associated with decreased acid
production, which in turn permits a greater number and variety of microbial species to enter
and subsequently colonize the intestinal tract. It has been proposed that shifts in the
composition and type of colonic microflora may facilitate the growth of bacteria such as B.
fragilis, E. faecalis and others linked to the development of CRC [119-124]. Supporting this
hypothesis are several studies showing an increase in the incidence of CRC following gastric
surgery for benign peptic ulcer disease [125, 126], although other studies failed to confirm the
association [127, 128].
H. pylori-Mediated Chronic Inflammation in the Colon by Direct Colonization

Inspired by the well-established proinflammatory and carcinogenic effects in the
stomach, a similar “H. pylori-induced chronic inflammation → dysplasia → neoplasia”
sequence has been suggested to occur in the colon. In this regard, a research group from
Greece demonstrated the presence of H. pylori organisms in colonic polyps and CRC tissues
by immunohistochemistry [129]. Based on their findings, the authors suggested that
established H. pylori infection in the colonic mucosa might be associated with a selectively
increased risk for left-sided adenoma and CRC.
Using 16S rDNA PCR amplification and pyrosequencing, Grahn et al showed that
Helicobacter DNA sequences are present in almost one third of tissue samples collected from
patients with CRC [130]. The specificity of the PCR primers used in the study, however, puts
the proposed direct association of H. pylori with CRC in question. First, one of the 16S rDNA
variable V3 region sequences matched to Helicobacter mustelae, which has not been found in
humans. Second, an earlier publication by the same group indicated that the primer (HJ-
JBS.V3.SE) used in their studies was found to target the non-primer specific DNA sequences
from Bilophila spp., Haemophilus spp., and uncultured proteobacteria, in the absence of H.
pylori [130, 131]. More importantly, according to the authors, they had no access to control
specimens and thus were unable to establish whether the detection of H. pylori DNA in CRC
tissue samples had any statistical significance.
While the mechanisms of H. pylori-induced gastric cancer are fairly well-described, the
role of H. pylori in colorectal carcinogenesis is not easy to understand. As pointed out by
Soylu et al [132], simply identifying H. pylori in colorectal polyps or CRC tissue samples
does not suffice for the assertion of a definitive association. H. pylori organisms are likely to
be transmitted through a fecal-oral route and are shed in the stool of infected individuals
[133-135]. Thus, it is not a surprise that some H. pylori organisms, which are passively
moving with the digested contents within the intestinal tract, can be detected in colonic tissue
samples. Furthermore, while H. pylori-associated gastric cancer is known to be the
consequence of active and chronic gastritis with resulting intestinal metaplasia and dysplasia,
there are no reports of active or chronic colitis due to H. pylori infection in the colon [136].
In summary, colorectal carcinogenesis is a multivariable process influenced and
facilitated by complex interactions between environmental factors, intestinal microflora,
individual immune status and genetic background. H. pylori infection in the stomach may
indirectly influence CRC development in a variety of ways. Once the gastric acid barrier and
normal digestive functions are impaired, intestinal eubiotic microflora in some individuals
may undergo dramatic changes to promote the growth of species that might have carcinogenic
effects on colonic mucosa. These effects may be more prominent with more virulent H. pylori
strains, which cause more severe mucosal damage and atrophy in the stomach. In addition,
toxins such as CagA and VacA produced by more virulent H. pylori species can enhance the
production of proinflammatory cytokines that may possess proliferative effect on colonic

mucosa. Once a dysplastic/neoplastic process is initiated, gastrins may function as autocrine

growth factors to promote further tumor progression.
Conclusion
While there is a noticeable decrease in the prevalence of H. pylori in some parts of the
world, the incidence of other non-gastric digestive disorders is also changing. Meta-analysis
studies have suggested that H. pylori infection may protect against eosinophilic esophagitis,
Barrett’s esophagus and inflammatory bowel disease. Esophageal adenocarcinoma also shows
a strong inverse association with H. pylori infection, in particular with CagA-positive strains.
The relationship between H. pylori infection and colorectal cancer is controversial with some
reports showing positive association and others none. The exact mechanisms for these
associations are unclear. It has been suggested that H. pylori-induced atrophic gastritis leads
to a decrease in acid production resulting in reduced esophageal injury and changes in
intestinal microflora. H. pylori microorganisms also have immunomodulating effects on the
host T-cell response and production of various cytokines, which may in turn affect the
development of immune-mediated disorders or even carcinogenesis. Further examination of
the link between H. pylori infection and extragastric disorders may provide additional insights
into the physiology, immunology and pathology of the GI tract.
References
[1] Prasad GA, Alexander JA, Schleck CD, Zinsmeister AR, Smyrk TC, Elias RM, Locke
GR 3rd, Talley NJ, (2009). Epidemiology of eosinophilic esophagitis over three
decades in Olmsted County, Minnesota. Clin. Gastroenterol. Hepatol., Oct;7(10):1055-
61.
[2] Ronkainen J, Talley NJ, Aro P, Storskrubb T, Johansson SE, Lind T, Bolling-
Sternevald E, Vieth M, Stolte M, Walker MM, Agréus L, (2007). Prevalence of
oesophageal eosinophils and eosinophilic oesophagitis in adults: the population-based
Kalixanda study. Gut., May;56(5):615-20.
[3] Rothenberg ME, (2009). Biology and treatment of eosinophilic esophagitis.
Gastroenterology., 137:1238–1249.
[4] Mishra A, (2009). Mechanism of eosinophilic esophagitis. Immunol. Allergy Clin.
North Am., Feb;29(1):29-40.
[5] D'Elios MM, Codolo G, Amedei A, Mazzi P, Berton G, Zanotti G, Del Prete G, de
Bernard M, (2009). Helicobacter pylori, asthma and allergy. FEMS Immunol. Med.
Microbiol., Jun;56(1):1-8.
[6] Amedei A, Codolo G, Del Prete G, de Bernard M, D'Elios MM, (2010). The effect of
Helicobacter pylori on asthma and allergy. J. Asthma Allergy., Sep 29;3:139-47.
[7] Dellon ES, Peery AF, Shaheen NJ, Morgan DR, Hurrell JM, Lash RH, Genta RM,
(2011). Inverse association of esophageal eosinophilia with Helicobacter pylori based
on analysis of a US pathology database. Gastroenterology., Nov;141(5):1586-92.

[8] Ronkainen J, Talley NJ, Aro P, Storskrubb T, Johansson SE, Lind T, Bolling-
Sternevald E, Vieth M, Stolte M, Walker MM, Agréus L, (2007). Prevalence of
oesophageal eosinophils and eosinophilic oesophagitis in adults: the population-based
Kalixanda study. Gut., May;56(5):615-20.
[9] Kalach N, Huvenne H, Gosset P, Papadopoulos S, Dehecq E, Decoster A, Creusy C,
Dupont C, (2011). Eosinophil counts in upper digestive mucosa of Western European
children: variations with age, organs, symptoms, Helicobacter pylori status, and
pathological findings. J. Pediatr. Gastroenterol. Nutr., Feb;52(2):175-82.
[10] Strachan DP, (1989). Hay fever, hygiene, and household size. BMJ., Nov
18;299(6710):1259-60.
[11] Mosmann TR, Coffman RL, (1989). TH1 and TH2 cells: different patterns of
lymphokine secretion lead to different functional properties. Ann. Rev. Immunol.,
7:145-173.
[12] Abbas AK, Murphy KM, Sher A, (1996). Functional diversity of helper T lymphocytes.
Nature, 383:787-793.
[13] D'Elios MM, Manghetti M, De Carli M, Costa F, Baldari CT, Burroni D, Telford JL,
Romagnani S, Del Prete G, (1997). T helper 1 effector cells specific for Helicobacter
pylori in the gastric antrum of patients with peptic ulcer disease. J. Immunol., Jan
15;158(2):962-7.
[14] Guiney DG, Hasegawa P, Cole SP, (2003). Helicobacter pylori preferentially induces
interleukin 12 (IL-12) rather than IL-6 or IL-10 in human dendritic cells. Infect.
Immun., Jul;71(7):4163-6.
[15] Hafsi N, Voland P, Schwendy S, Rad R, Reindl W, Gerhard M, Prinz C, (2004). Human
dendritic cells respond to Helicobacter pylori, promoting NK cell and Th1-effector
responses in vitro. J. Immunol., Jul 15;173(2):1249-57.
[16] Falk GW, (2002). Barrett's esophagus. Gastroenterology., May;122(6):1569-91.
[17] Cameron AJ, Lomboy CT, Pera M, Carpenter HA, (1995). Adenocarcinoma of the
esophagogastric junction and Barrett's esophagus. Gastroenterology., Nov;109(5):1541-
6.
[18] Hvid-Jensen F, Pedersen L, Drewes AM, Sørensen HT, Funch-Jensen P, (2011).
Incidence of adenocarcinoma among patients with Barrett's esophagus. N. Engl. J.
Med., Oct 13;365(15):1375-83.
[19] Fischbach LA, Nordenstedt H, Kramer JR, Gandhi S, Dick-Onuoha S, Lewis A,El-
Serag HB, (2012). The association between Barrett's esophagus and Helicobacter pylori
infection: a meta-analysis. Helicobacter., Jun;17(3):163-75.
[20] Wang C, Yuan Y, Hunt RH, (2009). Helicobacter pylori infection and Barrett's
esophagus: a systematic review and meta-analysis. Am. J. Gastroenterol.,
Feb;104(2):492-500.
[21] Rokkas T, Pistiolas D, Sechopoulos P, Robotis I, Margantinis G, (2007). Relationship
between Helicobacter pylori infection and esophageal neoplasia: a meta-analysis. Clin,
Gastroenterol. Hepatol., Dec;5(12):1413-7.
[22] Zhuo X, Zhang Y, Wang Y, Zhuo W, Zhu Y, Zhang X, (2008). Helicobacter pylori
infection and oesophageal cancer risk: association studies via evidence-based meta-
analyses. Clin. Oncol. (R Coll Radiol)., Dec;20(10):757-62.
[23] Islami F, Kamangar F, (2008). Helicobacter pylori and esophageal cancer risk: a meta-
analysis. Cancer Prev Res (Phila)., Oct;1(5):329-38.

[24] Corley DA, Kubo A, Levin TR, Block G, Habel L, Zhao W, Leighton P, Rumore G,
Quesenberry C, Buffler P, Parsonnet J, (2008). Helicobacter pylori infection and the
risk of Barrett's oesophagus: a community-based study. Gut., Jun;57(6):727-33.
[25] Anderson LA, Murphy SJ, Johnston BT, Watson RG, Ferguson HR, Bamford KB,
Ghazy A, McCarron P, McGuigan J, Reynolds JV, Comber H, Murray LJ, (2008).
Relationship between Helicobacter pylori infection and gastric atrophy and the stages of
the oesophageal inflammation, metaplasia, adenocarcinoma sequence: results from the
FINBAR case-control study. Gut., Jun;57(6):734-9.
[26] Ronkainen J, Aro P, Storskrubb T, Johansson SE, Lind T, Bolling-Sternevald E, Vieth
M, Stolte M, Talley NJ, Agréus L, (2005). Prevalence of Barrett's esophagus in the
general population: an endoscopic study. Gastroenterology., Dec;129(6):1825-31.
[27] Rex DK, Cummings OW, Shaw M, Cumings MD, Wong RK, Vasudeva RS, Dunne D,
Rahmani EY, Helper DJ, (2003). Screening for Barrett's esophagus in colonoscopy
patients with and without heartburn. Gastroenterology., Dec;125(6):1670-7.
[28] Rajendra S, Ackroyd R, Robertson IK, Ho JJ, Karim N, Kutty KM, (2007).
Helicobacter pylori, ethnicity, and the gastroesophageal reflux disease spectrum: a
study from the East. Helicobacter., Apr;12(2):177-83.
[29] Rugge M, Russo V, Busatto G, Genta RM, Di Mario F, Farinati F, Graham DY, (2001).
The phenotype of gastric mucosa coexisting with Barrett's oesophagus. J. Clin. Pathol.,
Jun;54(6):456-60.
[30] Vicari JJ, Peek RM, Falk GW, Goldblum JR, Easley KA, Schnell J, Perez-Perez GI,
Halter SA, Rice TW, Blaser MJ, Richter JE, (1998). The seroprevalence of CagA-
positive Helicobacter pylori strains in the spectrum of gastroesophageal reflux disease.
Gastroenterology., Jul;115(1):50-7.
[31] Vaezi MF, Falk GW, Peek RM, Vicari JJ, Goldblum JR, Perez-Perez GI, Rice TW,
Blaser MJ, Richter JE, (2000). CagA-positive strains of Helicobacter pylori may protect
against Barrett's esophagus. Am J Gastroenterol., Sep;95(9):2206-11.
[32] Ferrández A, Benito R, Arenas J, García-González MA, Sopeña F, Alcedo J, Ortego J,
Sainz R, Lanas A, (2006). CagA-positive Helicobacter pylori infection is not associated
with decreased risk of Barrett's esophagus in a population with high H. pylori infection
rate. BMC Gastroenterol., Feb 16;6:7.
[33] Blaser MJ, Perez-Perez GI, Lindenbaum J, Schneidman D, Van Deventer G, Marin-
Sorensen M, Weinstein WM, (1991). Association of infection due to Helicobacter
pylori with specific upper gastrointestinal pathology. Rev Infect Dis., Jul-Aug;13 Suppl
8:S704-8.
[34] Loffeld RJ, Ten Tije BJ, Arends JW, (1992). Prevalence and significance of
Helicobacter pylori in patients with Barrett's esophagus. Am J Gastroenterol.,
Nov;87(11):1598-600.
[35] Spechler SJ, Jain SK, Tendler DA, Parker RA, (2002). Racial differences in the
frequency of symptoms and complications of gastro-oesophageal reflux disease.
Aliment Pharmacol Ther., Oct;16(10):1795-800.
[36] Lieberman D, Fennerty MB, Morris CD, Holub J, Eisen G, Sonnenberg A, (2004).
Endoscopic evaluation of patients with dyspepsia: results from the national endoscopic
data repository. Gastroenterology., Oct;127(4):1067-75.
[37] Islami F, Kamangar F, Aghcheli K, Fahimi S, Semnani S, Taghavi N, Marjani HA,
Merat S, Nasseri-Moghaddam S, Pourshams A, Nouraie M, Khatibian M, Abedi B,

Brazandeh MH, Ghaziani R, Sotoudeh M, Dawsey SM, Abnet CC, Taylor PR,
Malekzadeh R, (2004). Epidemiologic features of upper gastrointestinal tract cancers in
Northeastern Iran. Br. J. Cancer., Apr 5;90(7):1402-6.
[38] Hongo M, Nagasaki Y, Shoji T, (2009). Epidemiology of esophageal cancer: Orient to
Occident. Effects of chronology, geography and ethnicity. J. Gastroenterol. Hepatol.,
May;24(5):729-35.
[39] Perez-Perez GI, Salomaa A, Kosunen TU, Daverman B, Rautelin H, Aromaa A, Knekt
P, Blaser MJ, (2002). Evidence that cagA(+) Helicobacter pylori strains are
disappearing more rapidly than cagA(-) strains. Gut., Mar;50(3):295-8.
[40] Lakatos PL, (2006). Recent trends in the epidemiology of inflammatory bowel diseases:
up or down? World J. Gastroenterol., Oct 14;12(38):6102-8.
[41] Loftus EV Jr, (2004). Clinical epidemiology of inflammatory bowel disease: Incidence,
prevalence, and environmental influences. Gastroenterology., May;126(6):1504-17.
[42] Thia KT, Loftus EV Jr, Sandborn WJ, Yang SK, (2008). An update on the
epidemiology of inflammatory bowel disease in Asia. Am. J. Gastroenterol.,
Dec;103(12):3167-82.
[43] Luther J, Dave M, Higgins PD, Kao JY, (2010). Association between Helicobacter
pylori
[44] infection and inflammatory bowel disease: a meta-analysis and systematic review of the
literature. Inflamm Bowel Dis., Jun;16(6):1077-84.
[45] Kato S, Furuyama N, Ozawa K, Ohnuma K, Iinuma K, (1999). Long-term follow-up
study of serum immunoglobulin G and immunoglobulin A antibodies after
Helicobacter pylori eradication. Pediatrics., Aug;104(2):e22.
[46] Fanti L, Ieri R, Mezzi G, Testoni PA, Passaretti S, Guslandi M, (2001). Long-term
follow-up and serologic assessment after triple therapy with omeprazole or lansoprazole
of Helicobacter-associated duodenal ulcer. J. Clin. Gastroenterol., Jan;32(1):45-8.
[47] Hirschl AM, Brandstätter G, Dragosics B, Hentschel E, Kundi M, Rotter ML, Schütze
K, Taufer M, (1993). Kinetics of specific IgG antibodies for monitoring the effect of
anti Helicobacter pylori chemotherapy. J. Infect. Dis., Sep;168(3):763-6.
[48] Perez-Perez GI, Maw AM, Feingold-Link L, Gunn J, Bowers AL, Minano C, Rautelin
H, Kosunen TU, Blaser MJ, (2010). Longitudinal analysis of serological responses of
adults to Helicobacter pylori antigens. J. Infect. Dis., Sep 15;202(6):916-23.
[49] Sonnenberg A, Genta RM, (2012). Low prevalence of Helicobacter pylori infection
among patients with inflammatory bowel disease. Aliment Pharmacol Ther.,
Feb;35(4):469-76.
[50] Rad R, Brenner L, Bauer S, Schwendy S, Layland L, da Costa CP, Reindl W,
Dossumbekova A, Friedrich M, Saur D, Wagner H, Schmid RM, Prinz C, (2006).
CD25+/Foxp3+ T cells regulate gastric inflammation and Helicobacter pylori
colonization in vivo. Gastroenterology., Aug;131(2):525-37.
[51] Maloy KJ, Salaun L, Cahill R, Dougan G, Saunders NJ, Powrie F, (2003). CD4+CD25+
T(R) cells suppress innate immune pathology through cytokine-dependent mechanisms.
J. Exp. Med., Jan 6;197(1):111-9.
[52] Li MO, Wan YY, Flavell RA, (2007). T cell-produced transforming growth factor-
beta1controls T cell tolerance and regulates Th1- and Th17-cell differentiation.
Immunity., May;26(5):579-91.

[53] Arnold IC, Hitzler I, Müller A, (2012). The Immunomodulatory Properties of

Helicobacter pylori Confer Protection Against Allergic and Chronic Inflammatory
Disorders. Front Cell Infect. Microbiol., 2:10.
[54] Leach MW, Davidson NJ, Fort MM, Powrie F, Rennick DM, (1999). The role of IL-10
in inflammatory bowel disease: "of mice and men". Toxicol. Pathol., Jan-
Feb;27(1):123-33.
[55] Higgins PD, Johnson LA, Luther J, Zhang M, Sauder KL, Blanco LP, Kao JY, (2011).
Prior Helicobacter pylori infection ameliorates Salmonella typhimurium-induced
colitis: mucosal crosstalk between stomach and distal intestine. Inflamm. Bowel Dis.,
Jun;17(6):1398-408.
[56] De Winter H, Cheroutre H, Kronenberg M, (1999). Mucosal immunity and
inflammation. II. The yin and yang of T cells in intestinal inflammation: pathogenic and
protective roles in a mouse colitis model. Am, J, Physiol., Jun;276(6 Pt 1):G1317-21.
[57] Parente F, Molteni P, Bollani S, Maconi G, Vago L, Duca PG, Rembacken B, Axon
AT, Bianchi Porro G, (1997). Prevalence of Helicobacter pylori infection and related
upper gastrointestinal lesions in patients with inflammatory bowel diseases. A cross-
sectional study with matching. Scand. J. Gastroenterol., Nov;32(11):1140-6.
[58] Pearce CB, Duncan HD, Timmis L, Green JR, (2000). Assessment of the prevalence of
infection with Helicobacter pylori in patients with inflammatory bowel disease. Eur. J.
Gastroenterol., Hepatol., Apr;12(4):439-43.
[59] Mantzaris GJ, Archavlis E, Zografos C, Zavos K, Petraki K, Triadaphyllou G, (1995).
Low prevalence of Helicobacter pylori in inflammatory bowel disease: association with
sulfasalazine. Am. J. Gastroenterol., Oct;90(10):1900.
[60] Triantafillidis JK, Gikas A, Apostolidiss N, Merikas E, Mallass E, Peros G, (2003). The
low prevalence of Helicobacter infection in patients with inflammatory bowel disease
could be attributed to previous antibiotic treatment. Am.. J. Gastroenterol.,
May;98(5):1213-4.
[61] Guslandi M, Fanti L, Testoni PA, (2002). Helicobacter pylori seroprevalence in Crohn's
disease: lack of influence by pharmacological treatment. Hepatogastroenterology., Sep-
Oct;49(47):1296-7.
[62] Väre PO, Heikius B, Silvennoinen JA, Karttunen R, Niemelä SE, Lehtola JK, Karttunen
TJ, (2001). Seroprevalence of Helicobacter pylori infection in inflammatory bowel
disease: is Helicobacter pylori infection a protective factor? Scand. J. Gastroenterol.,
Dec;36(12): 1295-300.
[63] Luther J, Owyang SY, Takeuchi T, Cole TS, Zhang M, Liu M, Erb-Downward J,
Rubenstein JH, Chen CC, Pierzchala AV, Paul JA, Kao JY, (2011). Helicobacter pylori
DNA decreases pro-inflammatory cytokine production by dendritic cells and attenuates
dextran sodium sulphate-induced colitis. Gut., Nov;60(11):1479-86.
[64] Vaira D, Miglioli M, Menegatti MK, et al. (1994). Prevalence of Helicobacter pylori
infection in gastric and non-gastric cancer: a multicenter study (abstract). Ital. J.
Gastroenterol., 26(suppl 2):161.
[65] Meucci G, Tatarella M, Vecchi M, Ranzi ML, Biguzzi E, Beccari G, Clerici E, de
Franchis R, (1997). High prevalence of Helicobacter pylori infection in patients with
colonic adenomas and carcinomas. J. Clin. Gastroenterol., Dec;25(4):605-7.

[66] Aydin A, Karasu Z, Zeytinoglu A, Kumanlioglu K, Ozacar T, (1999). Colorectal

adenomatous polyps and Helicobacter pylori infection. Am. J/ Gastroenterol.,
Apr;94(4): 1121-2.
[67] Fujimori S, Kishida T, Kobayashi T, Sekita Y, Seo T, Nagata K, Tatsuguchi A, Gudis
K, Yokoi K, Tanaka N, Yamashita K, Tajiri T, Ohaki Y, Sakamoto C, (2005).
Helicobacter pylori infection increases the risk of colorectal adenoma and
adenocarcinoma, especially in women. J, Gastroenterol., Sep;40(9):887-93.
[68] Lin YL, Chiang JK, Lin SM, Tseng CE, (2010). Helicobacter pylori infection
concomitant with metabolic syndrome further increase risk of colorectal adenomas.
World J. Gastroenterol., Aug 14;16(30):3841-6.
[69] Mizuno S, Morita Y, Inui T, Asakawa A, Ueno N, Ando T, Kato H, Uchida M,
Yoshikawa T, Inui A, (2005). Helicobacter pylori infection is associated with colon
adenomatous polyps detected by high-resolution colonoscopy. Int. J. Cancer., Dec
20;117(6):1058-9.
[70] Inoue I, Mukoubayashi C, Yoshimura N, Niwa T, Deguchi H, Watanabe M, Enomoto
S, Maekita T, Ueda K, Iguchi M, Yanaoka K, Tamai H, Arii K, Oka M, Fujishiro M,
Takeshita T, Iwane M, Mohara O, Ichinose M, (2011). Elevated risk of colorectal
adenoma with Helicobacter pylori-related chronic gastritis: a population-based case-
control study. Int, J. Cancer., Dec 1;129(11):2704-11.
[71] Moss SF, Neugut AI, Garbowski GC, Wang S, Treat MR, Forde KA, (1995).
Helicobacter pylori seroprevalence and colorectal neoplasia: evidence against an
association. J. Natl. Cancer Inst., May 17;87(10):762-3.
[72] Thorburn CM, Friedman GD, Dickinson CJ, Vogelman JH, Orentreich N, Parsonnet J,
(1998). Gastrin and colorectal cancer: a prospective study. Gastroenterology.,
Aug;115(2):275-80.
[73] Siddheshwar RK, Muhammad KB, Gray JC, Kelly SB, (2001). Seroprevalence of
Helicobacter pylori in patients with colorectal polyps and colorectal carcinoma. Am. J.
Gastroenterol., Jan;96(1):84-8.
[74] Bae RC, Jeon SW, Cho HJ, Jung MK, Kweon YO, Kim SK, (2009). Gastric dysplasia
may be an independent risk factor of an advanced colorectal neoplasm. World J.
Gastroenterol., Dec 7;15(45):5722-6.
[75] Abbass K, Gul W, Beck G, Markert R, Akram S, (2011). Association of Helicobacter
pylori infection with the development of colorectal polyps and colorectal carcinoma.
South Med. J., Jul;104(7):473-6.
[76] Hong SN, Lee SM, Kim JH, Lee TY, Kim JH, Choe WH, Lee SY, Cheon YK, Sung IK,
Park HS, Shim CS, (2012). Helicobacter pylori infection increases the risk of colorectal
adenomas: cross-sectional study and meta-analysis. Dig. Dis. Sci., Aug;57(8):2184-94.
[77] Robertson DJ, Sandler RS, Ahnen DJ, Greenberg ER, Mott LA, Cole BF, Baron JA,
(2009). Gastrin, Helicobacter pylori, and colorectal adenomas. Clin. Gastroenterol.
Hepatol., Feb;7(2):163-7.
[78] Zumkeller N, Brenner H, Zwahlen M, Rothenbacher D, (2006). Helicobacter pylori
infection and colorectal cancer risk: a meta-analysis. Helicobacter., Apr;11(2):75-80.
[79] Zhao YS, Wang F, Chang D, Han B, You DY, (2008). Meta-analysis of different test
indicators: Helicobacter pylori infection and the risk of colorectal cancer. Int. J.
Colorectal Dis., Sep;23(9):875-82.

[80] Sirinek KR, Levine BA, Moyer MP, (1985). Pentagastrin stimulates in vitro growth of
normal and malignant human colon epithelial cells. Am. J. Surg., Jan;149(1):35-9.
[81] Watson SA, Durrant LG, Morris DL, (1988). Growth-promoting action of gastrin on
human colonic and gastric tumour cells cultured in vitro. Br. J. Surg., Apr;75(4):342-5.
[82] Watson S, Durrant L, Morris D, (1989). Gastrin: growth enhancing effects on human
gastric and colonic tumour cells. Br. J. Cancer., Apr;59(4):554-8.
[83] Sirinek KR, Levine BA, Moyer MP, (1985). Pentagastrin stimulates in vitro growth of
normal and malignant human colon epithelial cells. Am. J. Surg., Jan;149(1):35-9.
[84] Wang TC, Koh TJ, Varro A, Cahill RJ, Dangler CA, Fox JG, Dockray GJ, (1999).
Processing and proliferative effects of human progastrin in transgenic mice. J. Clin.
Invest., Oct 15;98(8):1918-29.
[85] Koh TJ, Dockray GJ, Varro A, Cahill RJ, Dangler CA, Fox JG, Wang TC, (1999).
Overexpression of glycine-extended gastrin in transgenic mice results in increased
colonic proliferation. J. Clin. Invest., Apr;103(8):1119-26.
[86] Wang TC, Dockray GJ, (1999). Lessons from genetically engineered animal models. I.
Physiological studies with gastrin in transgenic mice. Am. J. Physiol., Jul;277(1 Pt
1):G6-11.
[87] Koh TJ, Goldenring JR, Ito S, Mashimo H, Kopin AS, Varro A, Dockray GJ, Wang TC,
(1997). Gastrin deficiency results in altered gastric differentiation and decreased
colonic proliferation in mice. Gastroenterology., Sep;113(3):1015-25.
[88] Smith JP, Wood JG, Solomon TE, (1998). Elevated gastrin levels in patients with colon
cancer or adenomatous polyps. Dig. Dis. Sci. Feb;34(2):171-4.
[89] Wong K, Beardshall K, Waters CM, Calam J, Poston GJ, (1991). Postprandial
hypergastrinaemia in patients with colorectal cancer. Gut., Nov;32(11):1352-4.
[90] Thorburn CM, Friedman GD, Dickinson CJ, Vogelman JH, Orentreich N, Parsonnet J,
(1998). Gastrin and colorectal cancer: a prospective study. Gastroenterology.,
Aug;115(2):275-80.
[91] Pinson DM, Havu N, Sztern MI, Mattsson H, Looney GA, Kimler BF, Hurwitz A,
(1995). Drug-induced hypergastrinemia: absence of trophic effects on colonic
carcinoma in rats. Gastroenterology., Apr;108(4):1068-74.
[92] Hurwitz A, Sztern MI, Looney GA, Pinson DM, Bauer KD, Kimler BF, (1995). Effects
of omeprazole on cell kinetics of carcinogen-induced colon tumours in rats. Cell Prolif.,
Oct;28(10):525-31.
[93] Baldwin GS, Zhang QX, (1992). Measurement of gastrin and transforming growth
factor alpha messenger RNA levels in colonic carcinoma cell lines by quantitative
polymerase chain reaction. Cancer Res., Apr 15;52(8):2261-7.
[94] Van Solinge WW, Nielsen FC, Friis-Hansen L, Falkmer UG, Rehfeld JF, (1993).
Expression but incomplete maturation of progastrin in colorectal carcinomas.
Gastroenterology., Apr;104(4):1099-107.
[95] Finley GG, Koski RA, Melhem MF, Pipas JM, Meisler AI, (1993). Expression of the
gastrin gene in the normal human colon and colorectal adenocarcinoma. Cancer Res.,
Jun 15;53(12):2919-26.
[96] Xu Z, Dai B, Dhruva B, Singh P, (1994). Gastrin gene expression in human colon
cancer cells measured by a simple competitive PCR method. Life Sci., 54(10):671-8.
[97] Singh P, Dai B, Wu H, Owlia A, (2000). Role of autocrine and endocrine gastrin-like
peptides in colonic carcinogenesis. Curr. Opin/ Gastroenterol., Jan;16(1):68-77.

[98] Bombski G, Gasiorowska A, Orszulak-Michalak D, Neneman B, Kotynia J, Strzelczyk

J, Janiak A, Malecka-Panas E, (2003). Elevated plasma gastrin, CEA, and CA 19-9
levels decrease after colorectal cancer resection. Int. J. Colorectal Dis., Mar;18(2):148-
52.
[99] Wong K, Beardshall K, Waters CM, Calam J, Poston GJ, (1991). Postprandial
hypergastrinaemia in patients with colorectal cancer. Gut., Nov;32(11):1352-4.
[100] Orbuch M, Venzon DJ, Lubensky IA, Weber HC, Gibril F, Jensen RT, (1996).
Prolonged hypergastrinemia does not increase the frequency of colonic neoplasia in
patients with Zollinger-Ellison syndrome. Dig. Dis. Sci., Mar;41(3):604-13.
[101] Freston JW, Borch K, Brand SJ, Carlsson E, Creutzfeldt W, Håkanson R, Olbe L,
Solcia E, Walsh JH, Wolfe MM, (1995). Effects of hypochlorhydria and
hypergastrinemia on structure and function of gastrointestinal cells. A review and
analysis. Dig. Dis. Sci., Feb;40(2 Suppl):50S-62S.
[102] Creutzfeldt W, Lamberts R, (1991). Is hypergastrinaemia dangerous to man? Scand. J.
Gastroenterol., Suppl. ;180:179-91.
[103] Graffner H, Singh G, Chaudry I, Milsom JW, (1992). Omeprazole-induced
hypergastrinemia does not influence growth of colon carcinoma. Dig. Dis. Sci.,
Apr;37(4):485-9.
[104] Hurwitz A, Sztern MI, Looney GA, Pinson DM, Bauer KD, Kimler BF, (1995). Effects
of omeprazole on cell kinetics of carcinogen-induced colon tumours in rats. Cell Prolif.,
Oct;28(10):525-31.
[105] Singh M, Dhindsa G, Friedland S, Triadafilopoulos G, (2007). Long-term use of proton
pump inhibitors does not affect the frequency, growth, or histologic characteristics of
colon adenomas. Aliment. Pharmacol. Ther., Oct 1;26(7):1051-61.
[106] Lamberts R, Wartenberg T, Creutzfeldt W, (1999). Role of circulating gastrin in
colorectal adenomas and carcinomas. Digestion., Mar-Apr;60(2):101-9.
[107] Suzuki H, Matsumoto K, Terashima H, (1998). Serum levels of gastrin in patients with
colorectal neoplasia. Dis. Colon. Rectum., Sep;31(9):716-7.
[108] Penman ID, el-Omar E, Ardill JE, McGregor JR, Galloway DJ, O'Dwyer PJ, McColl
KE, (1994). Plasma gastrin concentrations are normal in patients with colorectal
neoplasia and unaltered following tumor resection. Gastroenterology., May;106
(5):1263-70.
[109] Kikendall JW, Glass AR, Sobin LH, Bowen PE, (1992). Serum gastrin is not higher in
subjects with colonic neoplasia. Am. J. Gastroenterol., Oct;87(10):1394-7.
[110] Parsonnet J, Friedman GD, Orentreich N, Vogelman H, (1997). Risk for gastric cancer
in people with CagA positive or CagA negative Helicobacter pylori infection. Gut.,
Mar;40(3):297-301.
[111] Shmuely H, Passaro D, Figer A, Niv Y, Pitlik S, Samra Z, Koren R, Yahav J, (2001).
Relationship between Helicobacter pylori CagA status and colorectal cancer. Am. J.
Gastroenterol., Dec;96(12):3406-10.
[112] Fan XG, Yakoob J, Fan XJ, Keeling PW, (1996). Enhanced T-helper 2 lymphocyte
responses: immune mechanism of Helicobacter pylori infection. Ir. J. Med. Sci., Jan-
Mar;165(1):37-9.
[113] Francipane MG, Alea MP, Lombardo Y, Todaro M, Medema JP, Stassi G, (2008).
Crucial role of interleukin-4 in the survival of colon cancer stem cells. Cancer Res., Jun
1;68(11):4022-5.

[114] Kim JM, Kim JS, Lee JY, Kim YJ, Youn HJ, Kim IY, Chee YJ, Oh YK, Kim N, Jung
HC, Song IS, (2007). Vacuolating cytotoxin in Helicobacter pylori water-soluble
proteins upregulates chemokine expression in human eosinophils via Ca2+ influx,
mitochondrial reactive oxygen intermediates, and NF-kappaB activation. Infect.
Immun., Jul;75(7):3373-81.
[115] Kuipers EJ, Pérez-Pérez GI, Meuwissen SG, Blaser MJ, (1995). Helicobacter pylori and
atrophic gastritis: importance of the cagA status. J. Natl. Cancer Inst., Dec 6;87(23):
1777-80.
[116] Kanno T, Matsuki T, Oka M, Utsunomiya H, Inada K, Magari H, Inoue I, Maekita T,
Ueda K, Enomoto S, Iguchi M, Yanaoka K, Tamai H, Akimoto S, Nomoto K, Tanaka
R, Ichinose M, (2009). Gastric acid reduction leads to an alteration in lower intestinal
microflora. Biochem Biophys Res Commun., Apr 17;381(4):666-70.
[117] Macfarlane GT, Macfarlane LE, (2009). Acquisition, evolution and maintenance of the
normal gut microbiota. Dig. Dis., 27 Suppl 1:90-8.
[118] Husebye E, (2005). The pathogenesis of gastrointestinal bacterial overgrowth.
Chemotherapy., 51 Suppl 1:1-22.
[119] Batt RM, Rutgers HC, Sancak AA, (1996). Enteric bacteria: friend or foe? J. Small
Anim. Pract., Jun;37(6):261-7.
[120] Cuevas-Ramos G, Petit CR, Marcq I, Boury M, Oswald E, Nougayrède JP, (2010).
Escherichia coli induces DNA damage in vivo and triggers genomic instability in
mammalian cells. Proc. Natl. Acad. Sci. USA., Jun 22;107(25):11537-42.
[121] Wu S, Morin PJ, Maouyo D, Sears CL, (2003). Bacteroides fragilis enterotoxin induces
c-Myc expression and cellular proliferation. Gastroenterology., Feb;124(2):392-400.
[122] Toprak NU, Yagci A, Gulluoglu BM, Akin ML, Demirkalem P, Celenk T, Soyletir G,
(2006). A possible role of Bacteroides fragilis enterotoxin in the aetiology of colorectal
cancer. Clin. Microbiol. Infect., Aug;12(8):782-6.
[123] Sobhani I, Tap J, Roudot-Thoraval F, Roperch JP, Letulle S, Langella P, Corthier G,
Tran Van Nhieu J, Furet JP, (2011). Microbial dysbiosis in colorectal cancer (CRC)
patients. PLoS One., Jan 27;6(1):e16393.
[124] Wang X, Allen TD, May RJ, Lightfoot S, Houchen CW, Huycke MM, (2008).
Enterococcus faecalis induces aneuploidy and tetraploidy in colonic epithelial cells
through a bystander effect. Cancer Res., Dec 1;68(23):9909-17.
[125] Moore WE, Moore LH, (1995). Intestinal floras of populations that have a high risk of
colon cancer. Appl. Environ. Microbiol., Sep;61(9):3202-7.
[126] Stemmermann GN, Nomura AM, Chyou PH, (1991). Cancer incidence following
subtotal gastrectomy. Gastroenterology., Sep;101(3):711-5.
[127] Bundred NJ, Whitfield BC, Stanton E, Prescott RJ, Davies GC, Kingsnorth AN, (1985).
Gastric surgery and the risk of subsequent colorectal cancer. Br. J. Surg., Aug;72(8):
618-9.
[128] Toftgaard C, (1987). Risk of colorectal cancer after surgery for benign peptic
ulceration. Br. J. Surg., Jul;74(7):573-5.
[129] Thiruvengadam R, Hench V, Melton LJ 3rd, DiMagno EP, (1998). Cancer of the
nongastric hollow organs of the gastrointestinal tract after gastric surgery. Arch. Intern.
Med., Feb; 148 (2):405-7.

[130] Kapetanakis N, Kountouras J, Zavos C, Polyzos SA, Katsinelos P, Romiopoulos I,

(2012). Helicobacter pylori infection and colorectal carcinoma: pathologic aspects. J.
Gastrointest Oncol., 3(4):377-9.
[131] Grahn N, Hmani-Aifa M, Fransén K, Söderkvist P, Monstein HJ, (2005). Molecular
identification of Helicobacter DNA present in human colorectal adenocarcinomas by
16S rDNA PCR amplification and pyrosequencing analysis. J. Med. Microbiol.,
Nov;54(Pt 11):1031-5.
[132] Monstein HJ, Olsson C, Nilsson I, Grahn N, Benoni C, Ahrné S, (2005). Multiple
displacement amplification of DNA from human colon and rectum biopsies: bacterial
profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based
TTGE and pyrosequencing analysis. J. Microbiol. Methods., Dec;63(3):239-47.
[133] Soylu A, Ozkara S, Alis H, Dolay K, Kalayci M, Yasar N, Kumbasar AB, (2008).
Immunohistochemical testing for Helicobacter Pylori existence in neoplasms of the
colon. BMC Gastroenterol., Aug 14;8:35.
[134] Kelly SM, Pitcher MC, Farmery SM, Gibson GR, (1994). Isolation of Helicobacter
pylori from feces of patients with dyspepsia in the United Kingdom. Gastroenterology.,
Dec;107(6):1671-4.
[135] Kabir S, (2001). Detection of Helicobacter pylori in faeces by culture, PCR and
enzyme immunoassay. J, Med, Microbiol., Dec;50(12):1021-9.
[136] Parsonnet J, Shmuely H, Haggerty T, (1999). Fecal and oral shedding of Helicobacter
pylori from healthy infected adults. JAMA., Dec 15;282(23):2240-5.
[137] Tatishchev SF, VanBeek C, Wang HL, (2012). Helicobacter pylori infection and
colorectal carcinoma: is there a causal association? J. Gastrointest Oncol., 3(4):380-5.

Chapter 10
Biliary and Liver Diseases
Peng Zhu1, Xun Li2, Yuming Wang*1 and Liang Qiao†3

1
Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University,
Chongqing, China
2
Hepatopancreatobiliary Surgery Insititute of Gansu Province, Department of Surgery,
First Hospital of Lanzhou University, Clinical Medical College Cancer Center
of Lanzhou University, Lanzhou, Gansu Province, China
3
Storr Liver Unit, University of Sydney at Westmead Millennium Institute,
Westmead NSW, Australia
Abstract
At the present stage of knowledge, the participation of the Helicobacter pylori (H.
pylori) in the pathology of hepatobiliary diseases in human has not been univocally
documented. H. pylori could be a risk factor for the progression of liver disease to
cirrhosis and hepatocellular carcinoma, which has been observed in animal models.
Overall, H. pylori infection usually aggravates the primary hepatobiliary diseases.
Meanwhile, research in this area has been limited by the lack of a gold standard in the
diagnosis in the hepatobiliary system. While current treatment of H. pylori in patients
with hepatobiliary disease is still based on the therapy of stomach infection, the need and
efficacy are controversial. Along with the in-depth research of H. pylori infection in this
area, we may know H. pylori more comprehensive one day.
Keywords: Helicobacter pylori, hepatobiliary disease, diagnosis, eradication therapy
*
Correspondence to: Yuming Wang, Institute of Infectious Diseases, Southwest Hospital, The Third Military
Medical University, Chongqing 400038, China, Telephone: +86-23-65334998, Fax: +86-23-68754858, E-
mail:wym417@163.com
†
Correspondence to: Liang Qiao, PhD, MD, Storr Liver Unit, Westmead Millennium Institute, The University of
Sydney at Westmead Hospital, Westmead, NSW 2145, Australia, Telephone: +612 98459132, Fax: +612 9845
9103 Email: liang.qiao@sydney.edu.au

186 Peng Zhu, Xun Li, Yuming Wang et al.
Introduction
Helicobacter pylori (H. pylori) is a spiral-shaped, flagellated, microaerophilic Gram-
negative bacillus that colonizes the gastric mucosa of more than 50% of the human
population, with the highest prevalence in the developing countries [1]. Human being is not
the only host for H. pylori as the bacteria have been identified in experimental animal models
such as mice, mongolian gerbils and Rhesus monkeys [2, 3]. Stomach is the primary natural
habitat for H. pylori and its colonization is usually without any symptoms. In developing
countries such as India, Pakistan, Latin America and Africa, the infection rate of H. pylori is
approximately 80% of the population by 20 years of age. In contrast, this rate is as low as 10–
20% in developed countries [4]. The prevalence of H. Pylori ranges from 60 to 80% in
patients with gastric ulcer and 90–100% in those with duodenal ulcer. Spreading of H. pylori
may be through the contaminated drinking water and/or food, just like hepatitis A virus
(HAV) and hepatitis E virus (HEV). Age, socio-economic status, education level, health
conditions, living environment and occupation of patients are significantly related to H. pylori
infection. H. pylori is very stubborn because once infected, no therapeutic intervention can
achieve a lifelong eradication, and its self-clearance rate is low.
Since its isolation from human gastric mucosa in 1982, H. pylori has been recognized as
a causative agent for many gastrointestinal diseases, including peptic ulcers, gastric cancer
and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Furthermore, chronic H.
pylori infection is related to various other extragastric diseases, including idiopathic
thrombocytopenic purpura (ITP), cardiovascular diseases and some forms of hepatobiliary
diseases. Available data about the prevalence of H. pylori in hepatobiliary diseases is limited
and controversial, and our understanding of H. pylori in the pathology of the hepatobiliary
diseases has undergone major changes in recent years.
In this article we aimed to review the currently data on the role of H. pylori in common
hepatobiliary diseases and the underlying mechanisms.
Effect of H. pylori Infection in Various

Hepatobiliary Diseases
H. Pylori Infection in Hepatitis
The identification of H. pylori DNA in liver tissues indicats that this bacteria may play a
role in the development and evolution of liver diseases. However, the effect of H. pylori
infection in patients with pre-existing chronic hepatitis has not yet been completely
determined. Previous animal studies and epidemiological data have showed that co-infection
of H. pylori with HBV or HCV may accelerate the natural course of HBV and HCV induced
hepatitis and associated liver diseases. Clinical epidemiological data showed that patients
with chronic viral hepatitis and cirrhosis have a much higher rate of H. pylori infection than
healthy controls [5], as serum antibody for H. pylori was found in 57-77% of hepatitis C virus
(HCV) infected patients, 68% of hepatitis B virus (HBV) infected patients, and 83% of
patients with liver cirrhosis, compared to sex- and age-matched control group in which anti-
H. pylori was positive in 50-59% of the subjects tested [6]. The overall prevalence of H.

Biliary and Liver Diseases 187
pylori infection in all types of liver diseases studied was 47.1% whereas its prevalence rate in
control cases was 16.6% (Table 10.1). Despite these studies, the role of H. pylori in chronic
hepatitis is still controversial.
Table 10.1. Studies addressing the association between H. pylori infection

and liver diseases
Some studies have suggested that co-existence of acute viral hepatitis and H. pylori may
represent two independent diseases that co-incidentally occur together. This might be due to
the different immune response patterns to acute hepatitis and H. pylori. In general, many
epidemiological investigations showed that HAV sero-prevalence was lower than H. pylori in
early life, and then became higher in later life. Anti-HAV IgG is long-lived thus once formed
it is generally detectable for life. In contrast, H. pylori IgG titers decline following
elimination of the infection, often to undetectable levels within a year or two [6]. Acute
hepatitis virus co-infection with H. pylori seem to be two independent diseases together,
which acute hepatitis is self-limiting but H. pylori persistent infection will cause chronic
stomach disease. Jeggli et al [20] and Tschopp et al [21] estimated the rate of H. pylori and
HEV infection in sewage cleaners, and did not find significantly increased rate of acute
hepatitis E and upper gastrointestinal ulcer.
H. pylori Infection in Cirrhosis and its Complications
Goo et al [22] previously reported the development of primary biliary cirrhosis in a

C57BL/6 mouse model infected with H. pylori over 1 year. In another study by Goo et al [23]
H. pylori infection for 4 months could cause functional and morphological degenerative
changes in hepatocytes, such as the depletion of glycogen particles, hydropic degeneration,

binucleation and accumulation of numerous small fat droplets. But there was no severe
hepatitis present which indicated the H. pylori may not cause heavy hit on the liver. This is
also the first study in vivo to investigate the association between the presence of H. pylori
DNA in the liver and hepatic fibrosis.
There is an increased trend of H. pylori infection in patients with liver cirrhosis [15]. It
has been reported that peptic ulcer disease is present in approximately 5-20% of patients with
liver cirrhosis, whereas in general population, only 2-4% of patients have peptic ulcer
diseases [24, 25]. In addition, a meta-analysis pooling data from seven studies showed that H.
pylori infection increased the risk of peptic ulcer disease in patients with cirrhosis, with an
estimated odds ratio of 2.70 [26]. In animal studies, H. pylori infection has been shown to
promote hepatic fibrosis [2, 23]. H. pylori has been successfully cultured from human liver
biopsy specimen [27]. The underlying mechanism why patients with liver cirrhosis are
susceptible to H. pylori infection, and how H. pylori contribute to the pathogenesis of
hepatobiliary diseases remain largely unknown. Therefore, functional changes such as the
depletion of hepatic glycogen could point to impending liver damage during H. pylori
infection. It has been reported that lipopolysaccharide (LPS) produced by H. pylori was able
to stimulate Kupffer cells to produce TNF-α and TGF-β1, which can active the hepatic
stellate cell (HSC), there by facilitating liver fibrosis [2].
Some studies have suggested that CagA positive strain of H. pylori is associated with
more severe form of liver diseases [28], including HCV-related chronic hepatitis and liver
cirrhosis with or without HCC [19]. H. pylori infection was suggested to raise the plasma
ammonia and endotoxin levels in patients with decompensated liver cirrhosis [29]. H. pylori
infection could aggravate liver cirrhosis by promoting the development of minimal hepatic
encephalopathy (MHE). For example, 63% (22/35) of the patients with MHE was H. pylori
infected whereas only 37% (11/30) of those without MHE were H. pylori positive, and among
the patients with MHE, fasting blood ammonia level was significantly higher in the H. pylori
infected individuals (1.80 ± 0.34 μg/ml) than in those without H. pylori infection (1.39 ± 0.14
μg/ml) (P<0.001) [30].
However, other studies could not demonstrate a contributing role of H. pylori infection in
severe liver diseases, especially in decompensated type [31]. Thus, the definite impact of H.
pylori infection in hepatic encephalopathy (HE) is still controversial. On the other hand, liver
cirrhosis particularly decompensated liver cirrhosis may contribute to the development of
peptic ulcers through several mechanisms such as a reduction in mucosal prostaglandin E2,
increased serum gastrin concentration [32], impaired mucus secretion [33], and portal
hypertensive gastropathy [34]. Therefore, the role of H. pylori infection in the development of
decompensated liver cirrhosis and its complications require further validation.
H. pylori Infection in Alcoholic Liver Disease and Non-Alcoholic Fatty Liver

Disease (NAFLD)
Alcohol is mainly metabolized by hepatic alcohol dehydrogenase (ADH). The gastric

isoenzyme of ADH localized in the gastric mucosal cells is responsible for the alcohol
metabolism in about 10% of the patients. H. pylori could decrease the activity of the gastric
ADH in the gastric mucosal cells [35], thus increasing the “accessibility” of alcohol for the
liver. This may create an additional risk factor for the alcoholic damages of the liver [36].

Consequently, in patients with chronic alcoholic liver disease, H. pylori infection may
enhance alcoholic liver injury. The high prevalence of both H. pylori infection and ADH
renders further research linking them necessary.
The role of H. pylori infection in patients with NAFLD has not been determined.
Nonalcoholic fatty liver disease (NAFLD) is considered to be the hepatic manifestation of
insulin resistance (IR) syndrome [37], given that IR plays a key role in its pathogenesis. There
is possibly one study reporting in Japanese that H. pylori infection was one of the
independent risk factors for the development of NAFLD [38]. As suggested in a very recent
study, H. pylori infection may represent an extra hit contributing to the pathogenesis of
NAFLD, but may have little effect on the disease progression from simple steatosis to non-
alcoholic steatohepatitis (NASH) [39]. It could be hypothesized that H. pylori infection may
contribute to the pathogenesis of NAFLD, mainly adding to the first hit of a disease
considered to develop through multiple hits. H. pylori infection may trigger TNF-α, whereas
adiponectin is secondarily increased to counterbalance the pro-inflammatory cascade.
However, these results need to be further verified.
H. pylori Infection and Cholelithiasis
Since H. pylori was successfully isolated from human liver [27], the DNA fragments of
H. pylori, H. bilis, H. hepaticus and other Helicobacter spp were found in liver, biliary tract
tissues and stone specimens in patients with various diseases [40,41]. Evidences supporting
the association between the H. pylori infection and biliary lithiasis could be found in either
bile, biliary tissue or stone samples, but no bacteria could be cultured in these cells
successfully. For examples, by using immunohistochemistry and PCR, 16S rDNA which was
identified as H. pylori by sequencing and denaturing gradient gel electrophoresis and
immunohistochemistry was detected in bile. In a study involving 22 Ukraine patients with
chronic cholecystitis, 16 patients (73%) showed positive 16S rDNA in the gall bladder neck
tissues [42].
A bacterium closely resembling H. pylori was detected once immunohistologically and
by PCR in resected gallbladder tissue, and a possible association with cholelithiasis was
hypothesized [43]. Another study showed that H. pylori infection in the bile was correlated
with the formation of subtypes of gall stone [44]. The cholelithiasis with the H. pylori
infection is usually cholesterol stones. The patients with cholelithiasis have a 130 kDa protein
exhibiting the activity of aminopeptidase and promoting the crystallization of cholesterol
[45]. In excess of 90% of the H. pylori strains contains the similar enzyme which can promote
the crystallization of cholesterol [44]. Clinical epidemiological data from East Asia, South
Asia and South America (where H. pylori infection are generally highly endemic) [46, 47],
and a recently published meta-analysis [48] have supported a causative link between H. pylori
infection and cholelithiasis. In this meta-analysis, a significantly higher presence of H. Pylori
was observed in the lithiasis group than in the control group (35.82% versus 26.75%,
P=0.01), implicating a potential association between this bacterial infection and lithiasis.

H. pylori Infection and Liver Cancer
H. pylori was classified as a typeⅠcarcinogen by the International Agency for Research

on Cancer in 1994 [49], and being responsible for 5.5% of all cancers. H. pylori infection
occurs with a high frequency in patients with cirrhosis and hepatocellular carcinoma (HCC),
which may explain the frequent occurrence of gastroduodenal ulcer in these patients.
Numerous epidemiological evidences have suggested that association, but research of the
mechanism is still very scarce and scattered. Wang et al [50] demonstrated the presence of H.
pylori bacteria inside the HCC tissue by immunohistochemical stain for H. pylori.
Nevertheless, whether H. pylori plays any role in the development of liver cancer remains
undetermined. A finding indicated by Ward et al [51] is that H. pylori infection of the liver in
healthy A/JCr male mice is capable of inducing a strong inflammatory change in the
parenchyma (for example, hepatitis) leading to HCC, this may explain the role of H. pylori
infection in the HCC. Co-infection of H. pylori with HBV or HCV, the key etiological factors
for liver cancer, may accelerate hepatic carcinogenesis [19]. The CagA and CagE genes have
been found to be associated with cholangiocarcinoma. In vitro studies have showed that the
CagA (+) strain of H. pylori had significantly stronger effect on inducing the production of
IL-8, iNOS, p53 and Bax, but decreasing the expression of Bcl-2 than the CagA (-) strain in
cholangiocarcinoma cell lines [52]. However, exactly how H. pylori might be involved in
liver cancer formation is unknown. It has been suggested that γ-glutamyltranspeptidase
(GGT) of H. pylori may be involved in facilitating the malignant transformation of
hepatobiliary cells by altering cell kinetics and promoting inflammation.
Pathogenic Mechanisms of H. pylori Infection

in Hepatobiliary Diseases
How does H. pylori colonize to the liver and biliary tract remains undetermined. It is now
suggested that H. pylori may get into the liver through the portal vein and biliary tract, of
which retrograde infection through the biliary tract may be a more important mechanism
(Figure 10.1).
Biliary epithelium can easily be colonized by H. pylori from the duodenum because of
the anatomical proximity. Increased permeability of the portal vein system and intestinal
mucosa during liver cirrhosis can facilitate a shift of the normal gut flora and bacterial
products to enter the portal circulation. However, it is unclear how H. pylori could escape the
normal immune barrier and colonize in the liver.
In a recent study, the levels of several important inflammatory cytokines were compared
between H. pylori infected and non-infected liver biopsy specimens [53]. The study revealed
a higher level of IL-10, lower or absent level of IFN-α and a decreased level of IL-17A in H.
pylori positive liver tissues than in H. pylori negative counterparts. The study hypothesized
that gastric H. pylori can reach the liver by retrograde transfer from the duodenum when the
cytokine pattern of the host is of more regulatory type than pro-inflammatory type, indicating
that the presence of H. pylori in the liver could be a consequence rather than a cause of
hepatic diseases. It is thought that H. pylori by itself and its toxins in the blood stream can
damage the liver. One study suggested that H. pylori infection may be related to increasing

endotoxins (i.e. LPS) found in Gram-negative bacteria in patients with cirrhosis [29].
However, despite the successful detection of H. pylori DNA in the liver, isolation and culture
of H. pylori from the liver in human beings has been difficult and rare [54].
Figure 10.1 Two proposed pathways for Helicobacter spp (including H. pylori) colonization of the
liver. The bacterium pass from the stomach to the liver through the duodenum and biliary tract, or may
arrive in the liver from the circulation through the hepatic portal vein. (Pellicano et al. 2008).
Diagnostic Challenges of H. pylori

in Hepatobiliary Diseases
The isolation and culture of H. pylori are very important in basic and clinical studies,
which can be used for the assessment of new diagnostic methods and drug eradication effect.
Non-invasive tests such as serology detection were carried out in patients to assess whether
H. pylori was present. Thus, it could avoid the endoscopy which was discomfort and
expensive. H. pylori always infects and colonizes in the stomach first, so that current clinical
diagnosis and laboratory research of H. pylori infection are almost based on detecting the
bacteria in the stomach. Sensitivity and specificity of these methods for H. pylori detecting
were different. Urease is a characteristic enzyme produced by the H. pylori. The activity of
urease is the strongest in the currently known H. pylori, which can decompose the acid into
NH4 and CO2 in urea (normal concentration of 3-4 mM). By this way the increased local pH
value can neutralize stomach acid to facilitate the colonization of H. pylori. The serology is

the preferred technique in epidemiological surveys, but its reliability is lower than other
diagnostic tools such as histology or the urea breath test.
Currently, the diagnosis of H. pylori infection is based on several approaches: (1)
identification of bacteria or their products (mainly for urease) in the gastric mucosa following
endoscopic procedures; (2) detection of the bacterial metabolites in the breath (e.g., 13C breath
test); (3) detection of specific antibody against the bacterial protein in the blood; (4) detection
of bacterial antigen in the stool. The sensitivity and specificity of the commonly used
diagnostic approaches for H. pylori are summarized in Table 10.2.
Table 10.2. The sensitivity and specificity of the detection methods for H. pylori
It must be noted that colonization with H. pylori in the gastric mucosa is not a disease by
itself. Rather, it is a condition that is associated with an increased incidence of several
disorders of the upper gastrointestinal tract and hepatobiliary system. Thus, testing for H.
pylori is only recommended in individuals who have (1) active peptic ulcer diseases, (2) a
past history of peptic ulcers, (3) a low grade gastric MALT lymphoma, (4) undergone
endoscopic resection of early gastric cancer, (5) a family history, i.e., first degree relatives
with gastric cancer, (6) persistent dyspepsia, and (7) an ethnic background of certain
nationalities such as Chinese, Korean, Japanese, or Central American descent, as these groups
tend to have a higher incidence of H. pylori infection and stomach cancer.
So far, there is no clear guideline on whether H. pylori screening is necessary in patients
with chronic liver diseases. Because sensitivity and specificity differ greatly among various
detecting techniques, the gold standard for the identification of H. pylori in hepatobiliary
system has yet to be established. Up till now, only a few studies have confirmed the existence
of H. pylori in biliary system by immunohistochemistry. The routine hematoxylin-eosin stain
is not well suited for H. pylori detection because of the weak contrast between the bacteria
and the mucus. The Warthin-Starry stain provides a better visualization of the bacteria, but
the procedure is difficult to carry out. This technique is time-consuming and requires instant
preparation of the relevant reagents [55]. Notably, some studies using Warthin-Starry stain
demonstrated a higher presence of H. pylori in lithiasis patients (P = 0.007). Culture remains
the definitive method to prove the viability of H. pylori. Goo et al. [23] revealed that no
grown H. pylori from bile could be observed in and only 3.02% (6/199) of the tissue samples
were positive in cultivation of this bacterium. Why this occurs might be due to the use of
frozen bile as sample and the strongly inhibitory effects of bile acid, and further research on
exploring the optimal conditions for growing H. pylori in vitro is necessary.

Clinically, H. pylori infection in hepatobiliary system usually follows stomach infection

but the effective non-invasive detection methods for H. pylori infection in hepatobiliary
system are not available. The validity of applying the standard diagnostic approaches for H.
pylori infection as used for the upper gastrointestinal tract disorders may exhibit different
sensitivity and specificity in cirrhotic patients, and some tests (e.g., the rapid urease test) is
not suitable for detecting H. pylori infection in the hepatobiliary system.
Even through invasive procedures such as ERCP, the detection rate for H. pylori in
gallstone, liver or biliary tract tissues is very low [56]. On the other hand, presence of H.
pylori in the hepatobiliary tissues does not necessarily confirm that these tissues had been
infected with this bacterium. Because the culture of the organism from the liver is extremely
difficult, the culture of H. pylori in the hepatocyte is not feasible. Indeed, the only strain
cultured (H. pylori) was from a patient with Wilson’s disease [27]. It has not yet been
determined whether the presence of H. pylori DNA in the liver represents a true colonization
or only enterohepatic circulation of H. pylori and its DNA fragments. To even complicate the
issue, H. pylori sequences had been found in the liver tissues in those with negative H. pylori
serology [57]. Thus, the significance and cost-effectiveness of identifying H. pylori in the
liver and biliary tract through invasive approaches requires further verification.
Eradication Therapy
To date, the eradication therapy of H. pylori infection in the stomach is more important
than in other organs, because the gastric infection is usually thought to be the main source of
H. pylori. Since more than a decade ago, combination of a PPI (standard dose, b.i.d.),
clarithromycin (CAM) (500 mg, b.i.d.) and amoxicillin (1,000 mg, b.i.d.) or metronidazole
(MZN) 500 mg (or 400 mg in England, q.i.d.), given for 14 days remains the mainstay for H.
pylori eradication worldwide, and can yield more than 90% of eradication rate [58, 59].
The treatment of H. pylori in patients with hepatobiliary disease still based on the therapy
of stomach infection, just like the stomach outside various diseases (such as asthma,
cardiovascular disease, ITP, etc.) caused by H. pylori. No specific treatment approaches are
available for H. pylori infection in those with hepatobiliary diseases. The presence of
decreased liver function in patients with hepatobiliary diseases could influence the clearance
rate and pharmacokinetics of various medicines. For example, a prolonged elimination of
metronidazole in cirrhotic patients may increase the frequency of side effects. In addition, the
concentrations of the active metabolite of clarithromycin were found to be lower in patients
with hepatic impairment. Therefore, the severity of the chronic liver disease might affect the
H. pylori eradication rate.
So far, there have been few clinical trails of H. pylori eradication therapy in hepatobiliary
diseases, and the results varied considerably. In a study by Jung et al. [60], the improvement
of serum ALT level was significantly correlated with successful H. pylori eradication (84.7%)
by standard triple therapy in patients with liver cirrhosis and chronic hepatitis. In another
study, it was found that standard triple therapy based eradication of H. pylori infection in
patients with liver cirrhosis and peptic ulcer disease could be helpful but did not protect all
cirrhotic patients from peptic ulcer recurrence, and most relapsed ulcers were gastric ulcers in
H. pylori negative patients [61]. However, the study of Lo et al. [62] showed that the

prevalence of H. pylori in patients with cirrhosis and duodenal ulcer was only 52%, and
suggested that eradication of H. pylori have not effectively reduced the recurrence risk for
ulcers. But the limitation of sample size in this research is relatively small.
However, in patients with decompensated liver cirrhosis, successful eradication of H.
pylori infection may be beneficial. E.g., eradication of H. pylori in patients with HE led to a
significant decrease in arterial blood ammonia levels and a rapid improvement of HE [29].
When the standard triple therapy was coupled with lactulose therapy, a significant and rapid
improvement in fasting blood ammonia levels and psychometric tests in patients with MHE
were observed, irrespective of H. pylori status before treatment [61]. Meanwhile, the standard
triple treatment in these patients are effective without significant side effects. Further
prospective studies are needed to elucidate the role of H. pylori eradication and associated
complications in patients with chronic liver disease.
Conclusion
Although a cause-and-effect association between H. pylori infection and certain human
hepatobiliary diseases has been suggested, the evidence was mostly based on the extrapolated
studies of the role of H. pylori infection in gastroduodenal diseases. Thus, there remains much
to explore in the exact role of H. pylori in hepatobiliary diseases. The benefit of standard
eradication therapy for H. pylori infection in patients with chronic hepatobiliary diseases
warrants large scale clinical trials.
References
[1] Frenck RW, Jr., Clemens J,(2003). Helicobacter in the developing world. Microbes
Infect,5:705-713.
[2] Ki MR, Goo MJ, Park JK, Hong IH, Ji AR, Han SY, et al.(2010). Helicobacter pylori
accelerates hepatic fibrosis by sensitizing transforming growth factor-beta1-induced
inflammatory signaling. Lab Invest,90:1507-1516.
[3] Boonjakuakul JK, Canfield DR, Solnick JV,(2005). Comparison of Helicobacter pylori
virulence gene expression in vitro and in the Rhesus macaque. Infect Immun,73:4895-
4904.
[4] Brown LM,(2000). Helicobacter pylori: epidemiology and routes of transmission.
Epidemiol Rev,22:283-297.
[5] Konturek SJ, Gonciarz M, Gonciarz Z, Bielanski W, Mazur W, Mularczyk A, et
al.(2003). Progastrin and its products from patients with chronic viral hepatitis and liver
cirrhosis. Scand J Gastroenterol,38:643-647.
[6] Ponzetto A, Pellicano R, Leone N, Cutufia MA, Turrini F, Grigioni WF, et al.(2000).
Helicobacter infection and cirrhosis in hepatitis C virus carriage: is it an innocent
bystander or a troublemaker? Med Hypotheses,54:275-277.
[7] Avenaud P, Marais A, Monteiro L, Le Bail B, Bioulac Sage P, Balabaud C, et
al.(2000). Detection of Helicobacter species in the liver of patients with and without
primary liver carcinoma. Cancer,89:1431-1439.

[8] Nilsson HO, Mulchandani R, Tranberg KG, Stenram U, Wadstrom T,(2001).

Helicobacter species identified in liver from patients with cholangiocarcinoma and
hepatocellular carcinoma. Gastroenterology,120:323-324.
[9] Dore MP, Realdi G, Mura D, Graham DY, Sepulveda AR,(2002). Helicobacter
infection in patients with HCV-related chronic hepatitis, cirrhosis, and hepatocellular
carcinoma. Dig Dis Sci,47:1638-1643.
[10] Coppola N, De Stefano G, Marrocco C, Scarano F, Scolastico C, Tarantino L, et
al.(2003). Helicobacter spp. and liver diseases. Infez Med,11:201-207.
[11] Pellicano R, Mazzaferro V, Grigioni WF, Cutufia MA, Fagoonee S, Silengo L, et
al.(2004). Helicobacter species sequences in liver samples from patients with and
without hepatocellular carcinoma. World J Gastroenterol,10:598-601.
[12] Ito K, Nakamura M, Toda G, Negishi M, Torii A, Ohno T,(2004). Potential role of
Helicobacter pylori in hepatocarcinogenesis. Int J Mol Med,13:221-227.
[13] Zhang SQ, Bao Y, Zu MH, 123,(2004). The correlation between Helicobacter infection
and hepatocellular carcinoma. Zhongguo Zhongliu Linchuang,31 761–764.
[14] Huang Y, Fan XG, Zhou JH,(2004). Immunohistochemistry of Helicobacter pylori in
primary liver carcinoma tissues. Zhong Nan Da Xue Xue Bao Yi Xue Ban,29:15-17.
[15] Rocha M, Avenaud P, Menard A, Le Bail B, Balabaud C, Bioulac-Sage P, et al.(2005).
Association of Helicobacter species with hepatitis C cirrhosis with or without
hepatocellular carcinoma. Gut,54:396-401.
[16] Li N, Zhang SH, Xuan SY, Qiang X,(2006). Study on Helicobacter infection in liver
tissue from hepatocellular carcinoma]. Zhonghua Liu Xing Bing Xue Za Zhi,27:894-
896.
[17] Vivekanandan P, Torbenson M,(2008). Low frequency of Helicobacter DNA in benign
and malignant liver tissues from Baltimore, United States. Hum Pathol,39:213-216.
[18] Abu Al-Soud W, Stenram U, Ljungh A, Tranberg KG, Nilsson HO, Wadstrom
T,(2008). DNA of Helicobacter spp. and common gut bacteria in primary liver
carcinoma. Dig Liver Dis,40:126-131.
[19] Esmat G, El-Bendary M, Zakarya S, Ela MA, Zalata K,(2012). Role of Helicobacter
pylori in patients with HCV-related chronic hepatitis and cirrhosis with or without
hepatocellular carcinoma: possible association with disease progression. J Viral
Hepat,19:473-479.
[20] Jeggli S, Steiner D, Joller H, Tschopp A, Steffen R, Hotz P,(2004). Hepatitis E,
Helicobacter pylori, and gastrointestinal symptoms in workers exposed to waste water.
Occup Environ Med,61:622-627.
[21] Tschopp A, Joller H, Jeggli S, Widmeier S, Steffen R, Hilfiker S, et al.(2009). Hepatitis
E, Helicobacter pylori and peptic ulcers in workers exposed to sewage: a prospective
cohort study. Occup Environ Med,66:45-50.
[22] Goo MJ, Ki MR, Lee HR, Hong IH, Park JK, Yang HJ, et al.(2008). Primary biliary
cirrhosis, similar to that in human beings, in a male C57BL/6 mouse infected with
Helicobacter pylori. Eur J Gastroenterol Hepatol,20:1045-1048.
[23] Goo MJ, Ki MR, Lee HR, Yang HJ, Yuan DW, Hong IH, et al.(2009). Helicobacter
pylori promotes hepatic fibrosis in the animal model. Lab Invest,89:1291-1303.
[24] Siringo S, Burroughs AK, Bolondi L, Muia A, Di Febo G, Miglioli M, et al.(1995).
Peptic ulcer and its course in cirrhosis: an endoscopic and clinical prospective study. J
Hepatol,22:633-641.

[25] Tsai CJ,(1998). Helicobacter pylori infection and peptic ulcer disease in cirrhosis. Dig
Dis Sci,43:1219-1225.
[26] Vergara M, Calvet X, Roque M,(2002). Helicobacter pylori is a risk factor for peptic
ulcer disease in cirrhotic patients. A meta-analysis. Eur J Gastroenterol
Hepatol,14:717-722.
[27] de Magalhaes Queiroz DM, Santos A,(2001). Isolation of a Helicobacter strain from the
human liver. Gastroenterology,121:1023-1024.
[28] Argent RH, Zhang Y, Atherton JC,(2005). Simple method for determination of the
number of Helicobacter pylori CagA variable-region EPIYA tyrosine phosphorylation
motifs by PCR. J Clin Microbiol,43:791-795.
[29] Abdel-Hady H, Zaki A, Badra G, Lotfy M, Selmi C, Giorgini A, et al.(2007).
Helicobacter pylori infection in hepatic encephalopathy: Relationship to plasma
endotoxins and blood ammonia. Hepatol Res,37:1026-1033.
[30] Agrawal A, Gupta A, Chandra M, Koowar S,(2011). Role of Helicobacter pylori
infection in the pathogenesis of minimal hepatic encephalopathy and effect of its
eradication. Indian J Gastroenterol,30:29-32.
[31] Chang SS, Hu HY,(2012). Helicobacter pylori is not the predominant etiology for liver
cirrhosis patients with peptic ulcer disease. Eur J Gastroenterol Hepatol.
[32] Furuta T, Baba S, Takashima M, Futami H, Arai H, Kajimura M, et al.(1998). Effect of
Helicobacter pylori infection on gastric juice pH. Scand J Gastroenterol,33:357-363.
[33] Suzuki H, Ishii H,(2004). Peptic ulcer disease complicated with liver cirrhosis. Nihon
Rinsho,62:532-540.
[34] Chen CT, Wang TF, Chan CC, Lee FY, Chang FY, Lin HC, et al.(2002). Role of
chronic Helicobacter pylori infection in hyperdynamic circulation of cirrhotic patients.
Hepatogastroenterology,49:208-212.
[35] Jelski W, Chrostek L, Laszewicz W, Szmitkowski M,(2007). Alcohol dehydrogenase
(ADH) isoenzyme activity in the sera of patients with Helicobacter pylori infection.
Dig Dis Sci,52:1513-1516.
[36] Gonciarz M, Wloch M, Gonciarz Z,(2006). Helicobacter pylori in liver diseases. J
Physiol Pharmacol,57 Suppl 3:155-161.
[37] Chen SH, He F, Zhou HL, Wu HR, Xia C, Li YM,(2011). Relationship between
nonalcoholic fatty liver disease and metabolic syndrome. J Dig Dis,12:125-130.
[38] Takuma Y,(2011). Helicobacter pylori infection and liver diseases. Gan To Kagaku
Ryoho,38:362-364.
[39] Polyzos SA, Kountouras J, Papatheodorou A, Patsiaoura K, Katsiki E, Zafeiriadou E, et
al.(2013). Helicobacter pylori infection in patients with nonalcoholic fatty liver disease.
Metabolism,62:121-126.
[40] Tiwari SK, Khan AA, Ibrahim M, Habeeb MA, Habibullah CM,(2006). Helicobacter
pylori and other Helicobacter species DNA in human bile samples from patients with
various hepato-biliary diseases. World J Gastroenterol,12:2181-2186.
[41] Chen W, Li D, Cannan RJ, Stubbs RS,(2003). Common presence of Helicobacter DNA
in the gallbladder of patients with gallstone diseases and controls. Dig Liver
Dis,35:237-243.
[42] Apostolov E, Al-Soud WA, Nilsson I, Kornilovska I, Usenko V, Lyzogubov V, et
al.(2005). Helicobacter pylori and other Helicobacter species in gallbladder and liver of

patients with chronic cholecystitis detected by immunological and molecular methods.

Scand J Gastroenterol,40:96-102.
[43] Kawaguchi M, Saito T, Ohno H, Midorikawa S, Sanji T, Handa Y, et al.(1996).
Bacteria closely resembling Helicobacter pylori detected immunohistologically and
genetically in resected gallbladder mucosa. J Gastroenterol,31:294-298.
[44] Figura N, Cetta F, Angelico M, Montalto G, Cetta D, Pacenti L, et al.(1998). Most
Helicobacter pylori-infected patients have specific antibodies, and some also have H.
pylori antigens and genomic material in bile: is it a risk factor for gallstone formation?
Dig Dis Sci,43:854-862.
[45] Offner GD, Gong D, Afdhal NH,(1994). Identification of a 130-kilodalton human
biliary concanavalin A binding protein as aminopeptidase N. Gastroenterology,
106:755-762.
[46] Shimoyama T, Takahashi R, Abe D, Mizuki I, Endo T, Fukuda S,(2010). Serological
analysis of Helicobacter hepaticus infection in patients with biliary and pancreatic
diseases. J Gastroenterol Hepatol,25 Suppl 1:S86-89.
[47] Yakoob J, Khan MR, Abbas Z, Jafri W, Azmi R, Ahmad Z, et al.(2011). Helicobacter
pylori: association with gall bladder disorders in Pakistan. Br J Biomed Sci,68:59-64.
[48] Zhou D, Zhang Y, Gong W, Mohamed SO, Ogbomo H, Wang X, et al.(2011). Are
Helicobacter pylori and other Helicobacter species infection associated with human
biliary lithiasis? A meta-analysis. PLoS One,6:e27390.
[49] Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the
Evaluation of Carcinogenic Risks to Humans. Lyon, 7-14 June 1994. IARC Monogr
Eval Carcinog Risks Hum,61:1-241.
[50] Wang X, Willen R, Svensson M, Ljungh A, Wadstrom T,(2003). Two-year follow-up
of Helicobacter pylori infection in C57BL/6 and Balb/cA mice. APMIS,111:514-522.
[51] Ward JM, Fox JG, Anver MR, Haines DC, George CV, Collins MJ, Jr., et al.(1994).
Chronic active hepatitis and associated liver tumors in mice caused by a persistent
bacterial infection with a novel Helicobacter species. J Natl Cancer Inst,86:1222-1227.
[52] Boonyanugomol W, Chomvarin C, Baik SC, Song JY, Hahnvajanawong C, Kim KM,
et al.(2011). Role of cagA-positive Helicobacter pylori on cell proliferation, apoptosis,
and inflammation in biliary cells. Dig Dis Sci,56:1682-1692.
[53] Silva LD, Rocha AM, Rocha GA, de Moura SB, Rocha MM, Dani R, et al.(2011). The
presence of Helicobacter pylori in the liver depends on the Th1, Th17 and Treg
cytokine profile of the patient. Mem Inst Oswaldo Cruz,106:748-754.
[54] Boonyanugomol W, Chomvarin C, Song JY, Kim KM, Kim JM, Cho MJ, et al.(2012).
Effects of Helicobacter pylori gamma-glutamyltranspeptidase on apoptosis and
inflammation in human biliary cells. Dig Dis Sci,57:2615-2624.
[55] Megraud F, Lehours P,(2007). Helicobacter pylori detection and antimicrobial
susceptibility testing. Clin Microbiol Rev,20:280-322.
[56] Roosendaal R, Kuipers EJ, Vandenbroucke-Grauls CM, Kusters JG,(2002).
Helicobacter species are not detectable by 16S rDNA PCR in bile from Dutch patients
with common bile duct stones. Digestion,66:89-91.
[57] Castera L, Pedeboscq A, Rocha M, Le Bail B, Asencio C, de Ledinghen V, et al.(2006).
Relationship between the severity of hepatitis C virus-related liver disease and the
presence of Helicobacter species in the liver: a prospective study. World J
Gastroenterol,12:7278-7284.

[58] Malfertheiner P, Megraud F, O'Morain CA, Atherton J, Axon AT, Bazzoli F, et

al.(2012). Management of Helicobacter pylori infection--the Maastricht IV/ Florence
Consensus Report. Gut,61:646-664.
[59] Malfertheiner P, Megraud F, O'Morain C, Bazzoli F, El-Omar E, Graham D, et
al.(2007). Current concepts in the management of Helicobacter pylori infection: the
Maastricht III Consensus Report. Gut,56:772-781.
[60] Jung SW, Lee SW, Hyun JJ, Kim DI, Koo JS, Yim HJ, et al.(2009). Efficacy of
Helicobacter pylori eradication therapy in chronic liver disease. Dig Liver Dis,41:134-
140.
[61] Mitrica D, Plesa A, Constantinescu R, Drug V, Stanciu C,(2011). Efficacy of
Helicobacter pylori eradication therapy in cirrhotic patients with peptic ulcer disease.
Rev Med Chir Soc Med Nat Iasi,115:367-374.
[62] Lo GH, Yu HC, Chan YC, Chen WC, Hsu PI, Lin CK, et al.(2005). The effects of
eradication of Helicobacter pylori on the recurrence of duodenal ulcers in patients with
cirrhosis. Gastrointest Endosc,62:350-356.

Associated Extra-Gastrointestinal
Diseases

Chapter 11
Neurological Disorders and

Georgia Deretzi¹, Christos Zavos², Stergios A. Polyzos²,

Evangelia Giartza-Taxidou², Emmanouel Gavalas²,
Iakovos Tsiptsios¹, Theocharis Tsironis,1 Marina Boziki2
and Jannis Kountouras*²
¹Department of Neurology, Multiple Sclerosis Unit,
Papageorgiou General Hospital, Thessaloniki, Greece
²Department of Medicine, Second Medical Clinic,
Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece
Abstract
Neuroinflammation is considered a characteristic and critical component in the
preliminary phase and progression of nearly all neuroinflammatory disorders and could
be initiated by peripheral inflammatory conditions. Helicobacter pylori (H. pylori), a
potential gastrointestinal pathogen, induces humoral and cellular immune responses that,
owing to the sharing of homologous epitopes (molecular mimicry), cross-react with
nervous system components thereby contributing and possibly perpetuating neural tissue
damage. The current Chapter summarizes the implication of H. pylori in different
neurological disorders of the central nervous system (cerebrovascular diseases, mild
cognitive impairment, Alzheimer's disease, Parkinson's disease, seizure disorders,
migraine, multiple sclerosis and neuromyelitis optica), the peripheral nervous system
(Guillain-Barré syndrome) and ophthalmic disorders (glaucoma); additionally, it provides
recent data on understanding the possible immune properties of H. pylori in the
pathogenetic mechanisms of neurological diseases. More clinical and experimental
studies are required to establish H. pylori as a neurological pathogen.
* Correspondence to: Jannis Kountouras, MD, PhD, Professor of Medicine, Gastroenterologist, 8 Fanariou St.,
Byzantio, 551 33, Thessaloniki, Macedonia, Greece, Tel: +30-2310-892238, Fax: +30-2310-892744, E-mail:
jannis@auth.gr.

202 Georgia Deretzi, Christos Zavos, Stergios A. Polyzos et al.
Keywords: Helicobacter pylori, multiple sclerosis, Alzheimer's disease, Parkinson's disease,

glaucoma
Introduction
Although Helicobacter pylori (H. pylori) is the most frequently encountered cause of
gastroduodenal disorders, in the recent years interesting associations have been established
between H. pylori and several extragastric conditions [1-3].
Particularly, a high H. pylori frequency has been reported in different neurological
disorders of the central nervous system (CNS) including cerebrovascular diseases, mild
cognitive impairment, Alzheimer's disease (AD), Parkinson's disease (PD), seizure disorders,
migraine or multiple sclerosis (MS) and ophthalmic disorders (glaucoma (defined as “ocular
AD”), anterior optic ischemic neuropathy). H. pylori has also been postulated in autoimmune
diseases of peripheral nervous system (PNS) such as acute immune polyneuropathies; it is a
factor responsible for the etiology of Guillain-Barré syndrome (GBS) [2,4-6].
The pathogenetic role of H. pylori and the pathophysiological mechanisms behind
clinical observations linking this bacterium with neurological disorders still remain unclear
and under investigation. H. pylori has long been described to induce extraintestinal diseases
by means of molecular mimicry, a mechanism that may also be implicated in nerve damage
[7,8]. H. pylori infection induces humoral and cellular immune responses that, owing to the
sharing of homologous epitopes, cross-react with components of nervous system, thereby
contributing and possibly perpetuating neural tissue damage [9,10].
Neuroinflammation and neurodegeneration could be potentially initiated by peripheral
inflammatory conditions. H. pylori infection could indirectly affect neural and brain tissue by:
disrupting the blood-brain barrier (BBB) or blood-nerve barrier (BNB); and releasing
numerous vasoactive substances and proinflammatory cytokines [interleukin (IL)-1β, IL-6,
tumor necrosis factor (TNF)-α], acting at a distance and being involved in pathogenesis of
CNS and PNS disorders. Activated monocytes (possibly infected with H. pylori due to
defective autophagy resulting in H. pylori replication in autophagic vesicles) might also enter
the brain due to BBB disruption contributing to neurological H. pylori-related disorders.
Furthermore, this pathogen influences the pathophysiology of neurodegenerative and
neuroinflammatory disorders by promoting platelet and platelet-leukocyte aggregation;
releasing various pro-inflammatory and vasoactive substances; producing reactive oxygen
metabolites and circulating lipid peroxides; influencing the apoptotic process; and increasing,
through induction of atrophic gastritis, homocysteine, which contributes to endothelial
damage and neurodegeneration [7]. Expectantly, eradicating H. pylori infection might alter
the process of neurodegenerative disorders [10-13].
This review focuses on the potential pathogenic role of H. pylori implicated in
neurological and ophthalmological neurodegenerative diseases. Gaining a better
understanding of the relationship between H. pylori infection and nervous system could
provide an insight on the pathogenesis and therapeutic strategies reducing the burden of the
aforementioned neurological diseases and their associated high costs, morbidity and
mortality; and could have clinical and public health implications for the prevention, detection
and eradication of H. pylori infection.

Neurological Disorders and Helicobacter pylori Infection 203
Neuroimmunologic Disorders and H. pylori

A common characteristic of the CNS neurodegenerative disorders is neuroinflammation,
marked by augmented numbers of activated and primed microglia, increased inflammatory
cytokines and decreased anti-inflammatory molecules. CNS neuroinflammation is a critical
component in the progression of several neurodegenerative diseases which sensitize the brain
to produce an exaggerated response to immune stimuli in the periphery [14,15].
Neuroinflammation might initiate from the periphery and systemic conditions through
disrupted BBB or BNB powerfully influence various pathologies. Gastrointestinal tract (GIT)
represents a vulnerable area through which pathogens influence the brain and induce CNS
neuroinflammation. The pathogens including H. pylori may access the CNS through blood,
the nasal olfactory pathways and the GIT. GIT is strictly connected to the CNS and a bi-
directional communication exists between them. The brain is involved in regulating the
immune and gut system. The GIT is the primary immune organ with specialized
immunoregulatory and anti-inflammatory functions, represented by the GI immune system.
A series of H. pylori-related factors have been implicated in inducing BBB disruption,
including inflammatory mediators (e.g., cytokines, chemokines induced by H. pylori
infection) and oxidative stress [16]. H. pylori could indirectly affect the brain and other target
organs, e.g., the heart, through the release of numerous cytokines such as TNF-α acting at a
distance [16]. TNF-α is involved in BBB disruption through a mechanism involving matrix
metalloproteinases’ upregulation. TNF-α and IL-6 (TNF-α is the main trigger for the
production of IL-6 by a variety of cells) play important roles in the regulation of the synthesis
of other acute phase proteins which are established risk factors for atherosclerosis, such as
fibrinogen and factor VIII. These cytokines also have profound effects on lipid metabolism
directly at the site of the atherosclerotic lesion, but could influence the atheroma process
through blood circulating levels, distant production of cytokines, or through stimulating
circulating white blood cells to produce them, thereby contributing to BBB disruption and
pathogenesis of heart and brain pathologies [16-20]. H. pylori may be involved in the
pathophysiology mechanisms of BBB disruption including the release of large amounts of
vasoactive substances and pro-inflammatory cytokines, such as IL-6, -8, and TNF-α,
eicosanoids (leukotrienes, prostaglandins catalyzed by cyclo-oxygenase enzymes), and acute
phase proteins (fibrinogen, C-reactive protein) involved in a number of disorders including
MS [11,21], AD [7,12,22-25], and idiopathic parkinsonism [26-28], which can lead to long-
term neurologic deficits. In addition, H. pylori-induced vacuolating A (VacA) cytotoxin
exhibits chemotactic activities to the bone marrow-derived mast cells (BMD-MCs) and
induces BMD-MCs to produce proinflammatory cytokines including TNF-α [29]; BMD-MCs
reside adjacent to blood and lymphatic channels, mainly under epithelial surfaces including
the BBB and GIT [30]. H. pylori stimulates MCs directly or via gastrin induction and MCs
are actively involved in the pathogenesis of H. pylori-associated pathologies [31]. Apart from
activated MCs, vascular endothelial growth factor (VEGF), IL-8, chymase or tryptase (a
serine endoprotease released by MCs) and MC growth factor linked to H. pylori infection
[30,32], MCs themselves can be stimulated by corticotropin releasing hormone, secreted
under stress, to release mediators including histamine, IL-8, tryptase and VEGF, which
disrupt the BBB [32].

New findings provide an important suggestion as to why H. pylori causes a broad

spectrum of diseases [7,11,12,18,24-28,31,33,34]. H. pylori-induced VacA cytotoxin
promotes intracellular survival of the bacterium, modulates host immune responses and
induces autophagy [35,36]. Consequently, H. pylori as intracellular microorganism invades
and replicates in the cells. The autophagy induction by H. pylori is not only found in
macrophages, but also in DCs and gastric epithelial cells. The bacterium's residence inside
infected cells will increase its resistance to antimicrobial treatment, avoid neutralization by
anti-H. pylori antibodies, impair antigen presentation, and alter the cellular immune response
[37,38]. In turn, the potential influx of activated monocytes infected with H. pylori through
the disrupted BBB in the brain might lead to microglia activation and the development of
brain pathologies. In addition, recent studies showed that from the oral cavity, a permanent
reservoir of H. pylori [39,40], H. pylori may reach, through the nasal cavity [41] the anterior
surface of the eye, causing blepharitis or worsening it, at least on the basis of cytological
criteria [42]. This bacterium through oral-nasal-olfactory pathway, might also access brain,
thereby leading to the development of degenerative diseases. However, further studies are
needed to elucidate these fields.
Infectious stimuli of H. pylori may also participate in the development of autoimmune
disorders by inducing an increased expression of heat shock proteins (HSPs), chaperones, and
transplantation antigens, which results in abnormal processing and presentation of self
antigens [43]. Specifically, superantigens appear to be one of the most effective bacterial
components to induce inflammatory reactions and to take part in the development and course
of autoimmune mechanisms; defective immune system is associated with a higher risk of a
development of autoimmune disease, including MS, associated with H. pylori infection
[11,34,44]. Remarkably, current infections such as H. pylori infection induce the mentioned
humoral and cellular immune responses that, owing to the sharing of homologous epitopes
(molecular mimicry), cross-react with components of CNS thereby contributing neural tissue
damage in neurodegenerative disorders [11,34,44-46]. Interestingly, molecular mimicry of
host structures by the saccharide portion of LPS of the GIT pathogens Campylobacter jejuni
(C. jejuni) and H. pylori are thought to be connected with the development of autoimmune
sequelae observed in PNS inflammatory autoimmune neuropathies such as GBS syndrome
[9,47].
Central Nervous System Neurological

Disorders and H. pylori
Cerebrovascular Diseases
Ischemic stroke is the rapid loss of brain function due to failure of blood supply to a part
of the brain. This can be due to ischemia (lack of blood flow) caused by blockage (thrombosis
or arterial embolism). It is the second leading cause of death worldwide. It mainly involves
the large and middle cerebral arteries, which often leads to stroke, and intermittent
claudication due to vessel destruction or plaque split and subsequent thrombosis.
An association between H. pylori infection and stroke has been recently suggested.
Studies of clinical and epidemiological materials have identified inflammatory markers and

immunoregulatory genes as contributors of risk for stroke. Notably, atherosclerosis, a major

cause of stroke, is mainly an inflammatory process [48,49]. Chronic infection with various
microorganisms, particularly, Chlamydia pneumoniae, Cytomegalovirus and H. pylori may
play a role in etiological factors, linking inflammation and atherogenesis [50-54]. H. pylori
infection may contribute directly to atherosclerosis through damage effect of the pathogen on
atherosclerotic plaques. Signaling of inflammatory mediators, molecular mimicry, viral
dysregulation of cell activity and platelet H. pylori interactions may be immunological
mechanisms implicating indirectly in pathogenesis of atherosclerosis. Particularly,
inflammatory markers such as fibrinogen, IL-8 levels, leukocyte’s amount, and C-reactive
protein, are raised in seropositive H. pylori patients, promoting blood clot formation. Free
radical formation resulting in lipid peroxidation, cross reacting antibodies to HSPs and
hyperhomocysteinemia are possible pathogenic mechanisms linking H. pylori with stroke. H.
pylori-induced chronic gastric inflammation could result in malabsorption particularly of
vitamin B12 and secondary hyperhomocysteinemia [50,55-57].
Progression of carotid lesions associated with H. pylori infection may explain the onset of
carotid plaques and stroke [58,59]. Accumulation of low-density lipoproteins is the initial
phenomenon of the atherosclerotic leading to activation of the endothelium, and promoting
recruitment of monocytes and T cells. Monocytes in lesions differentiating into macrophages,
internalize modified lipoproteins. T cells recognize local antigens and accumulate T helper-1
responses contributing to local inflammation and plaque growth. Inflammatory activation
may lead to local proteolysis, plaque rupture, thrombus formation, ischemia and infarction
[49].
Specifically, higher prevalence of H. pylori infection was found particularly amongst
stroke patients in CagA-positive strains [60] due to pro-inflammatory cytokines leading to
accelerated atherosclerosis [61]. A potential mechanism of intima-media thickness of vessels
and carotid plaques is mainly linked with antibodies against CagA cross-reacting with
vascular endothelial antigens. [62,63]. Moreover, a higher prevalence of both CagA and
VacA-positive H. pylori was found in patients with idiopathic dysrhythmia consisting risk
factor for stroke [64]. Associations have been demonstrated between anti-H. pylori antibodies
and autoimmune conditions such as anti-phospholipid syndrome and giant cell arteritis
relating to pathogenesis of ischemic stroke. Whether there is causative role of H. pylori in
these autoimmune diseases or they represent epiphenomena remains doubtful [65].
H. pylori infection is more likely to be related to large or small vessel stroke as compared
to cardioembolic stroke and mainly in early stages of coronary atherosclerosis rather than
advanced coronary atherosclerosis [66]. Interestingly, two recent meta-analyses indicated that
chronic H. pylori infection was significantly associated with an increased risk of ischemic
stroke especially for non-cardioembolic origin [59,67]. Furthermore, case-control studies
reported an association between H. pylori infection and stroke. There is H. pylori
seropositivity associated with carotid plaques and cerebrovascular and cardiovascular events
in Italy [68] and Africa [69]; however, carotid atherosclerosis is not related to H. pylori
infection in a case-control study in the UK [70,71]. Importantly, it is suggested that treatment
of chronic infections as causal contributors to stroke could offer potential therapeutic targets
for stroke prevention [59,72].

Alzheimer's Disease (AD) and Mild Cognitive Impairment
AD is the most prevalent type of age-related dementia, and its effect on society increases
exponentially as the population ages. The exactly cause for AD is still essentially
unknown. Among several existing hypotheses trying to explain the cause of the disease
(Cholinergic, Amyloid, Tau hypothesis and genetic susceptibility) systemic infections has
also been proposed to play a causative role in AD’s neuroinflammation [73]. Recent
experimental data indicate that neuroinflammation, mediated by the brain’s innate immune
system, contributes to AD neuropathology and exacerbates the course of the disease [74].
This option is supported by positron emission tomography imaging studies, which have
shown that cognitive status in patients with AD is correlated with microglial activation
[75]. Moreover, there is a beneficial role of non-steroidal anti-inflammatory drugs in the
initial asymptomatic stages of the disease supporting the inflammatory face of the disease
[76]. Among systemic infections (herpes simplex virus type 1, picornavirus,
Borna disease virus, Chlamydia pneumoniae, spirochete), H. pylori has been proposed as
potential causative contributor to the pathogenesis or pathophysiology of brain cognitive
impairment, dementia, and sporadic AD. In this regard, recent case-control studies reported
an association between H. pylori infection and AD. Neuroinflammation and cerebrovascular
lesion load in a group of cognitive impairment and AD patients according to their H. pylori
status [77]. A high prevalence of H. pylori infection was found in patients with AD using
histology for diagnosis of H. pylori infection and detected higher levels of anti-H. pylori IgG
in the cerebrospinal fluid (CSF) of patients with AD compared to controls. Moreover, CSF
anti-H. pylori IgG antibodies correlated with the degree of severity of the neurological disease
[12,21,25,77]. Additionally, H. pylori infection might influence the apoptotic process,
promote platelet-leukocyte aggregation, increase homocysteine levels and damage the
endothelial lining of blood vessels influencing the pathophysiology of AD. The elevated
levels of homocysteine are linked to the mentioned atrophic gastritis which can lead to
malabsorption of vitamin B12 and folate resulting in failure of methylation by 5-methyl-
tetrahydrofolate [78]. A recently proposed causative mechanism is AD’s association with
Hpn, a histidine-rich protein abundant in H. pylori bacterium forming amyloid-like oligomers
in physiologically conditions, and possibly in AD [79]. Even European studies indicated the
association between H. pylori infection and AD [12,21,77]; in a Japanese cohort of patients
no association was found between H. pylori infection and AD [80] that may be due to the
relatively high H. pylori infection prevalence in the general Japanese population [80].
Importantly, H. pylori eradication may positively influence AD leading to a significant
improvement in the clinical manifestations, thereby suggesting a possible common link
between H. pylori and AD [22,23]; H. pylori infection seems to contribute to AD decline by
several mechanisms and H. pylori eradication regimen may improve the long-term survival
rate of the disease.
Parkinson's Disease (PD)
PD is a degenerative movement disorder of the CNS affecting more seriously the basal
ganglia, a group of brain structures innervated regularly by the dopaminergic system. Tremor,
rigidity, slowness in movement, and postural instability are the primarily motor symptoms

[81]. A number of studies have suggested that H. pylori may be accountable for idiopathic
parkinsonism [13,82]. H. pylori eradication showed improved stride length and maintenance
of gait, is prognostic indicator in established idiopathic parkinsonism [27,82] and is
implicated in Levo(L)-dopa absorption; H. pylori infection affects the absorption of L-dopa in
patients with PD and thereby worsening patients’ symptomatology [83]. Improvement in
motor function is found when H. pylori is eradicated due to better absorption of L-dopa [84].
Moreover, stable and long-lasting plasma L-dopa concentration was found in H. pylori-
eradicated patients by improving the pharmacokinetic and clinical response to L-dopa [84].
The above-mentioned studies suggest that H. pylori eradication may represent an
excellent therapeutic opportunity and can play an effective clinical role in PD patients by
improving symptomatology and by increasing the prolonged concentration of L-dopa [13,85].
Demyelinating Disorders
One of the most interesting neurological associations with H. pylori infection has been
with demyelinating diseases of the CNS such as MS and neuromyelitis optica (NMO).
MS is an autoimmune-mediated inflammatory disease of CNS, destroying the myelin and
the axon in variable degrees and producing significant physical disability. The hallmark of
MS is symptomatic episodes affecting different anatomic locations. Disease onset usually
occurs in young adults, and it is more common in women [86]. Some studies have suggested
that H. pylori may be a protective factor against MS in Japanese population [87]. Others have
associated H. pylori with NMO [88]. The variable role played by H. pylori in the
pathogenicity of MS may be predicated on ethnicity [8]. In this regard, by using histology,
recognized as the practical gold standard for the diagnosis of current H. pylori infection, our
series showed a strong association between H. pylori infection and MS Caucasian patients
[11]. Moreover, H. pylori may be the common denominator between systemic sclerosis and
MS [34,89]. It was recently shown that microbial peptides with limited sequence homology to
myelin basic protein can induce MS-like disease in mice humanized for a T cell receptor
derived from an MS patient [90]. H. pylori infection seems to be one of the risk factors for the
development of anti-aquaporin-4(+) (anti-AQP4) MS [44]. Genetics factors and H. pylori
infection are risk factors only for anti-AQP4 antibody positive NMO / NMO spectrum
disorder (NMOSD) but not for anti-AQP4 antibody negative NMO/NMOSD [89,91]. In this
regard, the mentioned H. pylori-induced BBB disruption might permit access of AQP4-
specific antibodies and T lymphocytes to the CNS, thereby playing a major role in NMO
pathogenesis [44]. In addition, H. pylori neutrophil-activating protein (NAP), as a virulence
factor, recruits leukocytes from the vascular lumen, and activates neutrophils, monocytes and
MCs. Moreover, an influx of mentioned H. pylori-infected monocytes owing to defective
autophagy resulting in H. pylori replication in autophagic vesicles, through the disrupted
BBB might lead to degeneration in the brain partially by potential activation of natural killer
cells and interferon-γ production, detrimental for MS. Therefore, H. pylori NAP and H. pylori
infection itself, by inducing several mediators, may influence MS/NMO pathophysiology,
thereby raising possible concerns regarding even the C-terminal region of H. pylori NAP use
as a candidate vaccine. Accordingly, relative studies are also needed to clarify the
aforementioned concerns [92].

Immunomodulatory properties of H. pylori (namely the H. pylori-SS1 antigen, common

in humans) was recently detected in mice in an experimental model of MS. This could be a
significant development in the understanding of the role played by H. pylori infection in
neurological diseases [93].
Migraine
Migraine is a chronic neurological disorder characterized by moderate to severe headache

and nausea. It affects 14% of women and 6% of men, and causes severe economic burdens
[94]. It has been shown that patients with H. pylori have a higher risk of migraine headache
compared to those without infection [95,96]. Eradication of H. pylori resulted in a significant
improvement in the clinical symptoms of migraine, a significant reduction in frequency,
density and duration of migraine attacks [97-99].
Several mechanisms have been proposed linking H. pylori infection with migraine
headache, including persistent inflammation and oxidative stress. Alterations of vascular
permeability, as a result of released vasoactive substances and superoxide radicals and nitric
oxide result in migraine headache [100-102]. Particularly, CagA-positive H. pylori strains are
strongly associated with migraine with aura. This was explained by a higher inflammatory
response of the gastric mucosa to more virulent strains, which release substances that may act
as triggers of vasospasm in peculiar cerebral arterial districts, probably implicated in the
“aura” phenomenon. This may explain the improvement in migraine headache following H.
pylori eradication [103,104].
Epilepsy
Epilepsy is a common and diverse group of chronic neurological disorders characterized

by epileptic seizures [105]. Several clinical and experimental data have implicated the failure
in BBB function in triggering chronic or acute seizures [106]. Other studies implicate the
interactions between a common blood protein, albumin and astrocytes [107]. It is speculated
that H. pylori infection, by inducing proinflammatory cytokines (mainly production of TNF-
α), leads to upregulation of matrix metalloproteinases that causes disruption of the BBB
[108,109]. TNF-α may lead to neuroinflammation and neuronal damage in epilepsy, thereby
triggering seizure induction and epilepsy progression [109-112]. Relative recent clinical data
suggest a probable association between H. pylori infection and epilepsy, influencing the
prognosis of epileptic patients. The epileptic patients with H. pylori showed poor prognosis as
compared with non-infected patients [113-115], although other studies do not support the role
of H. pylori infection in patients with epilepsy [111]. Eradication of H. pylori may lead to
improvement in neurological symptoms in epileptic patients [113]. However, further large-
scale studies are needed to elucidate in depth the role of H. pylori infection in the
pathophysiology of epilepsy.

Peripheral Nervous System (PNS) Neurological

Disorders and H. pylori
The PNS is protected by the BNB, which is more vulnerable than BBB of the CNS to
antibody mediated disorders because it is not as tight as the BBB. Anti-VacA IgG in the CSF
not only changes the permeability of the cell membrane through ion channel dysfunction, but
may also involve the axonal Schwann cell plasmalemma in some patients with GBS [116]. A
high rate of antibodies, particularly against native VacA were detected in the CSF obtained
from patients with GBS [5,117]. Moreover, the sequence homology found between H. pylori
VacA and human Na+/K+-ATPase A subunit suggests that antibodies to VacA involve ion
channels in axonal Schwann cell plasmalemma resulting in demyelination in some patients
[118].
H. pylori may play an important role in acute inflammatory demyelinating polyradiculo-
neuropathy and particularly in axonal type of GBS. H. pylori infection seems to be one of the
risk factors for the development of autoimmune neuropathies and molecular mimicry of host
structures by the saccharide portion of lipopolysaccharide of the GI pathogens Campylobacter
jejuni (C. jejuni) and H. pylori is considered to be associated with the development of
autoimmune sequelae in inflammatory neuropathies. Chemical analyses of the core
oligosaccharides of neuropathy-associated C. jejuni strains have revealed structural homology
with human gangliosides. H. pylori infection may be involved in the pathogenesis of
autoimmune neuropathies, and molecular mimicry serves as a possible underlying mechanism
[9,10,119,120]. Gangliosides can directly bind to H. pylori VacA. Even though their roles as
target receptors for the adhesion and entry of H. pylori remain to be investigated, it is clear
that the binding of gangliosides to VacA inhibits internalization of VacA and cell
intoxication [121].
Glaucomatous Neuropathy and H. pylori
Glaucoma is recognized as an optic neuropathy and is defined as “ocular” AD; it is a

chronic neurodegeneration of the optic nerve, the second cause, after cataract, of world
blindness and of major public health importance [7,122,123]. Selective death of retinal
ganglion cells (RGC) is the hallmark of glaucoma, also associated with structural changes in
the optic nerve head; RGCs’ death after axonal injury can be induced by a variety of different
stimuli [124]. The process of RGC death is thought to be biphasic: a primary injury
responsible for initiation of damage followed by a slower secondary degeneration related to
noxious environment surrounding the degenerating cells. A working knowledge of the
environmental risk factors for the induction and progression of the disease is essential to our
clinical practices and helps those patients at greater risk of disease progression and blindness;
a major priority is to achieve a better understanding of the risk factors, likely to involve gene-
environment interactions [16].
Evidence for the possibility of an infectious agent comes from reports in younger patients
with exfoliative glaucoma (XFG) after intraocular surgery or trauma with iris surgery in
infancy and childhood or after penetrating keratoplasty from elderly donors [125]. Moreover,
in the middle Norway eye-screening study, the prevalence of XFG in both members of

married couples is significantly higher than expected [125], thereby suggesting a common
environmental (probably infectious) agent, which may be of etiological significance for the
XFG development [126]. In this respect, Koch's postulates regarding a causal association with
a disease seem to apply in glaucoma [127], but, until recently, no one had associated any
microorganisms with glaucoma. Specifically, although degenerative diseases, including
glaucoma or AD, have an increasingly high impact on aged population, their association with
H. pylori infection has not been thoroughly researched. Current H. pylori infection, (via
molecular mimicry), cross-reacts with components of nerves, thereby contributing and
possibly perpetuating the apoptotic neural tissue damage observed in glaucoma. Moreover,
eradicating H. pylori infection might delay glaucoma progression, particularly at early disease
stages. However, before antibiotic therapy for H. pylori infection becomes an established step
in the management of glaucoma, sufficient evidence must be provided that glaucoma
parameters are positively influenced by the eradication of H. pylori infection.
In 2001, by using histology, representing the mentioned practical gold standard for H.
pylori infection diagnosis, we documented for the first time a high prevalence of H. pylori
infection in Greek patients with primary open-angle glaucoma (POAG) and XFG,
establishing a relationship between H. pylori infection and glaucoma [128]. These results may
indicate either a common factor that causes susceptibilities to both glaucoma and H. pylori
infection or that H. pylori may be a causal factor for developing glaucoma. In a subsequent
study [129], we reported a beneficial effect of H. pylori eradication on glaucoma progression,
suggesting a possible causal link between the bacterium and glaucoma; at the 2-year clinical
endpoint, glaucoma parameters (mean intraocular pressure and mean visual field parameters)
improved in the subgroup of patients where H. pylori eradication was successful (P<0.001 for
intraocular pressure; P≤0.01 for visual field parameters), but not in the rest of the patients.
Moreover, we reported an increased H. pylori-specific IgG antibody concentration in the
aqueous humor of patients with POAG and XFG; the concentration of this antibody
correlated with the degree of vertical cupping, possibly indicating the severity of
glaucomatous damage [130].
In a new study published in 2012 [131], we showed, for the first time, the significant
finding of the existence of H. pylori bacteria in the trabeculum and iris specimens of
glaucoma patients, thereby further supporting the role for H. pylori infection in the
pathophysiology of POAG. Specifically, the study included 51 consecutive patients who
underwent trabeculectomy for POAG not responsive to topical anti-glaucoma therapy. The
presence of H. pylori was established through an upper GI endoscopy and histology, or a urea
breath test in 8 patients, who were deemed not suitable endoscopy candidates or refused to
undergo an endoscopy. All the patients underwent a trabeculectomy surgical procedure in
their eyes, during which tissue samples from trabeculum, conjunctiva, and iris were
immediately obtained, were placed in tubes containing 10% formalin and submitted for
histologic examination. These specimens were stained with Cresyl fast violet stain (for
detection of H. pylori organisms). In 5 patients, in whom gastric H. pylori histology was
positive, we managed to identify for the first time histologically with Cresyl fast violet stain
H. pylori bacteria in the trabeculum and iris specimens. The reason why H. pylori was not
found in the trabeculum and iris of all POAG patients who were tested positive for H. pylori
status could be explained, apart from H. pylori absence in the eye, by the very small size of
the sample of tissue obtained and submitted for histopathology during the trabeculectomy, or
possibly by the standard local antisepsy prior to surgery. Despite the small number of cases,

the findings of this study are important because for the first time H. pylori bacteria have been
detected in the trabeculum and iris of POAG patients, proving that the bacterium is present
locally, and is possibly directly implicated in glaucomatous damage.
In our most recent study published in Immuno-Gastroenterology [132], we obtained
biopsy specimens on upper GI endoscopy from 43 POAG patients, which were then evaluated
for H. pylori presence, for expression of genes involved in cell proliferation and apoptosis
(Ki-67, p53, Bcl-2), and for indices of cellular immune surveillance [Τ-lymphocytes (TLs)
and B-lymphocytes (BLs)]. Of the 43 patients eligible for upper GI endoscopy, 90.7% were
tested positive for H. pylori infection. Ki-67 was positively expressed in 81.25% patients with
H. pylori infection and in 1 patient without H. pylori infection. p53 was positively expressed
in 31.25% patients with H. pylori infection but not in the patients without H. pylori infection.
Bcl-2 was positively expressed in 68.75% patients with H. pylori infection and in 1 patient
without H. pylori infection. Ki-67, p53 and Bcl-2 were over-expressed in 19%, 25% and
37.5%, respectively, of the patients with H. pylori infection, but none of them was over-
expressed in the patients without H. pylori infection. TLs marker was positively expressed in
all patients with H. pylori infection and in the one patient without H. pylori infection. BLs
marker was positively expressed only in one patient with this infection. Therefore, we
provided further insight that the H. pylori-induced oncogenes Ki-67, p53 and Bcl-2, and TLs
are involved in cell proliferation and apoptotic pathways thereby contributing to
glaucomatous neuropathy, with an additional oncogenic potential. In this respect, and apart
from the apoptotic processes involved in the pathophysiology of glaucomatous neuropathy,
recent experimental data indicate that cell proliferation rather than astrocyte hypertrophy,
characterizes early pressure-induced optic nerve head injury; optic nerve head is the principal
site of initial axonal injury in glaucoma. Furthermore, at the eye level, Ki-67 has been used as
an index of melanoma proliferation and as an index showing response to therapy with agents
that inhibit the proliferation of Tenon’s fibroblasts, decreasing the excessive scarring after
trabeculectomy [132].
Regarding the potential pathogenic mechanisms involved in H. pylori-related
glaucomatous neuropathy, we have provided an overview of the mentioned various
pathophysiologic mechanisms underlying the H. pylori infection and POAG association
[133,134], which include: promoting platelet and platelet-leukocyte aggregation also involved
in the pathophysiology of glaucoma; releasing proinflammatory and vasoactive substances,
including cytokines (IL -1, -6, -8, -10, -12, TNF-α, interferon-γ), eicosanoids (leukotrienes,
prostaglandins) and acute phase proteins (fibrinogen, C-reactive protein), involved in vascular
disorders and glaucoma; stimulating mononuclear cells to induce a tissue factor-like
procoagulant activity that converts fibrinogen into fibrin; causing the development of cross
mimicry between endothelial and H. pylori antigens; producing oxidative stress and
circulating lipid peroxides; and influencing mainly the apoptotic process, parameters that may
also exert their own effects in the induction and/or glaucoma progression and other
neurodegenerative disorders (GBS, AD, PD) associated both with H. pylori infection and
glaucoma. Importantly, H. pylori infection and glaucoma share the Fas/FasL and the
mitochondria-mediated apoptotic pathways, thereby proposing an apoptotic link in the
pathophysiology of both diseases. In particular, increased endothelin-1 (a potent constrictor of
arterioles and venules), nitric oxide (NO) and inducible NO synthase (iNOS) levels are
associated with H. pylori infection. Endothelin-1-induced anterior optic nerve vessels
vasoconstriction and NO vascular tone modulation in the ophthalmic artery may produce

glaucomatous damage. Moreover, NO, a rapidly diffusing gas, is a potent neurotoxin that may
facilitate the apoptotic RGC death in glaucomatous optic neuropathy. Support for the
consideration of NO neurotoxicity in glaucoma is provided by experimental evidence
demonstrating that RGC apoptosis is attenuated by neutralizing antibodies against TNF-α or
by selective inhibitors of iNOS, thereby suggesting that the inhibitors of TNF-α or of the
inducible isoform NOS2 may provide novel therapeutic targets for neuroprotection in the
treatment of glaucomatous optic neuropathy. In addition, systemic H. pylori-induced
oxidative damage may be the mechanism which links oxidative stress, H. pylori infection and
the apoptotic damage to the trabecular meshwork and optical nerve head that results in
glaucoma. In this regard, oxidative stress is an essential underlying cause of neuroin-
flammatory and neurodegenerative diseases including glaucoma and BBB damage connected
to them; oxidative stress activates protein tyrosine kinase and matrix metalloproteinases
resulting in BBB dysfunction [133,134].
Specifically, a series of factors have been implicated in inducing blood ocular barrier
(BOB) disruption, including inflammatory mediators (e.g., cytokines and chemokines induced
by H. pylori infection) and the mentioned oxidative stress [31,135]; TNF-α is involved in
BBB/BOB disruption through a mechanism involving matrix metalloproteinases’
upregulation [136]; BBB/BOB disruption contributes to brain neurodegenerative diseases
including glaucoma [20,21,137]. In addition, MCs, linked to H. pylori infection [30,31,138],
release mediators including histamine, IL-8, tryptase and VEGF, which disrupt the BBB [32].
Consequently, H. pylori-circulating antibodies might enter the aqueous circulation due to
BBB/BOB disruption possibly contributing to glaucoma pathophysiology; when serum-
specific antibodies access the brain, they are capable of killing RGCs [25,139], thereby
possibly contributing to glaucoma development and progression. BBB/BOB disruption could
also play an important role in promoting entry of immune cell infiltration and pathogens into
the brain resulting in the development of brain pathologies [140]; an influx of H. pylori-
infected monocytes, owing to the mentioned defective autophagy resulting in H. pylori
replication in autophagic vesicles, through the disrupted BBB/BOB might lead to glaucoma
neuropathy [31,134]. Besides, since the oral cavity might act as H. pylori permanent
reservoir, this bacterium may reach the eye through the nasal cavity, causing ophthalmic
pathologies possibly including glaucoma [31,134].
It is important to note that reports from other ethnic populations also showed a
relationship between glaucoma and H. pylori infection [141], although this has not been
confirmed by all the relevant studies published so far [141] and thus this association applies
to a world's sub-population of glaucoma patients; consistent associations with the Greek data
were shown in studies from China, Korea, India, Turkey, and Iran [142].
In view of the aforementioned data, eradication of H. pylori infection may benefit the
glaucomatous optic neuropathy (which accounts for approximately 90.8 million affected
individuals) by ameliorating mainly the apoptotic loss of RGCs and their axons, and the
progressive loss of visual field sensitivity, thereby suggesting its future role in glaucoma
neuroprotection [133].

Conclusion
Neurological diseases, in general, are multifactorial disorders; genetic, epigenetic and
environmental factors contribute for their clinical phenotypes. The illustration of immune
properties of H. pylori in the pathogenic mechanism of neurological diseases with an
immunological background may offer clarity. Until now it remains under investigation
whether H. pylori participates positively as causative factor in diverse neurological diseases
(AD, cerebrovascular disease, demyelinating disorders, migraine, parkinsonism, GBS, and
glaucoma) or immunological implications of this enigmatic bacterium is an epiphenomenon.
Even in the recent Maastricht IV consensus guidelines for the management of H. pylori
infection published last year, it is stated that the evidence available shows unclear causative
association between H. pylori and other extragastric disorders [143,144], the possible role
of H. pylori as a trigger for some extragastric diseases has been largely investigated and there
are definitely many studies concerning neurological disorders with interesting results. The
positive associations could possibly support the notion that in susceptible individuals H.
pylori infection may influence the process of diseases. The final acceptance of H. pylori as a
neurological pathogen requires more clinical studies as well as in vivo and in vitro
experimental studies to evaluate the potential role of this gastric bacterium. The long-standing
effects of H. pylori eradication therapy on the course of neurological disorders merit further
investigation.
References
[1] Banić M, Franceschi F, Babić Z, et al. (2012). Extragastric manifestations of
Helicobacter pylori infection. Helicobacter.,17:49-55.
[2] Suzuki H, Franceschi F, Nishizawa T, et al. (2011). Extragastric manifestations of
Helicobacter pylori infection. Helicobacter., Sep;16 Suppl 1:65-9.
[3] Malfertheiner P, Selgrad M, Bornschein J, (2012). Helicobacter pylori: clinical
management. Curr Opin Gastroenterol., 28:608-14.
[4] Chiba S, Sugiyama T, Matsumoto H, et al. (1998). Antibodies against Helicobacter
pylori were detected in the cerebrospinal fluid obtained from patients with Guillain-
Barre syndrome. Ann Neurol., 44:686-8.
[5] Chiba S, Sugiyama T, Yonekura K, et al. (2002). An antibody to VacA of Helicobacter
pylori in cerebrospinal fluid from patients with Guillain-Barre syndrome. J Neurol
Neurosurg Psych., 73:76-8.
[6] Annunziata P, Figura N, Galli R, et al. (2003). Association of anti-GM1 antibodies but
not of anti-cytomegalovirus, Camphylobacter jejuni and Helicobacter pylori IgG, with
a poor outcome in Guillain-Barre syndrome. J Neurol Sci., 213:55-60.
[7] Kountouras J, Gavalas E, Zavos C, et al. (2007). Alzheimer’s disease and Helicobacter
pylori infection: defective immune regulation and apoptosis as proposed common links.
Med Hypotheses., 8:378-88.
[8] Kountouras J, Zavos C, Gavalas E, et al. (2008). Helicobacter pylori may hold a
variable role in multiple sclerosis based on ethnicity. Med Hypotheses., 71:614-5.

[9] Kountouras J, Deretzi G, Zavos C, et al. (2005). Association between Helicobacter

pylori infection and acute inflammatory demyelinating polyradiculoneuropathy. Eur J
Neurol., 12:139-43.
[10] Kountouras J, Deretzi G, Grigoriadis N, et al. (2008). Guillain-Barré syndrome. Lancet
Neurol., 7:1080-1.
[11] Gavalas E, Kountouras J, Deretzi G, et al. (2007). Helicobacter pylori and multiple
sclerosis. J Neuroimmunol., 188:187-9.
[12] Kountouras J, Tsolaki M, Gavalas E, et al. (2006). Relationship between Helicobacter
pylori infection and Alzheimer disease. Neurology., 66:938-40.
[13] Nielsen HH, Qiu J, Friis S, et al. (2012). Treatment for Helicobacter pylori infection
and risk of Parkinson's disease in Denmark. Eur J Neurol., 19:864-9.
[14] Deretzi G, Kountouras J, Polyzos SA, et al. (2011). Gastrointestinal immune system
and brain dialogue implicated in neuroinflammatory and neurodegenerative diseases.
Curr Mol Med., 11:696-707.
[15] Deretzi G, Kountouras J, Grigoriadis N, et al. (2009). From the "little brain"
gastrointestinal infection to the "big brain" neuroinflammation: a proposed fast axonal
transport pathway involved in multiple sclerosis. Med Hypotheses., 73:781-7.
[16] Kountouras J, (2009). Helicobacter pylori: an intruder involved in conspiring
glaucomatous neuropathy. Br J Ophthalmol., 93:1413-5.
[17] Perry VH, (2010). Contribution of systemic inflammation to chronic
neurodegeneration. Acta Neuropathol., 120:277-8.
[18] Diomedi M, Stanzione P, Sallustio F, et al. (2008). Cytotoxin-associated Gene-A-
positive Helicobacter pylori strains infection increases the risk of recurrent
atherosclerotic stroke. Helicobacter., 13:525-31.
[19] Guerreiro RJ, Santana I, Brás JM, et al. (2007). Peripheral inflammatory cytokines as
biomarkers in Alzheimer's disease and mild cognitive impairment. Neurodegener Dis.,
4:406-12.
[20] Tan ZS, Beiser AS, Vasan RS, et al. (2007). Inflammatory markers and the risk of
Alzheimer disease: the Framingham Study. Neurology., 68:1902-8.
[21] Honjo K, van Reekum R, Verhoeff NP, (2009). Alzheimer's disease and infection: do
infectious agents contribute to progression of Alzheimer's disease? Alzheimers Dement.,
5:348-60.
[22] Kountouras J, Boziki M, Gavalas E, et al. (2009) Eradication of Helicobacter pylori
may be beneficial in the management of Alzheimer's disease. J Neurol., 256:758-67.
[23] Kountouras J, Boziki M, Gavalas E, et al. (2010). Five-year survival after Helicobacter
pylori eradication in Alzheimer disease patients. Cogn Behav Neurol., 23: 199-204.
[24] Kountouras J, Tsolaki M, Boziki M, et al. (2007). Association between Helicobacter
pylori infection and mild cognitive impairment. Eur J Neurol., 14:976-82.
[25] Kountouras J, Boziki M, Gavalas E, et al. (2009). Increased cerebrospinal fluid
Helicobacter pylori antibody in Alzheimer's disease. Int J Neurosci., 119:765-77.
[26] Dobbs RJ, Dobbs SM, Weller C, et al. (2008). Helicobacter hypothesis for idiopathic
parkinsonism: before and beyond. Helicobacter., 13:309-22.
[27] Dobbs SM, Dobbs RJ, Weller C, et al. (2010). Differential effect of Helicobacter pylori
eradication on time-trends in brady/hypokinesia and rigidity in idiopathic parkinsonism.
Helicobacter., 15:279-94.

[28] Charlett A, Dobbs RJ, Dobbs SM, et al. (2009). Blood profile holds clues to role of
infection in a premonitory state for idiopathic parkinsonism and of gastrointestinal
infection in established disease. Gut Pathog., 1:20.
[29] Supajatura V, Ushio H, Wada A, et al. (2002). Cutting edge: VacA, a vacuolating
cytotoxin of Helicobacter pylori, directly activates mast cells for migration and
production of proinflammatory cytokines. J Immunol., 168:2603-7.
[30] Kountouras J, Zavos C, Diamantidis MD, et al. (2009). A concept of Helicobacter
pylori and stress-secreted mast cells' potential involvement in brain metastases. J
Neuroimmunol., 209:121-2.
[31] Kountouras J, Zavos C, Chatzopoulos D, et al. (2008). New aspects of Helicobacter
pylori infection involvement in gastric oncogenesis. J Surg Res., 146:149-58.
[32] Theoharides TC, Rozniecki JJ, Sahagian G, et al. (2008). Impact of stress and mast cells
on brain metastases. J Neuroimmunol., 205:1-7.
[33] Pfeiffer RF, (2009). Neuroinflammation and Parkinson disease: the silent battleground.
Neurology., 73:1434-5.
[34] Kountouras J, Gavalas E, Deretzi G, et al. (2010). Helicobacter pylori with or without
its neutrophil-activating protein may be the common denominator associated with
multiple sclerosis and neuromyelitis optica. Mult Scler., 16:376-7.
[35] Terebiznik MR, Raju D, Vázquez CL, et al. (2009). Effect of Helicobacter pylori's
vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells. Autophagy.,
5:370-9.
[36] Raju D, Jones NL, (2010). Methods to monitor autophagy in H. pylori vacuolating
cytotoxin A (VacA)-treated cells. Autophagy., 6:138-43.
[37] Wang YH, Wu JJ, Lei HY, (2009). When Helicobacter pylori invades and replicates in
the cells. Autophagy., 5: 540-2.
[38] Deen NS, Huang SJ, Gong L, et al. (2013). The impact of autophagic processes on the
intracellular fate of Helicobacter pylori: more tricks from an enigmatic pathogen?
Autophagy., 9:639-52
[39] Momtaz H, Souod N, Dabiri H, et al. (2012). Study of Helicobacter pylori genotype
status in saliva, dental plaques, stool and gastric biopsy samples. World J
[40] Niv Y, (2008). H. pylori recurrence after successful eradication. World J
[41] Včeva A, Danić D, Včev A, et al. (2012). The significance of Helicobacter pylori in
patients with nasal polyposis. Med Glas (Zenica)., 9:281-6.
[42] Saccà SC, Pascotto A, Venturino GM, et al. (2006). Prevalence and treatment of
Helicobacter pylori in patients with blepharitis. Invest Ophthalmol Vis Sci., 47:501-8.
[43] Targosz A, Brzozowski T, Pierzchalski P, et al. (2012). Helicobacter pylori promotes
apoptosis, activates cyclooxygenase (COX)-2 and inhibits heat shock protein HSP70 in
gastric cancer epithelial cells. Inflamm Res., 61:955-66.
[44] Li W, Minohara M, Piao H, et al. (2009). Association of anti-Helicobacter pylori
neutrophil-activating protein antibody response with anti-aquaporin-4 autoimmunity in
Japanese patients with multiple sclerosis and neuromyelitis optica. Mult Scler.,
15:1411-21.
[45] Thounaojam MC, Kaushik DK, Basu A, (2013). MicroRNAs in the brain: its regulatory
role in neuroinflammation. Mol Neurobiol., (in press)

[46] Sfriso P, Ghirardello A, Botsios C, et al. (2010). Infections and autoimmunity: the
multifaceted relationship. J Leukoc Biol., 87:385-95.
[47] Moran AP, Prendergast MM, (2001). Molecular mimicry in Campylobacter jejuni and
Helicobacter pylori lipopolysaccharides: contribution of gastrointestinal infections to
autoimmunity. J Autoimmun., 16:241-56.
[48] Ross R, (1999). Atherosclerosis--an inflammatory disease. N Engl J Med., 340:115-26.
[49] Hansson GK, (2009). Atherosclerosis--an immune disease: the Anitschkov Lecture
2007. Atherosclerosis., 202:2-10.
[50] Elkind MS, Luna JM, Moon YP, et a, (2010)l. Infectious burden and carotid plaque
thickness: the northern Manhattan study. Stroke., 41:e117-22.
[51] Kountouras J, Zavos C, Deretzi G, et al. (2011). Helicobacter pylori may be involved in
stroke pathophysiology by altering tumor necrosis factor-α and matrix
metalloproteinases. Eur J Neurol., 18:e76.
[52] Carter AM, Braunholtz D, Catto AJ, (2003). Helicobacter pylori infection in subjects
with acute ischaemic stroke. Dig Liver Dis., 35:16-9.
[53] Grau AJ, Buggle F, Lichy C, et al. (2001). Helicobacter pylori infection as an
independent risk factor for cerebral ischemia of atherothrombotic origin. J Neurol Sci.,
186:1-5.
[54] Heuschmann PU, Neureiter D, Gesslein M, et al. (2001). Association between infection
with Helicobacter pylori and Chlamydia pneumoniae and risk of ischemic stroke
subtypes: results from a population-based case-control study. Stroke., 32:2253-8.
[55] Nazmi A, Diez-Roux AV, Jenny NS, et al. (2010). The influence of persistent
pathogens on circulating levels of inflammatory markers: a cross-sectional analysis
from the Multi-Ethnic Study of Atherosclerosis. BMC Public Health., 10:706.
[56] Farsak B, Yildirir A, Akyon Y, et al. (2000). Detection of Chlamydia pneumoniae and
Helicobacter pylori DNA in human atherosclerotic plaques by PCR. J Clin Microbiol.,
38:4408-11.
[57] Haider AW, Wilson PW, Larson MG, et al. (2002). The association of seropositivity to
Helicobacter pylori, Chlamydia pneumoniae, and cytomegalovirus with risk of
cardiovascular disease: a prospective study. J Am Coll Cardiol., 40:1408-13.
[58] Corrado E, Rizzo M, Tantillo R, et al. (2006). Markers of inflammation and infection
influence the outcome of patients with baseline asymptomatic carotid lesions: a 5-year
follow-up study. Stroke., 37:482-6.
[59] Grau AJ, Urbanek C, Palm F, (2010). Common infections and the risk of stroke. Nat
Rev Neurol., 6:681-94.
[60] De Bastiani R, Gabrielli M, Ubaldi E, et al. (2008). High prevalence of Cag-A positive
H. pylori strains in ischemic stroke: a primary care multicenter study. Helicobacter.,
13:274-7.
[61] Mayr M, Kiechl S, Mendall MA, et al. (2003) Increased risk of atherosclerosis is
confined to CagA-positive Helicobacter pylori strains: prospective results from the
Bruneck study. Stroke., 34:610-5.
[62] Franceschi F, Sepulveda AR, Gasbarrini A, et al. (2002). Cross-reactivity of anti-caga
antibodies with vascular wall antigens: possible pathogenic link between Helicobacter
pylori infection and atherosclerosis. Circulation., 106:430-4.
[63] Pietroiusti A, (2002). Role of CagA positive Helicobacter pylori strains in ischemic
heart disease and in ischemic stroke. Ital Heart J., 3:626-8.

[64] Franceschi F, Brisinda D, Buccelletti F, et al. (2013). Prevalence of virulent

Helicobacter pylori strains in patients affected by idiopathic dysrhythmias. Intern
Emerg Med., (in press)
[65] Ram M, Barzilai O, Shapira Y, et al. (2013). Helicobacter pylori serology in
autoimmune diseases - fact or fiction? Clin Chem Lab Med., 51:1075-82.
[66] Park MJ, Choi SH, Kim D, et al. (2011). Association between Helicobacter pylori
seropositivity and the coronary artery calcium score in a screening population. Gut
Liver., 5:321-7.
[67] Wang ZW, Li Y, Huang LY, et al. (2012). Helicobacter pylori infection contributes to
high risk of ischemic stroke: evidence from a meta-analysis J Neurol., 259:2527-37.
[68] Spence JD, Norris J, (2003). Infection, inflammation, and atherosclerosis. Stroke.,
34:333-4.
[69] Longo-Mbenza B, Nsenga JN, Mokondjimobe E, et al. (2012). Helicobacter pylori
infection is identified as a cardiovascular risk factor in Central Africans. Vasc Health
Risk Manag., 6:455-61.
[70] Mayr M, Kiechl S, Tsimikas S, et al. (2006). Oxidized low-density lipoprotein
autoantibodies, chronic infections, and carotid atherosclerosis in a population-based
study. J Am Coll Cardiol., 47:2436-43.
[71] Yang X, Gao Y, Zhao X, et al. (2011). Chronic Helicobacter pylori infection and
ischemic stroke subtypes. Neurol Res., 33:467-72.
[72] van de Beek D, (2013). Stroke: long-term effect of infections after stroke. Nat Rev
Neurol., 9:126-7.
[73] Itzhaki RF, Wozniak MA, (2008). Herpes simplex virus type 1 in Alzheimer's disease:
the enemy within. J Alzheimers Dis., 393-405.
[74] Krstic D, Madhusudan A, Doehner J, et al. (2012). Systemic immune challenges trigger
and drive Alzheimer-like neuropathology in mice. J Neuroinflammation., 9:151.
[75] Edison P, Archer HA, Gerhard A, et al. (2008). Microglia, amyloid, and cognition in
Alzheimer's disease: an [11C](R)PK11195-PET and [11C]PIB-PET study. Neurobiol
Dis.,32:412-9.
[76] Breitner JC, Baker LD, Montine TJ, et al. (2011). ADAPT Research Group. Extended
results of the Alzheimer's disease anti-inflammatory prevention trial. Alzheimers
Dement., 7:402–11.
[77] Roubaud-Baudron C, Krolak-Salmon P, Quadrio I, et al. (2012). Impact of chronic
Helicobacter pylori infection on Alzheimer's disease: preliminary results. Neurobiol
Aging., 33:1009.e11-9.
[78] Ozer B, Serin E, Gumurdulu Y, et al. (2005). Helicobacter pylori eradication lowers
serum homocysteine level in patients without gastric atrophy. World J Gastroenterol.,
11:2764-7.
[79] Ge R, Sun X, (2011). The in vivo functions of a histidine-rich protein Hpn in
Helicobacter pylori: linking gastric and Alzheimer's diseases together? Med
Hypotheses., 77:788-90.
[80] Kountouras J, Zavos C, Boziki M, et al. (2011). Association between Helicobacter
pylori infection and Alzheimer’s disease in Japan. J Neurol., 258:2086.
[81] Obeso JA, Rodríguez-Oroz MC, Benitez-Temino B, et al. (2008). Functional
organization of the basal ganglia: therapeutic implications for Parkinson's disease. Mov
Disord., 23 (Suppl 3):S548-59.

[82] Dobbs RJ, Charlett A, Dobbs SM, et al. (2012). Towards defining a rigidity-associated
pathogenic pathway in idiopathic parkinsonism. Neurodegener Dis., 10:183-6.
[83] Lyte M, (2010). Microbial endocrinology as a basis for improved L-DOPA
bioavailability in Parkinson's patients treated for Helicobacter pylori. Med Hypotheses.,
74:895-7.
[84] Pierantozzi M, Pietroiusti A, Brusa L, et al. (2006). Helicobacter pylori eradication and
l-dopa absorption in patients with PD and motor fluctuations. Neurology., 66:1824-9.
[85] Kountouras J, Zavos C, Polyzos SA, et al. (2012). Helicobacter pylori infection and
Parkinson's disease: apoptosis as an underlying common contributor. Eur J Neurol.,
19:56.
[86] Compston A, Coles A, (2008). Multiple sclerosis. Lancet., 372:1502-17.
[87] Li W, Minohara M, Su JJ, et al. (2007). Helicobacter pylori infection is a potential
protective factor against conventional multiple sclerosis in the Japanese population. J
Neuroimmunol., 184:227-31.
[88] Long Y, Gao C, Qiu W, et al. (2013). Helicobacter pylori infection in neuromyelitis
optica and multiple sclerosis. Neuroimmunomodulation., 20:107-12.
[89] Kountouras J, Zavos C, Gavalas E, et al. (2011). Helicobacter pylori may be a common
denominator associated with systemic and multiple sclerosis. Joint Bone Spine., 78:222-
3.
[90] Harkiolaki M, Holmes SL, Svendsen P, et al. (2009). T cell-mediated autoimmune
disease due to low-affinity cross reactivity to common microbial peptides. Immunity.,
30:348-57.
[91] Yoshimura S, Isobe N, Matsushita T, et al. (2013). South Japan Multiple Sclerosis
Genetics Consortium. Distinct genetic and infectious profiles in Japanese neuromyelitis
optica patients according to anti-aquaporin 4 antibody status. J Neurol Neurosurg
Psychiatry., 84:29-34.
[92] Kountouras J, Zavos C, Deretzi G, et al. (2012). Potential implications of Helicobacter
pylori-related neutrophil-activating protein. World J Gastroenterol., 18:489-90.
[93] Boziki M, Grigoriadis N, Deretzi G, et al. (2012). Helicobacter pylori
immunomodulative properties in a mouse model of multiple sclerosis.
Immunogastroenterology., 1:34-9.
[94] Kemper RH, Meijler WJ, Korf J, et al. (2001). Migraine and function of the immune
system: a meta-analysis of clinical literature published between 1966 and 1999.
Cephalalgia., 21:549-57.
[95] Hosseinzadeh M, Khosravi A, Saki K, et al. (2011). Evaluation of Helicobacter pylori
infection in patients with common migraine headache. Arch Med Sci., 7:844-9.
[96] Ciancarelli I, Di Massimo C, Tozzi-Ciancarelli MG, et al. (2002). Helicobacter pylori
infection and migraine. Cephalalgia., 22:222-5.
[97] Gasbarrini A, De Luca A, Fiore G, et al. (1998). Beneficial effects of Helicobacter
pylori eradication on migraine. Hepatogastroenterology., 45:765-70.
[98] Hong L, Zhao Y, Han Y, et al. (2007). Reversal of migraine symptoms by Helicobacter
pylori eradication therapy in patients with hepatitis-B-related liver cirrhosis.
[99] Faraji F, Zarinfar N, Zanjani AT, et al. (2012). The effect of Helicobacter pylori
eradication on migraine: a randomized, double blind, controlled trial. Pain Physician.,
15:495-8.

[100] Kim YJ, Kim EH, Hahm KB, (2012). Oxidative stress in inflammation-based
gastrointestinal tract diseases: challenges and opportunities. J Gastroenterol Hepatol.,
27:1004-10.
[101] Tunca A, Ardicoglu Y, Kargili A, et al. (2007). Migraine, Helicobacter pylori, and
oxidative stress. Helicobacter., 12:59-62.
[102] Ashina M, (2012). Vascular changes have a primary role in migraine. Cephalalgia.,
32:428-30.
[103] Gasbarrini A, Gabrielli M, Fiore G, et al. (2000). Association between Helicobacter
pylori cytotoxic type I CagA-positive strains and migraine with aura. Cephalalgia.,
20:561-5.
[104] Gasbarrini G, Racco S, Franceschi F, Miele L, Cammarota G, et al. (2010).
Helicobacter pylori infection: from gastric to systemic disease. Recenti Prog Med.,
101:27-33.
[105] Chang BS, Lowenstein DH, (2003). Epilepsy. N Engl J Med., 349:1257-66.
[106] Oby E, Janigro D, (2006). The blood-brain barrier and epilepsy. Epilepsia., 47:1761-74.
[107] Ivens S, Kaufer D, Flores LP, et al. (2007). "TGF-beta receptor-mediated albumin
uptake into astrocytes is involved in neocortical epileptogenesis. Brain., 130(Pt 2):535-
47.
[108] Yin P, Yang L, Zhou HY, et al. (2011). Matrix metalloproteinase-9 may be a potential
therapeutic target in epilepsy. Med Hypotheses., 76:184-6.
[109] Deretzi G, Kountouras J, Gavalas E, et al. (2011). Multiple sclerosis and seizures:
possible role of Helicobacter pylori. Eur J Neurol., 18:116.
[110] Kountouras J, Deretzi G, Zavos C, et al. (2012). Helicobacter pylori's potential
association with epilepsy. Seizure., 21:151.
[111] Asadi-Pooya AA, Dehghani SM, Petramfar P, et al. (2012). Helicobacter pylori
infection in patients with epilepsy. Seizure., 21:21-3.
[112] Kountouras J, Zavos C, Deretzi G, et al. (2011). Helicobacter pylori might be a
potential therapeutic target in epilepsy. Med Hypotheses., 76:763.
[113] Ozturk A, Ozturk CE, Ozdemirli B, et al. (2007). Helicobacter pylori infection in
epileptic patients. Seizure., 16:147-52.
[114] Okuda M, Miyashiro E, Nakazawa T, et al. (2004). Helicobacter pylori infection and
idiopathic epilepsy. Am J Med., 16:209-10.
[115] Razeem M, Razak A, Tan HJ, et al. (2012). Prevalence of Helicobacter pylori infection
in epilepsy patients in a teaching hospital in Malaysia. Neurol Asia., 17:293-6.
[116] Chavada G, Willison HJ, (2012). Autoantibodies in immune-mediated neuropathies.
Curr Opin Neurol., 25:550-5.
[117] Chiba S, Sugiyama T, Yonekura K, et al. (2004). An antibody to VacA of Helicobacter
pylori in the CSF of patients with Miller-Fisher syndrome. Neurology., 63:2184-6.
[118] Ghabaee M, Ghanbarian D, Brujeni GN, et al. (2010). Could Helicobacter pylori play
an important role in axonal type of Guillain-Barré syndrome pathogenesis? Clin Neurol
Neurosurg., 112:193-8.
[119] Kountouras J, Zavos C, Deretzi G, et al. (2011). Helicobacter pylori may play an
important role in both axonal type Guillain-Barré syndrome and acute inflammatory
demyelinating polyradiculoneuropathy. Clin Neurol Neurosurg., 113:520.

[120] Kountouras J, Deretzi G, Zavos C, et al. (2011). Helicobacter pylori infection may
trigger Guillain-Barré syndrome, Fisher syndrome and Bickerstaff brainstem
encephalitis. J Neurol Sci., 305:167-8.
[121] Wada A, Hasegawa M, Wong PF, et al. (2010). Direct binding of gangliosides to
Helicobacter pylori vacuolating cytotoxin (VacA) neutralizes its toxin activity.
Glycobiology., 20:668-78.
[122] Johnson GJ, Foster PJ, (2005). Can we prevent angle-closure glaucoma? Eye., 19:1119-
24.
[123] McKinnon SJ, (2003). Glaucoma: ocular Alzheimer’s disease? Front Biosci., 8:s1140-
56.
[124] Kountouras J, Zavos C, Chatzopoulos D, (2004). Induction of apoptosis as a proposed
pathophysiological link between glaucoma and Helicobacter pylori infection. Med
Hypotheses., 62:378-81.
[125] Ritch R, (2001). Perspective on exfoliation syndrome. J Glaucoma., 10(5 Suppl 1):S33-
5.
[126] Ringvold A, (1999). Epidemiology of the pseudo-exfoliation syndrome. Acta
Ophthalmol Scand., 77:371-5.
[127] Van Buskirk EM, (2003). Aphorisms of the hero. J Glaucoma., 12:189-92.
[128] Kountouras J, Mylopoulos N, Boura P, et al. (2001). Relationship between
Helicobacter pylori infection and glaucoma. Ophthalmology., 108:599-604.
[129] Kountouras J, Mylopoulos N, Chatzopoulos D, et al. (2002). Eradication of
Helicobacter pylori may be beneficial in the management of chronic open-angle
glaucoma. Arch Intern Med., 162:1237-44.
[130] Kountouras J, Mylopoulos N, Konstas AG, et al. (2003). Increased levels of
Helicobacter pylori IgG antibodies in aqueous humor of patients with primary open-
angle and exfoliation glaucoma. Graefes Arch Clin Exp Ophthalmol., 241:884-90.
[131] Zavos C, Kountouras J, Sakkias G, Venizelos I, Deretzi G, Arapoglou S, (2012).
Histological presence of Helicobacter pylori bacteria in the trabeculum and iris of
patients with primary open-angle glaucoma. Ophthalmic Res., 47:150-6.
[132] Zavos C, Kountouras J, Sakkias G, et al. (2012). Oncogenes’ expression in Greek
patients with primary open-angle glaucoma in association with Helicobacter pylori
status. Immunogastroenterology., 1:40-6.
[133] Kountouras J, Zavos C, Deretzi G, et al. (2011). Neuroprotection in glaucoma: is there a
future role of Helicobacter pylori eradication? Exp Eye Res., 92:436-8.
[134] Zavos C, Kountouras J, (2012). Further data on the association between Helicobacter
pylori infection and primary open-angle glaucoma. Clin Ophthalmol., 6:243-5.
[135] Keep RF, Xiang J, Ennis SR, et al. (2008). Blood-brain barrier function in intracerebral
hemorrhage. Acta Neurochir., Suppl. 105:73-7.
[136] Candelario-Jalil E, Taheri S, Yang Y, et al. (2007). Cyclooxygenase inhibition limits
blood-brain barrier disruption following intracerebral injection of tumor necrosis factor-
alpha in the rat. J Pharmacol Exp Ther., 323:488-98.
[137] Mendall MA, Patel P, Asante M, et al. (1997). Relation of serum cytokine
concentrations to cardiovascular risk factors and coronary heart disease. Heart., 78:273-
7.
[138] Kountouras J, Zavos C, Chatzopoulos D, (2004). Primary open-angle glaucoma:
pathophysiology and treatment. Lancet., 364:1311-2.

[139] Itzhaki RF, Wozniak MA, Appelt DM, et al. (2004). Infiltration of the brain by
pathogens causes Alzheimer’s disease. Neurobiol Aging., 25:619-2.
[140] Izzotti A, Saccà SC, Bagnis A, et al. (2009). Glaucoma and Helicobacter pylori
infection. Correlations and controversies. Br J Ophthalmol., 93:1420-7.
[141] Kountouras J, Zavos C, Katsinelos P, Vardaka E, (2012). Glaucoma and Helicobacter
pylori Dig Liver Dis., 44:962-3.
[142] Kountouras J, Zavos C, Deretzi G, (2009). Primary open-angle glaucoma. N Engl J
Med., 360:2679.
[143] Malfertheiner P, Megraud F, O’Morain CA, et al. (2012). Management of Helicobacter
pylori infection--the Maastricht IV/Florence Consensus Report. Gut., 61:646-64.
[144] O’Connor A, O’Morain CA, (2012). Neurological manifestations of Helicobacter
pylori infection: epiphenomenon or immunologic incident? Immunogastroenterology.,
1:5-6.

Chapter 12

Autoimmune and Rheumatological
Disorders
Sarfaraz Ahmed Hasni*

National Institute of Arthritis, Musculoskeletal and Skin diseases,
National Institutes of Health, Bethesda, MD, US
Abstract
Helicobacter pylori (H. pylori) can colonize human gastric mucosa chronically and
evade eradication by its manipulation of host immune responses. This chronic immune
system modulation can potentially trigger an immune response against self-antigens
leading to autoimmunity in a genetically susceptible host. Multiple studies by using
animal models and in-vitro experiment have attempted to provide plausible mechanisms
through which H. pylori can cause autoimmunity. There is epidemiological data showing
a positive association between H. pylori infection with certain autoimmune diseases.
Eradication of H. pylori infection was shown to be associated with better disease
outcomes in case of some autoimmune diseases but the evidence is not very strong. There
is also evidence suggesting advantageous effects of chronic H. pylori infection. Presence
of H. pylori provides protection against allergic and autoimmune diseases as shown by
some population based studies.
Role of H. pylori infection in autoimmune diseases is intriguing but controversial
with some evidence implicating it as a trigger for autoimmunity whereas other studies
indicate its protective effect against autoimmune diseases.
Keywords: autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus,

antiphospholipid antibody syndrome, Sjögren’s syndrome, Systemic sclerosis, Vasculitis,
IgG4 associated diseases, polymyositis, dermatomyositis, fibromyalgia
*
email: hasnisa@mail.nih.gov.

224 Sarfaraz Ahmed Hasni
Introduction
Autoimmune diseases share a common underlying pathogenic mechanism of loss of self-
tolerance by the immune system. This activation of immune system against self-antigen
resulting in autoimmunity is triggered by environmental factors in a genetically susceptible
host [1]. Most of the studies looking at genetic susceptibility for autoimmune diseases reveal
that high risk alleles only confer a slight increase in risk of development of autoimmune
diseases. Genome wide association studies (GWAS) of the autoimmune diseases have
identified close to 400 single nucleotide polymorphisms (SNPs) associations with
autoimmune diseases [2]. However, these SNP associations only modestly increase the odds
of developing autoimmune diseases. Several epidemiological studies looking at the
prevalence of autoimmune diseases in cohorts of both identical and non-identical twins reveal
that the concordance rate of autoimmune diseases is much higher in the identical twins
compared to the non-identical twins [3]. The concordance rate among identical twins in
patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) is reported
to be 33% and 12.3% respectively; compared to the concordance rate of 0% in SLE and 3.5%
in RA among non-identical twins [4]. These findings suggest that the genetic susceptibility to
develop autoimmunity is based on a polygenic model.
Hence, genetic makeup of an individual plays a definite but small part in his/her
susceptibility to develop autoimmune disease. This led to the search for additional non-
genetic (environmental) triggers of autoimmunity. Recently an expert panel from the National
Institute of Environmental Health Sciences published their review of the role of various
environmental exposures in the development of human autoimmune diseases [5]. There are
host of environmental factors implicated in triggering of autoimmune diseases, ranging from
chemicals such as silica, asbestos, pesticides, smoking; physical factors such as ionizing
radiation and biologic agents diet, food and infections. The autoimmune diseases result from
interactions between these multiple environmental factors and the immune system in a
genetically high risk individual.
Role of Infectious Agents in Pathogenesis

of Autoimmune Diseases
Infectious diseases agents are ubiquitously present in our environment and extensively
interact with the immune system making them an ideal candidate to be considered in
pathogenesis of rheumatologic illnesses. Numerous microbial agents including viruses,
bacteria and fungi have all been implicated as possible triggers of autoimmunity. Studies done
in the animal models of autoimmune diseases provide strong indication of infections as
environmental triggers of autoimmunity [6]. In addition the significant role of interferon-α in
some autoimmune diseases is also suggestive of an infectious etiology [7]. There are several
proposed mechanisms through which these microbial agents can lead to autoimmunity, such
as molecular mimicry, bystander activation of autoreactive cells and epitope spreading [8].

Helicobacter pylori Infection and Autoimmune and Rheumatological Disorders 225
Helicobacter pylori in Pathogenesis

of Autoimmune Diseases
Helicobacter pylori (H. pylori) is probably one of the most studied bacteria since its
identification by Warren and Marshall [9]. It is reported to be present in our East African
ancestors more than 58,000 years ago and is now prevalent worldwide [10]. H. pylori is
usually acquired during childhood can persist lifelong, surviving the acidic stomach
environment and elimination by the host immune system. In addition to its physical and
biochemical characteristics, H. pylori are able to manipulate both the innate and acquired
immune system’s responses. These lifelong interactions of H. pylori with the host immune
system make it one of the prime suspects providing the non-genetic trigger for the
autoimmunity. At the same time some studies have even implicated a symbiotic role for H.
pylori, suggesting H. pylori’s interactions with immune system protects it’s host from certain
allergic and autoimmune diseases [11, 12].
Interplay between Helicobacter pylori

and Immune System
Large populations around the world are asymptomatic carriers of H. pylori but only a
small subset (10-15%) actually develop diseases caused by H. pylori [13]. This relatively low
degree of pathogenicity is due to interplay between multiple bacterial and host dependent
factors. However, the widespread colonization points to one of the most intriguing aspects of
H. pylori infection i.e. its ability to survive host defense mechanisms. H. pylori survives in
the nutrient-poor environment between gastric epithelium and mucus layer by stimulating the
immune system to cause mild degree of inflammation which leads to the release of nutrients
by damaged epithelial cells; while at the same time avoiding elimination by both the innate
and adaptive immune systems [14].
Helicobacter pylori and the Innate Immune System
The innate immune system and the epithelial cells are the first line of defense against any
invading microorganism. Their ability to detect invading pathogen is dependent on
recognizing conserved microbial structures such as microbial nucleic acids, peptidoglycans,
lipopolysaccharides, lipoproteins and flagellins [15]. These conserved microbial structures are
commonly referred as pathogen-associated molecular patterns (PAMPs). These PAMPs are
recognized by pattern recognition receptors (PRRs). In humans there at least four distinct
groups of PRRs, the Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) present on
the cytoplasmic and endosomal membranes; and nucleotide-binding oligomerization domain
(NOD) like receptors (NLRs) and retinoic acid inducible gene (RIG) like receptors (RLRs)
present in the cytosol [16]. Upon activation by their specific PAMP ligands these receptors
induce cytokine genes and release activated forms of pro-inflammatory cytokines. This
inflammatory response is balanced by simultaneous activation of anti-inflammatory
pathways. Chronic activation of PRRs by pathogens such as H. pylori can tip this balance in
favor of a more pro-inflammatory cytokine milieu. In addition, the PRRs can also be activated
by endogenous host molecules released as a result of cell damage caused by microbial
infections. Chronic damage to epithelial cells caused by indolent infection from H. pylori
releases host molecule in a pro-inflammatory environment, these self-antigens are then
recognized by PRRs eliciting an immune response against them. This is one of the proposed
mechanism for the pathogenesis of autoimmune diseases based on malfunctioning of innate
immune system triggered by infectious agents [17].
Different components of H. pylori are able to activate innate immune system. The
neutrophil activating protein (NAP) of Helicobacter activates TLR 2 leading to increased IL-
12 and IL-23 production. This cytokine milieu promotes a preferential T-helper 1 (Th-1)
response, characterized by high levels of interferon gamma and tumor necrosis factor alpha
(TNF-α) [18]. There are also studies indicating molecular mimicry between H. pylori NAP
and neural tissue, leading to high levels of anti-aquaporin (AQP)-4 antibodies formation in
patients infected with H. pylori [19]. These anti-AQP-4 antibodies are implicated in neural
damage in patients with multiple sclerosis (MS) and neuromyelitis optica. Other H. pylori
components such as vacuolating cytotoxin A (VacA), the cag pathogenicity island (PAI) and
heat shock protein 60 (HSP 60) also promote secretion of pro-inflammatory cytokines such as
IL-6, IL-8 and TNF-α and a Th-1 polarized immune response [20]. Another proinflammatory
cytokine, IL-18 which skews the immune response in favor of Th-1 is predominantly induced
by outer inflammatory protein (OipA) of H. pylori [21]. Flagella are potent activators of
innate immune system by binding to TLR5. H. pylori flagella are essential for its motility and
survival in the gastric mucosa, and do not cause activation of immune system after TLR 5
binding [22]. Similarly, lipopolysaccharide (LPS) from most gram negative bacteria is
recognized by TLR4 and causes a robust immune response by activating host innate immune
system. Compared to other gram negative bacteria, LPS from H. pylori is reported to have
100-10,000 fold decrease in endotoxin activity due to its unusual phosphorylation pattern in
the lipid A region [23].
Helicobacter pylori and Adaptive Immune System
Innate immune system not only acts as the first line of defense against invading
pathogens but also activates the adaptive immune system to create a more robust immune
response with long lasting memory cells. The adaptive immune response is dependent on
number of factors including but not limited to: the processing and presentation of antigens,
cytokine milieu and engagement of co-stimulatory molecules. The adaptive immune response
against pathogens is also tightly controlled by a balance between pro and anti-inflammatory
cytokines and mechanisms to prevent any response against self-antigens. The self-tolerance of
adaptive immune system is achieved by clonal deletion of auto-reactive cells in the thymus,
anergy against the self-antigens in the periphery and suppressive actions of regulatory T
lymphocytes (T regs) [24]. Breakdown in any of these control mechanisms can lead to
inappropriate immune response against self-antigens causing localized or systemic
autoimmune diseases.
Helicobacter pylori infection causes a Th-1 skewed response evidenced by experimental
as well as clinical data. Antral biopsies of patients with H. pylori infection showed a
predominance of CD4+ T-lymphocytes against H. pylori Cag-A protein [25]. Further

characterization of these lymphocytes revealed expression of high levels of Th-1 cytokines-

interferon-gamma and IL-2 and low levels of Th-2 cytokines-IL-4 and IL-5 [26]. Additional
work revealed that H. pylori induce increased IL-12 production from monocytes,
macrophages and dendritic cells leading to induction of interferon gamma producing Th1
cells [27]. However, this Th-1 mediated response is not vigorous enough to eliminate H.
pylori; in fact this insufficient response is associated with increased bacterial colonization and
increased gastritis. This chronic bacterial colonization associated with an inadequate
lymphocytic response creates a viscous cycle leading to a chronic Th-1 predominant
environment. This has implications for the pathogenesis of organ specific autoimmune
diseases, as evident by the central role played by Th-1 cells in animal models of autoimmune
uveitis and patients with insulin dependent diabetes [28].
Helicobacter pylori also manipulates immune suppressing activities of
CD4+CD25high/Foxp3+ T cells (T-reg) to avoid elimination by the immune system. Both the
gastric and duodenal mucosae of H. pylori positive individuals showed high percentage of T-
regs (about 5% of all CD4+ T cells) as compared to uninfected individuals [29]. Studies in
mouse model of H. pylori infection revealed that these T-reg cells attenuate H. pylori induced
Th-1 cell responses and facilitate increased bacterial colonization [30]. At the same time
depletion of T-regs resulted in reduced bacterial colonization [31]. Furthermore, in-vitro
studies on T-lymphocytes of H. pylori infected individuals showed that T-regs actively
suppresses memory CD4+ T-cell responses [32]. Peripheral blood of H. pylori-infected
patients has a significant decrease in Th-1/Th-2 ratio; this peripheral blood Th2 polarization is
postulated to be secondary to T-regs and may induce systemic autoimmune diseases [33].
Humoral immune response against H. pylori results in production of local and systemic
specific IgG and IgA antibodies but the influence of these antibodies on bacterial colonization
and gastric inflammation is controversial. In a cohort of 131 patients with Helicobacter
colonization gastric inflammatory changes were not related to the antibody response against
H. pylori; suggesting antibodies produced against H. pylori do not affect the natural history of
H. pylori related diseases [34]. Studies in the animal models suggest that IgA antibody
against H. pylori impair the elimination of bacteria and promote development of gastritis [35].
Hence, humoral responses against H. pylori result in production of non-protective antibodies
and do not seem to affect the pathogenesis of H. pylori related gastritis. However, chronic
activation of B lymphocyte by H. pylori has implications for autoimmune diseases and
development of MALT lymphoma. Splenic B lymphocytes were shown to have prolonged
survival in H. pylori infected mice due to their protection from spontaneous apoptosis [36].
This chronic activation and prolonged survival of the B lymphocytes can lead to production
of autoantibodies due to persistence of autoreactive B-lymphocytes; which are usually present
only in insignificant numbers in the secondary lymphoid organs. In addition, the urease
component of H. pylori was demonstrated to preferentially stimulate B-1 subpopulation of B
lymphocytes [37]. These B-1 lymphocytes are considered to be the primary source of
antibodies against self-antigens such as DNA and IgG-like rheumatoid factor, contributing to
the pathogenesis of various autoimmune diseases.

Helicobacter pylori and Autoimmune Diseases

Overview
Chronic colonization of gastric mucosa by H. pylori is possible due to its ability to

manipulate the innate and adoptive immune responses. This chronic immune modulation
serving as a trigger for autoimmune process had been researched extensively since the
discovery of H. pylori. Multiple studies over the last three decades were able to show
increased prevalence of H. pylori in various autoimmune diseases. Studies on animal models
of autoimmune diseases have suggested various mechanisms resulting in immune
dysregulation due to H. pylori infection. However, there is a lack of prospective and/or
mechanistic studies providing evidence to establish the role of H. pylori as an autoimmune
trigger.
Interestingly data from some recent studies suggest a protective role of H. pylori
infection against autoimmune and allergic diseases. Multiple cross sectional studies involving
a variety of populations showed a significant inverse relationship between atopy and asthma
with H. pylori. Two large studies based on US National Health and Nutrition Survey
(NHANES) conducted between 1988-1994 and NHANES conducted between 1999-2000
looked at the association between H. pylori serology and presence of asthma [38]. Data from
both of these studies revealed an inverse relationship between ever having had asthma with
having H. pylori positive serology, odds ratio=0.79 (95% CI=0.63 to 0.99). Using mouse
models of Helicobacter felis, B lymphocytes activated by Helicobacter TLR-2 ligands were
found to induce IL-10 (an anti-inflammatory cytokine) producing T-regs [39]. A recent
literature review and meta-analysis showed 27.1% of inflammatory bowel disease (IBD)
patients were H. pylori seropositive compared with 40.9% patients in the control group;
suggesting that H. pylori colonization has a protective effect against inflammatory bowel
disease [40]. Population based study from Japan showed an inverse relationship between
multiple sclerosis and H. pylori seropositivity [41].
Commonly cited explanation for this negative association is the so called “Hygiene
hypothesis”: cleaner conditions early in life lead to a higher incidence of allergic and
autoimmune diseases later in life as the natural development of immune system is suppressed.
There could be multiple mechanisms causing this increased risk of allergic and autoimmune
diseases in H. pylori negative patients as they have lower numbers of activated T-regs and
low levels of gastric hormones with immunomodualtory effects such as leptin and gastrin.
Helicobacter pylori and Rheumatoid Arthritis
Rheumatoid arthritis (RA) is a multisystem, chronic, autoimmune disease that primarily

presents as inflammatory synovitis involving peripheral joints with additional extra-articular
organ involvement in some cases. Various microbial agents such as gonococcus,
Mycobacterium species, Streptococci species were implicated in the pathogenesis of
rheumatoid arthritis over the last century [42]. Prevailing theory regarding the pathogenesis of
RA suggests interactions between multiple environmental agents in a genetically susceptible
host initiating immune response against self-antigens. Smoking, reproductive status, various

chemical exposures, in addition to infectious disease agents are shown to be associated with
risk of development of RA.
Presence of H. pylori seropositivity in the patients with RA had been reported to be
similar to healthy controls in various populations. In a cohort of 4500 patients with RA in
Japan 49.3% were found to be H. pylori seropositive which is very close to seropositivity of
50% in general Japanese population [43]. This prevalence rate of H. pylori seropositivity in
RA patients is similar to previously reported data from other groups [44]. Similarly serum
samples of 187 patients with RA and 140 healthy controls in Columbia were reported to have
similar percentage of H. pylori seropositivity (80.4% and 80.7%, respectively) [45].
Despite lack of epidemiological data supporting increased prevalence of H. pylori in RA
patients, results from some experimental and treatment studies seems to implicate H. pylori in
the pathogenesis of RA. H. pylori urease is expressed on the surface of bacterial membrane
and is critical for its attachment to the gastric mucosa, thus postulated to be important in
initiating immune response. Purified H. pylori urease was demonstrated to have a strong
capacity to stimulate immunoglobulin bearing B lymphocytes, specifically innate B-1
lymphocytes compared to acquired B-2 lymphocytes [37]. These B-1 lymphocytes tend to
produce non-antigen specific IgM and IgG3 antibodies, which are polyreactive against
multiple bacteria as well as self-antigens. These self-reacting antibodies may contribute to
pathogenesis of autoimmune diseases. Indeed Yamanishi and colleagues reported data from
in-vitro studies that when B-1 lymphocytes were stimulated by purified H. pylori urease
various autoantibodies including IgM rheumatoid factor (RF) were produced in the
supernatant [37]. A significantly higher percentage (23.9%) of CD5+ B-1 lymphocytes were
found to be present in patients with RA as compared to their healthy relatives (18.3%,
p<0.05) and healthy controls (16.1%, p<0.05) [46]. Furthermore, levels of RF showed a
statistically significant correlation to the percentage of CD5+ B-1 lymphocytes. Furthermore,
eradicating H. pylori infection in RA patients leads to a significant improvement of laboratory
and clinical parameters as reported by Seriolo and colleagues in a study of 34 RA patients
infected with H. pylori [47]. As compared to the baseline a progressive improvement of all
clinical indices was observed in H. pylori eradicated RA patients after 2 years, whereas H.
pylori negative RA patients remained substantially unchanged during the same time period
[48]. These data seem to suggest that H. pylori is implicated in the pathogenesis of
rheumatoid arthritis and its eradication is advantageous; but many other studies have been
unable to corroborate these findings. Results from a study of 184 rheumatoid arthritis patients
(113 seropositive and 71 seronegative for H. pylori) failed to reveal any association of H.
pylori infection with the clinical features or disease activity of RA [49]. In a single case
report, patient experienced worsening of RA symptoms on eradication of H. pylori [50].
Similarly, eradication of H. pylori did not affect levels of C-reactive protein in RA patients
[51].
In summary, there is lack of epidemiological evidence supporting an association between
H. pylori and RA but some experimental data supporting its role in pathogenesis and
conflicting data regarding any beneficial effect of H. pylori eradication.

Helicobacter pylori and Systemic Lupus Erythematosus
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease of unknown

etiology. Clinical manifestations of SLE are rather heterogeneous and range from mild
dermatological involvement to more severe internal organ involvement such as renal and
CNS lupus. Diagnosis of SLE is made in part by detection in serum of auto-antibodies such as
Anti-nuclear antibody (ANA) and Anti-double stranded DNA (Anti-ds-DNA) antibody.
Multiple infectious agents, such as cytomegalovirus, parvovirus B19 and EBV have been
proposed to be the triggers of autoimmunity in patients with SLE.
Urease from H. pylori was shown to induce production of anti-DNA antibodies in mice
[37]. Multiple population based studies have however revealed a negative association of SLE
with H. pylori seropositivity. A study from a Japanese cohort showed that seroprevelance of
H. pylori was indeed lower in SLE patients as compared to other autoimmune diseases and
was similar to the healthy controls (44). Data from a US study comparing 466 multiethnic
SLE patients with similar numbers of matched healthy controls revealed that SLE patients
were less frequently seropositive for H. pylori as compared to healthy controls (36.5% vs.
42.9% respectively; OR 0.8, p=0.045) [52]. Analysis of the same cohort showed that this low
prevalence of H. pylori seropositivity was mostly driven by subset of female African-
American SLE patients who had H. pylori seropositivity of only 38.1% compared to 60.2% of
controls; OR 0.41, p=0.0009. In comparison European-American and Hispanic sub-groups of
the same cohort had similar levels of seropositivity as compared to their respective matched
controls. Recently reported H. pylori seropositivity among European and Latin American
SLE cohorts was 47% and 71% respectively similar to their control population [53]. Among
the same group of patients, prevalence of anti-dsDNA antibodies was 21% in H. pylori
seropositive subjects which was significantly higher than that of H. pylori seronegative
subjects (16.2%, p<0.05). These findings are similar to those described earlier in animal
models where H. pylori infection leads to production of auto-antibodies. Evidence from
multiple SLE cohorts indicates a protective effect of H. pylori infection possibly as result of a
preferential Th-1 response and induction of T-regs. Further studies of H. pylori’s effects on
the immune system of patients with SLE may reveal pathways important in the pathogenesis
of SLE.
Helicobacter pylori and Antiphospholipid Antibody Syndrome
Antiphospholipid antibody syndrome (APS) is characterized by development of

unprovoked thrombosis resulting in deep vein thrombosis and pulmonary embolism. Patients
with APS test positive for lupus anticoagulant, IgG, IgM or IgA anticardiolipin antibodies and
anti-β2-glycoprotein-1 antibodies.
Role of infectious disease agents in the pathogenesis of APS is based on theoretical
model of a 2 hit phenomena where an initial bacterial infection produces APS related
antibodies and a subsequent infectious disease agent triggers thrombosis in a genetically host.
This theory is based on observation of circulating APS antibodies for long time in a
significant proportion of patients with a much smaller percentage among them developing
thrombosis. There is significant evidence suggestive of cross reactivity between anti-β2-
glycoprotein-1 antibodies and amino acids sequences of various infectious disease agents

leading to autoantibody production due to molecular mimicry [54]. One of the main
mechanisms triggering a prothrombotic state in APS patients is believed to be endothelial cell
activation by anti-β2-glycoprotein-1 antibodies; this activation is shown to be due to TLR-4
activation by infectious disease agents [55].
There is sequence homology between cell surface phospholipids and some of H. pylori
proteins raising the possibility of antibody production due to cross reactivity.
More than half (56%) of 95 patients with APS from Europe were tested positive for H.
pylori serology which was significantly higher than matched controls (p=0.007) [53]. Data
from the same study reported a statistically significant lower prevalence of IgG
anticardiolipin antibodies and anti-β2-glycoprotein-1 antibodies among the cohort of 1290
patients with various autoimmune diseases as compared to controls. In a case report by
Cicconi and colleagues eradication of H. pylori was associated with clinical and serological
resolution of APS [56].
Hence, there is some theoretical basis of development of APS secondary to infectious
triggers such as H. pylori with scant support from epidemiological and clinical observations.
Helicobacter pylori and Sjögren’s Syndrome
Sjögren’s syndrome develops as a result of lymphocyte infiltration causing destruction of

exocrine glands, mainly lacrimal and salivary glands. Sjögren’s syndrome specific anti-SSA
and anti-SSB antibodies are present in most of the patients.
Oral cavity acts as an extra-gastro-duodenal reservoir for H. pylori and is reported to be
present in up to 100% of general population [57]. H. pylori was detected in saliva of patients
without concurrent stomach colonization and presence of H. pylori antibodies in serum [58].
H. pylori adhesins such as BabA and SabA bind to human salivary glycoprotein such as
MUC5B (MG1) and MUC7 (MG2) and salivary agglutinin (gp-340) [59,60]. These salivary
glycoproteins are part of innate defense against orally ingested pathogens. Prolonged presence
of H. pylori in the oral cavity can promote its binding to salivary glycoproteins resulting in
innate immune response. It is believed that 60 kilo dalton heat shock protein (hsp-60) is
closely related to the pathogenesis of Sjögren’s syndrome; H. pylori was shown to produce a
similar protein hsp-62s in Sjögren’s syndrome patients [61].
There are conflicting data from epidemiological studies of H. pylori seroprevalence
among patients with Sjögren’s syndrome. Presence of H. pylori antibodies was 79.4% in a
group of 34 primary Sjögren’s syndrome patients, which was significantly higher than
patients with other autoimmune diseases and healthy controls (18.2% and 48.8% respectively)
[61]. This high prevalence of H. pylori in Sjögren’s syndrome patients was also reported by
Showji et. al [44]. In another cohort of 67 patients with Sjögren’s syndrome seroprevalence of
H. pylori was 80.6%; significantly higher than patients with autoimmune diseases without
sicca symptoms and healthy controls [62]. There was no association found between H. pylori
seropositivity and presence of Sjögren’s syndrome specific autoantibodies. A much lower
prevalence (45%) of H. pylori antibodies was reported in 145 Sjögren’s syndrome patients
from Sweden similar to healthy controls [63]. Again, there was no significant association
found between H. pylori seropositivity and presence of Anti-SSA, Anti-SSB antibodies or
focus score (a measure of inflammation) on lip biopsy in these patients.

Sjögren’s syndrome patients have an increased incidence of development of MALT

lymphoma similar to patients with H. pylori infection. The pathologic basis of this increased
incidence in both populations is considered to be chronic antigenic stimulation of
lymphocytes. In case reports of patient with parotid MALT lymphoma successful eradication
of H. pylori was associated with disappearance of lymphoma [64,65].
Helicobacter pylori and Systemic Sclerosis
Systemic sclerosis is a disorder of connective tissue characterized by vascular disease and

increased deposition of collagen in the skin and other affected organs. It is classified as
limited vs. diffuse based on extent of skin involvement and the presence of characteristic
auto-antibodies (anti-centromere and anti-SCL 70 antibodies respectively).
Prevalence of anti- H. pylori antibodies was significantly higher (55.6%) in a group of
European patients with Systemic sclerosis as compared to the healthy controls [53]. In
another group of 42 patients with Systemic sclerosis without dyspeptic symptoms prevalence
of H. pylori seropositivity was 62% [66]. In addition, H. pylori seropositive status was
associated with more severe skin, musculoskeletal and gastrointestinal involvement as well as
higher erythrocyte sedimentation rate (ESR). There are other studies indicating a higher
prevalence of H. pylori infection in patients with Systemic sclerosis, however this could also
be a result of disturbed gastrointestinal motility favoring an increased bacterial presence.
There are studies indicating no difference in prevalence of H. pylori between patients with
Systemic sclerosis and healthy controls [67]. A study done by Danese et. al revealed that
majority (90%) of patients with Systemic sclerosis were infected with the more virulent CagA
strain as compared to only 37% of healthy controls [68]. This increased presence of highly
virulent strain of H. pylori in Systemic sclerosis patients needs further investigation to
understand any possible pathogenetic link.
Raynaud’s phenomena is commonly present in association with various autoimmune
diseases, most frequently in Systemic sclerosis. Kountouras and colleagues by employing
histological criteria for the diagnosis of H. pylori detected it in 87% of patients with primary
or secondary Raynaud’s phenomena [69]. Furthermore, eradication of H. pylori was
associated with clinical improvement in 75% of patients who completed their study. Previous
studies revealed a complete resolution of episodes of Raynaud’s phenomena in 17% and
reduction in symptoms of 72% of patients after eradication of H. pylori infection [70].
Helicobacter pylori and Vasculitis
Systemic vasculitis is a group of heterogeneous autoimmune disorders marked by injury

to vasculature of various sizes. Clinical manifestations of specific disorder depend on the size
and organ system involved with presence of characteristic autoantibodies. The primary
pathogenetic pathway in vasculitis is injury to the endothelium. There are various proposed
mechanisms implicating microbial pathogens in the pathogenesis of vasculitis such as direct
microbial invasion of endothelial cells, immune complex (IC)-mediated vessel wall damage
and activation of autoreactive lymphocytes. There is evidence supporting role of Hepatitis B,
C and Staphylococcus aureus among other pathogens in various vasculitis.

The role of H. pylori in pathogenesis of vasculitis is less well defined. There are multiple
epidemiological studies exploring an association of H. pylori with various vasculitis.
In a cohort of 54 patients with Wagner’s granulomatosis (WG), H. pylori IgG antibodies
were more common among patients with WG, a significant finding compared with controls (p
= 0.002) [71]. In a cohort of 36 Polish patients with WG majority (71.4%) were positive for
H. pylori [72]. H. pylori patients had more severe disease based on clinical and laboratory
parameters.
A significant majority (82.8%) of patients with Giant Cell arteritis (GCA) were found to
be seropositive for H. pylori as compared to healthy controls (p<0.001) [53].
A meta-analysis of 10 studies involving 749 children with Henoch-Schonlein purpura
(HSP) and 560 controls reported 49.27% (369/749) of HSP children had evidence of H. pylori
infection compared with 23.39% (131/560) of children in the control group [73]. There was
reduced recurrence of HSP reported after eradication of H. pylori from some groups. In a
Turkish cohort of 40 patients with Behçet's disease (BD), H. pylori was found in 26 patients
(65%) with BD and in 28 controls (70%) (no significant difference by chi-squared test, p >
0.05) [74]. Another group of Turkish patients with BD also reported similar rates of H. pylori
infection in patients and control population [75].
Helicobacter pylori and IgG4 Related Diseases
Recently a group of clinical entities were classified as IgG4 related diseases as they all
considered to be sharing same underlying pathogenetic pathway. IgG4 related diseases are
characterized by elevated levels of serum IgG4, abundant infiltration of the affected tissue by
IgG4 positive plasma cells, storiform fibrosis, obliterative phlebitis and diffuse
lymphoplasmacytic infiltration. Some of the disease entities lumped under this name includes
retroperitoneal fibrosis, autoimmune pancreatitis and Mikulicz’s disease.
Patients with Autoimmune pancreatitis (AIP) develop auto-antibodies against carbonic
anhydrase II and lactoferrin. These auto-antibodies are suspected to have a pathogenic role in
the development of AIP. Role of H. pylori in pathogenesis of AIP is based on finding of
sequence homology between one of its enzymes alpha-carbonic anhydrase and human
carbonic anhydrase II [76]. Additionally, the homologous segments were found to contain the
binding motif of the HLA molecule DRB1*0405.
Frulloni and colleagues identified a peptide in 18 of the 20 AIP patients [77]. There is
amino acid sequence homology between this newly identified peptide with plasminogen-
binding protein (PBP) of H. pylori and with ubiquitin-protein ligase E3 component n-
recognin 2 (UBR2), an enzyme highly expressed in acinar cells of the pancreas. In addition,
19 out of the 20 AIP patients were found to have anti-bodies against PBP. Hence, molecular
mimicry of pancreatic peptides with H. pylori proteins may trigger auto-antibody production
in a genetically susceptible host leading to AIP.

Helicobacter pylori and Polymyositis and Dermatomyositis
Polymyositis (PM) and dermatomyositis (DM) are autoimmune disorders manifested by

skin and muscle pathology. As with other autoimmune diseases the etiology is elusive with
only limited data regarding the role of H. pylori.
Prevalence of H. pylori was found to be the same in patients with PM and DM as
compared to healthy controls [61, 78].
Helicobacter pylori and Fibromyalgia
Fibromyalgia (FMG) is a relatively common condition primarily affecting middle aged

women, characterized by generalized musculoskeletal pain and tenderness at specific
anatomical sites called tender points.
Various chronic infections have been implicated in the etiopathogenesis of FMG.
Antibodies against H. pylori IgG was found to be present in 44 out of 65 FMG patients,
which was significantly higher than controls (p=0.025) [79]. Amongst the FMG patients there
were no significant differences in clinical features between H. pylori IgG positive vs. H.
pylori IgG negative patients. In a separate study reported by Malt and colleagues one third of
the 28 patients were H. pylori seropositive, similar to healthy controls [80].
Conclusion
Autoimmune diseases are thought to develop due to interactions of various environmental
agents with the host immune system in a genetically susceptible individual. Chronic
infections such as H. pylori are considered to provide the trigger for autoimmunity. There is
conflicting evidence to support the role of H. pylori in pathogenesis of various autoimmune
diseases with some data implicating a protective effect.
Author Note
This research was supported [in part] by the Intramural Research Program of the National
Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of
Health
References
[1] Muniz Caldas CA, Freire de Carvalho J, (2012). The role of environmental factors in
the pathogenesis of non-organ-specific autoimmune diseases. Best Pract. Res. Clin.
Rrheumatol., 26(1):5-11.
[2] Rai E, Wakeland EK, (2011). Genetic predisposition to autoimmunity--what have we
learned? Sem. Immunol., 23(2):67-83.

[3] Leslie RD, Hawa M, (1994). Twin studies in auto-immune disease. Acta Genet. Med
Gemell., 43(1-2):71-81.
[4] Salvetti M, Ristori G, Bomprezzi R, Pozzilli P, Leslie RD, (2000). Twins: mirrors of
the immune system. Immunol. Today., 21(7):342-7.
[5] Miller FW, Alfredsson L, Costenbader KH, Kamen DL, Nelson LM, Norris JM, et al.
(2012). Epidemiology of environmental exposures and human autoimmune diseases:
findings from a National Institute of Environmental Health Sciences Expert Panel
Workshop. J Autoimmun., 39(4):259-71.
[6] Bach JF, (2005). Infections and autoimmune diseases. J. Autoimmun., 25 Suppl:74-80.
[7] Crow MK, Kirou KA, (2004). Interferon-alpha in systemic lupus erythematosus. Curr.
Opin. Rheumatol., 16(5):541-7.
[8] Getts MT, Miller SD, (2010). 99th Dahlem conference on infection, inflammation and
chronic inflammatory disorders: triggering of autoimmune diseases by infections. Clin.
Expe Immunol., 160(1):15-21.
[9] Marshall BJ, Warren JR, (1984). Unidentified curved bacilli in the stomach of patients
with gastritis and peptic ulceration. Lancet., 16;1(8390):1311-5.
[10] Linz B, Balloux F, Moodley Y, Manica A, Liu H, Roumagnac P, et al. (2007). An
African origin for the intimate association between humans and Helicobacter pylori.
Nature., 22;445(7130):915-8.
[11] Zevit N, Balicer RD, Cohen HA, Karsh D, Niv Y, Shamir R, (2012). Inverse
association between Helicobacter pylori and pediatric asthma in a high-prevalence
population. Helicobacter., 17(1):30-5.
pylori infection and inflammatory bowel disease: a meta-analysis and systematic review
of the literature. Inflamm. Bowel Dis., 16(6):1077-84.
[13] Delahay RM, Rugge M, (2012). Pathogenesis of Helicobacter pylori infection.
Helicobacter., 17 Suppl 1:9-15.
[14] Baldari CT, Lanzavecchia A, Telford JL, (2005). Immune subversion by Helicobacter
pylori. Trends Immunol., 26(4):199-207.
[15] Ishii KJ, Koyama S, Nakagawa A, Coban C, Akira S, (2008). Host innate immune
receptors and beyond: making sense of microbial infections. Cell Host Microbe.,
12;3(6):352-63.
[16] Hansen JD, Vojtech LN, Laing KJ, (2011). Sensing disease and danger: a survey of
vertebrate PRRs and their origins. Devel. Compar Immunol., 35(9):886-97.
[17] Mogensen TH, (2009). Pathogen recognition and inflammatory signaling in innate
immune defenses. Clin. Microbiology Rev., 22(2):240-73.
[18] D'Elios MM, Amedei A, Cappon A, Del Prete G, de Bernard M, (2007). The
neutrophil-activating protein of Helicobacter pylori (HP-NAP) as an immune
modulating agent. FEMS Immunol. Med. Microbiol., 50(2):157-64.
[19] Kountouras J, Zavos C, Chatzopoulos D, (2004). Induction of apoptosis as a proposed
pathophysiological link between glaucoma and Helicobacter pylori infection. Med.
Hypoth., 62(3):378-81.
[20] D'Elios MM, Andersen LP, (2009). Inflammation, immunity, and vaccines for
Helicobacter pylori. Helicobacter., 14 Suppl 1:21-8.

[21] Yamauchi K, Choi IJ, Lu H, Ogiwara H, Graham DY, Yamaoka Y, (2008). Regulation
of IL-18 in Helicobacter pylori infection. J. Immunol., (Baltimore, Md : 1950).
15;180(2):1207-16.
[22] Lee SK, Stack A, Katzowitsch E, Aizawa SI, Suerbaum S, Josenhans C, (2003).
Helicobacter pylori flagellins have very low intrinsic activity to stimulate human
gastric epithelial cells via TLR5. Microb. Infect., 5(15):1345-56.
[23] Taylor JM, Ziman ME, Huff JL, Moroski NM, Vajdy M, Solnick JV, (2006).
Helicobacter pylori lipopolysaccharide promotes a Th1 type immune response in
immunized mice. Vaccine., 5;24(23):4987-94.
[24] Fehervari Z, Sakaguchi S, (2004). CD4+ Tregs and immune control. J. Clin. Invest.,
114(9):1209-17.
[25] D'Elios MM, Manghetti M, De Carli M, Costa F, Baldari CT, Burroni D, et al. (1997).
T helper 1 effector cells specific for Helicobacter pylori in the gastric antrum of
patients with peptic ulcer disease. J. Immunol., 15;158(2):962-7.
[26] Bamford KB, Fan X, Crowe SE, Leary JF, Gourley WK, Luthra GK, et al. (1998).
Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper
cell 1 phenotype. Gastroenterology., 114(3):482-92.
[27] Haeberle HA, Kubin M, Bamford KB, Garofalo R, Graham DY, El-Zaatari F, et al.
(1997). Differential stimulation of interleukin-12 (IL-12) and IL-10 by live and killed
Helicobacter pylori in vitro and association of IL-12 production with gamma
interferon-producing T cells in the human gastric mucosa. Infect. Immun., 65(10):4229-
35.
[28] Singh VK, Mehrotra S, Agarwal SS, (1999). The paradigm of Th1 and Th2 cytokines:
its relevance to autoimmunity and allergy. Immunol. Res., 20(2):147-61.
[29] Lundgren A, Stromberg E, Sjoling A, Lindholm C, Enarsson K, Edebo A, et al. (2005).
Mucosal FOXP3-expressing CD4+ CD25 high regulatory T cells in Helicobacter
pylori-infected patients. Infect Immun., 73(1):523-31.
[30] Raghavan S, Fredriksson M, Svennerholm AM, Holmgren J, Suri-Payer E, (2003).
Absence of CD4+CD25+ regulatory T cells is associated with a loss of regulation
leading to increased pathology in Helicobacter pylori-infected mice. Clin. Exp.
Immunol., 132(3):393-400.
[31] Rad R, Brenner L, Bauer S, Schwendy S, Layland L, da Costa CP, et al. (2006).
CD25+/Foxp3+ T cells regulate gastric inflammation and Helicobacter pylori
colonization in vivo. Gastroenterology., 131(2):525-37.
[32] Lundgren A, Suri-Payer E, Enarsson K, Svennerholm AM, Lundin BS, (2003).
Helicobacter pylori-specific CD4+ CD25high regulatory T cells suppress memory T-
cell responses to H. pylori in infected individuals. Infect. Immun., 71(4):1755-62.
[33] Satoh Y, Ogawara H, Kawamura O, Kusano M, Murakami H, (2012). Clinical
Significance of Peripheral Blood T Lymphocyte Subsets in Helicobacter pylori-
Infected Patients. Gastroenterol Res Pract., 2012:819842. Epub 2012/04/27.
[34] Luzza F, Maletta M, Imeneo M, Marcheggiano A, Biancone L, Pallone F. Mucosal and
systemic antibody levels against Helicobacter pylori do not parallel gastric
inflammatory changes, (1998). Ital. J. Gastroenterol. Hepatol., 30(1):36-9.
[35] Akhiani AA, Stensson A, Schon K, Lycke NY, (2005). IgA antibodies impair resistance
against Helicobacter pylori infection: studies on immune evasion in IL-10-deficient
mice. J. Immunol., 15;174(12):8144-53.

[36] Bussiere FI, Chaturvedi R, Asim M, Hoek KL, Cheng Y, Gainor J, et al. (2006). Low
multiplicity of infection of Helicobacter pylori suppresses apoptosis of B lymphocytes.
Cancer. Res., 1;66(13):6834-42.
[37] Yamanishi S, Iizumi T, Watanabe E, Shimizu M, Kamiya S, Nagata K, et al. (2006).
Implications for induction of autoimmunity via activation of B-1 cells by Helicobacter
pylori urease. Infect. Immun., 74(1):248-56.
[38] Blaser MJ, Chen Y, Reibman J, (2008). Does Helicobacter pylori protect against
asthma and allergy? Gut., 57(5):561-7.
[39] Sayi A, Kohler E, Toller IM, Flavell RA, Muller W, Roers A, et al. (2010). TLR-2-
activated B cells suppress Helicobacter-induced preneoplastic gastric
immunopathology by inducing T regulatory-1 cells. J. Immunol., 15;186(2):878-90.
pylori infection and inflammatory bowel disease: a meta-analysis and systematic review
of the literature. Inflamm. Bowel Dis., 16(6):1077-84.
[41] Li W, Minohara M, Su JJ, Matsuoka T, Osoegawa M, Ishizu T, et al. (2007).
Helicobacter pylori infection is a potential protective factor against conventional
multiple sclerosis in the Japanese population. J. Neuroimmunol., 184(1-2):227-31.
[42] Benedek TG, (2006). The history of bacteriologic concepts of rheumatic fever and
rheumatoid arthritis. Semin. Arthritis. Rheum., 36(2):109-23.
[43] Tanaka E, Singh G, Saito A, Syouji A, Yamada T, Urano W, et al. (2005). Prevalence
of Helicobacter pylori infection and risk of upper gastrointestinal ulcer in patients with
rheumatoid arthritis in Japan. Mod. Rheumatol., 15(5):340-5.
[44] Showji Y, Nozawa R, Sato K, Suzuki H, 81996). Seroprevalence of Helicobacter pylori
infection in patients with connective tissue diseases. Microb. Immunol., 40(7):499-503.
[45] Meron MK, Amital H, Shepshelovich D, Barzilai O, Ram M, Anaya JM, et al. (2009).
Infectious aspects and the etiopathogenesis of rheumatoid arthritis. Clin. Rev. Allergy.
Immunol., 38(2-3):287-91.
[46] Youinou P, Mackenzie L, Katsikis P, Merdrignac G, Isenberg DA, Tuaillon N, et al.
(1990). The relationship between CD5-expressing B lymphocytes and serologic
abnormalities in rheumatoid arthritis patients and their relatives. Arthritis. Rheum.,
33(3):339-48.
[47] Seriolo B, Cutolo M, Zentilin P, Savarino V, (2001). Helicobacter pylori infection in
rheumatoid arthritis. J. Rheumatol., 28(5):1195-6.
[48] Zentilin P, Seriolo B, Dulbecco P, Caratto E, Iiritano E, Fasciolo D, et al. (2002).
Eradication of Helicobacter pylori may reduce disease severity in rheumatoid arthritis.
Aliment. Pharmacol. Ther., 16(7):1291-9.
[49] Ishikawa N, Fuchigami T, Matsumoto T, Kobayashi H, Sakai Y, Tabata H, et al.
(2002). Helicobacter pylori infection in rheumatoid arthritis: effect of drugs on
prevalence and correlation with gastroduodenal lesions. Rheumatology (Oxford).,
41(1):72-7.
[50] Matsukawa Y, Asai Y, Kitamura N, Sawada S, Kurosaka H, (2005). Exacerbation of
rheumatoid arthritis following Helicobacter pylori eradication: disruption of established
oral tolerance against heat shock protein? Med Hypotheses., 64(1):41-3.
[51] Steen KS, Lems WF, Visman IM, de Koning MH, van de Stadt RJ, Twisk JW, et al.
(2009). The effect of Helicobacter pylori eradication on C-reactive protein and the lipid

profile in patients with rheumatoid arthritis using chronic NSAIDs. Clin Exp
Rheumatol., 27(1):170.
[52] Sawalha AH, Schmid WR, Binder SR, Bacino DK, Harley JB, (2004). Association
between systemic lupus erythematosus and Helicobacter pylori seronegativity. J.
Rheumatol., 31(8):1546-50.
[53] Ram M, Barzilai O, Shapira Y, Anaya JM, Tincani A, Stojanovich L, et al. (2013).
Helicobacter pylori serology in autoimmune diseases - fact or fiction? Clin. Chem.
Laborat. Med., 1;51(5):1075-82.
[54] Shoenfeld Y, Blank M, Cervera R, Font J, Raschi E, Meroni PL, (2005). Infectious
origin of the antiphospholipid syndrome. Ann. Rheum. Dis., 65(1):2-6.
[55] Raschi E, Testoni C, Borghi MO, Fineschi S, Meroni PL, (2003). Endothelium
activation in the anti-phospholipid syndrome. Biomed. Pharm., 57(7):282-6.
[56] Cicconi V, Carloni E, Franceschi F, Nocente R, Silveri NG, Manna R, et al. (2001).
Disappearance of antiphospholipid antibodies syndrome after Helicobacter pylori
eradication. Am. J. Med., 111(2):163-4.
[57] Song Q, Haller B, Ulrich D, Wichelhaus A, Adler G, Bode G, (2000). Quantitation of
Helicobacter pylori in dental plaque samples by competitive polymerase chain reaction.
J. Clin. Pathol., 53(3):218-22.
[58] Burgers R, Schneider-Brachert W, Reischl U, Behr A, Hiller KA, Lehn N, et al. (2008).
Helicobacter pylori in human oral cavity and stomach. Eur. J. Oral Sci., 116(4):297-
304. PubMed PMID: 18705796. Epub 2008/08/19.
[59] Prakobphol A, Boren T, Ma W, Zhixiang P, Fisher SJ, (2005). Highly glycosylated
human salivary molecules present oligosaccharides that mediate adhesion of leukocytes
and Helicobacter pylori. Biochem., 15;44(6):2216-24.
[60] Walz A, Odenbreit S, Stuhler K, Wattenberg A, Meyer HE, Mahdavi J, et al. (2009).
Identification of glycoprotein receptors within the human salivary proteome for the
lectin-like BabA and SabA adhesins of Helicobacter pylori by fluorescence-based 2-D
bacterial overlay. Proteomics., 9(6):1582-92.
[61] Aragona P, Magazzu G, Macchia G, Bartolone S, Di Pasquale G, Vitali C, et al. (1999).
Presence of antibodies against Helicobacter pylori and its heat-shock protein 60 in the
serum of patients with Sjogren's syndrome. J. Rheumatol., 26(6):1306-11.
[62] El Miedany YM, Baddour M, Ahmed I, Fahmy H, (2005). Sjogren's syndrome:
concomitant H. pylori infection and possible correlation with clinical parameters. Joint,
Bone, Spine: Rev. Rhum., 72(2):135-41.
[63] Theander E, Nilsson I, Manthorpe R, Jacobsson LT, Wadstrom T, (2001).
Seroprevalence of Helicobacter pylori in primary Sjogren's syndrome. Clin. Exp.
Rheumatol., 19(6):633-8.
[64] Iwai H, Nakamichi N, Nakae K, Konishi M, Inaba M, Hoshino S, et al. (2009). Parotid
mucosa-associated lymphoid tissue lymphoma regression after Helicobacter pylori
eradication. Laryngosc., 119(8):1491-4.
[65] Suchy BH, Wolf SR. Bilateral mucosa-associated lymphoid tissue lymphoma of the
parotid gland. Archives of otolaryngology--head & neck surgery. 2000 Feb;126(2):224-
6. PubMed PMID: 10680876. Epub 2000/02/19.
[66] Radic M, Kaliterna DM, Bonacin D, Vergles JM, Radic J, Fabijanic D, et al. (2012). Is
Helicobacter pylori infection a risk factor for disease severity in systemic sclerosis?
Rheumatol. Int., Dec 6. Epub 2012/12/12.

[67] Sulli A, Seriolo B, Savarino V, Cutolo M, (2000). Lack of correlation between gastric
Helicobacter pylori infection and primary or secondary Raynaud's phenomenon in
patients with systemic sclerosis. J. Rheumatol., 27(7):1820-1.
[68] Danese S, Zoli A, Cremonini F, Gasbarrini A, (2000). High prevalence of Helicobacter
pylori type I virulent strains in patients with systemic sclerosis. J. Rheumatol.,
27(6):1568-9.
[69] Kountouras J, Zavos C, Gavalas E, Deretzi G, Katsinelos P, Boura P, et al. (2011).
Helicobacter pylori may be a common denominator associated with systemic and
multiple sclerosis. Joint, bone, spine: Rev Rhum., 78(2):222-3; author reply 3.
[70] Carloni E, Cremonini F, Di Caro S, Padalino C, Gerardino L, Santoliquido A, et al.
(2000). Helicobacter pylori-related extradigestive diseases and effects of eradication
therapy. Dig. Liv. Dis., Suppl 3:S214-6.
[71] Lidar M, Lipschitz N, Langevitz P, Barzilai O, Ram M, Porat-Katz BS, et al. (2009).
Infectious serologies and autoantibodies in Wegener's granulomatosis and other
vasculitides: novel associations disclosed using the Rad BioPlex 2200. Ann. Acad. Sci.,
1173:649-57.
[72] Zycinska K, Wardyn KA, Zycinski Z, Smolarczyk R, (2008). Correlation between
Helicobacter pylori infection and pulmonary Wegener's granulomacytosis activity. J.
Physiol. Pharmacol., 59 Suppl 6:845-51.
[73] Xiong LJ, Tong Y, Wang ZL, Mao M. Is Helicobacter pylori infection associated with
Henoch-Schonlein purpura in Chinese children? a meta-analysis. World J Ped.,
8(4):301-8.
[74] Cakmak SK, Cakmak A, Gul U, Sulaimanov M, Bingol P, Hazinedaroglu MS, (2009).
Upper gastrointestinal abnormalities and Helicobacter pylori in Behcet's disease. Int. J.
Dermatol., 48(11):1174-6.
[75] Ersoy O, Ersoy R, Yayar O, Demirci H, Tatlican S, (2007). H. pylori infection in
patients with Behcet's disease. World J. Gastroenterol., 7;13(21):2983-5.
[76] Guarneri F, Guarneri C, Benvenga S, (2005). Helicobacter pylori and autoimmune
pancreatitis: role of carbonic anhydrase via molecular mimicry? J. Cell Molec. Med.,
9(3):741-4.
[77] Frulloni L, Lunardi C, Simone R, Dolcino M, Scattolini C, Falconi M, et al. (2009).
Identification of a novel antibody associated with autoimmune pancreatitis. N. Eng. J.
Med., 26;361(22):2135-42.
[78] Kalabay L, Fekete B, Czirjak L, Horvath L, Daha MR, Veres A, et al. (2002).
Helicobacter pylori infection in connective tissue disorders is associated with high
levels of antibodies to mycobacterial hsp65 but not to human hsp60. Helicobacter.,
7(4):250-6.
[79] Akkaya N, Akkaya S, Polat Y, Turk M, Turk T, Turhal E, et al. (2011). Helicobacter
pylori seropositivity in fibromyalgia syndrome. Clin. Rheumatol., 30(1):43-9.
[80] Malt EA, Olafsson S, Ursin H, (2004). Fibromyalgia: a manifestation of Helicobacter
pylori infection? Scand J Rheumatol., 33:131–1131.

Chapter 13
The Impact of Helicobacter pylori

Colonization on Peptides Regulating
Appetite and Satiety:
Ghrelin, Leptin, and Obestatin
Víctor Rafael Coria Jiménez*, Carolina Romo González

and Patricia Chico Aldama
Laboratory of Experimental Bacteriology, National Institute of Pediatrics,
Mexico City, Mexico
Abstract
The stomach is an organ involved in neuroendocrine regulations that participates in
energy metabolism equilibrium of an individual. It is an autonomous ecosystem, a fact
that has long been established, with physiological characteristics that empower it to select
a particular microbial flora with the ability and capacity to colonize and inhabit a habitat
originally considered as free of microorganisms.
Helicobacter pylori (H. pylori) is capable of colonizing human stomach where it
could stay for a prolonged period, and sometimes decades, with induction of chronic
inflammatory process that substantially modifies the physiology of the organ.
The colonization and eradication of this bacterium are events capable of leading to
variations in weight and size of the host. These variations depend on the age on which
colonization by the microorganism took place, host genetic characteristics, strain
virulence, and the socioeconomic environment in which the host-parasite relationship is
developed.
It has been described that one of the mechanisms leading to the association of H.
pylori colonization to body weight index variation of the host is the induction of
modifications in the genetic expression and blood levels of ghrelin, acyl-ghrelin, and
leptin which are peptide hormones that intervene in regulating appetite.
*
Corresponding author. Laboratory of Experimental Bacteriology, National Institute of Pediatrics, Mexico City,
Mexico. Email: coria.rafael@gmail.com.

242 Víctor Rafael Coria Jiménez, Carolina Romo González and Patricia Chico Aldama
The aim of this chapter is to present and discuss the evidences trying to explain how
colonization by H. pylori affects the genetic expression of ghrelin, leptin, and obestatin,
the ghrelin o-acyltransferase enzymatic activity, and the effect of these changes in the
variations of weight, size, growth velocity, and body mass index of the colonized host.
Keywords: Helicobacter pylori, colonization, eradication, Ghrelin, Leptin, Obestatin, Body

Mass Index (BMI)
Introduction
The study of the role of microorganisms of gastrointestinal tracts in the physiology of the
host has elucidated the understanding on their function as important factors that affect in a
significant form metabolism in the humans.
The discovery of colonization of human stomach by Helicobacter pylori (H. pylori) and
its association with the development of gastritis, and gastric ulcer and cancer represented a
new paradigm in clinical Microbiology.
On studying with detailed the relation, host-parasite, between H. pylori and the humans,
new and interesting aspects of the dynamics, complexity, and antic relationship existing
between this bacterium and the humans were observed.
Some authors in developed countries have noticed that after the eradication of H. pylori,
there has been a significant increase in the prevalence rate of different types of pathologies
(reflux esophagitis, Barrett’s esophagus, adenocarcinoma of the esophagus, and overweight,
among others) in the people. This has led to the suggestion that this microorganism could be
functioning as comensal which in some way contributes in the physiology of the colonized
host.
Recently, the role of H. pylori on some of the mechanisms regulating appetite, especially
the expression and serial levels of peptide hormones that control food consumption, has been
studied. Also, it has been demonstrated that the presence and eradication of H. pylori is
significantly related with variations in ghrelin, leptin, and probably obestatin, which
represents a novel focus on the influence exercised by the microorganisms on the complex
mechanism of energetic balance in individuals.
Until date, the mechanisms by which H. pylori influence on the synthesis and function of
ghrelin, leptin, and obestatin is not precisely known. This constitutes a new and fascinating
field of research which in short or medium terms would surely yield fruits that could help in
developing new and better therapies for some metabolic disequilibrium based on the
understanding and manipulation of the relations between the humans and their intestinal
microbiota.
Bacterial Colonization
of the Gastrointestinal Tract
Currently, we face a serious and growing public health problem, as follows: the increased
overweight and obesity rates in our populations, mainly in children. Overweight and obesity

The Impact of Helicobacter pylori Colonization on Peptides ... 243
are the result of a series of dynamic interactions between various social, genetic, and
environmental factors, among which is the intestinal microbiota, which is defined as the set of
microorganisms that are present in the human intestine under normal conditions; this
heterogeneous microbial community plays an important role in the metabolism of the host
and even participates as a conditioning factor for the development of metabolic disorders such
as diabetes and various cardiovascular diseases [1 – 5]. Different authors have documented
that the composition of the microbiota of obese people is different from that of people with
normal BMI.. However, it has not been possible to establish in human studies whether the
microbiota promotes the increased body mass index (BMI) of the host or if it is the host who
selects his or her individual microbiota; nevertheless, in experiments with germfree mice
inoculated with the microbiota from obese mice, significant weight gain attributable to the
new intestinal microbiota was observed [6].
Figure 13.1 schematizes the possible relationships between the activity of the
microorganisms of the intestinal microbiota and variations in the weight and size of the
human host. It is clear that there is a dynamic equilibrium that is influenced by factors of the
host, of the microorganisms, and of the socioeconomic and cultural environment in which this
complex network of relationships evolves [2]. The composition of the gastric ecosystem has
been analyzed, demonstrating a high degree of inter-individual variability, which indicates
that the human stomach may be home to a particular microbial ecosystem. This finding
reinforces the concept that the gastric microbiota may in turn play important, yet
undiscovered, roles in health and in human disease [7].
Excess calories
Altered lipid metabolism
Signaling molecules:
SFA lipolysis, food intake
Bacterial TMAO Risk marker

for CVD
metabolites
Hippurate Risk marker for

diabetes and hypertension
Gut
Microbiota LPS : TLR4/CD14 Innate inflammatory
response
Flagellum : TLR5 Insulin resistance

Bacterial obesity
structures Estructural lipids:

Metabolic Syndrome
TLR2
Peptidoglycan: Type 2 CVD

NOD1, 2 diabetes
Figure 13.1. Relationships of the gut microbiota and the induced variations in the metabolism of the
host. [From: Harris K, et al. J Obes. 2012].
[SFA - short-chain fatty acids, TMAO - Trimethylamine-N-Oxide, LPS – Lypopolisaccharide, TLR -
Toll-like receptors 2, 4 and 5, NOD - Nucleotide oligomerization domain 1 and 2, CVD -
cardiovascular diseases]

Helicobacter pylori as Part

of the Gastric Microbiota
H. pylori is a bacterium that colonizes 50% of humans, occurring mainly in
underdeveloped countries and in lower socio-economic sectors. H. pylori can be maintained
throughout the life of the host, with colonization beginning in infancy [8,9]. The natural
history of an infection with H. pylori is influenced by host factors such as the age at
acquisition, time of evolution, presence of polymorphisms of interleukins, differences in the
levels of gastric acid production between individuals, stomach region colonized by H. pylori,
and, on the side of the bacterium, by the presence, functionality, and expression of virulence
factors, which is mainly the presence of the cagPAI (cag-pathogenicity island). The presence
of this island has allowed classification of H. pylori strains in two types: those with the island
(cagPAI+), associated with serious infectious processes and those that do not possess the
island (cagPAI-) and related to mild to moderate infections and asymptomatic individuals. It
is important to note that currently, there are many authors that suggest that the colonization by
H. pylori can be considered as an ancient and commensal relationship [10-17].
Table 13.1. Modulators of hunger and satiety from gastrointestinal tract,

pancreas and adipose tissue
Peptide Sites of synthesis Major actions

Gastrointestinal
tract
CCK I-cells of duodenum, jejunum promotes gallbladder contraction increases
widespread CNS expression secretion of pancreatic enzymes and bicarbonate
inhibits gastric acid secretion slows gastric
emptying reduces food intake
gastrin G-cells of gastric antrum increases gastric acid promotes gastric epithelial
cell proliferation no known effect on food intake
ghrelin A-cells of gastric fundus; promotes release of GH and other pituitary
small and large intestine; hormones increases food intake promotes gastric
hypothalamic nuclei motility promotes PP release inotropic effect on
heart vasodilatation
GIP K-cells of duodenum and incretin effect on insulin secretion increases fatty
Jejunum acid synthesis in adipose tissue enterogastrone
effect diminishes intestinal motility increases
mesenteric blood flow effects on food intake
unknown—fourth ventricle administration has
no effect
GLP-1 L-cells of distal small and incretin effect on insulin secretion suppresses
large intestine; glucagon release promotes pancreatic b-cell
immunoreactivity in growth inhibits gastric emptying inhibits gastric
hypothalamus, dorsovagal secretion inhibits energy intake effects on
complex, pituitary cardiovascular system
GLP-2 L-cells of distal small and promotes tissue repair and intestinal mucosal
large intestine; growth enhances digestive and absorptive
immunoreactivity in capacity of intestine inhibits gastric secretion
hypothalamus, dorsovagal inhibits feeding when administered centrally; no
complex, pituitary effect of peripheral administration

Peptide Sites of synthesis Major actions

Motilin proximal small intestine; some prokinetic action on gut, mediates migrating
reports of immunoreactivity in motor complexes stimulates gallbladder
CNS contraction promotes enzyme secretion in
stomach and pancreas stimulates PP release
effects on food intake equivocal
oxyntomodulin L-cells of distal small and inhibits gastric acid production reduces gastric
large intestine; motility role as incretin equivocal inhibits food
immunoreactivity in intake
hypothalamus, dorsovagal
complex, pituitary
PHI/PHV gastrointestinal tract; heart; main physiological effects unclear PHI injected
lungs; kidney; central and ICV inhibits feeding
peripheral nervous systems
PYY3-36 L-cells of distal small and inhibits food intake inhibits gallbladder secretion
large intestine; reduces gut motility inhibits pancreatic secretion
immunoreactivity in enterogastrone effect
hypothalamus, medulla, pons
Secretin S-cells of the duodenum stimulates pancreatic exocrine secretions inhibits
gastric secretion promotes PP secretion no
known effect on food intake
somatostatin multiple organ systems, multiple actions across numerous organ systems
notably the D-cells of the gut inhibits gastric secretions reduces gut motility
and pancreas; hypothalamus inhibits release of numerous other gut hormones,
including insulin, glucagon, CCK, gastrin,
OXM, PP reduces food intake when
administered peripherally—physiological
importance of this effect unclear
Adipose tissue
Leptin Adipose tissue and stomach Anorexigen effect on food intake
Adiponectin Adipose tissue Anorexigen effect on food intake
Pancreas
PP pancreatic islets of relaxation of gallbladder inhibition of pancreatic
Langerhans; some reports of exocrine secretion equivocal effect on gastric
expression in hypothalamus, emptying inhibits food intake
pineal gland, pituitary,
substantia nigra, hippocampus
Amylin Pancreatic islets (islet b- Anorexigen, inhibition of gastric emptying,
amyloid) reduction of meal size
(Adapted from Chaudri O et al. 2006).
The Gastrointestinal Tract as an Endocrine Organ

The gastrointestinal tract is currently considered to be an important endocrine organ due
to the production of diverse peptide hormones involved in digestion, nutrient absorption,
motility, and secretion [18,19]. Nevertheless, other peptide hormones have a radical
involvement in the control of the induction mechanisms of appetite, satiety, and energy
homeostasis in an individual. In addition, the adipose tissue is also a major producer of
peptides that are prominently involved in these mechanisms [20-23]. In the gastrointestinal

tract, endocrine cells are distributed throughout the mucous membrane of the stomach, small
intestine, colon, etc. These cells contain granules that allow the secretion of various peptides
[24], and, although the main stimulus for the release of their hormones is the presence of food
in the intestinal lumen, their secretion can also be induced or inhibited by nerve stimulation or
by their own hormones, leading to paracrine, neurocrine, and endocrine functions. Table 13.1
describes the modulators of hunger and satiety from gastrointestinal tract, pancreas and
adipose tissue.
Communication between the gastrointestinal tract and the central nervous system (where
stimuli regulating appetite and satiety mechanisms are focused) is carried out bidirectionally
by the parasympathetic (vagus nerve and pelvic nerves) and sympathetic nervous system
(splanchnic nerve). In this communication, the enteric nervous system also participates, and
comprises an intrinsic network of neurons formed by the submucosal plexus, which controls
secretory glandular activity, visceral circulation, and motor activity through the myoenteric
plexus [25].
As part of the gastrointestinal system, the stomach not only participates in its physiology
with acid secretion, intestinal motility, digestion, and blood flow but also has endocrine
functions with an important role in the regulation of appetite and satiety by producing peptide
hormones with anorexigenic and orexigenic effects, such as ghrelin, obestatin, and leptin,
although the stomach is not the main producer but can produce small amounts of this
hormone [26-29]. Table 13.2 describes the factors influencing circulating leptin and ghrelin
levels.
i) Ghrelin is a hormone composed of 28 amino acids, which is encoded by the GHRL

gene that is located on human chromosome 3 (3p25-26). The hormone binds to the
GHSR1a (growth hormone secretagogue receptor type 1a). Ghrelin is produced
mainly in the stomach, but small quantities are synthesized in the intestine, pancreas,
placenta, kidney, pituitary gland, and hypothalamus. In the stomach, ghrelin is
produced primarily by the P/D1 cells of the gastric fundus. This hormone suppresses
the secretion and activity of insulin, and its key role is to positively regulate appetite
(orexigenic effect). Therefore, this hormone is considered to be an important
modulator of energy balance and metabolism. It has been reported that ghrelin plays
a significant role in many physiological processes, including the regulation of
metabolism, anxiety, and other events associated with cognition. Ghrelin has also
been reported to be involved in the regulation of nitric oxide synthase (iNOS), which
is important for its involvement in the control of gastric inflammation in response to
an infection by H. pylori [30-32]. Ghrelin levels are increased in the plasma before
the intake of food and during inanition. However, ghrelin levels are reduced in
subjects with obesity and insulin resistance. Ghrelin isoforms are known, as follows:
the non-acylated form, which is localized primarily in the stomach, and the acylated
form, which is considered the active form of the hormone and is present mainly in
the serum; additionally, some studies show that GHSR genes and ghrelin variants are
implicated in the pathophysiology of obesity [33, 34].
ii) Leptin is a peripheral hormone with an anorexigenic effect that is encoded by the OB
gene located on human chromosome 7 (7q31, 3). Leptin is a peptide composed of
166 amino acids that acts through LEPR (leptin receptor), which is located in the
hypothalamus. The hormone is produced mainly in adipose tissue, although small

amounts are also produced in the stomach. It is known that circulating leptin levels
are inversely related to BMI [35].
iii) Obestatin is a peptide of gastric origin. Interestingly, it is encoded by the ghrelin
gene. Thus, it is produced mainly in the stomach but can also be found in the spleen,
mammary gland, breast milk, and plasma. Obestatin is composed of 23 amino acids
derived from pro-ghrelin. The physiological role of obestatin in humans is still
poorly understood. It appears that it is an important part of the complex network
between the brain and gastrointestinal tract, as it inhibits appetite (anorexigenic
effect), reduces gastric motility and weight gain, and increases the release of
pancreatic juice. It has also been observed to be involved in memory and sleep
regulation, and recent studies indicated that it could affect cell proliferation. The
obestatin receptor is not well understood, with a study reporting that its receptor is
GPR39 (G protein-coupled receptor 39); however, other authors proposed that it is
GLP-1R (glucagon-like peptide-1 receptor). Therefore, this point is currently still in
dispute. Simultaneous transcription of the ghrelin and obestatin genes is thought to
exist, which suggests that there is possibly differential processing of primary
transcripts to generate two products with antagonistic effects [36, 37].
Table 13.2. Factors influencing circulating leptin and ghrelin levels
Leptin Ghrelin
Food intake (+) (-)
With increasing in Body mass index (+) (-)
High serum glucose (+) (-)
Insulin (+) (-)
Carbohydrate rich diet (+) (-)
Fasting (-) (-)
With increasing Age (-) (-)
Gender (+) (+)
(in females) (in females)
Fat rich diet (-) (+)
Exercise (-) no change
Growth hormone no change (-)
( + ) = increase ; ( - ) = decline.
(Adapted from: Arslan N, et al. 2010).
Ghrelin, leptin, and obestatin constitute a central axis in the modulation of appetite,
among other functions. Figure 13.2 shows the peripherial regulators and hipothalamic center
involved in the control of satiety and hunger. However, further studies are needed to clarify
their role and interconnection to indicate them as endocrine markers that may reflect changes
in the acute or chronic nutritional status and to have more evidence to be applied in anti-
obesity therapies, as recently proposed. Recent studies showed the importance of ghrelin and
the role it plays in some endocrine and chronic degenerative diseases, such as diabetes
mellitus, insulin resistance, cardiovascular disease, myocardial infarction, atherosclerosis,
cancer, and cachexia [38]. Evidence showed that ghrelin might increase myocardial
contractility, improve vasodilation, and have a protective effect against myocardial damage.

Recent data showed that ghrelin might influence atherogenesis, and the role of GHS and
ghrelin should be further evaluated because it may be a new opportunity for the treatment of
cardiovascular diseases [39].
Figure 13.2. Peripherial regulators and hipothalamic center involved in the control of satiety and
hunger.
The peptides may enter the brain through the bloodstream, these may reach the hipothalamus through
the vagal nerve and nucleus tracus solitarus. Ghrelin, Leptin, Insulin and PYY stimulate (+) and
suppress (-) hypothalamic neurones containing various neuropeptides (AGRP= agouti-related
transcript; NPY= neuropeptide Y; POMC= proopiomelanocortin; CART= cocaine and amphetamine-
regulated transcript) resulting in anorexic or orexic effects on balance between food intake and energy
expediture, as well as stimulating other functions. The receptors (green) are represented by numbers: 1
and 7=YIR (NPY1 receptor); 2=MC4R (melacortin-4 receptor); 3=Y2R (NPY2 receptor); 4=GHSR
(Growth Hormone receptor); 5. and 9=LEPRB (leptin receptor); 6=MC3R (melanocortin-3 receptor);
8= insulin receptor. Zones in the brain, PVN= paraventricular nucleus; ARC= arcuate nucleus; NTS=
nucleus tract solitarius; DVC= dorsal vagal complex.
In a follow-up study in adults consuming a diet and phylloquinone [40], the results have
shown a greater reduction of ghrelin. This was associated with an improvement of cytokines
and markers associated with insulin resistance and diabetes mellitus. The phylloquinone and
diet had a protective effect on chronic inflammatory diseases. Additionally, in a study of
nightshift workers who present circadian rhythm disorders (such as poor sleep, and changes in
behavior and eating habits) that predispose individuals to obesity and metabolic disorders, it
was observed that leptin and ghrelin showed alterations associated with changes in feeding
behavior, sleep, increased adiposity, and metabolic disorders [41].
Considering the multiple effects of ghrelin and that its clinical applications have been
shown to be useful in treating diseases such as cancer and cachexia, ghrelin can improve
wasting syndrome through the beneficial effects of growth hormone. Moreover, ghrelin may

have an important role in the treatment of intestinal motility disorders and obesity and for
some treatments for patients with diabetes mellitus, infections, rheumatologic diseases, and
those presenting GH deficiency, and it may assist in the diagnosis of these hormonal disorders
[42-44].
Some studies suggest that there are opportunities to develop effective treatments for some
of these diseases, including obesity, Prader-Willi syndrome, nervous anorexia, and diabetes
mellitus. In addition, there is strong evidence that these hormones are associated with growth
and stature in children with conditions that can affect their development and have a
significant impact on the quality and outcome of health and life [45].
Helicobacter pylori Colonization

and Its Role in Endocrine Diseases
Infections by H. pylori influence a large number of gastric functions, among which are
the secretion and concentration of acid (which is lower in the gastric juice of infected
subjects), alterations in the production and concentration of neuroendocrine peptides such as
gastrin and somatostatin, and the synthesis and secretion of ghrelin, acyl-ghrelin, obestatin,
and leptin [46-50].
Some authors have studied the possible link between the infection with H. pylori and
certain diseases of endocrine origin. Papamichel et al. conducted a literature review and
concluded that there is no consensus on the relationship between H. pylori infections and the
presence of endocrine disease, even though this is still a subject of investigation [51]. Some
studies indicate that there is a high prevalence of infections by H. pylori in patients with
diabetes mellitus type 1 or type 2 and that this infection is related to the duration of diabetes,
presence of dyspeptic symptoms, cardiovascular neuropathy, age, sex, high body mass index,
blood pressure, fasting glucose concentration, and glycosylated hemoglobin levels. Moreover,
the association of metabolic syndrome and H. pylori colonization remains controversial,
although the high incidence of metabolic syndrome and H. pylori infections could explain this
non-significant association. In addition, it is interesting to note that the possible role of
specific CagA+ strains of H. pylori in endocrine pathologies, which are potentially linked to
the presence of the bacterium, has been poorly studied [52-57].
Helicobacter pylori Infection and Its Relationship

to Malnutrition and BMI
H. pylori colonization significantly influences the metabolism of colonized individuals,
and this effect depends heavily on the age of the host at colonization. In this sense, the effects
and consequences of H. pylori infections on children with an acute infection and on adults
with a chronic infection are different. In general, it is considered that an infection with H.
pylori in children is associated with a decrease in the growth rate of the host, anemia due to
an absence of Fe+++, and an increase in the frequency of episodes of diarrhea caused by
different types of enteropathogens [57-61].

Figure 13.3 diagrammatically shows the relationship between H. pylori colonization and
its effects on the host, according to the model proposed by Windle et al. In a previous study,
Kopácôvá et al. [62] found a significant association, depending on age, between an infection
with H. pylori and BMI because in those individuals under 15 years of age, colonization was
associated with low BMI, while in individuals over 15 years of age, the colonization was
associated with a higher BMI, overweight, and obesity.
IL-1β
polimorphisms
Increased enteric
infections and diarrhea
Hypochlorhydria
Decreased of specific
Infection by H. pylori growth rate,
in childhood malnutrition
Iron-deficiency
anemia
Figure 13.3. Relationship between H. pylori colonization and its effects on the pediatric host in
underdeveloped countries [Adapted from: Windle HJ et al. Pediatrics 2007].
Although reports can provide conflicting data, the authors generally agree that an
infection with H. pylori is associated with decline in growth rates in childhood, and variations
in appetite, food intake and BMI in adults. Reports differ on whether this association is
directly attributable to the bacterial infection or is an indirect consequence of it, with it being
the causal agent of anemia, dyspepsia, and recurrent abdominal pain, which are all influenced
by the socioeconomic environment of the host [63-67]. After studying a Peruvian pediatric
population, Goodman et al. concluded that infections with H. pylori were associated with a
significant decrease in the specific growth rate of colonized subjects and suggested that
changes in this variable are more accurate to describe the acute effects of an infection with H.
pylori [68].
In the case of adult hosts, the findings and conclusions regarding the relationship between
H. pylori infections and BMI are also contradictory and are strongly influenced by the study
design and methodology used. Some studies on the relationship between the colonization by
H. pylori and BMI are controversial and present evidence that this possible relationship
depends on the ethnicity, socio-economic conditions, and age of the subjects assessed. On one
hand, Thjodleifsson et al. Aíslan et al. and Al-Akwaa [69-71] reported that the prevalence of
infections with H. pylori is higher in overweight or obese subjects when compared to normal
weight individuals. On the other hand, Méndez-Sánchez et al. and Dutta et al. [72,73] found

no difference between the frequency of infections with H. pylori between obese and normal
weight individuals, and, similar to what is reported in children, a significant relationship
between BMI and the presence of gastritis, which may be related to H. pylori seropositivity,
has also been found [74,75].
Moreover, other studies draw attention to the observation that the prevalence of H. pylori
is lower in obese than in non-obese individuals [76-79]. One of the first groups to recognize
that the incidence of H. pylori in the stomachs of morbidly obese patients was lower than in
the subjects with a normal BMI was the group of Scapa and collaborators; additionally,
Renshaw et al. and Ramaswamy et al. documented that patients undergoing surgery for
morbid obesity had a lower percentage of colonization by H. pylori than non-colonized
patients [81-83]. In a study of 414 cases and 683 controls, Wu et al. demonstrated that the
absence of an H. pylori infection (established by the absence of antibodies directed against
the bacterium) was significantly related to the presence of morbid obesity and that the
absence of infections in childhood was associated more significantly with the development of
obesity in adulthood [84]. Chronic H. pylori infections have been linked to a low BMI. In a
literature review, Macadam et al. concluded that there is sufficient evidence to consider that
colonization by H. pylori could be a protective factor against the development of obesity and
associated pathologies [85]. In a review conducted by Li et al. the authors note the direct
relationship between obesity and H. pylori infections, which may explain the increased
prevalence of cancer in the obese population—mainly when infected with the VacA+/CagA+
strains that are associated with lower levels of circulating ghrelin [86].
Several authors have noted that the relationship between H. pylori and BMI variations
may be due to changes in the production of leptin and ghrelin. Reports from studies
conducted with adults have described that individuals infected with H. pylori have lower
ghrelin levels and higher leptin levels when compared to non-infected subjects. This result
may be due to chronic gastritis that can directly influence ghrelin-producing cells and
consequently affect the levels of this circulating hormone [87]. In a systematic review on H.
pylori infections and circulating levels of ghrelin and BMI, Nweneka and Prentice indicate
that in obese individuals, H. pylori infections are not related to the levels of ghrelin, whereas
this relationship does occur in normal weight individuals where an infection is associated
with decreased levels of the hormone, which demonstrates the association between this
hormone and chronic inflammatory processes induced by obesity or bacterial infections [48].
Relationship between Helicobacter pylori

Infection and Ghrelin, Leptin,
and Obestatin Levels
It is generally accepted that H. pylori colonization is significantly related to changes in
the serum levels of ghrelin; however, there is no consensus among research groups on the
impact of colonization by H. pylori on the increases in serum leptin levels nor on the activity
of GOAT (ghrelin o-acyl-transferase), and no reports have established the impact of the
presence of H. pylori on the synthesis of obestatin [67,88-90]. Stec-Machalska et al. analyzed
ghrelin levels and the presence of H. pylori in premenopausal women. This is one of a few
studies that relate the bacterial genotype (cagA) with ghrelin production, and one of the

conclusions was that an increase of the ghrelin levels in gastric H. pylori-positive cagA+
patients could exert a protective effect against the presence of cancer and obesity [88]. There
have been various studies on gastric ghrelin levels in colonized subjects, finding that mRNA
levels are low in infected individuals [91-93]. Other authors have identified a lower quantity
of cells producing ghrelin in colonized patients, and the decrease of these cells in the stomach
is proportional to the severity of the gastritis, thus suggesting that this infection can suppress
the expression of ghrelin in the stomach [72,94-96].
The discrepancies that may exist between the plasma and gastric ghrelin levels reported
by different authors may be due to the stomach area where the biopsy was taken because it
has been observed that the levels are increased in the fundus and body of the stomach but not
in the region of the antrum, where no change has ever been observed. However, we cannot
rule out the possibility that the infecting strain may influence hormone levels [88]. On this
particular aspect, there is scarce information. One study showed that individuals infected with
virulent strains (H. pylori CagA+ and VacA+) have circulating ghrelin levels that are lower
than those infected with less virulence strains [96].
The age of colonized individuals is considered to be a factor that significantly affects the
variations of leptin and ghrelin due to an H. pylori infection. Two studies found low levels of
ghrelin in colonized children, while a third study found no differences. The following
difference in the populations examined in these studies should be considered: healthy and sick
children with normal weight or healthy children but with different body weights. [48,97]. In
the case study by Konturek et al. they found that plasma ghrelin levels are reduced, while
leptin levels are increased, in children and adults as a result of H. pylori infections; however,
Salles et al. found that the expression and plasma ghrelin and leptin levels were decreased in
infected patients in a study conducted in a population of elderly subjects. Additionally, it is
possible that the gender of the individuals significantly affects the serum levels of these
hormones because Chuang et al. found that men showed significant variations of ghrelin but
not leptin that was related to an infection with H. pylori, as compared to the variations
reported for female patients [98-100].
In a recent systematic review on infections with H. pylori and serum ghrelin levels in
which the inclusion and exclusion criteria of the involved studies were homogenized (type of
population, number of subjects studied, and the different methods to determine the plasma
levels of this hormone), the authors concluded that circulating ghrelin was significantly lower
in infected individuals compared to uninfected individuals; nevertheless, in individuals with
ulcers associated with H. pylori, high serum ghrelin levels were observed, while this was not
seen in individuals with gastric atrophy, where the levels are usually low. Of the 24 studies
considered, the circulating ghrelin levels were lower in infected versus uninfected patients in
15 studies, while 9 studies found no differences [48].
Leptin variations associated with colonization by H. pylori have been described by
several authors. There is a general consensus that the plasma and mRNA levels of this
hormone are increased through colonization by H. pylori. Slomiany and Slomiany have
suggested that leptin levels increase during H. pylori colonization, as this peptide functions as
a negative modulator of the pro-inflammatory activity of bacterial LPS (lipopolysaccharide)
[101,102]. Moreover, Fetissov et al. [103] have discussed the possibility that the presence of
auto-antibodies directed against ghrelin and leptin are responsible for variations in the plasma
concentrations of this hormone; thus, the presence of some microorganisms of the intestinal

microbiota, including H. pylori, may be influencing the balance in the function of the
hypothalamic-pituitary-adrenal axis.
Figure 13.4. Model of the regulation by leptin and ghrelin on the inflammatory process induced by H.
pylori [Adapted from: Slomiany & Slomiany. J Physiol Pharmacol. 2007, Slomiany & Slomiany. ISRN
Gastroenterol. 2011, and Slomiany & Slomiany. Inflammopharmacol. 2013]
(GHSR – Growth hormone secretatogue receptor, NO2 – Nitric oxide synthase constitutive and induced,
NFκB – Nuclear Factor, ATF-2, Activating transcription factor 2, JNK – c Jun N-terminal kinase, COX
– Ciclooxygenase 1 and 2, C/EBP - CCAAT/enhancer binding protein, cPLA – Phospholipase A2,
PGH2 – Prostaglandin H2).
The peptides ghrelin and leptin modulate the inflammatory response of the host induced by H. pylori.
By interaction with GHSR, ghrelin activates SRC and AKT that up-regulate cNO2 with nitrosylation of
IKKβ and inhibition of NFκB activation and translocation, that originates the repression of iNO2;
additionally, ghrelin inhibits p38 and JNK, and the translocation of ATF2, cJUN and cEBP , that
originate repression of COX2. In other pathway, leptin inhibits the activity of cPLA2 and the synthesis
of arachidonic acid.
We report in Figure 13.4 the possible mechanisms for the negative modulation that
ghrelin and leptin exert on the inflammatory process induced by the presence of H. pylori.
Regarding leptin, it exerts an inhibitory effect on the activity of cytosolic phospholipase
(cPLA), which in turn negatively controls the arachidonic acid cascade; however, in the
ghrelin model, this hormone interacts with the GHSR1a receptor causing a cascade that
triggers the inhibition of the nuclear translocation of C/EPBγ, phosphorylation of JNK and
p38, and NFκB activation and the suppression of COX-2 and iNOS [101,102,104].

Moreover, the negative association between ghrelin and growth hormone has been
documented, which is perhaps mediated by competition for the membrane receptor and the
inverse relationship between the activity of ghrelin and interleukin-1 beta (IL-1B), which is
associated with a susceptibility to H. pylori colonization [105-108].
Association between Helicobacter pylori

Eradication and Ghrelin, Leptin,
and Obestatin Peptides
Different authors have demonstrated that H. pylori eradication is significantly associated
with increases in the synthesis of ghrelin and its serum concentrations in both the acylated
and non-acylated forms. The seminal work of Nwokolo et al. is considered to now be a
"classic", as it established the relationship between the eradication of H. pylori and ghrelin
increases and the effect of colonization by this bacterium in growth retardation due to
decreased appetite and intake [109]. This has been confirmed by other groups by comparing
infected and uninfected patients and measuring the expression of genes coding for ghrelin and
obestatin by real-time PCR, the serum levels of these hormones by immunoblotting, and the
density of gastric ghrelin-producing cells using immunohistochemical techniques [109-113].
In a study reported by Osawa et al. the plasma concentrations of ghrelin before and after
the eradication of H. pylori were compared. In 50 patients, the plasma concentration of
ghrelin increased, while it decreased in 84 patients. The preproghrelin expression increased
almost 4 times, and the number of ghrelin-positive cells increased or remained unchanged.
The ghrelin variations were inversely correlated to the observed changes in body weight and
the initial ghrelin plasma levels [113].
Osawa [114] has reported that in comparing patients infected with H. pylori to uninfected
healthy controls, the plasma levels and mRNA expression of ghrelin and the number of cells
producing ghrelin were decreased in colonized subjects, although the acyl-ghrelin/total-
ghrelin relationship was higher in this group. In individuals where the bacteria were
eradicated, the increased mRNA expression of preproghrelin and variations in plasma ghrelin
levels were inversely influenced by BMI and the initial concentration of plasma ghrelin.
Additionally, Osawa and Jang have established the importance of the type and degree of
gastric lesions on variations in ghrelin levels that are associated with the presence and
eradication of H. pylori [114,115].
The review conducted by Nweneka and Prentice [48] included study reports that analyzed
the relationship between the eradication of H. pylori and changes in ghrelin levels, as follows:
a) From 11 studies in adults, 6 of them found that ghrelin levels did not change, while
the levels increased in 4 studies and were decreased in 1 study after eradication of the
bacterium.
b) Two studies in children found that ghrelin levels decreased after eradication of H.
pylori.
In two recently published studies in pediatric populations, the direct relationship between
the eradication of H. pylori and increases in ghrelin and acyl-ghrelin were established, which

is consistent with the results reported in adults, and this could lead to weight gains in treated
patients in the long-term [116,117].
We found that the majority of published articles accurately establish that the colonization
and eradication of H. pylori are factors that significantly affect serum ghrelin levels, although
the precise mechanisms that are involved or modulate these variations are not known. The
presence or absence of the bacterium does not seem to significantly relate to leptin levels, and
the relationship between H. pylori infections and the expression and levels of obestatin has
been poorly explored.
One aspect that has received little attention is the possible effect of colonization with or
eradication of H. pylori on the activity of GOAT, which is the enzyme responsible for the
posttranslational modification of ghrelin to generate octanoyl-ghrelin, which is the active
form of the hormone. In a clinical study, the group of Ando et al. [118] has reported that the
colonization and eradication of H. pylori are associated with changes in the serum levels of
ghrelin and acyl-ghrelin and the expression levels of the ghrelin and GOAT genes.
Colonization is associated with reductions in the plasma concentrations of ghrelin and acyl-
ghrelin and the mRNA levels of ghrelin and GOAT, while eradication is linked to increases in
plasma ghrelin and acyl-ghrelin levels, increases in the mRNA levels of ghrelin and GOAT,
and variations in the acyl-ghrelin/ghrelin relationship.
The relationship between the eradication of H. pylori and leptin levels has been less
studied, and research groups have not yet reached a consensus. In a literature review, Blaser
and Atherton concluded that leptin levels are high in individuals colonized with H. pylori, as
compared to non-colonized individuals, and that eradication leads to significant decreases in
leptin and ghrelin [52]. Moreover, Kebapcilar et al. studied the effect of the eradication of H.
pylori on the levels of serum leptin, the TOS (total oxidant status), soluble CD40 ligand
(sCD40 L), and body composition in patients with dyspepsia. The results showed that the
sCD40 L levels and TOS decreased after the eradication of H. pylori. Additionally, the
percentages of body fat and muscle fat decreased, while the fat-free mass increased
subsequent to eradication. In this study, the leptin levels showed no difference after
eradication of the bacterium [119].
In an article that examined the relationship between an infection with H. pylori and the
ghrelin and leptin concentrations in a pediatric population, Pacífico et al. [120] developed a
longitudinal case-control study to determine the baseline levels of ghrelin and leptin in
prepubescent Hp+ and Hp- individuals and to evaluate the long-term effect of H. pylori
eradication on the serum levels of these hormones and on body composition. The authors
concluded that in prepubescent individuals, ghrelin levels are inversely associated with
mucosal damage. In these populations, the eradication of H. pylori was associated with
decreases in the ghrelin levels and significant increases in BMI, lean tissue, and body fat.
Similarly, in a pediatric population study, Yang et al. [121] studied a cohort of 1,122
children for 1 year and showed that colonization by H. pylori was associated with low levels
of acyl-ghrelin and growth retardation and that eradication was associated with increases in
the serum levels of acyl-ghrelin and growth rate of treated children.
The presence and eradication of H. pylori has been studied in relation to how it affects
the nutritional balance of the host, which is modulated by the activity of different regulatory
hormones. In this sense, the ghrelin/obestatin relationship could be crucial for energy balance.
This relationship in individuals infected with H. pylori and in the host response to the
eradication of the bacterium and the relationships between ghrelin and gastrin levels among

H. pylori-positive patients and their possible association with the development of different
gastric pathologies have not been fully explored [122,123].
Relationship between Helicobacter pylori

Eradication and BMI
In general, reports from different research groups agree that the presence and eradication
of H. pylori affect the balance of peptide hormones that modulate appetite. This may
eventually lead to changes in the host’s BMI, even when there is no consensus among the
various groups as to how these changes are reflected in increases or decreases in body-weight
events that are dependent on host factors such as age, sex, race, associated diseases of the
colonized individual, and the socioeconomic environment in which it develops. In Asian
populations, a significant increase in the BMI of colonized individuals has been reported after
the eradication of H. pylori [124,125]. Azuma et al. [126] established that there were
increases in the weight and BMI of the study subjects when they were followed for a period
of 12 months after the eradication of the bacterium. Moreover, a case-control study showed
that the eradication of H. pylori was significantly associated with increases in the BMI of
treated patients and found that people with a high BMI had a higher risk of developing
gastroesophageal reflux disease (GERD) after the eradication of an infection with H. pylori
[127]. A study involving 10,537 individuals with ages ranging from 20 to 59 years showed
that the BMI increased after eradication of H. pylori, which was attributed to a resolution of
the symptoms of dyspepsia [128]. Akanuma et al. demonstrated increases in the BMI of
colonized and treated individuals with type 1 diabetes without significant increases in
glycosylated hemoglobin levels [129].
The relationship between the eradication rate of H. pylori and BMI has been controversial
because while some authors mention that the rate decreases at a higher BMI, others
demonstrate a direct relationship between the BMI and the eradication rate of the bacteria
[130,131].
An attempt has been made to find relationships between the H. pylori eradication and
changes in BMI by analyzing the possible influence that different factors in the host may
exercise. In this regard, Suto et al. have postulated that the serum PG I/PG II (pepsinogen I/II)
ratio could influence the effect of bacterial eradication on the BMI [132].
Francois et al. published a paper showing that the eradication of H. pylori in adults is
significantly associated with increased levels of acyl-ghrelin and leptin and an increased BMI
when the subjects were followed for a period longer than 2 years [133]. They demonstrated
that eradication significantly influenced the physiology of these hormones and, therefore, the
metabolic balance of the treated individuals.
In Figure 13.5, we show a model where the colonization and eradication of H. pylori
relate to changes in ghrelin, leptin, and obestatin and modulation of GOAT.

Ghrelin 1. Decreased appetite

2. Decreased of specific
Leptin growth rate*
Colonization 3. Decreased BMI
( Hp+ vs Hp-) Obestatin (?) 4. Malnutrition, anemia*
GOAT (?)
Growth hormone
Food intake , lipolysis
energy expenditure
H. pylori
Ghrelin
1. Increased appetite
Leptin 2. Increased BMI
Eradication
3. Overweigth, Obesity (?)
( Hp+ Hp- ) Obestatin (?)
GOAT (?)
Growth hormone
Food intake , lipogenesis
energy expenditure
Figure 13.5. Effect of the colonization and eradication of H. pylori on ghrelin, leptin, obestatin and the
activity of GOAT
Conclusion
It has been recently shown that the composition of the intestinal microbiota is a key
element in human metabolism and significantly influences the physiology and variations in
the weight, height, and BMI of colonized individuals. An infection by H. pylori has diverse
effects that are dependent on the age of the colonized subjects, as follows: in childhood, it is
associated with weight loss, iron deficiency anemia, and decreased growth rate, whereas in
adulthood it is associated with gastritis, gastric ulcer cancer, gastric MALT (mucosa-
associated lymphoid tissue) lymphoma, and a low BMI.
In contrast to the opinion of other authors, M. Blaser of New York University has
suggested that there is sufficient evidence to consider H. pylori a normal component of the
human microbiome and that this bacterium is capable of playing a role as a pathogen only
when the delicate balance between the microorganism, the host, and the environment is
broken. Moreover, the eradication of H. pylori has been associated with increased synthesis
and serum concentrations of ghrelin and decreases in leptin, which in turn are possibly
associated with increased weight and BMI. Several authors have noted that there is sufficient
evidence in the literature to consider colonization by H. pylori to be a protective factor against
the development of overweight and that indiscriminate eradication of the bacterium alters the
host gastric equilibrium and promotes the pandemic of overweight and obesity in which we
find ourselves [134, 135].

Experimental evidence of the relationship between the presence and eradication of H.

pylori and variations in the serum levels of ghrelin are numerous and consistent, while they
are scarce and often conflicting for leptin and are extremely reduced concerning the levels
and expression of obestatin. Nevertheless, one must consider that the discrepancies observed
among the different research groups may be due to the great heterogeneity in experimental
designs in relation to what is being measured (gene expression or serum concentrations,
plasma or gastric ghrelin, and acyl-ghrelin, leptin, or obestatin), how it is measured [Real
time-PCR, immunohistochemistry, and ELISA (enzyme-linked immunosorbent assay)], in
whom it is measured (pediatric or adult populations of diverse ethnic and socioeconomic
backgrounds), what are the associated diseases (gastritis, reflux, gastric cancer, and diabetes),
and how the effect on the host is determined (variations in the growth rate, changes in BMI,
changes in the activity of GOAT, and abnormal hormone levels). Therefore, the findings of
each study present high diversity, although it is possible to establish consensual conclusions.
The vast majority of studies in this field have been conducted in adult populations in
developed countries and for short periods of time. Thus, longitudinal studies with similar
methodologies with which to compare the results and conclusions adequately in countries
with high rates of H. pylori infection and in pediatric populations, which are more susceptible
to the effects of infection and bacterial eradication, are required. In this sense, few studies
have specifically assessed the possible role of the virulence of the organism, as defined by the
presence of the pathogenicity island.
A large number of experimental studies and the development of animal models are
required to establish the extent to which there is a causal relationship between colonization by
H. pylori and the expression levels of the peptide hormones that control appetite and to
demonstrate the mechanisms regulating these hormones that are susceptible to modulation by
H. pylori and how these regulatory systems are modified by the effect of the bacterium and its
eradication.
References
[1] DiBiase JK, Zhang H, Crowell MD, Krajmalnik-Brown R, Decker GA, Rittmann BE,
(2008). Gut microbiota and its possible relationship with obesity. Mayo Clin Proc.,
83:460-9.
[2] Harris K, Kassis A, Major G, Chou CJ, (2012). Is the gut microbiota a new factor
contributing to obesity and its metabolic disorders?. J Obes., 2012:879151. doi:
10.1155/2012/879151.
[3] Song Y, Altarejos J, Goodarzi MO, Inoue H, Xiuqing Guo X, Berdeaux R, Kim JH,
Goode J, Igata M, Paz J, Hogan MF, Singh PK, Goebel N, Vera L, Millar N, Cui J,
Jones MR, CHARGE Consortium, GIANT Consortium, Yii-Der I. Chen YDI, Taylor
KD, Hsueh WA, Rotter JI, Montminy M, (2010). The CREB coactivator CRTC3 links
catecholamine signaling to energy balance. Nature. 2010, 468(7326):933-9. doi:
10.1038/nature09564.
[4] Ludwig J, Sanbonmatsu L, Gennetian L, Adam E, Duncan GJ, Katz LF, Kessler RC,
Kling JR, Lindau ST, Whitaker RC, McDade TW, (2011). Neighborhoods, obesity, and
diabetes — A randomized social experiment. N Engl J Med., 365:1509-19.

[5] Villalobos-Comparán M, Villamil-Ramírez H, Villarreal-Molina T, Larrieta- Carrasco

E, León-Mimila P, Romero-Hidalgo S, Jacobo-Albavera L, Liceaga-Fuentes AE,
Campos-Pérez FJ, López-Contreras BE, Tusié-Luna T, del Río-Navarro BE, Aguilar-
Salinas CA, Canizales-Quinteros S, (2012). PCSK1 rs6232 is associated with childhood
and adult class III obesity in the Mexican population. PLoS ONE., 7(6):e39037.
doi:10.1371/journal.pone.0039037.
[6] Bäckhed F, Manchester JK, Semenkovich CF, Gordon JI, (2007). Mechanisms
underlying the resistance to diet-induced obesity in germ-free mice. PNAS., 104: 979–
84.
[7] Bik EM, Eckburg PB, Gill SR, Nelson KE, Purdom EA, Francois F, Perez-Perez G,
Blaser MJ, Relman DA, (2006). Molecular analysis of the bacterial microbiota in the
human stomach. PNAS USA., 103:732-7.
[8] Warren JR, Marshall B, (1983). Unidentified curved bacilli on gastric epithelium in
active chronic gastritis. Lancet., i:1273-5.
[9] Tonkic A, Tonkic M, Lehours P, Mégraud F, (2012). Epidemiology and diagnosis of
Helicobacter pylori infection. Helicobacter., 17 (Suppl.1):1–8.
[10] Delahay RM, Massimo Rugge M, (2012). Pathogenesis of Helicobacter pylori
infection. Helicobacter., 17 (Suppl. 1):9–15.
[11] Forman D, Newell DG, Fullerton F, Yarnell JW, Stacy AR, Wald N, Sitas F, (1991).
Association between infection with Helicobacter pylori and risk of gastric cancer:
evidence from a prospective investigation. BMJ., 302:1302-5.
[12] IARC. Monographs on the evaluation of the carcinogenic risks to humans. Vol. 61.
Schistosomes, liver flukes and Helicobacter pylori. International Agency for Research
on Cancer. Lyon 1994.
[13] Blaser MJ, Perez-Perez GI, Kleanthous H, Cover TL, Peek RM, Chyou PH,
Stemmermann GN, Nomura A, (1995). Infection with Helicobacter pylori strains
possessing cagA is associated with an increased risk of developing adenocarcinoma of
the stomach. Cancer Res., 55: 2111-15.
[14] Blaser MJ, Berg DE, (2001). Helicobacter pylori genetic diversity and risk of human
disease. J Clin Invest., 107:767-73.
[15] Parsonnet J, Friedman GD, Orentreich N, Vogelman H, (1997). Risk for gastric cancer
in people with CagA positive or CagA negative Helicobacter pylori infection. Gut.,
40:297–301.
[16] Hirai I, (2010). Infection of less virulent Helicobacter pylori strains in asymptomatic
healthy individuals in Thailand as a potential contributing factor to the Asian enigma.
Microb Infect.,12:227-30.
[17] Atherton JC, Blaser MJ, (2009). Coadaptation of H. pylori and humans: ancient history,
modern implications. J Clin Invest., 119:2475-87.
[18] Ahlman H, Nilsson O. The gut as the largest endocrine organ in the body. Annals of
Oncology 2001;12 (Suppl. 2):S63-8.
[19] Furness JB, Wolfgang A. A. K, Clerc N, (1999). The intestine as a sensory organ:
neural, endocrine, and immune responses. Am J Physiol Gastrointest Liver Physiol.,
277:G922-8.
[20] Chaudhri O, Small C, Bloom S, (2006). Gastrointestinal hormones regulating appetite.
Phil Trans R Soc B., 361:1187-209.

[21] Chaudhri OB, Field BCT; Bloom SR, (2008). Gastrointestinal satiety signals. Int. J.
Obes., 32:S28–31.
[22] Gardiner JV, Jayasena CN, Bloom SR, (2008). Gut hormones: A weight off your mind.
J Neuroendocrinol., 20;834–41.
[23] Christian L, Roth MD, Thomas Reinehr M, (2010). Roles of gastrointestinal and
adipose tissue peptides in childhood obesity and changes after weight loss due to
lifestyle intervention. Darch Pediatr Adolesc.,164:131-8.
[24] Gunawardene AR, Corfe BM, Staton CA, (2011). Classification and functions of
enteroendocrine cells of the lower gastrointestinal tract. Int J Exp Path., 92; 219–31.
[25] Silveira Rodríguez MB, Gómez-Pan A, Carraro Casieri R, (2006). Nuevas perspectivas
en el tratamiento de la obesidad: el aparato digestivo como órgano endocrino. Med Clin
Barc., 127:300-5.
[26] Inui A, Asakawa A, Bowers CY, Mantovani G, Laviano A, Meguid MM, Fujimiya M,
(2004). Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an
endocrine organ. FASEB J., 18;439-56.
[27] Ataka K, Inui A, Asakawa A, Kato I, Fujimiya M, (2008). Obestatin inhibits motor
activity in the antrum and duodenum in the fed state of conscious rats. Am J Physiol
Gastrointest Liver Physiol,. 294:G1210-8.
[28] Neary NM, Small CJ, Bloom SR, (2003). Gut and mind. Gut., 52:918–21.
[29] Lacquaniti A, Donato V, Chirico V, Buemi A, Buemi M, (2011). Obestatin: An
interesting but controversial gut hormone. Ann Nutr Metab., 59:193–9.
[30] Hillman JB, Tong J, Tschop M, (2011). Ghrelin biology and its role in weight-related
disorders. Discov Med., 11:521-8.
[31] Heppner KM,Tong J, Kirchner H, Nass R, Tschop MH (2011). The ghrelin O-
acyltransferase-ghrelin system: a novel regulator of glucose metabolism. Curr Opin
Endocrinol Diabetes Obes., 18:50-5.
[32] Slomiany BL, Slomiany A, (2011). Role of ghrelin-induced cSrc activation in
modulation of gastric mucosal inflammatory responses to Helicobacter pylori.
Inflammopharmacol., 19:197-204.
[33] Hosoda H, Kojima M, Kangawa K, (2002). Ghrelin and the regulation of food intake
and energy balance. Mol Interv., 2:494-503.
[34] Scerif M, Goldstone AP, Korbonits M, (2011). Ghrelin in obesity and endocrine
diseases. Mol Cell Endocrinol., 340:15-25. doi: 10.1016/j.mce.2011.02.011.
[35] Morton GJ, Schwartz MW, (2011). Leptin and the central nervous system control of
glucose metabolism. Physiol Rev., 91:389-411.
[36] Zhang JV, Ren PG, Avsian-Kretchmer O, Luo ChW, Rauch R, Klein C, Hsueh AJW,
(2005). Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on
food intake. Science., 310:996-9.
[37] Chen CY, Asakawa A, Fujimiya M, Lee SD, Inui A, (2009). Ghrelin gene products and
the regulation of food intake and gut motility. Pharmacol Rev., 61:430–81.
[38] Sadetzki WI, Kanety SH, Kundel Y, Pariente C, Epstein N, Oberman B, Catone R,
Kaufman B, Shimn I, (2006). Adiponectin, ghrelin, and leptin in cancer cachexia in
breast and colon cancer patients. Cancer., 106:966-73.
[39] Rizzo M, Rizvi AA, Sudar E, Soskic S, Obradovic M, Montalto G, Boutidir M,
Mikhailidis DP, Senovic ER, (2012). A review of the cardiovascular and anti-
atherogenic effects of Ghrelin. Curr Pharm Des. [Epub ahead of print].

[40] Juanola–Falgarona M, Salas-Salvado J, Estruch R, Miranda J, Martinez-Gonzalez MA,

Bulló M, (2013). Association between dietary phylloquinone intake and peripheral
metabolic risk markers related to insulin resistance and diabetes in ederly subset at high
cardiovascular risk. Cardiovasc Diabetol.,12:7. doi: 10.1186/1475-2840-12-7.
[41] Shiavo-Cardozo D, Lima MM, Pareja JC, Geloneze B, (2012). Appetite-regulating
hormones from the upper gut disrupted control of xenin and ghrelin in night workers.
Clin Endocrinol (Oxf)., Dec 1. doi: 10.1111/cen.12114. [Epub ahead of print].
[42] Argiles JM. Stemmler B, (2013). The potential of ghrelin in the treatment of cancer
cachexia. Expert Opin Biol Ther., 13:67-76.
[43] Kamiji MM, Inui A, (2008). The role of ghrelin analogs in wasting disease. Curr Opin
Clin Nut Metab Care., 4:443-51.
[44] Angelidis G, Valotassiou V, Georgoulias P, (2010). Current and potential roles of
ghrelin in clinical practice. J Endrocrinol Invest., 33:823-38.
[45] Lim CT, Korbonits M, (2012). Pediatric endocrine aspects of ghrelin. Pediatr
Endocrinol Rev., 628-38.
[46] Jeffery PL, McGuckin MA, Linden SK, (2011). Endocrine impact of Helicobacter
pylori: Focus on ghrelin and ghrelin o–acyltransferase. World J Gastroenterol., 7:1249-
60.
[47] Weigt J, Malfertheiner P, (2009). Influence of Helicobacter pylori on gastric regulation
of food intake. Curr Opin Clin Nutr Metab Care., 12:522-5.
[48] Nweneka CV, Prentice AM, (2011). Helicobacter pylori infection and circulating
ghrelin levels - A systematic review. BMC Gastroenterol., 11:7. doi: 10.1186/1471-
230X-11-7.
[49] Jang EJ, Park SW, Park JS, Park SJ, Hahm KB, Paik SY, Sin MK, Lee ES, Oh SW,
Park CY, Baik HW, (2008). The influence of the eradication of Helicobacter pylori on
gastric ghrelin, appetite, and body mass index in patients with peptic ulcer disease. J
Gastroenterol Hepatol., ;23 (Suppl 2):S278-85.
[50] Chuang CH, Sheu BS, Yang HB, Lee SC, Kao AW, Cheng HC, Chang WL, Yao WJ,
(2009). Gender difference of circulating ghrelin and leptin concentrations in chronic
Helicobacter pylori infection. Helicobacter., 14:54-60.
[51] Papamichael KX, Papaioannou G,KargaH, Roussos A, Mantzaris GJ, (2009).
Helicobacter pylori infection and endocrine disorders: Is there a link? World J
[52] Blaser MJ, Atherton JC, (2004). Helicobacter pylori persistence: biology and disease. J
Clin Invest., 113:321-33.
[53] Ariizumi K, Koike T, Ohara S, Inomata Y, Abe Y, Iijima K, Imatani A, Oka Y,
Shimosegawa T, (2008). Incidence of reflux esophagitis and Helicobacter pylori
infection in diabetic patients. World J Gastroenterol., 14:3212-7.
[54] Albaker WI, (2011). Helicobacter pylori infection and its relationship to metabolic
syndrome. Is it a myth or fact? Saudi J Gastroenterol., 17:165-9.
[55] Chen Y, Blaser MJ, (2012). Association between gastric Helicobacter pylori
colonization and glycated hemoglobin levels. J Infect Dis., 205:1195-202.
[56] Oluyemi A, Anomneze E, Smith S, Fasanmade O, (2012). Prevalence of a marker of
Helicobacter pylori active infection among patients with type 2 diabetes mellitus in
Lagos, Nigeria. BMC Res Notes., 5:284. doi: 10.1186/1756-0500-5-284.

[57] Windle HJ, Séller D, Crabtree JE, (2007). Childhood Helicobacter pylori infection and
growth impairment in developing countries: A vicious cycle? Pediatrics., 119:754-9.
[58] Vilchis J, Duque X, Mera R, Morán S, Torres J, González-Cossío T, Kageyama-
Escobar ML, Navarro F, Mendoza ME, Correa P, (2009). Association of Helicobacter
pylori infection and height of Mexican children of low socioeconomic level attending
boarding schools. Am J Trop Med Hyg., 81:1091-6.
[59] Vitale G, Barbaro F, Ianiro G, Cesario V, Gasbarrini G, Franceschi F, Gasbarrini A,
(2011). Nutritional aspects of Helicobacter pylori infection. Minerva Gastroenterol
Dietol., 57:369-77.
[60] Harris PR, Serrano CA, Villagrán A, Walker MM, Thomson M, Duarte I, Windle
HJ, Crabtree JE, (2012). Helicobacter pylori-associated hypochlorhydria in children,
and development of iron deficiency. J Clin Pathol doi:10.1136/jclinpath-2012-201243.
[61] Noto JM, Gaddy JA, Lee JY, M. Blanca Piazuelo B, Friedman DB, Colvin DC,
Romero-Gallo J, Giovanni Suarez G, Loh J, Slaughter JC, Tan S, Morgan DR, Wilson
KT, Bravo LE, Correa P, Cover TL, Amieva MR, Peek Jr. RM, (2013). Iron deficiency
accelerates Helicobacter pylori–induced carcinogenesis in rodents and humans. J Clin
Invest., 123:479–92.
[62] Kopácôvá M, Bures J, Koupil I, Rejchrt S, Vorîs V, Seifert B, Pozler O, Zívný P,
Douda T, Palika V, Holcík J, & the European Society for Primary Care
Gastroenterology, (2007). Body indices and basic vital signs in Helicobacter pylori
positive and negative persons. Eur J Epidemiol., 22:67–75.
[63] Thjodleifsson B, Asbjörnsdottir H, Sigurjonsdottir RB, Gíslason D, Olafsson I, Cook E,
Gíslason T, Jogi R, Janson C, (2007). Seroprevalence of Helicobacter pylori and CagA
antibodies in Iceland, Estonia and Sweden. Scand J Infect Dis., 39:683-9.
[64] Soylu OB, Ozturk Y, (2008). Helicobacter pylori infection: effect on malnutrition and
growth failure in dyspeptic children. Eur J Pediatr., 167:557-62.
[65] Ferrara M, Capozzi L, Russo R. Influence of Helicobacter pylori infection associated
with iron deficiency anaemia on growth in pre-adolescent children. Hematology.,
14:173-6.
[66] Gulcan M, Ozen A, Karatepe HO, Gulcu D, Vitrinel A, (2010). Impact of H. pylori on
growth: is the infection or mucosal disease related to growth impairment? Dig Dis Sci.,
55:2878-86.
[67] Ozen A, Furman A, Berber M, Karatepe HO, Mutlu N, Saricoban HE, Buyukgebiz B,
(2011). The effect of Helicobacter pylori and economic status on growth parameters
and leptin, ghrelin, and insulin-like growth factor (IGF)-I concentrations in children.
[68] Goodman KJ, Correa P, Mera R, Yepez MC, Cerón C, Campo C, Guerrero N, Sierra
MS, Bravo LE, (2011). Effect of Helicobacter pylori infection on growth velocity of
School-age Andean children. Epidemiology., 22:118-126.
[69] Thjodleifsson B, Olafsson I, Gislason D, Gislason T, Jögi R, Janson C,, (2008).
Infections and obesity: A multinational epidemiological study. Scand J Infect Dis.,
40:381-6.
[70] Arslan E, Atilgan H, Yavasoglu I, (2009). The prevalence of Helicobacter pylori in
obese subjects. Eur J Intern Med., 20:695-7.

[71] Al-Akwaa AM; (2010). Prevalence of Helicobacter pylori infection in a group of

morbidly obese Saudi patients undergoing bariatric surgery: a preliminary report. Saudi
J Gastroenterol., 16:264-7.
[72] Méndez-Sánchez N, Pichardo-Bahena R, Vásquez-Fernández F, Lezama-Mora JI,
León-Canales AL, Barredo-Prieto B, González-Avila D, Ponciano-Rodríguez G, Uribe
M, (2007). Effect of Helicobacter pylori infection on gastric ghrelin expression and
body weight. Rev. Gastroenterol. Mex., 72:359-64.
[73] Dutta SK, Arora M, Kireet A, Bashandy H, Gandsas A, (2009). Upper gastrointestinal
symptoms and associated disorders in morbidly obese patients: a prospective study.
Dig. Dis. Sci., 54:1243-6.
[74] Torisu T, Matsumoto T, Takata Y, Ansai T, Soh I, Awano S, Nakamichi I, Kagiyama S,
Sonoki K,Yoshida A, Hamasaki T, Iida M, Takehara T, (2008). Atrophic gastritis, but
not antibody to Helicobacter pylori, is associated with body mass index in a Japanese
population. J. Gastroenterol., 43:762-6.
[75] Watabe H, Mitsushima T, Derakhshan MH, Yamaji Y, Okamoto M, Kawabe T, Omata
M, McColl KE, (2009). Study of association between atrophic gastritis and body mass
index: a cross-sectional study in 10,197 Japanese subjects. Dig. Dis. Sci., May;54:988-
95.
[76] Mayne ST, Navarro SA, (2002). Diet, obesity and reflux in the etiology of
adenocarcinomas of the esophagus and gastric cardia in humans. J Nutr., 132 (11
Suppl):3467S-3470S.
[77] Loffeld RJLF, (2005). Helicobacter pylori, obesity and gastroesophageal reflux disease.
Is there a relation? A personal view. J Med., 63:344-7.
[78] Choi JY, Hahm KB, (2006). Ghrelin; Influences on Helicobacter pylori-associated
gastric diseases. Korean J Gastroenterol., 48:75-81.
[79] Osawa H. Ghrelin and Helicobacter pylori infection. World J Gastroenterol.
2008;14:6327-33.
[80] Weight J, Malfertheiner P, (2009). Influence of Helicobacter pylori on gastric
regulation of food intake. Curr Opin Clin Nutr Metab Care., 12:522-5.
[81] Scapa E, Boldur II, Gluskin J, Broide E, Bogokowsky H, Eshchar J, (1992).
Helicobacter pylori before and after gastric reduction for morbid obesity. Obes Surg.,
2:371-4.
[82] Renshaw AA, Rabaza JR, Gonzalez AM, Verdeja JC, (2001). Helicobacter pylori
infection in patients undergoing gastric bypass surgery for morbid obesity. Obes Surg.,
11:281-3.
[83] Ramaswamy A, Lin E, Ramshaw BJ, Smith CD, (2004). Early effects of Helicobacter
pylori infection in patients undergoing bariatric surgery. Arch Surg., 139:1094-6.
[84] Wu MS, Lee WJ, Wang HH, Huang SP, Lin JT, (2005) A case-control study of
association of Helicobacter pylori infection with morbid obesity in Taiwan. Arch Intern
Med., 165:1552-5.
[85] Macadam RC, Borse V, Dodo I, Pollard SG, (2004). Helicobacter pylori, ghrelin, and
obesity. Gut. 53:315-6.
[86] Li Q, Zhang J, Zhou Y, Qiao L, (2012). Obesity and gastric cancer. Front Biosci.,
17:2383-90.

[87] Roper J, Francois F, Shue PL, Mourad MS, Pei Z, Olivares de Perez AZ, Perez-Perez
GI, Tseng C, Blaser MJ, (2008). Leptin and Ghrelin in relation to Helicobacter pylori
status in adult males. J Clin Endocrinol Metab., 93:2350-7.
[88] Stec-Machalska K, Malicki S, Michalski B, Peczek L, Wisniewska-Jarosinka M,
Nawrot B, (2009). Gastric Ghrelin in relation to gender, stomach topography and
Helicobacter pylori in dyspeptic patients. World J Gastroenterol., 15:5409-41.
[89] Jeffery PL, McGuckin MA, Linden SK. Endocrine impact of Helicobacter pylori: focus
on ghrelin and ghrelin o-acyltransferase. World J Gastroenterol. 2011;17: 1249-60.
[90] Shiotani A, Miyanishi T, Uedo N, Iishi H. Helicobacter pylori infection is associated
with reduced circulating ghrelin levels independent of body mass index. Helicobacter.
2005;10:373-8.
[91] Isomoto H, Ueno H, Saenko VA, Mondal MS, Nishi Y, Kawano N, Ohnita K, Mizuta
Y, Ohtsuru A, Yamashita S, Nakazato M, Kohno S, (2005). Impact of H. pylori
infection on gastric and plasma ghrelin dynamics in humans. Am J Gastroenterol.,
100:1711-20.
[92] Salles N, Menard A, Georges A, et al. (2006). Effects of Helicobacter pylori infection
on gut appetic pepide (leptin, grhelin) expression in elderly inpatients. J Gerontol A
Biol Sci Med Sci., 61:1144-50.
[93] Jun DW, Lee OY , Lee YY, Choi HS, Kim TH, Yoon BC, (2007). Correlation between
gastrointestinal symptoms and gastric leptin and ghrelin expression in patients with
gastritis. Dig Dis Sci. 52:2866-72.
[94] Tatsuguchi A, Miyake K, Gudis K, Futagami S, Tsukui T, Wada K, Kishida T, Fukuda
Y, Sugisaki Y, Sakamoto C, (2004). Effect of Helicobacter pylori infection on ghrelin
expression in human gastric mucosa. Am J Gastroenterol., 99: 2121-7.
[95] Maksud FA, Alves JS, Diniz MT, Barbosa AJ, (2011). Density of ghrelin-producing
cells is higher in the gastric mucosa of morbidly obese patients. Eur J Endocrinol.,
165:57-62.
[96] Isomoto H, Nishi Y, Ohnita K, Mizuta Y, Kohno S, Ueno H, Nakazato M, (2005). The
relationship between plasma and gastric ghrelin levels and strain diversity in
Helicobacter pylori virulence. Am J Gastroenterol., 100:1425-7.
[97] Plonka M, Bielanski W, Konturek SJ, Targosz A, Sliwowski Z, Dobrzanska M,
Kaminska A, Sito E, Konturek PC, Brzozowski T, (2006). Helicobacter pylori infection
and serum gastrin, ghrelin and leptin in children of Polish shepherds. Dig. Liver. Dis.,
38:91-7.
[98] Konturek PC, Cześnikiewicz-Guzik M, Bielanski W, Konturek SJ, (2006). Involvement
of Helicobacter pylori infection in neuro-hormonal control of food intake. J Physiol
Pharmacol., 57(Suppl 5):67-81.
[99] Salles N, Ménard A, Georges A, Salzmann M, de Ledinghen V, de Mascarel A,
Emeriau JP, Lamouliatte H, Megraud F, (2006). Effects of Helicobacter pylori infection
on gut appetite peptide (leptin, ghrelin) expression in elderly inpatients. J Gerontol A
Biol Sci Med Sci., 61:1144-50.
[100] Chuang CH, Sheu BS, Yang HB, Lee SC, Kao AW, Cheng HC, Chang WL, Yao WJ,
(2009). Gender difference of circulating ghrelin and leptin concentrations in chronic
Helicobacter pylori infection. Helicobacter., 14:54-60.

[101] Slomiany BL, Slomiany A, (2007). Interference by leptin with Helicobacter pylori
lipopolysaccharide-induced cytosolic phospholipase A2 activation in gastric mucosal
cells. J Physiol Pharmacol., 58:117-30.
[102] Slomiany BL, Slomiany A, (2011). Helicobacter pylori Induces disturbances in gastric
mucosal Akt activation through inducible Nitric Oxide Synthase-dependent S-
Nitrosylation: Effect of ghrelin. ISRN Gastroenterol., 2011:308727. doi: 10.5402
/2011/308727.
[103] Fetissov SO, Hamze Sinno M, Coëffier M, Bole-Feysot C, Ducrotté P, Hökfelt T,
Dechelotte P, (2008). Autoantibodies against appetite-regulating peptide hormones and
neuropeptides: putative modulation by gut microflora. Nutrition., 24:348-59.
[104] Slomiany BL, Slomiany A, (2013.) Involvement of p38 MAPK-dependent activator
protein (AP-1) activation in modulation of gastric mucosal inflammatory responses to
Helicobacter pylori by ghrelin. Inflammopharmacol., 21:67–78.
[105] El-Omar EM, Rabkin CS, Gammon MD, Vaughan TL, Risch HA, Schoenberg JB,
Stanford JL, Mayne ST, Goedert J, Blot WJ, Fraumeni JF Jr, Chow WH, (2003).
Increased risk of noncardia gastric cancer associated with proinflammatory cytokine
gene polymorphisms. Gastroenterol., 124:1193–1201.
[106] Tahara T, Shibata T, Yamashita H, Yoshioka D, Okubo M, Yonemura J, Kamiya Y,
Ishizuka T, Nakagawa Y, Nagasaka M, Iwata M, Nakamura M, Hirata I, Arisawa T,
(2012). Synergistic effect of IL-1β and TNF-α polymorphisms on the H. pylori-related
gastric pre-malignant condition. Hepatogastroenterol., 59:2416-20. doi:10.5754
/hge10605.
[107] Abiko Y, Suzuki H, Masaoka T, Nomura S, Kurabayashi K, Hosoda H, Kangawa K,
Hibi T, (2005). Enhanced plasma ghrelin levels in Helicobacter pylori-colonized,
interleukin-1-receptor type 1-homozygous knockout (IL-1R1-/-) mice. World J
[108] Gonzalez PV, Cragnolini AB, Schiöth HB, Scimonelli TN, (2006). Interleukin-1 beta-
induced anorexia is reversed by ghrelin. Peptides., 27:3220-5.
[109] Nwokolo CU, Freshwater DA, Hare PO, Randeva HS, (2003). Plasma ghrelin
following cure Helicobacter pylori. Gut., 52: 637-40.
[110] Isomoto H, Ueno H, Saenko VA, Mondal MS, Nishi Y, Kawano N, Ohnita K, Mizuta
Y, Ohtsuru A, Yamashita S, Nakazato M, Kohno S, (2005). Impact of H. pylori
Infection on gastric and plasma ghrelin dynamics in humans. Am J Gastroenterol.,
100:1711-20.
[111] Lee ES, Yoon YS, Park CY, Kim HS, Um TH, Baik HW, Jang EJ, Lee S, Park HS, Oh
SW, (2010). Eradication of Helicobacter pylori increases ghrelin mRNA expression in
the gastric mucosa. J Korean Med Sci., 25: 265-71.
[112] Choe YH, Lee JH, Lee HJ, Paik KH, Jin DK, Song SY, (2007). Ghrelin levels in gastric
mucosa before and after eradication of Helicobacter pylori. Gut. Liver., 1:132-7.
[113] Osawa H, Kita H, Ohnishi H, Nakazato M, Date Y, Bowlus CL, Ishino Y, Watanabe E,
Shiiya T, Ueno H, Hoshino H, Satoh K, Sugano K, (2006). Changes in plasma ghrelin
levels, gastric ghrelin production, and body weight after Helicobacter pylori cure. J
[114] Osawa H, (2008). Ghrelin and Helicobacter pylori infection. World J. Gastroenterol.,
14:6327-33.

[115] Jang EJ, Park SW; Park JS, Park JS, Hahm KB, Paik SY, Sin MK, Lee ES, Oh SW,
Park CY, Baik HW, (2008). The influence of the eradication of Helicobacter pylori on
gastric ghrelin, appetite, and body mass index in patients with peptic ulcer disease. J
Gastroenterol Hepatol., (Suppl 2):S278-85.
[116] Yao-Jong Yang, Bor-Shyang Sheu, Hsiao-Bai Yang, Cheng-Chan Lu, Ching-Chun
Chiang, (2012). Eradication of Helicobacter pylori increases childhood growth and
serum acylated ghrelin levels. World J Gastroenterol., 18: 2674-81.
[117] Zhao-Hui Deng, Bo Chu, Ya-Zhen Xu, Bin Zhang, Li-Rong Jiang, (2012). Influence of
Helicobacter pylori infection on ghrelin levels in children. World J Gastroenterol.,
18:5096-100.
[118] Ando T, Mizuno S, Ishida T, Kondo Y, Miki I, Yoshida M, Azuma T, Ishikawa T,
Takagi T, Yagi N, Kokura S, Naito Y, Yoshikawa T, Asakawa A, Inui A, (2012).
Plasma ghrelin isoforms and gastric ghrelin O-acyltransferase expression are influenced
by Helicobacter pylori status. Nutrition., 28:967-72.
[119] Kebapcilar L, Sari I, Renkal AH, Alacacioglu A, Yuksel A, Ilhan E, Alkan B, Yuksel
D, Kozaci DL, Gunay N, (2009). The influence of Helicobacter pylori eradication on
leptin, soluble CD40 ligand, oxidative stress and body composition in patients with
peptic ulcer disease. Inter Med., 48:2055-9.
[120] Pacifico L, Anania C, Osborn JF, Ferrara E, Schiavo E, Bonamico M, Chiesa C,
(2008). Long-term effects of Helicobacter pylori eradication on circulating ghrelin and
leptin concentrations and body composition in prepuberal children. Eur J Endocrinol.,
158: 323-32.
[121] Yang YJ, Sheu BS, Yang HB, Lu CC, Chuang CC, (2012) Eradication of Helicobacter
pylori increases childhood growth and serum acylated ghrelin levels. World. J.
[122] Hassouna R, Zizzari P, Tolle V, (2010). The ghrelin/obestatin balance in the
physiological and pathological control of grewth hormone secretion, body composition
and food intake. J Neuroendocrinol., 22:793-804.
[123] Zub-Pokrowiecka A, Rembiasz K, Konturek PC, Budzynski A,Konturek SJ, Winiarski
M, Bielanski W, (2011). Ghrelin and gastrin in advanced gastric cancer before and after
gastrectomy. World J. Gastroenterol., 17:449-58.
[124] Furuta T, Shirai N, Xiao F, Takashima M, Hanai H, (2002). Effect of Helicobacter
pylori infection and its eradication on nutrition. Aliment Pharmacol Ther., 16:799-806.
[125] Fujiwara Y, Higuchi K, Arafa UA, Uchida T, Tominaga K, Watanabe T, Arakawa T.,
et al. (2002). Long-term effect of Helicobacter pylori eradication on quality of life,
body mass index, and newly developed diseases in Japanese patients with peptic ulcer
disease. Hepatogastroenterology., 49:1298-302.
[126] Azuma T, Suto H, Ito Y, Muramatsu A, Ohtani M, Dojo M, Yamazaki Y, Kuriyama
M, Kato T, (2002). Eradication of Helicobacter pylori infection induces an increase in
body mass index. Aliment Pharmacol Ther., 16 (Suppl 2):240-4.
[127] Yang YJ, Sheu BS, Chang WL, Cheng HC, Yang HB., (2009). Increased body mass
index after H. pylori eradication for duodenal ulcer predisposes to erosive reflux
esophagitis. J Clin Gastroenterol., 43:705-10.
[128] Lane JA, Murray LJ, Harvey IM, Donovan JL, Mair P, Harvey RF, (2011).
Randomised clinical trial: Helicobacter pylori eradication is associated with a

significantly increased body mass index in a placebo-controlled study. Aliment

Pharmacol Ther., 33:922-9.
[129] Akanuma M, Yanai A, Sakamoto K, Hirata Y, Yamaji Y, Kawazu S, Maeda S, (2012).
Influence of Helicobacter pylori eradication on the management of type 2 diabetes.
Hepatogastroenterology.,59(114). doi: 10.5754/hge11960.
[130] Abdullahi M, Annibale B, Capoccia D, Tari R, Lahner E, Osborn J, Leonetti F, Severi
C, (2008). The eradication of Helicobacter pylori is affected by body mass index
(BMI). Obes Surg, 18:1450-4.
[131] Singh N, Deb R, Kashyap PC, Bhatia V, Ahuja V, Sharma MP, (2008). Body mass
index and capita income influence duodenal ulcer healing and H. pylori eradication
whilst dietary factors play no part. Trop Gastroenterol, 29:26-31
[132] Suto, H, Yamazaki Y, Yoshida I, Yamakawa A, Ohtani M, Ito Y, Kuriyama M, Kato
T, Azuma T, (2009). The effects of Helicobacter pylori eradication on body mass index
and dyspeptic symptoms. Digestion., 79:235-42.
[133] Francois F, Roper J, Joseph N, Pei Z, Shak JR, Olivares de Perez AZ, Perez-Perez GI,
Blaser MJ, (2011). The effect of H. pylori eradication on meal-associated changes in
plasma ghrelin and leptin. BMC Gastroenterol., 11:37-45.
[134] Graham DY, (1997). The only good Helicobacter pylori is a dead Helicobacter pylori.
Lancet., 350:70-1.
[135] Blaser MJ, (1997). Not all Helicobacter pylori strains are created equal: Should all be
eliminated? Lancet., 349:1020-2.
[136] Arslan N, Erdur B, Aydin A, (2010). Hormones and cytokines in childhood obesity.
Indian Pediatrics., 47:829-39.

Chapter 14
Role of Helicobacter pylori

Infection in Hematological Disorders
Wajid Ali1, Zhong Wu1, Jin Zhou2 and Bing Peng1,*

1
Department of Hepatopancreatobiliary Surgery, West China Hospital,
Sichuan University, Chengdu, China
2
Department of Gastrointestinal Surgery, West China Hospital, Sichuan University,
Chengdu, China
Abstract
Helicobacter pylori (H. pylori) is a Gram-negative spiral, microaerophilic bacterium
classified as a carcinogen of class I, according to the World Health Organization (WHO).
The infection is a major cause of gastric pathology and increase risk of gastric cancer and
also been implicated in several systemic or hematological disorders. The present review
focus on interpreting the role of bacterium in hematological disorders, increasing
awareness of medical practitioners, regarding the bacterium-associated hematological
disorders. Idiopathic thrombocytopenic purpura (ITP) is a common autoimmune disease
mediated by auto antibodies against platelet glycoproteins. The efficacy of H. pylori
eradication in increasing the platelet count in adult patients with primary ITP has been
confirmed. Moreover, the bacterium causes iron deficiency anemia (IDA), low-grade B-
cell gastric MALT lymphomas by several mechanisms. In fact, it has been widely
recognized that anti-H. pylori treatment causes their regression. Despite the well
established associations of H. pylori with the previously mentioned hematological
disorders we also highlight the possible role of infection to other hematological disorders
such as megaloblastic anemia and myelodysplastic syndromes, monoclonal gammopathy,
and non-Hodgkin lymphomas of the stomach. This chapter analyzes the current
knowledge on the interaction between H. pylori infection and hematological diseases.
*
Corresponding author: Bing Peng, Department of Hepatopancreatobiliary Surgery, West China Hospital, Sichuan
University, Chengdu 610041, China. Tel: +86-28-85422477 Fax: +86-28-85422474. E-mail: pengbing84@
hotmail.com.

270 Wajid Ali, Zhong Wu, Jin Zhou et al.
Keywords: Helicobacter pylori (H. pylori), immune thrombocytopenia (ITP), iron deficiency
anemia (IDA), hematology
Introduction
Helicobacter pylori (H. pylori) consists of a large diversity of strains, and the genomes of
three have been completely sequenced. Study of the H. pylori genome is centered on attempts
to understand pathogenesis, the ability of this organism to cause disease [1, 2]. H. pylori
infection is defined by some basic characteristics of bacterium: factors which promote the
colonization of host (motility, urease, flagella, adhesion, PATPase) and factors promoting
tissue lesion [lipopolysaccharide (LPS), Neutrophil-activating protein (HP-NAP), cytotoxin-
associated geneA (CagA), vacuolization cytotoxin A (VacA), heat shock proteins (Hsps)] [3].
Cag pathogenic island (a common gene sequence believed responsible for pathogenesis)
contains over 40 genes, which mainly code a complex type IV. The cagA gene codes for one
of the major H. pylori virulence proteins. Bacterial strains that have the cagA gene are
associated with an ability to cause ulcers. The CagA protein is frequently co-expressed with
the vacuolating cytotoxin VacA [4]. Inflammatory processes of H. pylori infections are also
mediated by highly disulfide-bridged proteins. Helicobacter cysteine-rich proteins (Hcp),
particularly HcpA (hp0211), trigger an immune response through the differentiation of human
myeloid Thp1 monocytes into macrophages. Moreover, a number of other virulence factors
have been identified, which modulate the host immune response.
The mode of transmission of H. pylori remains poorly understood. Person to person
transmission of bacteria from fecal-oral, oral-oral and gastro-oral exposure is probably the
main route of infection. H. pylori prevalence and incidence differs by geography and race.
The prevalence increase by age and to areas with low socioeconomic status (see Chapter 6).
However a decrease in incidence of the infection is noted worldwide, mainly due to the use of
antibiotics and to the improvement of hygiene [5]. The role of H. pylori infection in the
causation of peptic ulcer and gastric cancer has been subject of extensive research since its
discovery in the 1980s as well as many extra-gastric manifestation including hematological,
cardiovascular, neurological, and metabolic disease. To demonstrate the association between
H. pylori and extra-gastric diseases we have used to Bradford-Hill criteria [6]. First there has
to be a close association between H. pylori and extra-gastric diseases. Second, there has to be
a biologically plausible mechanism. Third, H. pylori infection should precede the
development of the disease and finally, the eradication of H. pylori should result in an
improvement in the condition or cure of disease.
More Ascertained Hematological Associations

Idiopathic Thrombocytopenic Purpura (see also chapter 17)
Idiopathic thrombocytopenic purpura (ITP) is an autoimmune blood disorder

characterized by the production of antibodies against antigens on the surface of platelets,
resulting in platelet destruction, thrombocytopenia and subsequent mucocutaneous

Role of Helicobacter pylori Infection in Hematological Disorders 271
hemorrhage. Majority of these ITP patients have their platelets coated with immunoglobin G
antibodies that bind with the glycoprotein’s including GPIIb-IIIa, GPIb-IX, etc., on the
surface of platelets. Tissue macrophages recognize these antibody-associated platelets and
phagocytose them ultimately causing a decline in platelet count. Studies [7] have shown an
apparently higher than expected prevalence of H. pylori infection in patients with ITP, but
many of them do not have a matched control group. Among those with control groups,
Campuzano-Maya demonstrated that the prevalence of H. pylori infection in patients with
ITP was significantly higher than that in age- and gender-matched controls [8]. The most
plausible mechanism so far is that antibody production cross-reacts with platelet glycoprotein
antigens and H. pylori eradication is associated with the disappearance of auto-antibodies in
most cases. This provides a good evidence of the link between H. pylori and ITP [9]. HLA-
DQB1*03, -DRB1*11 and -DRB1*14 histocompatibility antigens have also been correlated
with higher probability of platelet response after eradication.
The pathogenesis of ITP is complex and partially known. The principal mechanism
seems to be the increased destruction of platelets opsonized by auto-antibodies, especially
against IIb/IIIa glycoprotein’s, in the splenic and hepatic reticuloendothelial system. The
destruction of platelets in the reticuloendothelial system promotes the formation of new
antigenic epitopes, which are exposed by macrophages to T lymphocytes. B lymphocytes are
stimulated to produce antibodies, which provoke and maintain thrombocytopenia in time [10].
The CagA antigen of H. pylori could be responsible for the cross-mimicry between H. pylori
and platelet glycoprotein’s [11]. The disappearance of auto antibodies in most cases of ITP
patients after H. pylori eradication was reported by other authors, supporting the hypothesis
that the autoimmune process may be mediated by the bacterial infection [7]. Another factor
responsible for the molecular mimicry may be the babA gene (blood group antigen-binding
adhesin gene) expressed by some H. pylori strains, which codifies for antigenic epitopes
which recognize sequences of Lewis blood group (Le antigens). These antigens are adsorbed
by platelets and could become the target for anti-Le antibodies produced by some patients
with a susceptible background [12]. The development of thrombocytopenia in H. pylori
infection may also be dependent on genetic influence. In fact, H. pylori may interact with
platelets through von Willebrand factor and IgG anti-H. pylori antibody and their
corresponding platelet receptors GPIb and FcgRIIA. The interaction between platelets and H.
pylori may cause chronic platelet consumption, contributing to the pathogenesis of
thrombocytopenia [13]. The first case of association of H. pylori with ITP was reported in
1998 by Gasbarrini et al. [7] The efficacy of H. pylori eradication in increasing the platelet
count in adult ITP patients has been confirmed in several reports [14-22]. Furthermore, the
long-term prognosis of thrombocytopenia related to H. pylori infection has been evaluated
extensively, along with the possibility of recurrence of ITP after re-infection of H. pylori. The
prognosis of the patients who responded to eradication has been reported to be excellent in a
follow-up period of 8 years. No recurrence of the disease has been noted after the long-term
follow up [23].
In a systematic review of 25 studies involving 1,555 patients, Stasi et al. found a
favorable response in platelet count following H. pylori eradication, especially in those with
mild ITP [24]. Further studies with larger number of patients are necessary to identify the
crucial predictive factors responsible for platelet recovery in chronic ITP patients with the H.
pylori infection, since in such patients eradication does not seem to be effective. The

prevalence and platelet response after eradication therapy in ITP adult patients are shown in
Table 14.1.
Table 14.1. Platelet response after H. pylori eradication and prevalence

in ITP adult patients
Study Platelet response (%) Prevalence (%) of H. pylori
Gasbarrini et al. 8/8 (100) 11/18 (61)

Jarque et al. 3/23 (13) 40/56 (71)
Emilia et al. 6/12 (50) 13/30 (43)
Veneri et al. 11/15 (73) 25/35 (71)
Kohda et al. 12/19 (63) 25/40 (62)
Michel et al. 4/14 (29) 16/74 (22)
Suzuki et al. 6/13 (46) 25/36 (69)
Fujimura et al. 88/161(55) 300/435 (69)
Kodama et al. 27/44 (61) 67/116 (58)
Emilia et al. 25/34 (74) 38/75 (51)
Tag et al. 17/25 (68) 23/25 (92)
Platelet response was defined as complete or partial response among patients who
obtained a successful eradication.
However, several older studies found no important correlation between H. pylori
eradication therapy and increase in platelet counts ITP in adult patients [25-29]. Such
discrepancies between the various studies could be partly attributed to the differences in the
definition of response regarding the degree of the increase of the platelet number, the
heterogeneity of the studied populations, the previous subjection to treatment for ITP or not,
the variability concerning time intervals of follow-up, or the possibility of false positive
results in diagnosing H. pylori.
ITP has an annual incidence of 40 to 50 cases per million in children less than 15 years of
age. In childhood, the natural history of ITP is quite different from that observed in adult
patients: in fact, acute ITP in children resolves within 1 to 6 months in the large majority of
cases mostly spontaneously [30]. Chronic ITP, so defined when lasting more than 6 or 12
months, develops in 20% of children, mostly in adolescent than in younger patients [31].
Furthermore, spontaneous remission is described to occur in one-third of children with
chronic ITP from months to years after the onset of thrombocytopenia. The behavior of ITP in
childhood, in addition to the differences of treatment of the acute phase, of the geographical
prevalence of H. pylori infection, of the different distribution of H. pylori strains, and the
heterogeneity of published studies, makes it difficult to compare the results of eradication of
H. pylori in ITP of children with the results of eradication of H. pylori in ITP of adults.
However, further in vitro and clinical studies are needed to elucidate the many still unclear
issues of this association such as the pathogenic mechanism and the geographical discrepancy
of platelet response to eradication therapy. Further randomized controlled trials are also
awaited to better identify which subgroups of ITP patients will benefit from eradication as an
initial treatment approach.

Iron Deficiency Anemia (see also Chapter 17)
Iron deficiency anaemia (IDA) is a world wide public health problem affecting both
developing and developed countries, with major consequences for human health as well as
socio-economic development. Iron deficiency is the most common nutritional deficiency in
the world and results in impairment of immune, cognitive and reproductive functions, as well
as decreased work performance. Many risk factors for IDA have been identified, most of
which are related to dietary habits. IDA may be caused by chronic blood loss and inadequate
iron intake. Chronic blood loss may be due to gastrointestinal disorders, especially peptic
ulcer and gastric cancer, which might be associated with H. pylori infection. In the past
decades, the association between H. pylori infection and IDA has been controversial but more
recently, H. pylori infection has been implicated as a cause of unexplained IDA in the
absence of GI blood loss. Dufour et al. [32] were the first to report a possible relation
between H. pylori and IDA. This was supported by several epidemiological studies showing a
lower ferritin level among patients with H. pylori infection although there were also studies
with a negative association [33]. Many studies suggest a relationship between IDA and H.
pylori infection, but it is not clear the mechanism by which this occurs. One of the suggested
mechanisms occurs through H. pylori-mediated Lactoferrin (Lf) increase in gastric tissue via
neutrophils. Lf captures iron from transferrin. The iron, thus, bound to Lf is in turn picked up
by the bacterium, by means of its outer membrane receptors, for its own growth. As H. pylori
turnover is very rapid, the bacterial iron stores are rapidly lost in the stools, together with the
dead bacteria. This mechanism, or at least its template, could explain why an iron supply is no
longer available for hemopoiesis, which only enhances H. pylori proliferation [34]. H. pylori
causes IDA by several other mechanisms: increased iron loss due to active hemorrhage
secondary to gastritis, peptic ulcer or gastric cancer, achlorhydria induced by chronic
pangastritis resulting in reduced iron absorption, reduced secretion of ascorbic acid to the
gastric mucosa and finally, iron utilization by the bacterium for colonization to the host
environment through protein production. Boyanova recently proposed how virulent strains of
H. pylori, such as those harboring CagA and VacA, work concurrently to provide both iron
acquisition from interstitial holotransferrin and enhanced bacterial colonization of host cells
apically [35, 36]. In an initial report, a seven year-old boy with refractory unexplained IDA
had resolution of the disease after treatment of the H. pylori related pangastritis [37]. Young
female athletes with IDA were also investigated and it was shown that the group who
received eradication treatment for H. pylori exhibited faster improvement of the anemia,
while the control group treated with oral iron alone showed no significant changes regarding
the course of anemia [38]. Fagan et al. examined 219 children aged 7–11 yrs with concurrent
H. pylori infection and concluded that the resolution of the infection for >14 months modestly
reduced the prevalence of iron deficiency and substantially reduced the prevalence of iron
deficiency and anemia [39]. However, there are several studies showing the absence of a
positive association between iron stores and H. pylori infection among children [40]. Meta-
analysis demonstrated that H. pylori eradication accelerated the improvement of ferritin levels
in iron deficiency people in the overall analysis; hemoglobin levels in iron deficiency people
did not demonstrate a significant acceleration of improvement after H. pylori eradication.
Because the change of hemoglobin levels lags behind that of ferritin levels and ferritin plays a
central role in iron storing during iron metabolism [41]. The results yielded that the
eradication of H. pylori in patients with IDA and chronic H. pylori-related gastritis is

associated with reversal of dependence of iron treatment, normalization of hemoglobin levels

after 6 months and long-term recovery from IDA [42].
In summary, a growing body of evidence supports a clinically significant influence of H.
pylori infection on body iron stores. Epidemiological studies also support an association
between H. pylori and low iron stores and several reports have shown resolution of refractory
cases of anemia after H. pylori eradication.
Figure 14.1. MALT lymphoma. The histomorphology in this case closely resembles severe H. pylori
gastritis. Note the infiltration of the muscularis mucosae at the base of the mucosa (arrows). The
clinical information was crucial in this case, as diffusely thickened gastric folds were seen
endoscopically.
Figure 14.2. Lymphoepithelial lesions. This infiltration and destruction of the gastric gland epithelium
by lymphocytes is a characteristic, but not specific, feature of MALT lymphoma (400x original
magnification).

Gastric Mucosa-associated Lymphoid Tissue (MALT) Lymphoma (see also

Chapters 3 and 8)
MALT lymphoma (Figure 14.1) is a low-grade B-cell lymphoma composed

predominantly of small lymphocytes that arises from the mucosal lymphoid tissue and is
normally acquired as a reaction to H. pylori infection, first described around the same time as
H. pylori by Isaacson and Wright [43]. This disease is considered one of the best models
reflecting how genetic events lead to oncogenesis, determine tumor biology, dictate clinical
behavior and represent viable therapeutic targets. Morphologically, the lymphoma cells may
be centrocyte-like (with small nuclei and scant cytoplasm, resembling follicle center cells) or
monocytoid (with ample pale cytoplasm and indented nuclei), often with admixed
centroblast-like cells (large cells that may have prominent nucleoli) [44]. Gastric MALT
lymphoma pathogenesis is a multistep process initiated by infection with H. pylori, which
induces genetic abnormalities and subsequent malignant transformation. Gene alterations
include trisomy of chromosomes 3, 7, 12 or 18 and the disease-specific chromosome
translocations t(1;14)(p22;q32), t(14;18)(q32;q21), t(11;18)(q21;q21) and t(3;14)(p13;q32)
resulting in IGHBCL10, IGH-MALT1, API2-MALT1 and IGH-FOXP1 rearrangements
respectively. Notably, these events are associated with the activation of nuclear factor-kB
(NF-kB) [45]. H. pylori gastritis owes its appearance to the acquisition of a
lymphoplasmacytic infiltrate in the gastric lamina propria, which may or may not be
accompanied by B cell nodules and even germinal centers [46]. This type of tissue is termed
“acquired MALT” to distinguish it from “native MALT” of the type seen, for example, in the
distal ileum, where germinal centers make up the Peyer’s patches. H. pylori strains expressing
the CagA gene have been associated with significant morbidity, and this gene may play a role
in lymphomagenesis. The transformation of H. pylori-related MALT lymphoma to diffuse
large B-cell lymphoma (DLBCL) has been postulated, although the exact mechanism of such
a transformation remains obscure. The distinction between severe H. pylori gastritis and early
MALT lymphoma is often difficult and requires careful assessment of clinical findings as
well as histomorphology. Morphological features helpful in the diagnosis of MALT
lymphoma include bona fide epithelial and mucosal injury, typified by the so-called
“lymphoepithelial lesion”, a characteristic but nonspecific infiltration of epithelial structures
by lymphoma cells (Figure 14.2). Reactive germinal centers, common in the deeper mucosa
in H. pylori gastritis, may be colonized by lymphoma cells, with destruction of the mantle
zone and the appearance of so-called “naked” follicles.
Furthermore, biopsy specimens are usually small and easily exhausted, putting the slides
cut for adequate morphological and IHC analysis at a premium. Thus, the differential
diagnosis of severe gastritis and incipient lymphoma is best made based on a combination of
clinical information, histomorphology, and IHC. Once the diagnosis of MALT lymphoma has
been made and H. pylori eradication therapy initiated, the definition of treatment failure must
be considered. While the majority of cases will respond to conservative therapy, the timing of
rebiopsy for assessment of response is crucial in avoiding an inappropriate judgment of
failure. Complete resolution of the lymphoid infiltrate typically takes several months, and
periods as long as two years to complete resolution have been reported [47]. It has been
widely recognized that eradication of H. pylori causes regression of the low-grade B-cell
gastric MALT lymphoma and that anti-H. pylori treatment should be applied. The long-term
follow-up of gastric MALT lymphoma after H. pylori eradication has been evaluated in a
series of 120 patients. The median follow up was 75 months, while the 5-year overall survival
was 90%. Eighty percent of the patients achieved complete histologic remission. Among the
patients with complete histologic remission, eighty percent were in continuous complete
histologic remission. However, histologic residual disease detected by B-cell monoclonality
and t(11;18), was present in a considerable number of patients with initial complete
histological remission. Hence, a watch-and-wait strategy and close follow-up are essential
[48].
Interestingly, after the eradication of H. pylori in gastric mucosa, the density of the
lymphoplasma cellular infiltration in the lamina propria is significantly reduced. Moreover,
residual lymphoid follicles were found less often in high grade malignant than in low grade
malignant MALT lymphomas. The genotype of H. pylori in gastric adenocarcinoma and
MALT lymphoma has been compared. The presence of the Cag pathogenicity island was
studied, along with allelic variations of the VacA and iceA genes. The combined presence of
both VacA s1a and iceA1 genes had a 5.6 fold higher frequency in adenocarcinoma. The
VacA m2 allele was the predominant subtype in MALT lymphoma and the combination of
the VacA m2 subtypes with the VacA s1 and the iceA1 variants occurred in MALT
lymphoma nearly five times more often than in chronic active gastritis [49].
More Debated Hematological Associations

Non-Hodgkin Lymphomas of the Stomach
The diagnosis of an aggressive non-Hodgkin lymphoma following the regression of a

gastric MALT lymphoma after eradication therapy has been reported. In correlation, non-
Hodgkin lymphoma affecting the stomach has been linked with previous H. pylori infection.
A causative role for the organism is plausible, but remains unproved [50].
Monoclonal Gammopathy of Undetermined Significance
In a proportion of patients with monoclonal gammopathy of undetermined significance

(MGUS), H. pylori infection is involved through chronic antigenic stimulation [51].
However, no evidence of resolution of MGUS after H. pylori eradication has been noted.
Megaloblastic Anemia, Myelodysplastic Syndrome and Other Disorders

The possible implication of H. pylori infection in the pathogenesis of megaloblastic
anemia has not been fully clarified. It is postulated that the infection causes atrophy of the
gastric mucosa, leading to megaloblastic anemia, through auto-immunity [52]. In addition,
cobalamin malabsorption, due to decreased excretion of the intrinsic factor, as a result of the
destruction of parietal cells, is another mechanism leading to pernicious anemia.
Moreover, in an investigation to define the mechanisms of thrombocytopenia and anemia,
the length and the volume ratio of liver to spleen were evaluated. The liver-to-spleen ratio,
platelet-to-spleen ratio, mean platelet volume (MPV)-to-spleen ratio and the MPV-to-liver
ratio were significantly lower in the H. pylori positive group compared with the H. pylori

negative group [53]. Furthermore, H. pylori has been reported to induce thrombocytosis
clinically indistinguishable from essential thrombocythemia. Besides, improvement of anemia
after eradication treatment in a patient with myelodysplastic syndrome (MDS) has been
published [54]. Moreover, the increased reported prevalence of H. pylori infection among
patients with MDS is an interesting finding, suggesting that there might be a link between H.
pylori and MDS and deserving further investigation. However, there is no evidence of a
causal relationship between H. pylori and MDS, although such a possibility cannot be entirely
ruled out. The prospect of a chronic H. pylori infection causing continuous stimulation,
fatigue and distress of the bone marrow deposits, thereby leading to alterations facilitating the
induction of MDS, is intriguing. Finally, a high prevalence of the infection has been reported
in patients with chronic idiopathic neutropenia. These patients have splenomegaly, attributed
to H. pylori infection. Finally, complete regression of a primary gastric plasmacytoma [55]
and of a primary extragastric thyroid mucosa-associated lymphoid tissue lymphoma after
eradication of H. pylori infection have been described.
Increased Childhood Leukemia Risk
Maternal infection with H. pylori may be associated with an increased risk of childhood
leukemia in the offspring; an Icelandic and Finnish epidemiological study revealed that the
title of maternal anti-H. pylori antibodies is correlated with high leukemic risk in the
Icelandic children in contrast to the Finnish [56].
Risk of Hemorrhage in Patients with Coagulation Disorders
H. pylori is the main etiological factor for erosive gastritis and duodenal or gastric peptic
ulcers often complicated with life-threatening hemorrhage in patients with coagulation
disorders. Szczepanik et al. investigated 146 patients with hemophilia (129 with hemophilia
A and 13 with hemophilia B) and found high frequency of upper gastrointestinal bleeding
associated with H. pylori. Therefore, they suggested that screening and eradication therapy
for H. pylori are appropriate in hemophilia patients [57].
Moreover, Choe et al. evaluated children with hemophilia A and hematemesis regarding
the causes of gastrointestinal hemorrhage and concluded that H. pylori should be considered
as an important cause of gastrointestinal bleeding [58].
Conclusion
Among the extragastric manifestations of H. pylori infection, the most convincing data
regard the association with ITP. Indeed, long-term follow up studies have shown that
approximately half of the subjects with ITP may obtain a durable platelet response after H.
pylori eradication. For this reason, the recent guidelines of the American Society of
Hematology (ASH) have included the detection of H. pylori infection in the basic evaluation
of subjects with suspected ITP [59]. This chapter includes the novel developments of H.

pylori associated hematological disorders and shows the need for further investigation
regarding the better understanding of the relevant pathophysiological disease mechanisms,
particularly in the less established associations between the infection and hematological
diseases. Such debated associations are supported by a small number of reports and include
monoclonal gammopathy of undetermined significance, megaloblastic anemia, non-Hodgkin
lymphomas of the stomach, myelodysplastic syndromes.
Primary lymphomas of the GI tract may consist of mature B, T or NK/T cell neoplasm, of
which, the two most commonly encountered morphologic subtypes are extranodal MALT
lymphoma and DLBCL [60]. Moreover, association of primary GI lymphomas with H. pylori
infection, particularly observed in extranodal MALT lymphoma and in a few cases of
DLBCL has revolutionized treatment approach, with conservative antibiotic regimen as the
primary therapeutic method in low-grade, stage I diseases. However, further in vitro and
clinical studies are needed to elucidate the many still unclear issues of this association such as
the pathogenic mechanism and the geographical discrepancy of platelet response to
eradication therapy.
In addition, the public health value of eradication may be particularly important in
disorders such as gastric cancer, MALT lymphomas, IDA or ITP. Adequate rates of anti-H.
pylori treatment have been achieved in outpatients and those with formal follow-up, whereas
suboptimal aspects of care include treatment of inpatients and care following treatment [61].
Further studies are required to identify strategies to improve the care of patients infected with
H. pylori in relation to the intriguing associations between the infection and hematological
disorders.
References
[1] Tomb J. F., White O., Kerlavage A. R. et al. (1997). The complete genome sequence of
the gastric pathogen Helicobacter pylori. Nature 388 (6642): 539–47.
[2] Genome information for the H. pylori 26995 and J99 strains Institut Pasteur. 2002.
Retrieved 2008-09-01.
[3] Peura D. A., Crowe S. E., (2010). Helicobacter pylori. In: Feldman M., Friedman L. S.,
Brandt L. J., editors. Sleisenger & Fordtran's gastrointestinal and liver disease, 9th ed.
Philadelphia: WB Saunders Company., 833-43.
[4] Graham D. Y., Sung J. J. Y., (2006). Helicobacter pylori. In: Feldman, Friedman LS,
Sleisenger M. H., editors. Sleisenger & Fordtran’s gastrointestinal and liver disease. 8th
ed. Philadelphia: WB Saunders Company, 104966.
[5] Everhart J. E., (2000). Recent developments in the epidemiology of Helicobacter pylori
Gastroenterol Clin North Am., 29:55979.
[6] Hill A. B., (1965). The environment and disease: association or causation? Proc R Soc
Med., 58:295 C30.
[7] Gasbarrini A., Franceschi F., Tartaglione R., Landolfi R., Pola P., Gasbarrini G.,
(1998). Regression of autoimmune thrombocytopenia after eradication of Helicobacter
pylori. Lancet, 352:878.
[8] Campuzano-Maya G., (2007). Proof of an association between Helicobacter pylori and
idiopathic thrombocytopenic purpura in Latin America. Helicobacter., 12: 265–73.

[9] Kohda K., Kuga T., Kogawa K. et al. (2002). Effect of Helicobacter pylori eradication
on platelet recovery in Japanese patients with chronic idiopathic thrombocytopenic
purpura and secondary autoimmune thrombocytopenic purpura. Br J Haematol., 118:
584–8.
[10] Douglas B. C., Blanchette V. S., (2002). Immune thrombocytopenic purpura. N. Engl.
J. Med., 436:995–100.
[11] Takahashi T., Yujiri T., Shinohara K., Inoue Y., Sato Y., Fujii Y. et al. (2004).
Molecular mimicry by Helicobacter pylori CagA protein may be involved in the
pathogenesis of H. pylori-associated chronic idiopathic thrombocytopenic purpura, Br.
J. Haematol., 124: 91–96.
[12] Gerhard M., Rad R., Prinz C., Naumann M., (2002). Pathogenesis of Helicobacter
pylori infection. Helicobacter., (Suppl. 1): 17–23.
[13] Byrne M. F., Kerrogan, Corcoran P. A., Atherton J. C., Murray F. E., Fitzgerald D. J. et
al. (2003). Helicobacter pylori binds von Willebrand factor and interacts with GPIb to
induce platelet aggregation. Gastroenterology, 38:1023–1030.
[14] Emilia G., Longo G., Luppi M., Gandini G., Morselli M., Ferrara L. et al. (2001).
Helicobacter pylori eradication can induce platelet recovery in idiopathic
thrombocytopenic purpura. Blood, 97:812–4.
[15] Fujimura K., Kuwana M., Kurata Y., Imamura M., Harada H., Sakamaki H. et al.
(2005). Is eradication therapy useful as the first line of treatment in Helicobacter pylori
positive idiopathic thrombocytopenic purpura? Analysis of 207 eradicated chronic ITP
cases in Japan. Int J Hematol., 81:162–8.
[16] Arnold D. M., Bernotas A., Nazi I., Stasi R., Kuwana M., Liu Y. et al. (2009). Platelet
count response to H. pylori treatment in patients with immune thrombocytopenic
purpura with and without H. pylori infection: a systematic review. Haematologica,
94:850–6.
[17] Tag H. S., Lee H. S., Jung S. H., Kim B. K., Kim S. B., Lee A. et al. (2010). Effects of
Helicobacter pylori eradication in patients with immune thrombocytopenic purpura.
Korean J Hematol., 45:12732.
[18] Franchini M., Cruciani M., Mengoli C., Pizzolo G., Veneri D. (2007). Effect of
Helicobacter pylori eradication on platelet count in idiopathic thrombocytopenic
purpura: a systematic review and meta-analysis. J Antimicrob Chemother., 60:237–46.
[19] Kikuchi T., Kobayashi T., Yamashita T., Ohashi K., Sakamaki H., Akiyama H., (2011).
Eight-year follow-up of patients with immune thrombocytopenic purpura related to H.
pylori infection. Platelets., 22:61–4.
[20] Veneri D., Franchini M., Gottardi M., D'Adda M., Ambrosetti A., Krampera M. et al.
(2002). Efficacy of Helicobacter pylori eradication in enhancing platelet count in adult
patients with idiopathic thrombocytopenic purpura. Haematologica, 87:1777–9.
[21] Kodama M., Kitadai Y., Ito M., Kai H., Masuda H., Tanaka S., et al. (2007). Immune
response to CagA protein is associated with improved platelet count after Helicobacter
pylori eradication in patients with idiopathic thrombocytopenic purpura. Helicobacter.,
12:36–42.
[22] Emilia G., Luppi M., Zucchini P., Morselli M., Potenza L., Forghieri F. et al. (2007).
Helicobacter pylori infection and chronic immune thrombocytopenic purpura: long-
term results of bacterium eradication and association with bacterium virulence profiles.
Blood, 110:3833–41.

[23] Kohda K., Kuga T., Kogawa K., Kanisawa Y., Koike K., Kuroiwa G. et al. (2002).
Effect of Helicobacter pylori eradication on platelet recovery in Japanese patients with
chronic idiopathic thrombocytopenic purpura and secondary autoimmune
thrombocytopenic purpura. Br J Haematol., 118:584–8.
[24] Stasi R., Rossi Z., Stipa E., Amadori S., Newland A. C., Provan D. (2005).
Helicobacter pylori eradication in the management of patients with idiopathic
thrombocytopenic purpura. Am J Med., 118:414–9.
[25] Jarque I., Andreu R., Llopis I., de la Rubia J., Gomis F., Senent L. et al. (2001).
Absence of platelet response after eradication of Helicobacter pylori infection in
patients with chronic idiopathic thrombocytopenic purpura. Br J Haematol., 115:1002–
3.
[26] Michel M., Cooper N., Jean C., Frissora C., Bussel J. B. (2004). Does Helicobacter
pylori initiate or perpetuate immune thrombocytopenic purpura? Blood, 103:890–6.
[27] Suzuki T., Matsushima M., Masui A., Watanabe K., Takagi A., Ogawa Y. et al. (2005).
Effect of Helicobacter pylori eradication in patients with chronic idiopathic
thrombocytopenic purpura — a randomized controlled trial. Am J Gastroenterol.,
100:1265–70.
[28] Fukui O., Shimoya K., Shimizu T., Fukuda H., Wasada K., Murata Y. (2005).
Helicobacter pylori infection and platelet counts during pregnancy. Int J Gynaecol
Obstet., 89:26–30.
[29] Michel M., Khellaf M., Desforges L., Lee K., Schaeffer A., Godeau B. et al. (2002).
Autoimmune thrombocytopenic purpura and Helicobacter pylori infection. Arch Intern
Med., 62:1033–6.
[30] Zeller B., Helgestad J., Hellebostad M. et al. (2000). Immune thrombocytopenic
purpura in childhood in Norway: a prospective, population based registration. Pediatr
Hematol Oncol., 17(7):551–558.
[31] Rosthøj S., Hedlund-Treutiger I., Rajantie J. et al. (2003). NOPHO ITP Working
Group. Duration and morbidity of newly diagnosed idiopathic thrombocytopenic
purpura in children: A prospective Nordic study of an unselected cohort. J Pediatr.,
143(3): 302–307.
[32] Dufour C., Brisigotti M., Fabretti G., Luxardo P., Mori P. G., Barabino A. (1993).
Helicobacter pylori gastric infection and sideropenic refractory anemia. J Pediatr
Gastroenterol Nutr., 17: 225–7.
[33] Cardenas V. M., Mulla Z. D., Ortiz M., Graham D. Y. (2006). Iron deficiency and
Helicobacter pylori infection in the United States. Am J Epidemiol., 163: 127–34.
[34] Barabino A., Dufour C., Marino C. E., Claudiani F., de Alessandri A. (1999).
Unexplained refractory iron-deficiency anemia associated with Helicobacter pylori
gastric infection in children: further clinical evidence. J. Pediatr. Gastroenterol. Nutr.,
28(1):116–119.
[35] Realdi G., Dore M. P., Fastame L. (1999). Extradigestive manifestations of
Helicobacter pylori infection-fact and fiction. Dig Dis Sci., 44:229–36.
[36] Boyanova L. (2011). Role of Helicobacter pylori virulence factors for iron acquisition
from gastric epithelial cells of the host and impact on bacterial colonization. Future
Microbiol., 6:843–6.

[37] Dufour C., Brisigotti M., Fabretti G., Luxardo P., Mori P. G., Barabino A. (1993).
Helicobacter pylori gastric infection and sideropenic refractory anemia. J Pediatr
Gastroenterol Nutr., 17:225–7.
[38] Choe Y. H., Kwon Y. S., Jung M. K., Kang S. K., Hwang T. S., Hong Y. C. (2001).
Helicobacter pylori-associated iron-deficiency anemia in adolescent female athletes. J
Pediatr., 39:100–4.
[39] Fagan R. P., Dunaway C. E., Bruden D. L., Parkinson A. J., Gessner B. D. (2009).
Controlled, household randomized, open-label trial of the effect of treatment of
Helicobacter pylori infection on iron deficiency among children in rural Alaska: results
at 40 months. J Infect Dis., 199:652–60.
[40] Banić M., Franceschi F., Babić Z., Gasbarrini A. (2012). Extragastric manifestations of
Helicobacter pylori infection. Helicobacter., 17:49–55.
[41] Zimmermann M. B., Hurrell R. F. (2007). Nutritional iron deficiency. Lancet, 370: 511-
520.
[42] Annibale B., Marignani M., Monarca B., Antonelli G., Marcheggiano A., Martino G. et
al. (1999). Reversal of iron deficiency anemia after Helicobacter pylori eradication in
patients with asymptomatic gastritis. Ann Intern Med., 131:668–72.
[43] Isaacson P., Wright D. H. (1983). Malignant lymphoma of mucosa-associated lymphoid
tissue. A distinctive type of B-cell lymphoma, Cancer, 8:1410–1416.
[44] Isaacson P. G., Du M. Q. (2005). Gastrointestinal lymphoma: where morphology meets
molecular biology. J. Pathol., 205: 255-74.
[45] Eck M., Schmausser B., Haas R., Greiner A., Czub S., Müller-Hermelink H. K. (1997).
MALT-type lymphoma of the stomach is associated with Helicobacter pylori strains
expressing the CagA protein. Gastroenterology, 112: 1482-1486k.
[46] Greenson J. K. et al. (2010). MALT lymphoma, in Diagnostic Pathology:
Gastrointestinal, pp. 2:70–2:75, Amirsys, Inc., Salt Lake City, Utah, USA, 2010.
[47] Montalban C., Santon A., Boixeda D. et al. (2001). Treatment of low grade gastric
mucosa-associated lymphoid tissue lymphoma in stage I with Helicobacter pylori
eradication. Long-term results after sequential histologic and molecular follow-up.
Haematologica, 86: 609–617.
[48] Wündisch T., Thiede C., Morgner A., Dempfle A., Günther A., Liu H. et al. (2005).
Long-term follow-up of gastric MALT lymphoma after Helicobacter pylori eradication.
J. Clin. Oncol., 23:8018–24.
[49] Koehler C. I., Mues M. B., Dienes H. P., Kriegsmann J., Schirmacher P., Odenthal M.,
(2003). Helicobacter pylori genotyping in gastric adenocarcinoma and MALT
lymphoma by multiplex PCR analyses of paraffin wax embedded tissues. Mol. Pathol.,
56: 36–42.
[50] Ohashi S., Segawa K., Okamura S., Mitake M., Urano H., Shimodaira M. et al. (1998).
Aggressive non-Hodgkin's lymphoma after successful eradication of Helicobacter
pylori and regression of gastric lymphoma of mucosa-associated lymphoid tissue. J.
Gastroenterol., 33: 724–7.
[51] Malik A. A., Ganti A. K., Potti A., Levitt R., Hanley J. F., (2002). Role of Helicobacter
pylori infection in the incidence and clinical course of monoclonal gammopathy of
undetermined significance. Am J Gastroenterol., 97: 1371–4.

[52] Ηershko C., Ronson A., Souroujon M., Maschler I., Heyd J., Patz J. (2006). Variable
hematologic presentation of autoimmune gastritis: age-related progression from iron
deficiency to cobalamin depletion. Blood, 107:1673–9.
[53] Doğan Z., Filik L., Ergül B., Sarikaya M., Akbal E. (2013). Association between
Helicobacter pylori and liver-to-spleen ratio: a randomized-controlled single-blind
study. Eur. J. Gastroenterol. Hepatol., 25:107–10.
[54] Matsukawa Y., Sawada S., Sawada U. (2006). Improvement of anemia by H. pylori
eradication in a patient with myelodysplastic syndrome. Med. Hypotheses, 67: 423.
[55] Arima N., Tsudo M., (2003). Extragastric mucosa-associated lymphoid tissue
lymphoma showing the regression by Helicobacter pylori eradication therapy. Br. J.
Haematol., 120:790–2.
[56] Tedeschi R., Luostarinen T., Marus A., Bzhalava D., Ogmundsdottir H. M., Dillner J. et
al. (2009). No risk of maternal EBV infection for childhood leukemia. Cancer.
Epidemiol. Biomarkers. Prev., 18:2790–2.
[57] Szczepanik A. B., Zaleska M., Wiszniewski A., Wislawski S., Misiak A., Maryniak R.,
et al. (2005). Helicobacter pylori infection in patients with haemophilia in Poland:
prevalence and risk of upper gastrointestinal bleeding. Haemophilia, 11:376–9.
[58] Choe B. H., Kim J. Y., Lee J. H., Kim J. M., Chu M. A., Cho S. M. et al. (2010). Upper
gastrointestinal bleeding in children with haemophilia: a clinical significance of
Helicobacter pylori infection. Haemophilia, 16: 223–413.
[59] Provan D., Stasi R., Newland A. C., Blanchette V. S., Bolton-Maggs P., Bussel J. B.,
Chong B. H., Cines D. B., Gernsheimer T. B., Godeau B., Grainger J., Greer I., Hunt B.
J., Imbach P. A., Lyons G., McMillan R., Rodeghiero F., Sanz M. A., Tarantino M.,
Watson S., Young J., Kuter D. (2009). International consensus report on the
investigation and management of primary immune thrombocytopenia. Blood, 6:168–
186.
[60] Psyrri A., Papageorgiou S., Economopoulos T. (2008). Primary extranodal lymphomas
of stomach: clinical presentation, diagnostic pitfalls and management. Annals of
Oncology, 19(12):1992–1999.
[61] Jones N., Chiba N., Fallone C., Thompson A., Hunt R., Jacobson K. et al. (2012).
Helicobacter pylori in First Nations and recent immigrant populations in Canada. Can.
J. Gastroenterol., 26(2):97–103.

Chapter 15

and Cardiovascular Disorders
Benjamin Longo-Mbenza1 Jacqueline Nsenga Nkondi2,

Marco Manfredi3,4 and Bertrand Tchana5†
1
Faculty of Health Sciences, Walter Sisulu University, Mthatha, South Africa
2
Division of Gastroenterology, University of Kinshasa, Kinshasa,
Democratic Republic of the Congo
3
4
Azienda Ospedaliero-Universitaria, University Hospital, Parma, Italy
5
Pediatric Cardiology, Department of Pediatrics, “Pietro Barilla” Children's Hospital,
Azienda Ospedaliero-Universitaria, University Hospital, Parma, Italy
Abstract
Helicobacter pylori (H. pylori) infection is reported to be associated with many
extragastrointestinal manifestations such as cardiovascular disorders and especially
ischemic hearth disease. In fact several epidemiological studies have suggested that H.
pylori infection might be involved in the pathogenesis and progression of atherosclerosis
plaque through the stimulation of proinflammatory cytokines production.
However, the evidence of this association is often weak and many studies could have
erroneous results. Therefore although there are some plausible mechanisms on the role of
H. pylori infection in cardiovascular diseases, they are currently not conclusive.
This chapter tries to summarize the latest knowledge regarding the possible role of
H. pylori infection in cardiovascular diseases.
*
Corresponding author: Prof. Benjamin Longo-Mbenza, Faculty of Healthy Sciences, Walter Sisulu University,
Mthatha, South Africa. Email: longombenza@gmail.com.
†
Corresponding author: Bertrand Tchana, MD, Pediatric Cardiology, Department of Pediatrics “Pietro Barilla”
Children's Hospital, Azienda Ospedaliero-Universitaria, Parma University Hospital, Via Gramsci 14, 43100
Parma, Italy. Email:btchana@ao.pr.it

284 Benjamin Longo-Mbenza Jacqueline Nsenga Nkondi, Marco Manfredi et al.
Introduction
Since the discovery of Helicobacter pylori (H. pylori) infection, the immunological
properties expressed by the bacterium in relation to the host have been investigated. The main
objective of those studies was to demonstrate how H. pylori may cause gastric mucosal
damage and, at the same time, elude the immunological response evoked by the host. Data
collected from those studies showed that the immunological response caused by this
bacterium is both locally and systemically oriented and may virtually influence the clinical
course of other diseases, outside the stomach, therefore opening the field of extragastric
manifestations of H. pylori infection [1-4]. In several studies an association between viral and
bacterial infections, such as H. pylori infection, and cardiovascular diseases (CVDs) has been
shown. Among the extraintestinal manifestations of H. pylori infection, vascular dysfunction
and ischemic heart disease (IHD) rank among the first positions.
Helicobacter pylori and Inflammation

Infection with H. pylori may be associated with systemic and vascular inflammation.
Prevalence of seropositivity to H. pylori appears to be significantly increased in subjects with
high sensitive C-Reactive Protein (CRP), a sensitive marker of systemic inflammation [5, 6].
Chronic infection with H. pylori may be accompanied by persistently increased production of
inflammatory metabolites and some antigenic substances, as heat shock protein, urease, and
lipopolysaccharide, all of which can be taken up and processed by lamina propria
macrophages and active T-cells, being probably the cause of increased production of
inflammatory cytokines such as interleukine (IL)-1, IL-6, tumor necrosis factor alpha (TNF-
), and most important IL-8 [7-10]. Recently, different studies have suggested a role of
inflammation in the pathogenesis of endothelial dysfunction [7-11]. Chronic inflammation
leads to an increase in the generation of pro-inflammatory cytokines, cell adhesion molecules,
and growth factors that can elicit inflammatory and proliferative changes in the vessel walls,
which also affect blood vessel motility resulting in endothelial dysfunction [12, 13]. In some
studies a circulating soluble form of intercellular adhesion molecule-1 (ICAM-1), which is
expressed on the activated endothelium in response to inflammatory cytokines, has been
shown to be elevated in subjects positive to anti-H. pylori [12-14]. Leukocyte binding to
cellular adhesion molecules on the surface of vascular endothelium, in response to many
inflammatory cytokines and CRP, may probably be the earliest event in the vascular
inflammatory process.
H. pylori infection has been shown to be associated with an increase in the level of
fibrinogen, and in addition, it is also associated with a deterioration in lipid profiles, an event
which could promote atherosclerosis [15]. Moreover, a previous study showed that H. pylori
eradication was associated with a significant reduction in serum CRP levels and a significant
rise in serum high-density lipoprotein (HDL)-cholesterol [13, 15].
There are two main metastatic pathways for systemic effects of focal inflammations,
systemic bacteremia (direct effect) and spreading of inflammatory mediators, released in
response to the infection at the diseased site (indirect effect, or immunological sounding).

Helicobacter pylori Infection and Cardiovascular Disorders 285
The indirect effects of the mediators have been subject to recent reviews [16-19]; in this
chapter we will focus on the evidence of direct effects of bacteremias on vascular tissue.
Chronic Infection, Inflammation

and Endothelial Dysfunction
According to the response-to-injury hypothesis of atherosclerosis, endothelial
dysfunction is the first step in the atherosclerotic process [20, 21]. This hypothesis has been
supported by many studies, which indicate that endothelial dysfunction occurs in subjects
with classic risk factors such as hypertension, diabetes mellitus, hyper-cholesterolemia, and
smoking.
Atherosclerosis is a chronic inflammatory condition associated with the presence of
conventional cardiovascular risk factors such as hyper-cholesterolemia, hypertension,
diabetes, smoking, and genetic factors. As far as the incidence of atherosclerosis is not
completely explained by these risk factors, it is now becoming clear that, in addition to the
smooth muscle cells that play a major role in the progression of atherosclerotic lesions,
leukocytes, growth factors and inflammatory mediators participate in a dynamic and
progressive process, beginning with endothelial dysfunction and characterized by
inflammation, as a key regulatory process interacting with the standard risk factors, leading to
clinical symptoms of disease [20-22]. The recognition of the inflammatory characteristics of
atherosclerosis led to the successful application of CRP as an acute-phase marker for
cardiovascular risk assessment [23, 24]. The inflammatory character of the atherosclerotic
lesion has turned attention towards the origins of flogosis, specifically, the contribution of
bacterial pathogens as a potential cause.
The notion that atherosclerosis is related to infections dates back to the 19th century,
based on the results of an animal model, where infection of a rabbit resulted in the formation
of fatty streaks [25]. A century later, the interest was rekindled again by animal experiments
and other investigations and prompting this idea to reemerge [21, 26]. An abundance of
epidemiological evidence has been presented in support of this idea [27, 28]. A longitudinal
seroepidemiological study on 572 patients where the extent of atherosclerosis was measured
by coronary angiography, carotid duplex sonography and ankle-arm index, suggested that
elevated IgA and IgG titers to infectious agents are associated with the extent of
atherosclerosis and with CVD death. After adjustment for age, sex, classical risk factors and
high sensitivity CRP, infectious burden was significantly associated with advanced
atherosclerosis [29]. The evidence that the aggregate burden of chronic infections,
exacerbated by the host’s immune response, may contribute to atherosclerosis, led to the
recognition of what is now known as ‘pathogen burden’ [30].
In an oral infections-systemic inflammation model observational studies have shown a
particular association of systemic inflammation and endothelial dysfunction with periodontal
inflammations, and specific studies have been designed [31-35]. Indeed, the results from one
study indicate that chronic infections, including periodontitis, may predispose individuals to
CVDs, and demonstrated that periodontal bacterial burden was related to the carotid intima-
media thickness, a measure of subclinical atherosclerosis [35]. The majority of the

epidemiological studies linked periodontal disease to increased incidence of cardiovascular,

cerebrovascular and peripheral vascular disease [34].
The initial event in bacteremia is the entry of bacteria into the circulation through the
diseased site. The most studied model is transient bacteremia with oral pathogens entering
through the microvasculature following tooth brushing and other dental procedures [36-39].
For example, blood samples taken from 30 patients before and after a procedure demonstrated
bacteremia following ultrasonic scaling, periodontal probing and tooth brushing of 23%, 16%
and 13%, respectively, using PCR [38, 39]. In a study on 194 patients, it was shown that the
periodontal site bleeding after tooth brushing was associated with an approximate eight-fold
increase in bacteremia. The data suggest the possibility of repeated daily encounter between
invasive oral pathogens and endothelia, in particular in the areas of turbulent flow, which are
exactly where atheromatous lesions form [36, 39].
Atherosclerotic development, since it involves both the innate and adaptive arms of the
immune system, bears similarity to bacterial infection. The response to injury’ hypothesis of
atherogenesis describes an inflammatory process leading to endothelial activation, growth
factor release, monocyte adhesion and migration while maturing into macrophages, followed
by foam cell formation and smooth muscle cell proliferation. The inflammation is not only
thought to cause initiation and progression of the atheroma, but also the thrombosis and acute
ischemic events leading to death [36, 40, 41].
Infectious agents are implicated in the etiology of different vascular conditions by
multiple mechanisms, including direct microbial invasion of endothelial cells, host cell
activation and stimulation of leukocyte influx and of cross-reactive B and T cells [41, 42].
Bacterial invasion of vascular tissue leading to intracellular localization bears multiple
advantages for the pathogen: a ‘privileged niche’ with access to host protein (nutritional) and
iron substrates; a sequestration from the humoral and cellular immune response; and a means
for persistence that is essential for a chronic pathogen. In direct bacteremic dissemination,
bacteria spread in the circulation and gain access to the endothelia. Conversely, bacteria
internalized in monocytes/macrophages or in dendritic cells at the diseased site, disseminating
to endothelia, where they can find a way to the lamina and tunica media due to extravasation
of the carrier phagocytes [36, 43, 44]. The infection with invasive bacteria has been shown to
induce monocyte migration and significantly enhance the production of proinflammatory
cytokines. Certain bacteria have evolved to invade nonprofessional phagocytic cells [36]. The
process of bacterial invasion of host tissue begins with adherence to the target cell. Many
pathogenic bacteria have acquired a large and diverse set of adhesive moieties on their
surfaces in order to recognize and bind to specific host cell surface receptors [43, 44].
Different bacteria species detected in bacteremias, have been shown to invade coronary artery
endothelial cells [45, 46, 47]. The bacteria can transmit between the same, as well as between
different cell types. Cell–cell contact between infected cells and new host cells increases the
rate of transmission, although bacteria can cross significant distance in medium as well [36].
The presence in the vascular wall of bacterial pathogen-associated molecular patterns
stimulates the toll-like receptors of the leukocytes leading to release of proinflammatory
cytokines contributing to atherogenesis [42]. The contribution of bacteria to the most critical
moment in atherogenesis, plaque destabilization and rupture, is not limited to the
inflammatory process alone. Plaque rupture, leading to leakage of the prothrombotic plaque
core in the circulation and thrombus formation, can be due to the bacteria-dependent release
of matrix metalloproteinases with concomitant suppression of the matrix metalloproteinase

antagonist, tissue inhibitor of metalloproteinases [48, 49]. Animal models can serve as a link
between hypotheses and clinical practice. Some authors clearly showed that H. pylori
promotes atherogenesis in heterozygous APOE (+ ⁄-) LDLR (+ ⁄ -) mice. In particular, H.
pylori infected and noninfected mice were fed a high fat diet from the age of 6 weeks;
development of atherosclerotic lesions was observed in infected animals and correlated with
an elevation of Th1-immune response against H. pylori heat shock-protein 60. To confirm the
plausibility of this hypothesis, H. pylori eradication significantly reduced the progression of
atherosclerosis in infected mice [11].
There is strong evidence that some bacteria species are disseminated into large vessels,
since DNA can be detected in atheromas. Using PCR, it has been found that 1.5-2.2% of the
total DNA in the atheromatous samples are bacterial [36, 47].
Vascular cell activation is a pivotal moment in initiation of atherogenesis. The smooth
muscle cells respond to bacteria in a prothrombotic manner or in a proliferative manner. An
alternative plausible mechanism through which bacteremias (or bacteria present in ruptured
plaque) may contribute to vascular thrombosis is the triggering of the coagulation cascade, as
described in other studies [37, 50].
Helicobacter pylori and Ischemic Hearth

and Vascular Disease
Atherosclerosis, a very common vascular disease, is responsible of different clinical
syndromes, including coronary artery disease, ischemic stroke, and peripheral artery disease.
It is caused by endothelial dysfunction and inflammation, one of the most common
manifestations of atherosclerosis and powerful and independent predictor of cardiac and
cerebral ischemic events.
Viral and bacterial infections, such as Chlamydia pneumonia, Hepatitis A virus,
Cytomegalovirus and Herpes simplex virus have been implicated in affecting development
and course of coronary atherosclerotic diseases. Serological evidence of association of
bacteria with chronic coronary heart disease (CHD) and myocardial infarction was presented
for the first time in The Lancet [45].
The role of H. pylori in ischemic heart disease is a matter of concern and controversies.
H. pylori is similar to Chlamydia pneumonia, Hepatitis A virus, Cytomegalovirus and Herpes
simplex virus, as it is also an obligatory intracellular pathogen and causes life-long persistent
infection. H. pylori also establishes persistent antibodies targeted to it. In the last decade,
different authors have investigated the relationship between H. pylori seroprevalence and the
risk of CHD, and different epidemiological studies have suggested that H. pylori infection
might be involved in its pathogenesis [1-6, 16].
Plasma levels of circulating ICAM-1 and vascular adhesion molecule-1 have been shown
to be increased in patients with angina pectoris and positive response radionuclide or stress
tests, suggesting endothelial cell activation [13, 14]. In the same way, increased plasma
concentration of the powerful vasoconstrictor endothelin-1 (ET-1) has been reported in
peripheral blood of these patients and has been shown to correlate with endothelial
dysfunction [5, 51]. CRP levels are known to be associated with endothelial cell activation
and coronary endothelial dysfunction [5, 52].

H. pylori infection causes chronic inflammation and increases the generation of

proinflammatory cytokines, cell adhesion molecules, growth factors and acute-phase proteins.
An increase in these factors may affect vessel motility and can elicit inflammatory and
proliferative changes in the vessel walls by endothelial dysfunction [53, 54, 55]. Several
pathological mechanisms have been postulated to explain the effect of H. pylori infection on
atherosclerosis [7, 53-56]. It has been suggested to initiate an acute-phase response and to
activate TNF-α, IL-6 and fibrinogen, which are inflammatory cytokines that can directly or
indirectly propagate an inflammatory process in arterial walls [7, 56]. H. pylori has been also
shown to cause platelet aggregation, an important aspect of acute destabilization of
atherosclerotic disease [7, 53-56]. Another possible mechanism by which H. pylori may cause
endothelial damage is by causing aggravated autoimmune hormonal responses because of
antigenic mimicry, such as immunological cross reactivity between bacterial and human heat-
shock proteins, which can lead to coronary calcification and early atherosclerosis [7, 56, 57].
In addition, direct colonization of the arterial wall by H. pylori has been suggested, and H.
pylori has been found in atheromas using the polymerase chain reaction technique.
The suspicion about H. pylori involvement in the pathological lesions is based on the
following: local inflammation can have systemic effects; H. pylori gastric infection is a
chronic process that lasts for decades; and persistent infection induces chronic inflammatory
and immune responses that can induce lesions both local and remote sites from the primary
infection site, manifestation caused by potential direct or indirect actions [52-54, 57]. The
direct effects on the vascular wall could include endothelial injury and dysfunction through
circulating endotoxins, smooth muscle proliferation, and local inflammation. The indirect
effects are more often pronounced, including elevation of inflammatory mediators with
proinflammatory, procoagulant, and atherogenic action, production of cross-reactive
antibodies, as well as malabsorption and metabolic disturbances such as overproduction of
ammonia by the bacterium.
Other authors performed an interesting study on the association between H. pylori
infection and coronary artery calcification score, starting from the assumption that this score,
measured by computed tomography, has previously been used as a screening test for coronary
atherosclerosis. Among 2.029 subjects enrolled, 59.8% were positive for H. pylori infection
and multivariate analysis revealed a positive association between H. pylori seropositivity and
severity of coronary artery calcification score [58].
Furthermore a role of H. pylori has been proposed in cardiac syndrome X by
microvascular dysfunction [5, 6, 55, 56]. Cardiac syndrome X is a clinical definition,
characterized by three main features: angina like chest pain; ST segment depression on
treadmill exercise testing and normal coronary arteriography.
Studies focusing on the effect of virulent strain of H. pylori, such as those carrying the
cytotoxin associated gene-A (CagA) island of pathogenicity, gave more enthusiastic results.
A meta-analysis have shown a significant association between infection sustained by those
strains and vascular damage, whereas pathophysiological studies suggested a possible
involvement in the atherosclerotic process mediated by a molecular mimicry between the
CagA antigen and atherosclerotic plaque peptides [59, 60]. CagA-positive strains of H. pylori
are known to be associated with a more severe gastric disease, through a more pronounced
stimulation of the immune system. Several studies have reported CagA-positive strains of H.
pylori to increase the risk of myocardial infarction among healthy men, probably showing that
infection sustained by those strains may be considered as a marker of vascular damage for

both coronary and cerebral vessels [7, 59, 60, 61]. Another group performed a clinico-
pathological study on patients with stable angina, unstable angina and normal controls,
finding that the anti-CagA antibody titer was significantly higher in patients with unstable
angina compared to stable. Moreover, anti-CagA antibodies recognized antigens localized
inside coronary atherosclerotic plaques. In the meta-analysis, seropositivity to CagA was
significantly associated with the occurrence of acute coronary events with an adjusted odds
ratio (OR) of 1.34 (95% confidence interval (CI), 1.15–1.58, p = .003) [62]. Therefore, it
could be hypothesize that the cross-reactivity between anti-CagA antibodies and endothelial
cells, in the presence of the other atherosclerotic risk factors, may contribute to
atherosclerotic plaques initiation and growth. These findings suggest that in a subset of
patients with unstable angina, an intense immune response against CagA-positive strains may
act as a trigger for the precipitation of coronary instability by a molecular mimicry
mechanism [53, 59, 60, 62]. CagA-positive strains of H. pylori have also been found to play a
role in the destabilization of coronary plaques [53, 59, 60].
Infection by CagA-positive strains has been associated with aortic atherosclerosis and an
increased intima-medial thickness of the carotid artery [63].
H. pylori has also been shown to affect the vascular risks and complications in patients
with diabetes mellitus although data concerning the prevalence of H. pylori infection among
these patients are scanty and controversial [64, 65]. In a very interesting study, the prevalence
of H. pylori infection in patients with diabetes mellitus as well as the association between
diabetic vascular complications and H. pylori infection, and the influence of H. pylori
infection on atherosclerosis and inflammatory biomarkers was assessed. Prevalence of H.
pylori infection was higher in patients with diabetes mellitus compared to healthy controls.
Carotid artery intima-media thickness was significant higher in H. pylori-infected patients.
Inflammatory biomarkers, as IL-6, and TNF-α were significantly associated with H. pylori
infection. In a multivariate analysis, blood glucose, triglycerides, erythrocytic sedimentation
rate, IL-6, and TNF-α increased the odds for atherothrombotic cause of cerebral ischemia in
patients with H. pylori infection, suggesting that H. pylori infection is common in diabetes
mellitus and seems to be linked to the presence of atherosclerosis and ischemic
cerebrovascular stroke, effect probably mediated by increasing cytokine levels [65]. In a
prospective uncontrolled and unblinded clinical investigation performed in an African
population revealed a significant association between H. pylori seropositivity and higher
cardiometabolic risk. In these patients, greater intima-media thickness, hypercholesterolemia,
diabetes mellitus, hypo-HDL-cholesterolemia, lower HDL-cholesterol, abdominal obesity and
higher rate of hypertension were significantly associated with H. pylori seropositivity. After
adjusting for age and sex, H. pylori-seropositive males showed higher mean values for
triglycerides, and total cholesterol. The levels of uric acid, plasma glucose, total cholesterol,
fibrinogen, and blood pressure after 3 weeks antibiotics duration were lower than their
baseline levels, even though weight, waist girth, and triglyceride levels did not change after
H. pylori eradication [66].
A possible role of H. pylori, in particular both CagA and VacA-positive strains has been
claimed in idiopathic arrhythmia [67-69].

Controversies
Studies exploring the role of H. pylori in general in atherosclerosis have given conflicting
results. Different authors have investigated the link between individual pathogens, including
H. pylori, pathogen burden and subclinical atherosclerosis, without finding significant
associations, leading to the hypothesis that pathogen burden may be related to a subset of the
population prone to develop clinical manifestations of atherosclerosis. One group investigated
the relationship between H. pylori IgG antibody titers and severity and intensity of coronary
atherosclerosis and also clinical presentation of CAD by enrolling a group of patients with
angiographically proven coronary artery disease, which in turn consisted of three different
subgroups, such as patients with myocardial infarction, patients with unstable angina, and
patients with stable angina. Control group included subjects with angiographically proven
normal coronary arteries. Seropositivity rates for H. pylori were similar between patients and
controls as well as among different subgroups. Nevertheless, while H. pylori IgG titer seems
not to play an important role in the presentation of CAD, a significant correlation was found
between H. pylori positivity and extent score of CAD [70]. Similar results were obtained by
other authors in a similar study, and they conclude that H. pylori infection has a modest
influence on CAD and progressive atheroma [65]. In a prospective 5-year follow-up study,
one group included 150 patients with subclinical carotid atherosclerosis, evaluating at
baseline all established traditional cardiovascular risk factors, fibrinogen, CRP as well as
seropositivity to H. pylori, Chlamydia pneumoniae, and Cytomegalovirus. Among all the
above-mentioned factors, only elevated CRP levels showed a predictive role in multivariate
analysis, while null findings were obtained by including viral and bacterial antibody titers,
thus suggesting a major role of inflammation, but not infection, in the progression of
atherosclerosis [62, 71]. Some other authors valued the possible link between H. pylori-
associated atrophic gastritis and carotid intima-media thickness. The results did not show a
significant difference in cartotid intima-media thickness between groups: therefore carotid
intima-media thickness was not associated with H. pylori-induced atrophic gastritis [72]. In
another study, samples of aortic wall were studied, by protein chain reaction, for the presence
of Chlamydia pneumoniae, Mycoplasma pneumoniae, H. pylori, Herpes simplex, and
Cytomegalovirus. Specimens were obtained from patients with atherosclerotic three-vessel
stable CAD referred to surgical revascularization (coronary group) and controls referred to
aortic valve replacement (valve group). Results demonstrated that all these micro-organisms
can be easily detected in macroscopically healthy aortic wall of either coronary or valve
patients, thus denying any possible pathogenic role in atherosclerotic disease [73].
Conclusion
During the last years knowledge about the classical risk factors for CVDs and particularly
CAD has increased, however not all the differences in morbidity from this disease can be
explained. Research has take the challenge of looking for other causal mechanisms and
several studies focused on a possible link between ischemic CVDs and chronic infections,
leading some authors to considering such infections as risk factor. After the first report on the
association between H. pylori infection and CAD, several studies have investigated showing

conflicting results. While there is a general agreement on the inflammatory basis of

atherosclerosis, the the role of chronic infection, such as infection with H. pylori, sustaining
an atherosclerosis process leading to vascular diseases is still a matter of controversies
especially regarding IHD, even if experimental evidence from animal studies supports the
concept that the role of the bacterium might be more important in the acute events. At present
it is not possible to draw unquestionable conclusions. Further studies and trials are needed
and longer follow-up is required to better elucidate the role of H. pylori in the pathogenesis of
atherosclerosis.
References
[1] Suzuki H, Franceschi F, Nishizawa T, Gasbarrini A, (2011). Extragastric manifestations
of Helicobacter pylori infection. Helicobacter.,16(Suppl 1):65–9.
[2] Banić M, Franceschi F, Babić Z, Gasbarrini A, (2012). Extragastric manifestations of
Helicobacter pylori infection Helicobacter.,17 Suppl 1:49-55.
[3] Tan HJ, Goh KL, (2012). Extragastric manifestations of Helicobacter pylori acts or
myth? A critical review J. Dig. Dis., 13(7):342-9.
[4] Franceschi F, Roccarina D, Gasbarrini A, (2006). Extragastric manifestations of
Helicobacter pylori infection. Minerva. Med. 2006 Feb; 97(1):39-45.
[5] Rasmi Y, Raeisi S, (2009). Possible role of Helicobacter pylori infection via
microvascular dysfunction in cardiac syndrome X. Cardiol. J., 16(6):585-7.
[6] Celik T, Iyisoy A, Yuksel UC, (2010). Possible pathogenetic role of Helicobacter
pylori infection in cardiac syndrome X. Int. J. Cardiol., 9;142(2):193-4.
[7] .Oshima T, Ozono R, Yano Y et al. (1997). Association of Helicobacter pylori infection
with systemic inflammation and endothelial dysfunction in healthy male subjects. J.
Am. Coll. Cardiol., 45:1219–1222.
[8] De Meyer GR, Herman AG, (1997). Vascular endothelial dysfunction. Progress.
Cardiovasc. Dis., 39: 325–342.
[9] Blum A, Tamir S, Mualem K, Ben-Shushan RS, Keinan-Boker L, Paritsky M, (2011).
Endothelial dysfunction is reversible in Helicobacter pylori-positive subjects. Am. J.
Med., 124(12):1171-4.
[10] Ayada K, Yokota K, Kobayashi K, Shoenfeld Y, Matsuura E, Oguma K, (2009).
Chronic infections and atherosclerosis. Clin. Rev. Allergy. Immunol., 37(1):44-8
[11] Ayada K, Yokota K, Hirai K, Fujimoto K, Kobayashi K, Ogawa H, et al. (2009).
Regulation of cellular immunity prevents Helicobacter pylori-induced atherosclerosis.
Lupus., 18:1154–68.
[12] Gomez-Scotto E, Seigneur M, Renard M, Houbouyan-Reveillard LL, Boisseau MR,
(2000) Interest in variations in soluble ICAM-1 plasma levels. From physiology to
clinical applications. J. Mal. Vasc., 25(3):156-65.
[13] Tousoulis D, Davies GJ, Asimakopoulos G et al. (2001). Vascular cell adhesion
molecule-1 and intercellular adhesion molecule-1 serum level in patients with chest
pain and normal coronary arteries (syndrome X). Clin. Cardiol., 24: 301–304.

[14] Arroyo-Espliguero R, Mollichelli N, Avanzas P et al. (2003). Chronic inflammation and

increased arterial stiffness in patients with cardiac syndrome X. Eur. Heart. J.,
24:2006–2011.
[15] Kanbay M, Gur G, Yucel M, Yilmaz U, Boyacioglu S, (2005). Does eradication of
Helicobacter pylori infection help normalize serum lipidand CRP levels? Dig. Dis. Sci.,
50:1228–1231.
[16] Rasmi Y, Raeisi S, Seyyed Mohammadzad MH, (2011). Association of inflammation
and cytotoxin-associated gene A positive strains of Helicobacter pylori in cardiac
syndrome X. Helicobacter., 17:116–120.
[17] Hayashi C, Gudino CV, Gibson FC 3rd, Genco CA, (2010). Review: pathogen-induced
inflammation at sites distant from oral infection: bacterial persistence and induction of
cell-specific innate immune inflammatory pathways. Mol. Oral. Microbiol., 25(5):305–
316.
[18] Teles R, Wang CY, (2011). Mechanisms involved in the association between peridontal
diseases and cardiovascular disease. Oral. Dis., 17(5):450–461
[19] Van Dyke TE, Kornman KS, (2008). Inflammation and factors that may regulate
inflammatory response. J. Periodontol., 79 Suppl. 8:1503–1507.
[20] Ridker PM, Rifai N, Pfeffer MA et al. (1998). Inflammation, pravastatin,and the risk of
coronary events after myocardial infarction in patients with average cholesterol levels.
Cholesterol and Recurrent Events (CARE) Investigators. Circulation., 98: 839–844.
[21] Ross R, (1999). Atherosclerosis – an inflammatory disease. N. Engl. J. Med.,
340(2):115–126.
[22] Libby P, Ridker PM, Hansson GK, (2009). Inflammation in atherosclerosis: from
pathophysiology to practice. J. Am. Coll. Cardiol., 54(23):2129–2138.
[23] Cosin-Sales J, Pizzi C, Brown S, Kaski JC, (2003). C-reactive protein, clinical
presentation, and ischemic activity in patients with chest pain and normal coronary
angiograms. J. Am. Coll. Cardiol., 41:1468–1474.
[24] Libby P, Okamoto Y, Rocha VZ, Folco E, (2010). Inflammation in atherosclerosis:
transition from theory to practice. Circ. J., 74(2):213–220.
[25] Gilbert A, Lion G, (1889). Arterites infectieuses experimentales. CR. Hebd. Sciences.
Mem. Soc. Biol. 41:583–584.
[26] Fabricant CG, Fabricant J, Litrenta MM, Minick CR, (1978). Virus-induced
atherosclerosis. J. Exp. Med. 148(1):335–340.
[27] Grau AJ, Marquardt L, Lichy C, (2006). The effect of infections and vaccinations on
stroke risk. Export. Rev. Neurother., 6(2):175–183.
[28] Epstein SE, Zhu J, Najafi AH, Burnett MS, (2009). Insights into the role of infection in
atherogenesis and in plaque rupture. Circulation., 119(24):3133–3141.
[29] Espinola-Klein C, Rupprecht HJ, Blankenberg S, et al. 82002). Impact of infectious
burden on extent and long-term prognosis of atherosclerosis. Circulation., 105(1):15–
21.
[30] Elkind MS, (2010). Infectious burden: a new risk factor and treatment target for
atherosclerosis. Infect. Disord. Drug. Targets., 10(2):84–90.
[31] Tonetti MS, (2009). Periodontitis and risk for atherosclerosis: an update on intervention
trials. J. Clin. Periodontol., 36 Suppl. 10:15–19.

[32] Kebschull M, Demmer RT, Papapanou PN, (2010). “Gum bug leave my heart alone!” –
epidemiologic and mechanistic evidence linking periodontal infections and
atherosclerosis. J. Dent. Res., 89(9): 879–902.
[33] Demmer RT, Desvarieux M, (2006). Periodontal infections and cardiovascular disease:
the heart of the matter. J. Am. Dent. Assoc., 137 Suppl.:14S–20S. quiz 38S.
[34] Zoellner H, (2011). Dental infection and vascular disease. Semin. Thromb. Hemost.,
37(3):181–192.
[35] Desvarieux M, Demmer RT, Rundek T, et al. (2005). Periodontal microbiota and
carotid intima-media thickness: the oral infections and vascular disease epidemiology
study (INVEST). Circulation., 111(5):576–582.
[36] Kozarov E, (2012). Bacterial invasion of vascular cell types: vascular infectology and
atherogenesis. Future. Cardiol., 8(1): 123–138.
[37] Iwai T, (2009). Periodontal bacteremia and various vascular diseases. J. Periodontal
Res., 44(6):689–694.
[38] Kinane DF, Riggio MP, Walker KF, MacKenzie D, Shearer B, (2005). Bacteraemia
following periodontal procedures. J. Clin. Periodontol., 32(7):708–713.
[39] Lockhart PB, Brennan MT, Thornhill M, et al. (2009). Poor oral hygiene as a risk factor
for infective endocarditis-related bacteremia. J. Am. Dent. Assoc., 140(10):1238–1244.
[40] Libby P, (2007). Inflammatory mechanisms: the molecular basis of inflammation and
disease. Nutr. Rev., 65(12 Pt 2):S140, S146.
[41] Stassen FR, Vainas T, Bruggeman CA, (2008). Infection and atherosclerosis. An
alternative view on an outdated hypothesis. Pharmacol. Rep., 60(1):85–92.
[42] Lidar M, Lipschitz N, Langevitz P, Shoenfeld Y, (2009). The infectious etiology of
vasculitis. Autoimmunity., 42(5):432–438
[43] Chavakis T, Wiechmann K, Preissner KT, Herrmann M (2005). Staphylococcus aureus
interactions with the endothelium: the role of bacterial ‘secretable expanded repertoire
adhesive molecules’ (SERAM) in disturbing host defense systems. Thromb. Haemost.,
94(2):278–285.
[44] Nobbs AH, Lamont RJ, Jenkinson HF, (2009). Streptococcus adherence and
colonization. Microbiol. Mol. Biol. Rev., 73(3):407–450..
[45] Saikku P, Leinonen M, Mattila K, et al. (1988). Serological evidence of an association
of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute
myocardial infarction. Lancet., 2(8618):983–986.
[46] Elkind MS, Luna JM, Moon YP, et al. 82010). Infectious burden and carotid plaque
thickness: the northern Manhattan study. Stroke., 41(3):e117–e122
[47] Ott SJ, El Mokhtari NE, Musfeldt M, et al. (2006). Detection of diverse bacterial
signatures in atherosclerotic lesions of patients with coronary heart disease.
Circulation., 113(7):929–937.
[48] Sato Y, Kishi J, Suzuki K, Nakamura H, Hayakawa T, (2009). Sonic extracts from a
bacterium related to periapical disease activate gelatinase A and inactivate tissue
inhibitor of metalloproteinases TIMP-1 and TIMP-2. Int. Endod. J., 42(12):1104–1111.
[49] Guan SM, Shu L, Fu SM, Liu B, Xu XL, Wu JZ, (2009). Prevotella intermedia
upregulates MMP-1 and MMP-8 expression in human periodontal ligament cells.
FEMS. Microbiol. Lett., 299(2): 214–222.

[50] Herzberg MC, Nobbs A, Tao L, et al. (2005). Oral streptococci and cardiovascular
disease: searching for the platelet aggregation-associated protein gene and mechanisms
of Streptococcus sanguisinduced thrombosis. J. Periodontol., 76 Suppl. 11:2101–2105.
[51] Lanza GA, (2007). Cardiac syndrome X: A critical overview and future perspectives.
Heart., 93: 159–166.
[52] Fichtlscherer S, Rosenberger G, Walter DH, Breuer S, Dimmeler S, Zeiher AM, (2000).
Elevated C-reactive protein levels and impaired endothelial vasoreactivity in patients
with coronary artery disease. Circulation., 102: 1000–1006.
[53] Franceschi F, Navarese EP, Mollo R, Giupponi B, De Marco G, Merra G, et al. (2009).
Helicobacter pylori and atherosclerosis. A review of the literature. Recenti. Prog. Med.,
100:91–6.
[54] Suzuki H, Suzuki M, Imaeda H, Hibi T, (2009). Helicobacter pylori and
microcirculation. Microcirculation., Aug 25:1–12.
[55] Rasmi Y, Raeisi S, (2009). Possible role of Helicobacter pylori infection via
microvascular dysfunction in cardiac syndrome X. Cardiol. J., 2009;16:585–7.
[56] Assadi M, Saghari M, Ebrahimi A, Reza Pourbehi M, Eftekhari M, et al. (2009). The
relation between Helicobacter pylori infection and cardiac syndrome X: a preliminary
study. Int. J. Cardiol., 134:e124–5.
[57] Ozdogru I, Kalay N, Dogan A, et al. (2007). The relationship between Helicobacter
pylori IgG titre and coronary atherosclerosis. Acta. Cardiol., 62:501–5.
[58] Park MJ, Choi SH, Kim D, Kang SJ, Chung SJ, Choi SY, et al. (2011). Association
between Helicobacter pylori Seropositivity and the Coronary Artery Calcium Score in a
Screening Population. Gut. Liver., 5:321–7.
[59] Franceschi F, Niccoli G, Ferrante G, Gasbarrini A, Baldi A,Candelli M, et al. (2009).
CagA antigen of Helicobacter pylori and coronary instability: insight from a clinico-
pathological study and a meta-analysis of 4241 cases. Atherosclerosis., 202:535–42.
[60] Niccoli G, Franceschi F, Cosentino N, Giupponi B, De Marco G, Merra G, et al. (2010).
Coronary atherosclerotic burden in patients with infection by CagA-positive strains of
Helicobacter pylori. Coron. Artery. Dis., 21:217–21.
[61] Elkind MSV, Ramakrishnan P, Moon YP, et al. (2010). Infectious burden and risk of
stroke: the northern Manhattan study. Arch. Neurol., 67(1):33–38.
[62] Rizzo M, Corrado E, Coppola G, et al. (2008). Prediction of cerebrovascular and
cardiovascular events in patients with subclinical carotid atherosclerosis: the role of C-
reactive protein. J. Invest. Med., 56:32–40.
[63] Nyberg A, Skagius E, Nilsson I, et al. (2008). Abdominal aortic aneurysm and infection
with CagA positive strains of Helicobacter pylori. Scand. J. Infect. Dis., 40:204–7.
[64] Hamed SA, Amine NF, Galal GM, et al. (2008). Vascular risks and complications in
diabetes mellitus: the role of Helicobacter pylori infection. J. Stroke. Cerebrovasc. Dis.,
17:86–94.
[65] Moyaert H, Franceschi F, Roccarina D, Ducatelle R, Haesebrouck F, Gasbarrini A.
Extragastric manifestations of Helicobacter pylori infection: other Helicobacters.
Helicobacter. 2008 Oct;13 Suppl 1:47-57
[66] Longo-Mbenza B, Nkondi Nsenga J, Vangu Ngoma D, (2007). Prevention of the
metabolic syndrome insulin resistance and theatherosclerotic diseases in Africans
infected by Helicobacter pylori infection and treated by antibiotics. Int. J. Cardiol.,
121:229 38.

[67] Andrew P, Montenero AS, (2007). Is there a link between atrial fibrillation and certain
bacterial infections? J. Cardiovasc. Med. (Hagerstown). 2007; 8(12):990–996.
[68] Bunch TJ, Day JD, Anderson JL, et al. (2008). Frequency of Helicobacter pylori
seropositivity and C-reactive protein increase in atrial fibrillation in patients undergoing
coronary angiography. Am. J. Cardiol., 101:848–51.
[69] Badran HM, Mahfouz ME, (2007). Cytotoxin-associated gene-A bearing strains of
Helicobacter pylori and atrial fibrillation due to ischemic origin: is there a link? Eur. J.
Cardiovasc. Prev. Rehabil., 14:518–20.
[70] Ozdogru I, Kalay N, Dogan A, et al. (2007). The relationship between Helicobacter
pylori IgG titre and coronary atherosclerosis. Acta. Cardiol., 62:501–5.
[71] Katz JT, Shannon RP, (2006). Bacteria and coronary atheroma: more fingerprints but
no smoking gun. Circulation., 113(7):920–922.
[72] Hagiwara N, Toyoda K, Inoue T, et al. (2007). Lack of association between infectious
burden and carotid atherosclerosis in Japanese patients. J. Stroke. Cerebrovasc. Dis.,
16:145–52.
[73] Altintas E, Ucbilek E, Ulu O, et al. (2007). Helicobacter pylori-associated atrophic
gastritis and carotid intima-media thickness: is there a link? Int. J. Clin. Pract.,
61:810–4.

Chapter 16
Helicobacter pylori and Respiratory

Tract Diseases
Qiang Wang1*, Chaoran Yu1, Gian Luigi de’Angelis2,3

and Vincenzo Di Comite4
1
Second Clinical Department, Tongji Hospital, Tongji Medical College of Huazhong
University of Science and Technology. Wuhan, HuBei, China
2
Gastroenterology and Endoscopy Unit, Azienda Ospedaliero-Universitaria di Parma,
3
Department of Pediatrics, “Pietro Barilla” Children’s Hospital, Azienda Ospedaliero-
Universitaria di Parma, University Hospital, Parma, Italy
4
Internal Medicine, Department of Emergency, Azienda Ospedaliero-Universitaria di
Parma, University Hospital, Parma, Italy
Abstract
The relationship between Helicobacter pylori (H. pylori) and gastrointestinal
disorders has been extensively studied, finding solid relationships with chronic gastritis,
peptic ulcer disease, low-grade lymphoma of gastric mucosa-associated lymphoid tissue,
and gastric adenocarcinoma. H. pylori is not exclusively located in the stomach and its
effects are not limited locally to digestive system. The chronic infection of H. pylori leads
to activation of immune response with production of mediators like cytokines, which may
sustain pathogenetic mechanisms in extra gastrointestinal districts.
Starting from a revision of the relevant literature, in this chapter we discuss the role
of H. pylori infection in respiratory diseases, mainly focused on the most common
pathologies such as bronchial asthma, chronic obstructive pulmonary disease (COPD),
lung cancer, tuberculosis and bronchiectasis.
*
Corresponding author: Qiang Wang MD, the Second Clinical Department, Tongji Hospital, Tongji Medical
College of Huazhong University of Science and Technology. Wuhan, HuBei, 430030, China. E-
mail:806161440@qq.com.

298 Qiang Wang, Chaoran Yu, Gian Luigi de’Angelis et al.
Introduction
Helicobacter pylori (H. pylori) is a spiral, micro-aerophilic, gram-negative bacterium that
specifically colonizes the stomach mucosa of approximately 50% of the world population.
The chronic H. pylori infection has been clearly linked to peptic ulcer disease, chronic
gastritis, atrophic gastritis, mucosa associated lymphoid tissue (MALT) lymphoma and
gastric cancer [1, 2].
The mucosa of the gastrointestinal and respiratory tracts constitutes a large surface
exposed to external agents. A vast array of antigens is presented to the immune system, also
depending on the presence of microbial communities. In addition to recognition pattern and
secretion of antibody defenses, MALT — with the involvement of dendritic, T, B cell
populations — reacts via the “common mucosal immune system”. We know that once H.
pylori colonizes the gastric mucosa, it induces a local inflammatory response as well as
systemic immune response [3]
Several complex mechanisms are involved in this process. For instance, as a Gram-
negative bacterium, H. pylori contains lipopolysaccharides that stimulate the production of
many cytokines, including interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α that
may sustain the persistent inflammatory response. In addition, vascular disorders can also be
induced by immune mediators activating mechanisms as vasospasm or platelet aggregation
[4]. Moreover, we have to consider that H. pylori is not exclusively located in the human
stomach [1] and that many respiratory diseases have in common with H. pylori infection a
chronic inflammation and increased immune response [2].
For these reasons, it is no longer possible to consider the H. pylori infection as an
exclusive local disease of the gastrointestinal tract, but its involvement must also be
considered in systemic diseases and pathologies of extraintestinal organs.
In the last years the role of H. pylori has been investigated in the pathogenesis of
extradigestive diseases, including skin, neurological, vascular and autoimmune disorders, as
well as some respiratory diseases.
There are several even controversial results and, of course, their interpretation is not
univocal; for instance, even if most of the works showed a relationship between bronchial
asthma and H. pylori with epidemiological studies and investigation in animal and human
models, on the other hand a large cross-sectional study in 2009 failed to demonstrate the
association between H. pylori infection and chronic pulmonary diseases, allergic disease or
decline in lung function [5]. We know that the gastric content may easily reach the airways
through gastroesophageal reflux, therefore if H. pylori is present in the gastric fluid, it is able
to colonize the respiratory mucosa through the oral-pharyngeal-laryngeal route [6].
However all these studies offered interesting perspectives, encouraging to continue the
investigation of the role of H. pylori in respiratory diseases.
Helicobacter pylori and Bronchial Asthma

Asthma is a chronic inflammatory disorder of the airways in which many cells and
cellular elements play a role; it is characterized by chronic inflammation associated with

Helicobacter pylori and Respiratory Tract Diseases 299
airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness,

chest tightness, and coughing [7].
In recent years, the prevalence of asthma is increasing in developed countries; this fact
can easily be related to exogenous factors (tobacco smoke, pollution, allergen exposure,
microbial infections) but also the contribution of indigenous microbiota can be considered
[2]. According to the “hygiene hypothesis” increased exposure in the early life to microbes
may be protective against asthma and allergic diseases [5].
Many studies have been trying to demonstrate a relationship between asthma and H.
pylori infection with discordant results. A recent meta-analysis by Wang et al. observed a
weak inverse association between H. pylori and asthma both in adults and in children [8], but
some previous studies showed that H. pylori infection protects children under 10 years from
asthma [9-11], mainly in the subjects with CagA-positive H. pylori strains. On the other hand,
a large cross-sectional study did not demonstrate association between H. pylori and chronic
pulmonary diseases, allergic disease or decline in lung function [5]. Furthermore, a meta-
analysis in 2012 failed to suggest an association between H. pylori and asthma risk [12] but,
on the contrary, in a very recent work asthmatic patients have a significant lower rate of H.
pylori infection compared to control subjects [13].
A plausible mechanism of relationship between asthma and H. pylori is that inflammation
in asthma is sustained by cytokines promoting a Th2 response (involved in allergic diseases,
including asthma) and inhibiting Th1 response (involved in protective immunity), meanwhile
H. pylori leads to a mucosal immune response sustained by a Th1 cytokine pattern.
Bronchial asthma has a multifactorial pathogenesis in which complex mechanisms have
been investigated and related to H. pylori, starting from the hypothesis of mutual influence of
Th1 and Th2 patterns. Here we report some studies on neutrophil-activation protein of H.
pylori (HP-NAP), regulatory T cells and dendritic cells.
HP-NAP
The inflammation in bronchial asthma and allergic diseases is due to prevalent activation
of T helper type 2 lymphocytes (Th2), promoted by cytokines like IL-4 or IL-5.
On the contrary, H. pylori infection promotes a Th1 type response mediated by cytokines
like IFN-gamma and IL-12.
The neutrophil-activation protein of H. pylori (HP-NAP) is a bacterial protein able to
drive the Th1 response and inhibit Th2 response in vivo and in vitro [14]. HP-NAP acts via
agonistic interaction with toll-like receptor, eliciting IL-12 and IL-23 production. In a mouse
model of allergic asthma, HP-NAP systemic administration reduced lung eosinophilia,
concentration of IL-4 and IL-5 in bronchoalveolar lavage and total serum IgE levels. Mucosal
administration was equally effective. This findings are consistent with a potential role of HP-
NAP in prevention and treatment of allergic diseases [15].
Regulatory T Cells
Regulatory T cells (T-regs) are vital for keeping the immune system in check, helping to
avoid immune-mediated pathology and unrestricted expansion of effector T cell populations.

Accordingly, regulatory T cells have been the focus of extensive research over the past few
years, and this has revealed many roles for these cells in numerous diseases, including
autoimmunity, allergy, microbial infection and cancer.
People with peptic ulcer disease have a reduced T-reg response compared to those
without ulcers. H. pylori induces a regulatory T cell response with production of high level of
IL-10. This influence on immune response could contribute to coexistence with human host
[16].
Experimental models of allergic airways diseases revealed a direct link between H. pylori
infection and suppression of disease through the induction of T-regs [17].
In a mouse model of allergic asthma, H. pylori infection protects from airway hyper-
responsiveness, tissue inflammation and goblet cells metaplasia, and prevent eosinophilia and
Th2 cell infiltration of lungs. Protection was most effective in mice infected neonatally and
abrogated after eradication of H. pylori. These findings were associated with impaired
maturation of dendritic cells and accumulation of highly suppressive T-regs in the lungs [18].
Dendritic Cells
Dendritic cells are potent accessory cells that play an important role in initiating
bronchial immune responses by activation of T-lymphocytes via presentation of antigens and
production of cytokines [19]. H. pylori infection reprogram dendritc cells toward a tolerance-
promoting phenotype, via IL-18 production. These dendritic cells fail in T cell activation and
this condition confers protection against allergic asthma and contribute to evade the adaptive
immune response [20].
Helicobacter pylori and COPD

Chronic obstructive pulmonary disease (COPD) is a lung disease characterized by
chronic obstruction of lung airflow that interferes with normal breathing, not fully reversible
and, usually, progressive [21]. The association with peptic ulcer disease has been stated many
years ago and, successively, higher H. pylori sero-prevalence in COPD patients compared to
controls has been demonstrated [22].
H. pylori infection could have a pro-inflammatory role and co-trigger COPD, but a causal
role has not been established, yet; some difficulties in this field come from the fact that both
conditions have multifactorial ethiology and are related to older age, male sex, and low socio-
economical status. Also cigarette smoking, well-known cause of COPD and peptic ulcer, can
be a confounding factor, because studies on its relationship with H. pylori are controversial,
and smoking itself could be a bias in previous works [2].
One hypothesis on the link between COPD and H. pylori infection is the chronic release
of inflammatory mediators by H. pylori that could contribute to the pathogenesis of chronic
bronchitis. An increase of prevalence of CagA-positive H. pylori strains in COPD patients has
been demonstrated; these virulent strains cause release of proinflammatory cytokines
including IL-1, IL-8, TNF-α. After eradication of H. pylori infection, cytokine levels in serum
normalize [4].

Hence, COPD and H. pylori infection are characterized by chronic inflammation in which
common mediators, especially IL-8, are involved [2].
Recent findings are encouraging. Even if previous studies failed to demonstrate an
association between H. pylori infection and severity of COPD, a recent work found higher H.
pylori IgG seropositivity in COPD compared to controls and these H. pylori-specific
antibodies significantly correlated to severity of respiratory disease stated with functional test
(FEV1) [23].
Moreover, a paper from Siva et al. showed a strong and independent relationship between
the presence of peptic ulcer disease and functional tests measuring severity of COPD,
suggesting that there is more than just a shared susceptibility to different environmental
stimuli [24].
Helicobacter pylori and Bronchiectasis

Bronchiectasis are abnormal and permanent dilations of bronchi due to chronic
inflammation and destruction of bronchial walls. In airways secretion of patients with active
bronchiectasis there are pro-inflammatory cytokine and the neutrophil is the prevalent cell.
Bronchial damage is result of neutrophilic inflammatory response to bacterial infection.
Like COPD, bronchiectasis share some pathogenetic features with H. pylori infection.
Both in gastroduodenal ulcers and bronchiectasis there is an exposure to luminal bacteria that
leads to recruitment of neutrophils, T lymphocytes, and to cytokine release (IL-8, TNF-α, IL-
1β). Patients with bronchiectasis have higher seropositivity to H. pylori CagA strains than
controls.
However a pathogenetic role for H. pylori remains unclear; the inhalation of bacteria
from the stomach into respiratory tract leading to chronic bronchial inflammation has been
hypothesized. Nevertheless, neither the identification of H. pylori in human bronchial tissue
nor the isolation from bronchoalveolar lavage fluid has yet been demonstrated [25]. These
findings were confirmed in a following work and, in addition, anti-H. pylori IgG levels in
patients with bronchiectasis did not show statistically significant difference compared to
control subjects [26].
Helicobacter pylori and Lung Cancer

Lung cancer is the leading cause of cancer mortality worldwide. Several factors are
involved in pathogenesis such as tobacco smoke, air pollution, occupational agents. The lungs
develop from the same endodermal cells that form the lining of the gastrointestinal tract and
produce several hormonal peptides [27]. H. pylori is a well established risk factor for stomach
cancer, therefore it is hypothesized that H. pylori can induce lung carcinogenesis partially
through similar mechanisms as in gastric cancer [28]. Several studies have been investigated
the role of H. pylori infection in lung cancer pathogenesis with discordant results.
Seroprevalence of CagA-positive strains of H. pylori were higher in lung cancer patients
compared to controls [29] but this data have not been confirmed in a more recent work [30].

Gastrin is a peptide hormone that stimulates secretion of gastric acid by the parietal cells
of the stomach and acts on gastric motility. High gastrin levels have been found in serum and
broncoalveolar lavage of lung cancer patients and enhanced mRNA expression of gastrin and
its receptor has been demonstrated in tumoral tissue. The synthesis of gastrin induced by H.
pylori could contribute to carcinogenesis, causing increased mucosal cell proliferation of
bronchial epithelium and induction of cyclooxygenase-2 [2] .
In conclusion epidemiological studies show contrasting results [30, 31], suggesting
increased lung cancer risk in the population affected to H. pylori infection, but there are no
findings supporting a causal relationship. A very recent review about emerging hypothesis on
H. pylori-lung cancer relationship is focused on p130cas-related mechanisms and on
gastroesophageal reflux and inhalation of urease and gastrin as carcinogenic factors [28].
Helicobacter pylori and Tuberculosis

The mycobacterium tuberculosis (TB) infection is present in one third of world's
population; most of them are in developing countries where TB is a common health problem
and the infection, facilitated by socioeconomic and sanitary condition, occurs early in life.
This is a feature in common with H. pylori infection and the conditions of life can explain the
frequent coexistence of both pathogens in studies conducted in these countries.
In the first studies, H. pylori infection was associated to active pulmonary TB and history
of peptic ulcer was related to positive tuberculin skin test. Successively, most of the works is
focused on H. pylori seroprevalence in TB infected patients and results have been mixed,
confirming or denying this association. Moreover, the antibiotic therapy of mycobacterium
tuberculosis can also be effective in eradicating H. pylori infection [32], and this fact must be
considered in reviewing epidemiological studies [22].
It has also been observed that genetic factor, HLA-DQ serotype, may contribute to
enhanced survival and replication of TB and is also associated to increased susceptibility to
H. pylori infection [2].
In conclusion, the association between tuberculosis and H. pylori infection is based on
serological case-control studies and even if coexistence has been observed, pathogenetic
mechanisms remain unclear.
Other Respiratory Diseases

Sarcoidosis
Sarcoidosis is a systemic disease primarily affecting lungs in which the exposure to

environmental or infective agents trigger an abnormal immune response in genetically
susceptible subjects. Gastrointestinal sarcoidosis is rare, but H. pylori infection has been
associated to gastric involvement and higher levels of IgG anti-H. pylori have been found in
sarcoidosis patients [33].

Upper Respiratory Tract
There are several studies confirming the colonization by H. pylori of the adjacent sites of
intestinal cavity, with or without stomach bacteria. As this organism is independent of the
stomach, capable of living in the oral cavity, including beyond the digestive tract, it may be
assumed that H. pylori can colonize in areas within the larynx and pharynx [34]. In fact,
recent investigations showed the presence of H. pylori DNA in nasal polyps, concha bullosa
and benign larynx diseases. CagA-positive H. pylori strains were observed in laryngeal
tissues alone [35]. Moreover H. pylori has been isolated in the tonsillar tissues of chronic
tonsillitis patients [36] and Abdel-Monem et al stated that adenotonsillar tissue can be an
extra-gastric reservoir for H. pylori in children with chronic adenotonsillitis [37].
Laryngopharyngeal Reflux
Even if H. pylori has been isolated in saliva and more generally in the ear-nose-throat
district [1], and gastroesophageal reflux, causing the passage of the bacteria from the stomach
to the airways, has been considered as potential mechanism for respiratory pathologies, there
is no significant association between laryngopharyngeal reflux and H. pylori infection, even
analyzing subgroups on the basis of presence of benign or malignant-premalignant lesions
[38].
Cystic Fibrosis
Cystic fibrosis (CF) is a multisystem disease characterized by pancreatic insufficiency,

malabsorption, liver disease, chronic sinusitis, diabetes, osteoporosis, male infertility in which
chronic pulmonary infections and inflammation cause most of the morbidity and mortality.
Although there are few studies available, the prevalence of H. pylori in CF patients seems
lower than control subjects. Potential influencing factors are the effect of the frequent
antibiotic therapy in CF patient respect to controls and the cross-reactivity between H. pylori
antigens and anti-Pseudomonas antibodies [2].
Instead, a recent paper concludes that prevalence of H. pylori infection in CF patients
does not differ from healthy subjects [39].
Conclusion
Over recent years, great efforts and consequent progresses in prevention and treatment of
respiratory pathologies have been made, mainly regarding asthma, COPD and lung cancer.
Anyway, the impact of respiratory diseases on public health, measured not only with
morbidity and mortality, but also in terms of socio-economical costs, remains huge. H. pylori
lives in the gastrointestinal tract due to its selective advantage of protection against highly
acidic environments. Even if several studies have showed the existence of H. pylori in the
mucosa of the upper respiratory tract and its potential role in the development of upper
respiratory diseases, it is still unclear whether H. pylori has the microbiological advantage in
the upper or lower respiratory tract. The research in these fields are emergent and numerous,
but at now, the most of studies show contrasting results, messages are not univocal, and there
are no findings supporting causal relationship between H. pylori infection and pathologies of
the respiratory tract. For these reasons we need of more extensive, rigorous, case-controlled
trials to better understand the role of H. pylori infection in respiratory diseases.
References
[1] Kurtaran H, Uyar ME, Kasapoglu B, Turkay C, Yilmaz T, Akcay A, Kanbay M,
(2008). Role of Helicobacter pylori in pathogenesis of upper respiratory system
diseases. J. Natl. Med. Assoc., 100(10):1224-30.
[2] Malfertheiner MV, Kandulski A, Schreiber J, Malfertheiner P, (2011). Helicobacter
pylori Infection and the Respiratory System: A Systematic Review of the Literature.
Digestion., 84(3):212-20.
[3] Underwood MA, Bevins CL, (2010). Defensin-Barbed Innate Immunity: Clinical
Associations in the Pediatric Population. Pediatrics., 125(6): 1237-1247.
[4] Kanbay M, Kanbayb A, Boyacioglu S, (2007). Helicobacter pylori infection as a
possible risk factor for respiratory system disease: A review of the literature.
Respiratory. Medicine., 101: 203–209.
[5] Fullerton D, Britton JR, Lewis SA, Pavord ID, McKeever TM, Fogarty AW, (2009).
Helicobacter pylori and lung function, asthma,atopy and allergic disease-A population-
based cross-sectional study in adults. Int. J. Epidemiol., 38: 419- 426.
[6] Suzuki H, Franceschi F, Nishizawa T, Gasbarrini A, (2011). Extragastric manifestations
of Helicobacter pylori infection. Helicobacter., 16 (suppl. 1): 65-69
[7] from the Global Strategy for Asthma Management and Prevention, 2012 update.
Definition and overview, pag. 3. GINA reports are available on www.ginasthma.org.
[8] Wang Q, Yu C, Sun Y (2013). The association between asthma and Helicobacter
pylori: a meta-analysis. Helicobacter., 18:41-53.
[9] Chen Y, Blaser MJ, (2008). Helicobacter pylori colonization is inversely associated
with childhood asthma. J. Infect. Dis., 4: 553-60.
[10] Chen Y, Blaser MJ, (2007). Inverse associations of Helicobacter pylori with asthma
and allergy. Arch. Intern. Med. , 167: 821-7.
[11] Jaber SM, (2006). Helicobacter pylori seropositivity in children with chronic disease in
Jeddah, Saudi Arabia. Saudi. J. Gastroenterol., 12: 21-6.
[12] Wang Y, Bi Y, Zhang L, Wang C, (2012). Is Helicobacter pylori infection associated
with asthma risk? A meta-analysis based on 770 cases and 785 controls. Int. J. Med.
Sci., 9(7):603-10.
[13] Zhou X, Wu J, Zhang G, (2013). Association between Helicobacter pylori and asthma:
a meta-analysis. Eur. J. Gastroenterol. Hepatol., 25(4):460-8..
[14] D'Elios MM, Codolo G, Amedei A, Mazzi P, Berton G, Zanotti G, Del Prete G, de
Bernard M, (2009). Helicobacter pylori, asthma and allergy. FEMS. Immunol. Med.
Microbiol., 56(1):1-8.

[15] Codolo G, Mazzi P, Amedei A, Prete GD, Berton G, D'Elios MM, Bernard MD, (2008).
The neutrophil-activating protein of Helicobacter pylori down- modulates Th2
inflammation in ovalbumin-induced allergic asthma. Cell. Microbiol., 10, 2355-63.
[16] Robinson K, Kenefeck R, Pidgeon EL, Shakib S, Patel S, Polson RJ, Zaitoun AM,
Atherton JC, (2008). Helicobacter pylori induced peptic ulcer disease is associated with
inadequate regulatory T cell responses. Gut., 57, 1375–1385.
[17] Taube C, Müller A, (2012). The role of Helicobacter pylori infection in the
development of allergic asthma. Expert. Rev. Respir. Med., 6(4):441-9.
[18] Arnold IC, Dehzad N, Reuter S, Martin H, Becher B, Taube C, Müller A, (2011).
Helicobacter pylori infection prevents allergic asthma in mouse models through the
induction of regulatory T cells. J. Clin. Invest., 8, 3088-3093.
[19] Bocchino V, Bertorelli G, D'Ippolito R, Castagnaro A, Zhuo X, Grima P, Di Comite V,
Damia R, Olivieri D, (2000). The increased number of very late activation antigen-4-
positive cells correlates with eosinophils and severity of disease in the induced sputum
of asthmatic patients. J. Allergy. Clin. Immunol.,105(1 Pt 1):65-70
[20] Oertli M, Müller A, (2012). Helicobacter pylori targets dendritic cells to induce
immune tolerance, promote persistence and confer protection against allergic asthma.
Gut. Microbes., 3(6):566-71.
[21] From the Global Strategy for the Diagnosis, Management and Prevention of COPD,
Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2013. Available from:
http://www.goldcopd.org/.
[22] Roussos, A., Philippou, N., Mantzaris, G.J., Gourgoulianis, K.I., (2006). Respiratory
Diseases and Helicobacter pylori Infection: Is There a Link?. Respiration., 73, 708–
714.
[23] Gencer M, Ceylan E, Yildiz Zeyrek F, Aksoy N, (2007). Helicobacter pylori
seroprevalence in patients with chronic obstructive pulmonary disease and its relation to
pulmonary function tests. Respiration., 74(2):170-5.
[24] Siva R, Birring SS, Berry M, Rowbottom A, Pavord ID, (2013). Peptic ulceration,
Helicobacter pylori seropositivity and chronic obstructive pulmonary disease.
Respirology., 18(4):728-31.
[25] Tsang KW, Lam WK, Kwok E, Chan KN, Hu WH, Ooi GC, Zheng L, Wong BC, Lam
SK, (1999). Helicobacter pylori and upper gastrointestinal symptoms in bronchiectasis.
Eur. Respir. J., 14(6):1345-50.
[26] Gülhan M, Ozyilmaz E, Tarhan G, Demirağ F, Capan N, Ertürk A, Canbakan S,
Ayaşlioğlu E, Gülhan E, Ahmed K, (2007). Helicobacter pylori in bronchiectasis: a
polymerase chain reaction assay in bronchoalveolar lavage fluid and bronchiectatic
lung tissue. Arch. Med. Res., 38(3):317-21.
[27] Koshiol J, Flores R, Lam TK, Taylor PR, Weinstein SJ, Virtamo J, Albanes D, Perez-
Perez G, Caporaso NE, Blaser MJ, (2012). Helicobacter pylori seropositivity and risk
of lung cancer. PloS. One., 7(2):e32106.
[28] Deng B, Li Y, Zhang Y, Bai L, Yang P, (2013). Helicobacter pylori infection and lung
cancer: a review of an emerging hypothesis. Carcinogenesis., May 16.
[29] Gocyk W, Nikliński T, Olechnowicz H, Duda A, Bielański W, Konturek PC, Konturek
SJ, (2000). Helicobacter pylori, gastrin and cyclooxygenase-2 in lung cancer. Med. Sci.
Monit., 6(6):1085-92.

[30] Najafizadeh K, Falah Tafti S, Shiehmorteza M, Saloor M, Jamali M, (2007). H. pylori

seroprevalence in patients with lung cancer. World. J. Gastroenterol., 28;13(16):2349-
51.
[31] Zhuo WL, Zhu B, Xiang ZL, Zhuo XL, Cai L, Chen ZT, (2009). Assessment of the
relationship between Helicobacter pylori and lung cancer: a meta-analysis. Arch. Med.
Res., 40(5):406-10.
[32] Sanaka M, Kuyama Y, Yamanaka M, Iwasaki M, (1999). Decrease in serum
concentrations of Helicobacter pylori IgG antibodies during antituberculosis therapy:
the possible eradication by rifampicin and streptomycin. Am. J. Gastroenterol.,
94(7):1983-4.
[33] Herndon BL, Vlach V, Dew M, Willsie SK, (2004). Helicobacter pylori-related
immunoglobulins in sarcoidosis. J. Investig. Med., 52(2):137-43.
[34] Najafipour R, Naserpour Farivar T, Pahlevan AA, Johari P, Safdarian F, Asefzadeh M,
(2012). Agreement rate of rapid urease test, conventional PCR, and Scorpion real-time
PCR in detecting Helicobacter pylori from tonsillar samples of patients with chronic
tonsillitis. J. Glob. Infect. Dis., 4 (2): 106-109.
[35] Burduk PK, Kaczmarek A, Budzynska A, Kazmierczak W, Gospodarek E, (2011).
Detection of Helicobacter pylori and cagA gene in nasal polyps and benign laryngeal
diseases. Arch. Med. Res., 42(8):686-9.
[36] Wibawa, T., Surono, A., Widodo, I., (2011). Isolation of viable Helicobacter pylori in
the tonsillar tissues of chronic tonsillitis patients. J. Infect. Dev. Ctries., 5, 561-564.
[37] Abdel-Monem, M.H., Magdy, E.A., Nour, Y.A., Harfoush, R.A., Ibreak, A., (2011).
Detection of Helicobacter pylori in adenotonsillar tissue of children with chronic
adenotonsillitis using rapid urease test, PCR and blood serology: A prospective study.
Int. J. Pediatr. Otorhinolaryngol., 75, 568–572.
[38] Cekin, E., Ozyurt, M., Erkul, E., Ergunay, K., Cincik, H., Kapucu, B., Gungor, A.,
(2012). The association between Helicobacter pylori and laryngopharyngeal reflux in
laryngeal pathologies. ENT-Ear, Nose & Throat J., 91, E6-E9.
[39] Drzymała-Czyż S, Kwiecień J, Pogorzelski A, Rachel M, Banasiewicz T, Pławski A,
Szczawińska-Popłonyk A, Herzig KH, Walkowiak J, (2013). Prevalence of
Helicobacter pylori infection in patients with cystic fibrosis. J. Cyst. Fibros., 31. pii:
S1569-1993(13).

Chapter 17

Gynecological and Reproductive
System Diseases
Ghada M. Mansour1,* and Marco Manfredi2,3

1
Ain Shams University, Cairo, Egypt
2
“Pietro Barilla” Children’s Hospital, Department of Pediatrics
Azienda Ospedaliero-Universitaria di Parma
University of Parma, Parma, Italy
3
Gastroenterology and Endoscopy Unit
Azienda Ospedaliero-Universitaria di Parma
University of Parma, Parma, Italy
Abstract
Helicobacter pylori (H. pylori) infection can be related to some gynecological
pathologies, especially unexplained infertility. Also during pregnancy H. pylori infection
can play a role in triggering several disorders. It is related to iron deficiency anemia, as in
the normal population, but it does not seem to be associated with gestational
thrombocytopenia. Moreover H. pylori infection may be the cause of severe hyperemesis
gravidarum and pre-eclampsia which can adversely affect the health of pregnant women
and cause fetal growth retardation. In fact several studies showed that the H. pylori
eradication improve these conditions with restoration of normal health of the pregnancy.
Although H. pylori infection has been proved in the first trimester of pregnancy,
there is no strong evidence until now, about teratogenicity neither maternal-fetal
transinfection.
Instead, the H. pylori transmission from mother to infant through breastfeeding has
been demonstrated.
*
Corresponding author. Ghada M. Mansour; Associate Consultant of Obstetrics and Gynecology, Ain Shams
University, Cairo, Egypt. Email: gmansour@hotmail.com; info@ghadamansour.com.

308 Ghada M. Mansour and Marco Manfredi
Keywords: Helicobacter pylori, female infertility, cervical mucosa, iron deficiency anemia,
pregnancy, thrombocytopenia, pre-eclampsia, and hyperemesis gravidarum
Introduction
Helicobacter pylori (H. pylori) is a very common pathogen found in nearly 55% of
population, 90% of peptic ulcer patients, and in 60–80% in gastritis with no ulcers [1]. The
prevalence rate is higher in developing countries than developed countries [2]. The possible
transmission route may be oral–oral, faeco–oral, iatrogenic transmission and vectorial spread
[3].
H. pylori infection can be related to gynaecological diseases or other associated medical
disorders during pregnancy. Although Lancier et al have suggested that pregnancy may by
itself increase the susceptibility to H. pylori infection due to changes in the humoral and cell
mediated immunity, the prevalence of H. pylori infection in pregnant women varies according
to the geographic areas around the world [4, 5].
The gold standard for diagnosis of H. pylori infection is by an invasive test, histological
examination, and a urea breath test by use of non-invasive methodology [6, 7]. In pregnancy,
however, non-invasive diagnostic methods, IgG antibodies seropositivity, and stool antigen
test are considered to be eligible alternatives [8].
This chapter tried to highlight the actual knowledge about the possible role of H. pylori
infection in some gynaecological pathologies and disorders which can occur during
pregnancy.
H. pylori Infection and Gynaecological

Pathologies
H. pylori and Cervical Mucosa
H. pylori infection was believed to play a role in several gynecological pathologies since
the cervical mucosa resembles the gastric environment. This microorganism was expected to
infect the upper genital tract via the oral-genital and fecal-genital routes. Kaya et al. studied
35 women with benign, pap-smear results. The presence of H. pylori in the uterine cervix and
active H. pylori infection were investigated with the H. pylori stool antigen test. Biopsy
specimens were performed to find H. pylori in cervical tissue. Seroprevalence was
investigated by using ELISA for H. pylori IgG and IgA. The H. pylori seroprevalence was
65.7%; further, 17.1% of the cases had an active infection. H. pylori was not found nor in the
cervix neither in the cervicovaginal secretions. These authors concluded that the cervix is not
a reservoir for H. pylori, and it does not appear to be transmitted through the fecal-genital
route [9].

Helicobacter pylori Infection and Gynecological … 309
H. pylori and Female Infertility
The role of infections as cause of infertility, a frequent problem among European

couples, is well recognised [10]. One cause of infertility is the presence in cervical mucus of
secretory IgA antibodies, which can react with some sperm structures, impairing their
motility. Figura et al. hypothesised that antibodies produced against H. pylori block the sperm
tail. They found that seropositivity for H. pylori infection was higher in women of couples
with infertility than in controls (44.8% vs 29.7%, p<0.001). Moreover they demonstrated that
anti-H. pylori IgG antibodies reacted with the tail, centrioles and equatorial zone of humans
sperm without affecting sperm motility. They considered that anti-H. pylori antibodies in
ovarian follicular fluid of women with infertility should have a negative effect on oocyte
quality [11]. In another study Kurotsuchi et al. analyzed 204 patients with idiopathic
infertility and they found that 22.1% of these patients were seropositive for anti-H. pylori IgG
antibodies in serum and follicular fluids. They concluded that H. pylori infection may
increase the risk of infertility [12].
The composition of cervical mucus during the ovulatory period is a key factor in the
sperm vitality but the evaluation of anti-H. pylori antibodies in vaginal samples may be
difficult because of the variability and heterogeneous composition of vaginal secretions. For
these reasons and in view of the importance of cervical factor and the role of the interaction
between mucus and sperm in infertility, Ambrosini et al. measured anti-H. pylori antibodies
in cervical mucus and they concluded that their presence in the cervical mucus may therefore
be considered a new local factor in female infertility, interfering with sperm progression.
Considering the close correlation found between anti-H. pylori antibody in serum and cervical
mucus, measuring serum antibodies could become an additional test, in particular in couples
with unexplained infertility [13].
H. pylori infection and Pregnancy

H. pylori Infection, Iron Deficiency Anemia and Thrombocytopenia in
Pregnancy (see also chapter 14)
The WHO estimates that 35% to 75% of pregnant women in developing countries and
approximately 20% of industrialized countries, are anemic during pregnancy [14]. Iron
deficiency anemia is the most common cause of anemia during pregnancy. Moderate-to-
severe maternal anemia has been associated with an increased risk of poor reproductive
outcomes, including low birth weight and preterm birth deliveries [15]. The relationship
between H. pylori infection and iron deficiency anemia has been shown [16]. Weyermann and
others found that pregnant women infected with H. pylori had lower mean hemoglobin level
at the beginning of pregnancy and a greater decrease in the mean hemoglobin level at the end
of pregnancy with higher chance of fetal growth retardation [17]. Various mechanisms have
been hypothesized for the development of iron deficiency anemia in H. pylori infection; some
of which are low gastric pH, low vitamin C levels in stomach, sequestration of serum iron and
ferritin by gastric H. pylori [18]. In this study these authors showed that the response to oral
folic acid supplementation was significantly better after H. pylori eradication therapy.

Mulayim and coworkers tried to determine whether there is a relationship between H.

pylori infection and iron deficiency anemia and thrombocytopenia in pregnant women
measuring hemoglobin and ferritin levels and platelet counts during the third trimester of
pregnancy. H. pylori infection was determined using a 14C-urea breath test (14C-UBT) after
delivery. In this study, 72 of 117 women had positive results on the 14C-UBT. Overall, 27 of
117 pregnant women had anemia (23.1%), and all them were in the H. pylori-positive group;
18 of 27 (66.7%) had iron deficiency anemia. Serum hemoglobin and ferritin levels and
neonatal body weight were found to be lower in the anemic group compared with the non-
anemic group among H. pylori-infected women. These authors found no statistically
significant differences with regard to gestational thrombocytopenia between the H. pylori-
positive and H. pylori-negative groups. [19] Also a study by Fukuia et al. failed to find an
association between thrombocytopenia and H. pylori infection during pregnancy [ 20].
However, several studies reported an association between H. pylori infection and
idiopathic thrombocytopenic purpura in a non-pregnant population most of whom
demonstrated a good therapeutic response and an increase of the platelet count after H. pylori
eradication. Although low platelet count in pregnancy may be idiopathic, more often it
clinically requires differential diagnosis between several diseases, such as severe pre-
eclampsia, HELLP syndrome, placental abruption and lupus erythematosus. Gestational
thrombocytopenia is a physiological reduction in platelet concentration in normal pregnancy,
usually in the third trimester but it is a diagnosis of exclusion [20]. The incidence of
gestational thrombocytopenia defined as platelet count less than 150,000/µl ranges between
4% and 7%. The decrease in platelet counts appears to occur from hemodilution as well as
platelet trapping. The exact mechanism of gestational thrombocytopenia, however, is still
unknown [21].
In a study, Epstein and colleagues concluded that there is no association between H.
pylori and thrombocytopenia in the pregnant hispanic population [22]. It was a secondary
analysis of 82 pregnant hispanic women with and without hyperemesis gravidarum who
underwent serologic evaluation for H. pylori IgG. Results of complete blood counts obtained
in the third trimester were analysed for thrombocytopenia. Out of the 82 subjects who had H.
pylori testing, 54 women had both serum H. pylori IgG results and third trimester platelet
levels. The prevalence of thrombocytopenia was 11.1%. Thirty-six subjects were seropositive
for H. pylori IgG and 18 subjects were seronegative. Out of the 36 subjects who were H.
pylori seropositive, four (11.1%) developed thrombocytopenia compared to three of 18
(16.7%) H. pylori-seronegative subjects (p = 0.67). There was no difference between the
groups in their mean platelet [22].
H. pylori and Hyperemesis Gravidarum
Hyperemesis gravidarum complicates from 0.3 to 2% of all pregnancies [23, 24]. It is

defined as vomiting during pregnancy that is severe enough to cause weight loss, dehydration,
acidosis from starvation, alkalosis from loss of hydrochloric acid, and hypokalemia. Mild to
moderate ketonuria may be detected by urine analysis. The cause of hyperemesis gravidarum
is unknown, but a variety of mechanisms related to endocrine and immunologic factors,
gastrointestinal conditions such as H. pylori infection and metabolic enzymes have been
proposed as possible underlying causes [25].

Sometimes symptoms of hyperemesis gravidarum become alarming; patients who

manifest continuous weight loss and electrolyte disturbances may be at risk for growth
restriction, fetal anomalies and decreased neonatal birth weight [26, 27].
Dehydration, electrolyte imbalance, and acid–base disturbances may lead to maternal
renal and hepatic injury [28, 29]. Many theories postulated the aetiology of hyperemesis
gravidarum. Eliakim et al. [28], stated that hyperemesis gravidarum involves pregnancy
induced by hormonal changes with concurrent gastrointestinal dysmotility and possible H.
pylori infection.
Many other studies correlated H. pylori infection to hyperemesis gravidarum [30-36].
The increased serum steroid and human chorionic gonadotropin levels that cause changes
in the pH of the gastrointestinal tract in addition to pregnancy-induced gastrointestinal tract
dysmotility and altered humoral and cell-mediated immunity in pregnancy, can favor the
activation of H. pylori infection.
Mansour and Nashaat, evaluated the role of H. pylori infection in the pathogenesis of
hyperemesis gravidarum and the effectiveness of a non-teratogenic regimen for its treatment
in intractable cases of eighty hyperemesis gravidarum women recruited from Ain Shams
University, in Egypt. A complete history was taken including history of medical disorders and
chronic medications intake as non steroidal anti-inflammatory drugs. After general and local
examination, ultrasound was done for all cases to exclude obstetric causes of hyperemesis.
Eighty normal pregnant women acted as control. Serum test for H. pylori IgG antibodies was
done for all patients and controls [31]. Seventy-one out of the 80 hyperemesis gravidarum
patients and 24 out of the 80 controls were H. pylori positive. Eight hyperemesis gravidarum
patients developed severe intractable vomiting. Three of them developed attacks of
hematemesis. Gastroscopy revealed antral gastritis and duodenitis. Gastric and duodenal
erosions were found in two cases. The eight H. pylori-positive patients with intractable
hyperemesis gravidarum received a non-teratogenic eradication therapy. Attacks of vomiting
decreased and pregnancy continued till delivery of healthy newborns. The study concluded
that screening for H. pylori should be performed in hyperemesis gravidarum patients and non-
teratogenic treatment can be considered in intractable cases. All the eight patients who
received the treatment in that study had passed 12 weeks of pregnancy. The therapy consisted
of ranitidine (class b of teratogenicity) 150 mg twice daily, plus metronidazole (class b) 500
mg twice daily and ampicillin (class b) 1000 mg twice daily for 2 weeks. Ampicillin and
ranitidine were given by parenteral route and metronidazole by rectal route, till patients could
receive oral therapy. Six patients showed marked improvement with reduced number of
vomiting attacks which became from 0 to 2 attacks per day and reduced epigastric pain. The
other two patients showed less improvement of the vomiting and pain; for these reasons doses
of antacids were increased and they also continued their pregnancy safely until delivery [31].
In this study, incidence of H. pylori was significantly higher in hyperemesis gravidarum
patients than normal group. Many studies found an increased incidence of H. pylori infection
in patients with hyperemesis gravidarum [27, 28, 37, 38]. It is well known that nausea and
vomiting are associated changes with hormonal levels following gestation, but the
pathogenesis of hyperemesis gravidarum is still unclear. With a rise in serum HCG level,
symptoms of nausea and vomiting appear, and when serum HCG levels decrease, nausea and
vomiting disappear. High incidence of hyperemesis is found in cases of molar pregnancies,
and in twin pregnancies exhibiting high serum HCG concentrations, and nausea and vomiting
immediately disappear when gestation is interrupted. Thus, most researchers believed that

elevated serum HCG levels are associated with hyperemesis gravidarum. However, it is not
easy to explain the fact that serum HCG levels are not directly proportional to the severity of
the symptoms. Another proposition is that hyperemesis gravidarun is caused by rapidly
increased estrogen levels. As a result of an increased accumulation of fluid caused by
elevated steroid hormones in pregnant women, a shift in pH may occur. A simple change of
pH in the gastrointestinal tract could hypothetically result in the appearance of manifestation
of a subclinical H. pylori infection, which can exacerbate gastrointestinal symptoms. Some
researchers mentioned that H. pylori infects the gastric mucosa of more than half of all
humans worldwide, but only 15% of those affected have clinical symptoms. It has been
reported that the pathogenicity of H. pylori is related not only to its virulence but also to host
gene susceptibility and environmental factors [39]. Bagis et al. obtained histopathologic
diagnosis of H. pylori infection by endoscopic evaluation in 20 pregnant women with severe
hyperemesis gravidarum and 10 pregnant women without gastric complaints. H. pylori was
diagnosed in 90% of hyperemesis gravidarum patients and 50% of control subjects. They
stated that the severity of H. pylori infection was higher in hyperemesis gravidarum patients.
It was suggested that the degree of gastric complaints may be associated with the density of
H. pylori infection [40]. Also the humoral and cellular immunity may differ from one
pregnant woman to another. Various epidemiologic studies have proved that the major risk
factor of H. pylori infection is low socioeconomic level [41]. A potential mechanism whereby
H. pylori infection may increase the risk of hyperemesis gravidarum might be explained by
physiological changes in pregnancy and the virulence of the bacterium. First, gastric
emptying and intestinal transit time may be prolonged during pregnancy due to elevated
serum concentration of steroid hormones and their relaxant effects on gastrointestinal smooth
muscle. Second, it has been assumed that pregnancy increases women’s susceptibility to
infections, and latent infections might be reactivated in pregnancy. Hypothetically, decreased
gastrointestinal motility and a depressed immune defense during pregnancy could result in the
manifestation of a sub-clinical H. pylori infection, which in turn irritates the gastrointestinal
tract. Finally, the inflammatory response to H. pylori might be determined by the virulence of
the infecting strain and the human host [22].
However, a recent case-control study showed that the association between H. pylori and
hyperemesis gravidarum is weaker than previously expected [42].
H. pylori and Pre-eclampsia
Pre-eclampsia (PE) is a severe hypertensive pregnancy-related disorder that affects 5%-

8% of women worldwide, thus representing the main cause of feto-maternal mortality and
morbidity. PE is characterized by excessive maternal inflammatory response, with high
circulating levels of pro-inflammatory cytokines and endothelial injury. The etiopathogenetic
mechanisms underlying are not still very clear [43]. Several lines of evidence suggest that
sub-clinical infections could play a role in the onset of PE [44].
While endothelial and trophoblast dysfunction has been shown to play a key role in the
occurrence of PE, together with the presence of an exaggerated inflammatory response and
hypercoagulative state, the primary trigger is still unknown [45, 46].
PE is often associated with fetal growth retardation (FGR), defined as failure of the fetus
to achieve its genetically determined growth potential [47]. FGR is commonly considered a

severe complication of PE, but whether or not PE and FGR are manifestations of the same
disorder, or two distinct pathologies, still remains unclear.
It has been demonstrated that H. pylori infection enhances platelets activation and
thrombus formation, thus inducing endothelial inflammation and injury. Therefore, H. pylori
could directly cause or intensify the generalized inflammation and endothelial dysfunction
typical of PE [48]. Furthermore, it was recently observed that H. pylori seropositive PE
subjects are characterized by a more severe inflammatory status [49, 50].
In a study, Cardaropoli et al , reported a direct association between H. pylori virulence
and the onset of PE complicated by FGR. Moreover, by investigating seropositivity for H.
pylori virulence factors, they were able to distinguish PE and FGR without hypertension as
different pathologies [51].
H. pylori strains carrying the CagA antigen are known to be among the most virulent and
are associated with increased inflammation. VacA is a H. pylori toxin crucial to promote and
maintain bacterial colonization. Importantly, combined seropositivity for both CagA and
VacA directly correlates with elevated morbidity [52, 53]. A study reported a strong
association between CagA positive H. pylori infection and the onset of PE in Italian women
[54]. These authors also found that CagA/VacA dual seropositivity is specifically associated
with PE and, in particular, with PE complicated by FGR [51]. In contrast, the absence of both
anti-CagA and anti-VacA antibodies is associated with a lower risk of PE. Interestingly, the
association with CagA-/VacA+ was found only in controls and normotensive women with
FGR pregnancies, while CagA+/VacA- patients belong to the PE groups CagA antigen is
associated with a more severe pattern, while VacA alone is not sufficient to cause the severe
systemic inflammation typical of PE. Therefore, chronic and severe H. pylori infections could
contribute not only to the exacerbated maternal inflammatory response leading to PE, but also
to the abnormal placentation typical of FGR [51].
Although a direct relationship between severe and persistent H. pylori infection and the
onset of PE complicated by FGR has been demonstrated, the only effective therapeutic option
of PE remains a timed, programmed delivery. It is known that women who experienced PE in
the course of their life have a higher probability to develop ischemic heart disease as well as
an increased mortality for cardiovascular diseases [55].
Patients with PE were shown to have a higher prevalence of CagA-positive strains of H.
pylori, which modulate the release of IL-18 and increase C reactive protein (CRP) and tumor
necrosis factor (TNF)-α and malondialdehyde levels, all known to be associated with PE. As
anti-CagA antibodies are able to cross-react with antigens of endothelial cells and
cytotrophoblast cells show an endothelial origin, Franceschi et al. studied a possible
relationship between H. pylori and pathological conditions of trophoblast. Anti-CagA
antibodies are able to recognize b-actin on the surface of trophoblast cells and may produce a
biological effect involved in PE [56].
Another study compared CRP, TNF-α, Chlamydia pneumonia IgG, IgM and plasma H.
pylori IgA levels between pre-eclamptic and normal pregnant women and concluded that
there were higher levels of serum CRP and TNF-α in pre-eclamptic H. pylori-seropositive
than in H. pylori seronegative women [57].

Transmission to the Fetus
No evidence of transmission of the H. pylori infection from the pregnant mother to her
fetus has been reported in the literature until now.
Several years ago, a study evaluated the mother-child transmission of anti-H. pylori
antibodies, and the authors concluded that IgG antibodies against H. pylori cross the placental
barrier and that, despite the present H. pylori infection in the mothers, infants born from these
H. pylori-positive women do not appear to have an increased risk of developing a H. pylori-
associated gastritis during the first year of life [58].
Teratogenicity
Neural tube defects (NTDs) are comprised of a group of congenital malformations that
include spina bifida, anencephaly, and encephalocele, which arise during the process of
neurulation between the third and fourth weeks of human pregnancy. The incidence of NTDs
varies in different parts of the world [59, 60].
H. pylori could cause nutrient loss to the fetus. Folate, vitamin B12, and ferritin can be
depleted in H. pylori infection; these same deficiencies are related to NTDs risk. Felkner et al.
concluded that H. pylori can play a role in NTDs causation by reducing folate and vitamin
B12 concentrations [61]. Several studies have found folic acid and vitamin B12 deficiency in
NTD-affected pregnancies compared with normal pregnancies [62]. Other studies reported
that serum/plasma vitamin B12 ratio and folate levels are lower in persons with H. pylori
infections compared with uninfected persons [63, 64]. Moreover, some authors stated that
vitamin B12, and folate levels improve after H. pylori eradication [65].
Golalipour et al. in Iran studied a possible relationship between H. pylori infection, folate
and vitamin B12 deficiency and NTDs. They concluded that H. pylori infection preventing
micronutrient absorption and consequently, reducing folate levels and B12, deficiency
pregnant women can increase the risk of the occurrence of NTDs in offsprings up to 2 times,
although these results were not statistically significant. Therefore the role of H. pylori on
causing NTDs if exists, seems to be indirect, by determining micronutrients deficiency (e.g.
folate and B12 malabsorption) [66]. But, in this regard, further studies need to be made.
Maternal Transmission of H. pylori in the Perinatal Period
In a study on mother-to-child transmission, Kitagawa et al. investigated maternal H.

pylori infection status to determine the potential mother-to-child transmission in the perinatal
period. After obtaining informed consent from 1,588 pregnant women, mother's blood and
cord blood were collected at delivery to measure H. pylori antibody. Gastric contents from the
neonates were cultured to isolate H. pylori. Vaginal discharge (73 women) and dental plaque
scraping swabs (48 women) were collected before delivery, and milk (66 women) was
collected after delivery from 212 H. pylori antibody-positive pregnant women to detect H.
pylori by PCR. The H. pylori antibody-positive rate for the pregnant women was 29.2%. H.
pylori was not detected in the vaginal discharge from H. pylori antibody-positive pregnant
women, but dental plaque scraping swabs from 4 women and milk from 4 women was

positive. H. pylori may have survived on nipples or fingers to contaminate milk. When the
nipples and hands of H. pylori-positive women were adequately sterilized or washed, the milk
subsequently obtained from these 4 women was negative when examined by PCR. Thus it is
important for H. pylori-positive mothers to sterilize and wash nipples and hands to prevent
horizontal infection. In that study, they considered that vertical infection during pregnancy or
at delivery is unlikely as a route of mother-to-child H. pylori antibody infection. Although the
possibility of vertical infection from mother-to-child in the perinatal period is low because H.
pylori is mainly transmitted orally, they found that horizontal infection could occur via breast
feeding [67].
Another study by Baldassarre et al. showed that in the perinatal period a positive finding
of H. pylori infection by stool antigen test is a sporadic event which equally occur in babies
from either positive and negative mothers and it disappears spontaneously within 1 year [68].
Conclusion
In recent years, H. pylori infection has been related to many extra-gastric diseases such as
some gynaecological pathologies and disorders during pregnancy.
Regarding to gynaecological system the most frequent H. pylori-associated disease is
unexplained female infertility; in fact anti-H. pylori antibodies present in the cervical mucus
can interfere with sperm progression.
During pregnancy H. pylori infection can increase dyspepsia, aggravate gastrointestinal
symptoms and cause/worsen iron deficiency anemia and hyperemesis gravidarum. Also PE
with FGR can be related to H. pylori infection in pregnant women. It seems that mainly anti-
CagA antibodies are able to react with cytotrophoblast and produce the typical alterations
characterizing PE . Indeed, several studies showed an improvement of these disorders after H.
pylori eradication treatment.
Various therapeutic regimens for H. pylori infection were mentioned in the literature with
different eradication rates [69-82]. During pregnancy, especially in the first trimester, H.
pylori eradication therapy should be used with real cautious for the risk of drug teratogenicity.
Therefore the eradication treatment should be reserved in only very selected cases such as in
women affected by intractable hyperemesis gravidarum and in third trimester of pregnancy.
The H. pylori eradication treatment in study by Mansour & Nashaat was given to the eight
complicated cases and although a significant response was noticed, further studies with larger
numbers of patients are needed to corroborate such findings. Also more studies on the safety
of proton pump inhibitors during pregnancy should be done, because proton pump inhibitors
will give better results in these cases. Patients’ education for food safety is important. Careful
food handling and hand washing are important to prevent introduction of food-borne
pathogens to the diet of pregnant women [31].
Some studies indicated a possible causal link between H. pylori infection and NTDs
through a secondary malabsorption of folic acid and vitamin B12.
A horizontal H. pylori infection via breastfeeding can occur and it can be prevented by
accurate hygiene of mothers.
On the other hand, a vertical transmission of H. pylori infection has not been
demonstrated, yet.

References
[1] Wu CY, Tseng JJ, Chou MM et al. (2000). Correlation between Helicobacter pylori
infection and gastrointestinal symptoms in pregnancy. Adv. Ther. 17:152–158.
[2] Soll AH, (1996) Helicobacter pylori induced gastritis. In: Bennet JC, Plum F (eds)
Cecil textbook of medicine, 20th edn. Philadelphia, WB Saunders, pp 659–660.
[3] Cave D, (1997). How is Helicobacter pylori transmitted? Gastroenterology., 113
(supp):S9–S14.
[4] Kalach N, Desrame J, Bonnet C, Commegeille P, Couturier D, Chaussade S, (2002).
Helicobacter pylori seroprevalence in asymptomatic pregnant women in France. Clin.
Diagn. Lab. Immunol. 9, 736-737.
[5] Lanciers S, Despinasse B, Mehta DI, Blecker U, (1999). Increased susceptibility to
Helicobacter pylori infection in pregnancy. Infect. Dis. Obstet. Gynecol., 7(4):195-8.
[6] McNulty CAM, Lehours P, Megraud F, (2011). Diagnosis of Helicobacter pylori
infection. Helicobacter., 16:10–8.
[7] Cevrioglu AS, Yilmazer M, Fenkci IV, Ellidokuz E, Kose S, (2004). Efficient and non-
invasive method for investigating Helicobacter pylori in gravida with hyperemesis
gravidarum: Helicobacter pylori stool antigen test. J. Obst. Gynaecol. Res., 30:136–41.
[8] Guven MA, Ertas IE, Coskun A, Ciragil P, (2011). Serologic and stool antigen assay of
Helicobacter pylori infection in hyperemesis gravidarum: which test is useful during
early pregnancy? Taiwan. J. Obst. Gynecol., 50:37–41.
[9] Kaya T, Ozan H, Ozakin C, (2009).The presence of Helicobacter pylori in cervical
preinvasive lesions. Clin. Exp. Obst. Gynecol., 36(2),113-115.
[10] Pellati D, Mylonakis I, Bertoloni G, Fiore C, Andrisani A, Ambrosini G, Armanini D,
(2008). Genital tract infections and infertility. Eur. J. Obstet. Gynecol. Reprod. Biol.
140, 3-11.
[11] Figura N, Piomboni P, Ponzetto A, et al. (2002). Helicobacter pylori infection and
infertility. Eur. J. Gastroenterol. Hepatol., 14. 663-9.
[12] Kurotsuchi, S, Hisao Ando H, Iwase A, Ishida Y, Hamajima N, Kikkawa F, (2008). The
plausibility of Helicobacter pylori-related infertility in Japan. Fertil. Steril., 90: 866-8.
[13] Ambrosini G, Andrisani A, Fiore C, Fggiani D, Antona D, Ragazzi E, Plebani M,
Armanini D, (2011). Anti-Helicobacter pylori antibodies in cervical mucus: a new
cause of infertility. Eur. J. Obst. Gynecol. Repro. Biol., 155, 157-160.
[14] World Health Organization, 1992. The prevalence of anemia in women: a tabulation of
available information, 2nd Edn. Geneva: World Health Organization.
[15] Farag TH, Stoltzfus RJ, Khalfan SS, Tielsch JM, (2007). Helicobacter pylori infection
is associated with severe anemia of pregnancy on Pemba Island, Zanzibar. Am. J. Trop.
Med. Hyg., 76(3), 541–548.
[16] DuBois S, Kearney DJ, (2005). Iron-deficiency anemia and Helicobacter pylori
infection: a review of the evidence. Am. J. Gastroenterol. 100,453-459.
[17] Weyermann M, Rothenbacher D, Gayer L, Bode G, Adler G, Grab D, Flock F, Brenner
H, 2005. Role of Helicobacter pylori infection in iron deficiency during pregnancy. Am.
J. Obstet. Gynecol. 192: 548–553..

[18] Malik R, Guleria K, Kaur I, Sikka M, Radhakrishnan G, (2011). Effect of Helicobacter

pylori eradication therapy in iron deficiency anaemia of pregnancy – A pilot study,
Indian. J. Med. Res., 134(2): 224–231.
[19] Mulayim B, Celik NY, Yanik FF, (2008). Helicobacter pylori infection detected by
14C-urea breath test is associated with iron deficiency anemia in pregnant women. J.
Obstet. Gynaecol. Res., 34(6), 980-5.
[20] Fukui O, Shimoya K, Shimizu T, Fukuda H, Wasada K, Murata Y, (2005).
Helicobacter pylori infection and platelet counts during pregnancy. Int. J. Gynaecol.
Obstet, 89, 26-30.
[21] Hashino S, Mori A, Suzuki S, Izumiyama K, Kahata K, Yonezumi M, et al. (2002).
Platelet recovery in patients with idiopathic thrombocytopenic purpura after eradication
of Helicobacter pylori. Int. J. Hematol., 77:188 – 91.
[22] Epstein A, Wing DA, Ouzounian JG, Miller DA, Lee RH, (2012). Helicobacter pylori
and thrombocytopenia in the pregnant hispanic population. J. Matern. Fetal. Neonatal.
Med., 25(12):2588-90.
[23] Broussard CN, Richter JE, (1998). Nausea and vomiting of pregnancy. Gastroenterol
Clin North Am., 27:123–151.
[24] Hod M, Orvieto R, Kaplan B, (1994). Hyperemesis gravidarum: a review. J. Reprod.
Med., 39(8):605–612.
[25] Sandven I, Abdelnoor M, Nesheim BJ, Melby KK, (2009). Helicobacter pylori
infection and hyperemesis gravidarum: a systematic review and meta-analysis of case–
control studies. Acta. Obstet. Gynecol. Scand., 88:1190-1200.
[26] Gross S, Librach C, Cecutti A, (1989). Maternal weight loss association with
hyperemesis gravidarum: a predictor of fetal outcome. Am. J. Obstet. Gynecol.,
160:906–909.
[27] Tincello DG, Johnstone MJ, (1996). Treatment of hyperemesis gravidarum with the 5-
HT3 antagonist ondansetron (Zofran). Postgrad. Med. J., 72(853):688–689.
[28] Eliakim R et al. (2000). Hyperemeiss gravidarum: a current review. Am. J. Perinatol.,
17:207.
[29] Sylvia E, (2008).Vomiting, pernicious. In: Sylvia Escott-Stump (ed) Nutrition and
Diagnosis Related care, 6th edn. Lippincott Williams & Wilkins, Philadelphia, p 384.
[30] Mansour Ghada M, Nashaat Ehab H, (2009). Helicobacter pylori and hyperemesis
gravidarum. Int. J. Gynaecol Obst., 106(1):63-4.
[31] Mansour GM, Nashaat EH, (2011). Role of Helicobacter pylori in the pathogenesis of
hyperemesis gravidarum. Arch. Gynecol. Obstet., 284(4):843-7.
[32] Frigo P, Lang C, Reisenberger K, Kolbl H, Hirschl AM, (1998). Hyperemesis
gravidarum associated with Helicobacter pylori seropositivity. Obstet. Gynecol.,
91(4):615–7.
[33] Jacoby EB, Porter KB, (1999). Helicobacter pylori infection and persistent hyperemesis
gravidarum. Am. J. Perinatol., 16(2):85–8.
[34] Wu CY, Tseng JJ, Chou MM et al. (2000). Correlation between Helicobacter pylori
infection and gastrointestinal symptoms in pregnancy. Adv. Ther., 17:152–158.
[35] Kocak I, Akcan Y, Ustun C, Demirel C, Cengiz L, Yanik FF, (1999). Helicobacter
pylori seropositivity in patients with hyperemesis gravidarum. Int. J. Gynaecol. Obstet.,
66(3):251–254.

[36] Jamal A, Pooransari P, Andansari R, (2004) Relationship between Helicobacter pylori

seropositivity and hyperemesis gravidarum. Acta. Med. Iran., 42(5):375–378.
[37] Flook NW, (1998). Helicobacter pylori primary care management from symptoms to
cure. Can. Fam. Physician., 44:1429–1430.
[38] Hayakawa S, Nakajima N, Karasaki-Suzuki M, Yoshinaga H, Arakawa Y, Satoh K,
Yamamoto T, (2000). Frequent presence of Helicobacter pylori genome in the saliva of
patients with hyperemesis gravidarum. Am. J. Perinatol., 17(5):243–247.
[39] Meijer BC, Thijs JC, Kleibeuker JH et al. (1997) Evaluation of eight enzyme
immunoassays for detection of immunoglobulin G against Helicobacter pylori. J. Clin.
Microbiol., 35:292–294.
[40] Bagis T, Gumurdulu Y, Kayaselcuk F et al. (2002) Endoscopy in hyperemesis
gravidarum and Helicobacter pylori infection. Int. J. Gynaecol. Obstet., 79:105–109.
[41] Karadeniz RS, Ozdegirmenci O, Altay MM et al. (2006). Helicobacter pylori
seropositivity and stool antigen in patients with hyperemesis gravidarum. Infect. Dis.
Obstet. Gynecol.,2006: 73073.
[42] Vikanes AV, Stoer NC, Gunnes N, Grjibovski AM, Samuelsen SO, Magnus P, Melby
KK, (2013). Helicobacter pylori infection and severe hyperemesis gravidarum among
immigrant women in Norway: a case–control study. Eur. J. Obstet. Gynecol. Reprod.
Biol., 167(1):41-6.
[43] Roberts JM, Gammill HS, (2005). Preeclampsia: recent insights. Hypertension., 46:
1243-1249.
[44] Rustveld LO, Kelsey SF, Sharma R, (2008). Association between maternal infections
and preeclampsia: a systematic review of epidemiologic studies. Matern. Child. Health.
J., 12:223-242.
[45] James JL, Whitley GS, Cartwright JE, (2010). Pre-eclampsia: fitting together the
placental, immune and cardiovascular pieces. J. Pathol., 221:363–78.
[46] Macey MG, Bevan S, Alam S, et al. (2010). Platelet activation and endogenous
thrombin potential in pre-eclampsia. Thromb. Res., 125:e76–81.
[47] Cetin I, Foidart JM, Miozzo M, Raun T, Jansson T, Tsatsaris V, Reik W, Cross J,
Hauguel-de-Mouzon S, Illsley N, Kingdom J, Huppertz B, (2004). Fetal growth
restriction: a workshop report. Placenta., 25:753-757.
[48] Ponzetto A, Cardaropoli S, Piccoli E, Rolfo A, Gennero L, Kanduc D, Todros T,
(2006). Pre-eclampsia is associated with Helicobacter pylori seropositivity in Italy. J.
Hypertens., 24:2445-2449.
[49] Redman CW, Sargent IL, (2003). Pre-eclampsia, the placenta and the maternal systemic
inflammatory response--a review. Placenta., 24 Suppl A: S21-S27.
[50] Ustun Y, Engin-Ustun Y, Ozkaplan E, Otlu B, Tekerekoglu MS, (2009). Association of
Helicobacter pylori infection with systemic inflammation in preeclampsia. J. Matern.
Fetal. Neonatal. Med., 1-4.
[51] Cardaropoli S, Rolfo A, Piazzese A, Ponzetto A, Todros T, (2011). Helicobacter
pylori's virulence and infection persistence define pre-eclampsia complicated by fetal
growth retardation. World. J. Gastroenterol., 21;17(47):5156-65.
[52] Kuipers EJ, Pérez-Pérez GI, Meuwissen SG, Blaser MJ, (1995). Helicobacter pylori
and atrophic gastritis: importance of the cagA status. J. Natl. Cancer. Inst., 87: 1777-
1780

[53] Van Doorn LJ, Figueiredo C, Mégraud F, Pena S, Midolo P, Queiroz DM, Carneiro F,
Vanderborght B, Pegado MD, Sanna R, De Boer W, Schneeberger PM, Correa P, Ng
EK, Atherton J, Blaser MJ, Quint WG, (1999). Geographic distribution of vacA allelic
types of Helicobacter pylori. Gastroenterology., 116: 823-830
[54] Ponzetto A, Cardaropoli S, Piccoli E, Rolfo A, Gennero L, Kanduc D, Todros T,
(2006). Pre-eclampsia is associated with Helicobacter pylori seropositivity in Italy. J.
Hypertens., 24: 2445-2449.
[55] Bellamy L, Casas JP, Hingorani AD, et al. (2007). Pre-eclampsia and risk of
cardiovascular disease and cancer in later life: systematic review and meta-analysis.
BMJ., 335:974.
[56] Franceschi F, Di Simone N, D'Ippolito S, Castellani R, Di Nicuolo F, Gasbarrini G,
Yamaoka Y, Todros T, Scambia G, Gasbarrini A, (2012). Antibodies anti-CagA cross-
react with trophoblast cells: a risk factor for pre-eclampsia? Helicobacter., 17(6):426-
34.
[57] UstUn Y; Engin-Ust Un Y; Ozkaplan E; Otlu B; Sait Tekereko Glu M, (2010).
Association of Helicobacter pylori infection with systemic inflammation in
preeclampsia. J. Matern. Fetal. Neonatal. Med., 23(4):311-4.
[58] Blecker U, Lanciers S, Keppens E, Vandenplas Y., (1994). Evolution of Helicobacter
pylori positivity in infants born from positive mothers. J. Pediatr. Gastroenterol. Nutr.,
19(1):87-90.
[59] Boyles AL, Billups AV, Deak KL, Siegel DG, Mehltretter L, Slifer SH, et al. (2006).
Neural tube defects and folate pathway genes: familybased association tests of gene-
gene and gene-environment interactions. Environ. Health. Perspect., 114: 1547-1552.
[60] Ray JG, Wyatt PR, Thompson MD, Vermeulen MJ, Meier C, Wong PY, et al. (2007).
Vitamin B12 and the risk of neural tube defects in a folic-acid-fortified population.
Epidemiology., 18: 362-366.
[61] Felkner M, Suarez L, Liszka B, Brender JD, Canfield M, (2007). Neural tube defects,
micronutrient deficiencies, and Helicobacter pylori: a new hypothesis. Birth. Defects.
Res. A. Clin. Mol. Teratol., 79: 617-621.
[62] Molloy AM, Kirke PN, Brody LC, Scott JM, Mills JL, (2008). Effects of folate and
vitamin B12 deficiencies during pregnancy on fetal, infant, and child development.
Food. Nutr. Bull., 29 (2 Suppl): S101-S111.
[63] Serin E, Gümürdülü Y, Ozer B, Kayaselçuk F, Yilmaz U, Koçak R, (2002). Impact of
Helicobacter pylori on the development of vitamin B12 deficiency in the absence of
gastric atrophy. Helicobacter., 7: 337-341.
[64] Tamura A, Fujioka T, Nasu M, (2002). Relation of Helicobacter pylori infection to
plasma vitamin B12, folic acid, and homocysteine levels in patients who underwent
diagnostic coronary arteriography. Am. J. Gastroenterol., 97: 861-866.
[65] Marino MC, de Oliveira CA, Rocha AM, Rocha GA, Clementino NC, Antunes LF, et
al. (2007). Long-term effect of Helicobacter pylori eradication on plasma homocysteine
in elderly patients with cobalamin deficiency. Gut., 56: 469-474.
[66] Golalipour MJ,Sedehi M, Qorbani M, (2012). Does maternal Helicobacter pylori
infection increase the risk of occurrence of neural tube defects in newborns in Northern
Iran? Neurosciences., Vol. 17 (3): 219-225.

[67] Kitagawa M, Natori M, Ktoh M, Sugimoto K, Omi H, Akiyama Y, Sago H, (2001).

Maternal transmission of Helicobacter pylori in the perinatal period. J. Obstet.
Gynaecol.Res., 27:225-230.
[68] Baldassarre ME, Monno R, Laforgia N, Fumarola L, Fanelli M, Sgobba M, Hassan C,
Panella C, Ierardi E, (2008). Helicobacter pylori detection by stool antigen test in the
perinatal period: an inadequate approach to establish maternal transmission, J. Ped.
Gastroenterol. Nutr., 47(5), 673-674.
[69] Manfredi M, Bizzarri B, de'Angelis GL, (2012). Helicobacter pylori infection:
sequential therapy followed by levofloxacin-containing triple therapy provides a good
cumulative eradication rate. Helicobacter., 17(4):246-53.
[70] Manfredi M, Bizzarri B, Sacchero RI, maccari S, Calabrese L, Fabbian F, de'Angelis
GL, (2012). Helicobacter pylori infection in clinical practice: probiotics and a
combination of probiotics + lactoferrin improve compliance, but not eradication, in
sequential therapy. Helicobacter., 17(4):254-63.
[71] Gisbert JP, Castro-Fernandez M, Perez-Asia et al. (2012). Fourth-line rescue therapy
with rifabutin in patients with three Helicobacter pylori eradication failures. Aliment.
Pharmacol. Ther., 35: 941.
[72] Malfertheiner P, Bazzoli F, Delchier JC et al. (2011). Helicobacter pylori
eradicationwith a capsule containing bismuth subcitrate potassium, metronidazole, and
tetracycline given with omeprazole versus clarithromycin-based triple therapy: a
randomised, open-label, non-inferiority, phase 3 trial. Lancet, 377:905.
[73] Basu PP, Rayapudi K, Pacana T et al. (2011). A randomized study comparing
levofloxacin, omeprazole, nitazoxanide, and doxycycline versus triple therapy for the
eradication of Helicobacter pylori. Am. J. Gastroenterol., 106: 1970.
[74] Kim YS, Kim SJ, Yoon JH et al. (2011). Randomised clincial trial: the efficacy of a 10-
day sequential therapy vs. a 14-day standard proton pump inibitor-based triple for
Helicobacter pylori in Korea. Aliment. Pharmacol. Ther., 34: 1098.
[75] Greenberg ER, Anderson GL, Morgan DR et al. (2011). 14-day triple, 5-day
concomitant, and 10-day sequential therapies for Helicobacter pylori infection in seven
Latin American sites: a randomised trial. Lancet., 378:597.
[76] Albrecht P, Kotowska M, Szajewska H, (2011). Sequential therapy compared with
standard triple therapy for Helicobacter pylori eradication in children: a double-blind,
randomized, controlled trial. J. Pediatr., 159:45.
[77] Bontems P, Kalach N, Oderda G et al. (2011). Sequential therapy versus tailored triple
therapies for Helicobacter pylori infection in children. J. Pediatr. Gastroenterol. Nutr.,
53: 646.
[78] Song MJ, Park DJ, Park JH et al. (2010). The effect of probiotics and mucoprotective
agents on PPI-based triple therapy for eradication of Helicobacter pylori. Helicobacter.,
15:206.
[79] Wu DC, Hsu PI, Wu JY et al (2010). Sequential and concomitant therapy with four
drugs is equally effective for eradication of H. pyliori infection. Clin. Gastroenterol.
Hepatol., 8: 36.
[80] Szajewska H, Horvath A, Piwowarczyk A, (2010). Meta-analysis: the effects of
Saccharomyces boulardii supplementation on Helicobacter pylori eradication rates and
side effects during treatment. Aliment. Pharmacol. Ther., 32: 1069.

[81] Sun Q, Liang X, Zheng Q et al. (2010). High efficacy of 14-day triple therapy-based,
bismuth-containing quadruple therapy for initial Helicobacter pylori eradication.
Helicobacter., 15: 233.
[82] Romano M, Cuomo A, Gravina AG et al. (2010). Empirical levofloxacin-containing
versus clarithromycin-containing sequential therapy for Helicobacter pylori eradication:
a randomised trial. Gut., 59: 1465.

Chapter 18

and Skin Diseases
Zekayi Kutlubay, Ozge Karakus, Burhan Engin,

Server Serdaroğlu and Yalçın Tüzün
Department of Dermatology, Istanbul University,
Cerrahpaşa Medical Faculty, Istanbul, Turkey
Abstract
Some authors claim that there is a potential role of Helicobacter pylori (H. pylori) in
several extraintestinal diseases such as hematological, cardiovascular, autoimmune,
neurological and skin diseases. Recently, H. pylori infection has been thought to be
associated with chronic urticaria, angioedema, immune thrombocytopenic purpura,
rosacea, Behçet’s disease, psoriasis, lichen planus, Sweet syndrome, and prurigo
nodularis. Most of the studies have been done suggesting an association between H.
pylori infection and skin diseases such as chronic idiopathic urticaria and rosacea. The
role of H. pylori infection in the development of skin diseases has been addressed in a
variety of studies, but clear evidence for their pathogenic role in dermatological diseases
has not been demonstrated. It is proposed that H. pylori infection, through the production
of specific cytotoxins, immunological stimulation by chronic infection, production of
pathogenetic antibodies possibly by molecular mimicry, and the release of vascular
mediators like histamines might be the triggering factors for the development of
dermatological diseases. The results of studies investigating H. pylori prevalence in
certain dermatological patients and the effect of eradication are conflicting. For this
reason, more randomized and case control studies are necessary to prove an association
between H. pylori and skin pathologies. In this chapter, skin diseases that are thought to
be associated with H. pylori will be discussed in detail.
 Corresponding author is Zekayi KUTLUBAY, M.D. Address: Istanbul University, Cerrahpaşa Medical Faculty,
Department of Dermatology, Istanbul, Turkey. Telephone: 00902124143000-21546; Email:
zekayikutlubay@hotmail.com.

324 Zekayi Kutlubay, Ozge Karakus, Burhan Engin et al.
Keywords: Helicobacter pylori, chronic urticaria, rosacea, psoriasis, Behcet’s disease,

eradication therapy
Introduction
Helicobacter pylori (H. pylori) is a Gram negative, microaerophilic, spiral shaped
bacterium that colonizes the gastric mucosa, affecting nearly half of the world population. H.
pylori also has been implicated in a few diseases that are sometimes not even related to the
gastrointestinal tract [1, 2, 3]. Recent evidence suggests that H. pylori infections play a role in
the pathogenesis of certain skin diseases. There have also been many uncontrolled, small
sample studies suggesting an association between H. pylori infection and skin disorders. The
pathophysiological event in H. pylori infection is initiation and continuation of an
inflammatory response. An interaction between the main bacterial factors such as CagA and
host signal transduction pathways seems to be critical for mediating cell transformation, cell
proliferation, invasion, apoptosis/anti-apoptosis, and angiogenesis [4]. The organism induces
a strong inflammation with the release of urease, phospholipase, hemolysin, platelet
activating factor, alcohol dehydrogenase, vacuolating cytotoxin and mucolytic factor. These
enzymes and toxins cause damage on the stomach and duodenum, they also interact with
other tissues. Some authors suggest that there have been a potential role of H. pylori in
several extraintestinal diseases such as hematological, cardiovascular, autoimmune,
neurological and skin diseases [1, 5, 6].
Helicobacter pylori and Dermatological Diseases

Recently, H. pylori infection has been thought to be associated with chronic urticaria,
angioedema, immune thrombocytopenic purpura, rosacea, Behçet’s disease, psoriasis, lichen
planus, Sweet syndrome, angioedema and prurigo nodularis [1, 5, 7].
Chronic Urticaria
Urticaria is a skin disease consisting of a wheal and flare reaction in which localized
intracutaneous edema is surrounded by an area of erythema that is typically pruritic. An
urticarial lesion is the result of localized edema in the dermis following vasodilatation and
increased vascular permeability with diffusion of plasma and various mediators into the
tissue. It is similar to the triple response of Lewis after the injection of histamine into the skin.
Initially, there is an erythema at the injection site secondary to vasodilatation; next, the edema
leads to a hive or edematous plaque; in the final stage, there is an erythematous ring
surrounding the hive as the axonal reflex induces centrifugal progression of vasodilatation.
Acute urticaria is one of the most common skin disorders; 20-30% of individuals have at least
one attack in their lifetime. Chronic urticaria is defined as chronic recurrent wheals lasting for
more than 6 weeks together with a significant disturbance of the daily life. The frequency of
chronic urticaria is less clear; its prevalence has been estimated at 1-4% and an etiologic

Helicobacter pylori Infection and Skin Diseases 325
factor is seldom identified and thus considered idiopathic. Chronic urticaria may have an
autoimmune basis in 45% of patients. Chronic idiopathic urticaria has a complex
pathophysiological mechanism. There are lots of possible initiating factors such as bacterial,
viral, fungal, or protozoan infections, although this has not been clearly confirmed. Chronic
urticaria may have numerous causative factors but in one third of the cases the origin remains
unclear. Because of the high frequency of H. pylori infections in overall world’s population, it
has become also a highly suspected etiological factor in chronic urticaria [8, 9].
Treatment and eradication of H. pylori were reported to be associated with remission of
chronic urticaria. Also there are studies that suggest no association between H. pylori
infection and chronic urticaria [10]. Different pathogenic mechanisms have been proposed to
explain the relationship. An autoimmune mechanism has been mooted that mimicry between
H. pylori lipopolysaccharide and Lewis blood group antigens can occur in autoimmune type B
gastritis. In this regard, positive autologous serum skin tests have been associated with H.
pylori infection in chronic urticaria. In some patients, autologous serum skin tests became
negative after H. pylori eradication [9]. Another possible association has been proposed with
the pathogenic mechanism related to an increase in gastric vascular permeability during the
infection, this results in a greater exposure of the host to alimentary allergens [9, 11].
Duodenal ulcer patients were shown to have a higher incidence of allergic manifestations than
controls. IgE containing cells in gastric and duodenal mucosa may be a factor, but there is a
lack of evidence for H. pylori-spesific IgE. The possibility that patients with urticaria develop
specific IgE against H. pylori is an attractive pathogenic explanation that unfortunately has
not been confirmed yet [12].
Hellmig et al. compared H. pylori infected patients with chronic urticaria to an age and
sex matched H. pylori negative control group. Each group had 74 patients and all urticaria
patients underwent an extensive diagnostic researches for triggering factors. H. pylori
infected patients were treated with antibiotics. They all were followed up for 58 months.
These authors reported that neither the prevalance of H. pylori nor the eradication therapy had
an influence on the clinical course of chronic urticaria. In 81% of H. pylori infected patients
were found to have another infectious focus. In conclusion, in that study, no evidence that
eradication of H. pylori improves the outcome in patients with chronic urticaria. Other
infections and spontaneous remission affect the real result of any kind of antimicrobial
therapy [8].
In another study, 87 patients with chronic urticaria were checked for the positivity
autologous serum skin test (ASST) and H. pylori C-urea breath test (C-UBT). There were 2
groups A and B; group A had 21 patients with positive ASST and C-UBT, group B had 24
patients with negative ASST and positive C-UBT. All patients with positive 13C-UBT, were
treated with amoxicillin, clarithromycin, omeprazole for 14 days. H. pylori eradication was
assessed by a second C-UBT after 8 weeks and the effect of eradication on chronic urticaria
was evaluated by urticaria activity score (UAS). After 8 weeks treatment, baseline UAS
reduced from 4.7 to 2.4 (p=0.27) in group A and reduced from 4.3 to 2.3 (p=0.008) in group
B. That study showed a significant reducement in UAS in chronic urticaria patients with
positive and negative ASST, after the eradication of H. pylori by triple therapy [13].
Yadav et al. studied to assess the prevalence of H. pylori infection and effect of H. pylori
eradication on the course of skin lesions in patients of chronic idiopathic urticaria (CIU).
Ninety-eight patients and 68 controls were enrolled in the study. All patients were screened
by endoscopy and biopsied in the antrum for urease and histopathology to identify H. pylori-

associated gastritis. Infected patients were treated with triple antibiotic therapy. Eradication
was confirmed by fecal antigen assay. The course of skin lesions and patients’ subjective
response to treatment were assessed by using chronic urticaria quality of life questionnaire
while objective response to the treatment was assessed by need for other medications like
antihistamines. H. pylori associated gastritis was present in 48 (70.58%) patients, out of
which 39 (81.25%) patients responded to eradication therapy. Ten (50%) patients without H.
pylori associated gastritis showed response to symptomatic therapy. This showed significant
association between presence of H. pylori and response to eradication treatment [14].
In another study, patients who were resistant to antihistaminic therapy were searched for
H. pylori and for positive patients it was studied if H. pylori eradication would reverse
antihistaminic resistance. Twenty-nine of 46 patients that had positive 13C-urea breath test,
were treated with a 14-day-treatment with amoxicillin 1 gr twice daily, clarithromycin 500
mg twice daily and omeprazole 20 mg twice daily. The effect of H. pylori eradication on
chronic urticaria was evaluated by the UAS, measured at baseline and at 8, 16, and 28 weeks
after triple therapy. Patients with positive 13C-UBT, benefited from the antihistamines after
the eradication of H. pylori infection [15].
Moreira et al. studied to determine the prevalence of H. pylori infection in 21 patients
with chronic idiopathic urticaria, and see the effect of eradication therapy on the skin lesions.
But they failed to show a significant association between H. pylori and CIU. But they found
that those patients who had clinical remission of disease were the ones with greater UBT
titers suggesting a role for the amount of colonization by H. pylori in the pathogenesis of
chronic urticaria [16].
In a study reported by Wedi et al. the rate of remission of urticaria when H. pylori was
eradicated was escalated compared to when H. pylori was not eradicated [17]. After looking
over the studies about the association between H. pylori and chronic urticaria, the findings are
controversial. The mechanism of chronic urticaria still remains unclear, so does H. pylori as
an aetiological factor. Also the severity and exacerbation of urticarial symptoms might
depend on the density of H. pylori. It is recommended that patients with urticaria who are also
infected with H. pylori can be treated with traditional eradication therapy. Therefore, the
diagnostic tests that determine the H. pylori infection, should be included for the patients with
chronic urticaria [9, 14, 17, 18].
Rosacea
Rosacea is a common inflammatory cutaneous disorder characterized by facial erythema,

telangiectasia, papules, pustules and edema that are mostly seen on the central portion of face.
Rosacea is highly heteregenous entity with an unknown epidemiology and pathophysiology.
There is no laboratory benchmark test and for which etiopathogenesis and physiology are not
understood exactly [19, 20]. Rosacea is seen in people between 30-50 years of age, with
women being more affected than men. Although, rhinophyma, which is characterized by
irreversible connective tissue and sebaceous gland hypertrophy, occurs mostly in men. Men
also progress to the advanced stages earlier than women [20].
There are four stages; pre-rosacea then stages I through III, with several variants. There
are also four clinical types of rosacea; erythematotelangiectatic, papulopustular, ocular and
phymatous. The disease lasts for years, with episodes of improvement or exacerbation. A lot

of triggering factors like alcohol, sun exposure, spicy foods, caffeine, hot and cold weather,
wind may affect the course of the disease [21].
The cause of rosacea remains still unknown. Genetic and environmental factors are
thought to be effective in the etiology of rosacea. Pathological mechanisms of rosacea are due
to innate immunity, vascular changes, reactive oxygen species (ROS) released by neutrophils,
ultraviolet radiation, and microbes. Some of them are based on the evidence of scientific
investigation, others on anectodal observation. The role of microorganisms in the
development of rosacea has been showed in a variety of studies, but indisputable evidence for
their role in the pathogenic role in the disease has not been proved [20, 22].
The dysregulation of innate immune system in patients with rosacea can be accused.
Triggering the immune system leads an increased amount of cytokines and anti-microbial
molecules in the skin. One of the most important peptides, cathelicidin is expressed
abnormally high levels in rosacea patients [23]. Cathelicidin peptides lead to an angiogenic
activity, and promote leukocyte chemotaxis, and increase vascular permeability. Cathelicidin
is an vasoactive and inflammatory peptid that may be the most important factor in
pathogenesis of rosacea [24].
The role of microorganisms in the development of rosacea has been addresssed in a
variety of studies. H. pylori is one of the potential causative factor for rosacea. Recent studies
have shown that the prevalence of H. pylori infection is higher in rosacea patients than in
control groups. But the association between rosacea and H. pylori infection is still
controversial [22, 25].
It is thought that H. pylori, through the production of specific cytotoxins and the release
of vascular mediators such as histamine, prostaglandins, leukotrienes and cytokines might
trigger rosacea symptoms. The bacterium highly produces ROS including nitric oxide that is
shown higher levels in rosacea patients than control groups [22, 23].
In Szlachcic’s study, 60 subjects with rosacea skin symptoms and 60 age-gender-matched
control subjects with dyspeptic symptoms were compared. Fifty-three (88.3%) of 60 subjects
with rosacea, were diagnosed as H. pylori infection compared to only 39 (65%) of the
nonulcer dyspeptic controls, confirmed by at least two H. pylori tests. The result according to
this study showed that rosacea could be considered one of the extragastric manifestations of
H. pylori infection in the stomach. The prevalence of H. pylori infection in rosacea patients
was significantly higher than in age-gender-matched controls. This study also showed that the
eradication therapy of H. pylori, also healed the symptoms of rosacea [26].
Baz et al. showed that in the rosacea population, the seropositivity was higher for IgM
and lower for IgG antibodies against H. pylori compared to controls. Also plasma
malondialdehyde (MDA) was higher and antioxidant potential (AOP) levels were lower in
patients than controls, demonstrating that these patients have increased (ROS) activity.
Plasma MDA and AOP levels were not affected by the seropositivity of H. pylori for IgM and
IgG in either group. They suggested that rosacea was an oxidative stress condition, as
reflected by the increased ROS activity and decreased AOP, regardless of H. pylori infection
[27].
Gurer et al. evaluated 33 rosacea patients and 20 healthy controls to investigate the
seropositivity of H. pylori and the serum nitrate levels. The levels of IgG antibodies against
H. pylori in the serum samples were measured using ELISA. Nitrate levels were measured by
using chemiluminescence detection. The seropositivity of H. pylori in rosacea patients was

found to be high; however, the serum nitrate levels were found to be normal, which showed
that there was no role of nitric oxide (NO) in the inflammatory mechanism of rosacea [28].
In a study done by Abram et al. they investigated several possible risk factors for rosacea,
especially H. pylori. Famial predisposition was found the most significant factor but there
was no association between H. pylori and rosacea in current study. Improvement of rosacea
after treatment against H. pylori was explained by the antioxidant effects of metronidazole
and/or other antibiotics [25].
Herr and You evaluated 50 rosacea patients and 50 controls to compare the
seroprevalence of H. pylori and evaluate an effect of the eradication therapy on rosacea.
Forty-two (84%) of 50 patients with rosacea and 39 (78%) of 50 controls were found positive
for H. pylori, showing no significant difference. The numbers of papulopustules and the
intense of erythema demonstrated no significant difference between the baseline and the
follow-ups both active treatment and after that. There was no improvement with the
eradication therapy in rosacea symptoms [29].
In another similar study, 25 rosacea patients and 87 controls were evaluated. IgG and IgA
antibodies against H. pylori were detected in both groups. Triple eradication therapy was
administered to the patients. The severity of rosacea was scored before and after therapy. No
significant difference was found in seropositivity in either group but there was a significant
decrease in the severity of H. pylori infection in positive rosacea patients at the end of the
treatment as compared with the initial scores [30].
Argenziano et al. studied serum IgG and IgA antibodies and anti-CagA against H. pylori
in 48 rosacea patients. IgG antibodies were present in 81% of the rosacea patients with
dyspepsia and 16% of the rosacea patients without dyspeptic symptoms. IgA antibodies were
present in 62% of patients with dyspepsia and in 6% of patients with no symptoms. Anti-
CagA antibodies were seen to be present in 75% of patients with dyspeptic rosacea patients,
and were prevalent in patients affected by rosacea with papules than erythematous rosacea
[31].
There are various studies investigating the association between H. pylori and rosacea.
Nevertheless, many conflicting results still exist. Some studies showed that by the eradication
of H. pylori, symptoms of rosacea were improved. However, because many regimens
employed to eradicate H. pylori infection are based on the use of metronidazole, it is not
exactly ruled out a direct antioxidant effect of metronidazole or the eradication of H. pylori on
rosacea. Because of the capacity of H. pylori producing inflammatory cytokines and the
capacity of CagA to stimulate IL-8 production, it should be investigated in rosacea patients as
a possible etiological factor [22, 29, 31].
Psoriasis Vulgaris
Psoriasis vulgaris is a chronic, immune-mediated inflammatory disease that affects

around 2% of the population in USA and Western Europe. The prevalence varies depending
on geographic area and racial groups. The variances depend on both genetic background and
exogenous factors, such as diet, cigarette smoking, infectious diseases, sunlight exposure, and
other environmental factors. The course is chronic by periods of exacerbation and remission.
Clinically, erythematous and scaly plaques are the primary lesions. The classic plaques are
mostly seen on knees, elbows, scalp, umbilicus, and gluteal cleft. Psoriasis has been

conceptualised as a T lymphocyte mediated autoimmune disease. Series of linked cellular

changes are defined such as hyperplasia of epidermal keratinocytes, vascular hyperplasia and
ectasia, and infiltration of T lymphocytes, neutrophils, and other leukocytes [32, 33, 34].
Onsun et al. evaluated 300 psoriasis patients and 150 controls to determine the prevalence
of H. pylori in both groups and the relationship between PASI (Psoriasis Area and Severity
Index) scores and the infection, and eradication therapy. Fifty patients with H. pylori
infections were randomly assigned to one of two groups, one of which received acitretin with
H. pylori treatment and the other acitretin alone. H. pylori infection was determined in 184
patients (61.3%) and 89 of 150 controls (59.3%). No significant differences between two
groups were found in terms of the prevalence of H. pylori infection. Patients who received
acitretin and eradication triple therapy for H. pylori improved more rapid than those who
received acitretin alone. Acitretin treatment was effective in both groups, but the p- value
associated with co-treatment for H. pylori was statistically more significant. This study
suggested that H. pylori played a role in the severity of psoriasis and eradication therapy was
sovereign improving the skin lesions more rapidly [35].
In another study, 50 psoriasis patients and 50 controls were detected for H. pylori
antibodies. Twenty psoriasis patients (40%) and 5 controls (10%) had H. pylori antibodies. In
this study there was a significant difference in the seropositivity of two groups [36].
Fabrizi et al. evaluated 20 psoriasis patients and 29 controls in an age range of 5-19
years. All patients were tested with 13C-urea breath test. Two of 20 psoriasis patients and 5 of
29 controls had a positive result. This study showed that there was a low prevalence of H.
pylori infection in age 5-19 years, also there was no relationship between H. pylori infection
and psoriasis at least in children and teenagers [37].
In one case report, 35-year-old man, presenting with a severe, refractory palmoplantar
psoriasis received several treatments but did not respond well to those treatments. Due to the
patient’s history of dyspepsia, 13C-urea breath test was performed and found positive. After
the triple eradication therapy for H. pylori infection, symptoms of palmoplantar psoriasis
started to improve. One year later there was no symptoms of psoriasis and no reinfection with
H. pylori. Even this was just one case, H. pylori should be thought as an etiologic factor of
psoriasis and be treated [38].
Behçet’s Disease
Behçet’s disease is a chronic, multisystemic inflammatory disorder, consisting of oral

aphthous ulcers, ocular symptoms, genital ulceration, skin lesions, and relapsing vasculitis. It
is more prevalent in Far East, Middle East and the Mediterranean countries. Genetic factors,
immunological findings, environmental factors such as infectious agents, and endothelial
factors have been implicated in the etiopathogenesis of the disease [39]. Involvement of the
gastrointestinal system is called Entero-Behçet’s disease. The gastric mucosa appears to be
less involved than the other parts of gastrointestinal system [3].
Ersoy et al. evaluated 45 patients with Behçet’s disease and 40 controls to study the
prevalence of H. pylori. They found no significant difference between two groups in respect
to the prevalence of H. pylori (73% vs 75%), and eradication rate (75% vs 70%) [40].
In another study, 26 patients with Behçet’s disease and 28 controls were underwent
fiberoptic esophagogastroduodenoscopy and tested for identifying H. pylori with urea breath

test. H. pylori was found positive in 26 patients (65%) with Behçet’s disease and in 28
controls (70%) and showed that there was no significant difference between two groups.
They suggested that the routine endoscopy for H. pylori infection might not be necessary
in asymptomatic patients with Behçet’s disease [41].
Apan et al. included 91 patients with Behçet’s diseases and 83 controls with or without
any gastrointestinal complaints. H. pylori IgG, IgM, and Cag-A IgG antibodies were tested in
both groups. They also studied the effect of eradication therapy on clinical findings. There
was no association between H. pylori IgG and IgM on both samples of Behçet’s disease and
control group. The prevalence of Cag-A positivity was significantly higher in Behçet’s
disease compared to the controls (64.8% vs 38.5%). Eradication of H. pylori had also
improved oral and genital ulceration, arthritis/arthralgia, and cutaneous findings of Behçet’s
disease [42].
H. pylori may be involved in the pathogenesis of Behçet’s disease or clinical
manifestations may be improved by the means of eradication therapy. So especially patients
with the gastrointestinal symptoms should be tested for H. pylori.
Other Diseases
Alopecia areata is an immune-mediated form of hair loss characterised by peribulbar

lymphocytic infiltrate. It affects 1-2% of the population. The pathogenesis of alopecia areata
is still not fully understood. Clinical course is variable and any hair-bearing area can be
involved. There is an association between alopecia areata and other autoimmune diseases
such as autoimmune thyroiditis, Sjögren syndrome, lichen planus, and idiopathic
thrombocytopenic purpura [43, 44]. In a study, 31 patients with alopecia areata and 24
controls were evaluated for the presence of H. pylori stool antigen (HpSAg). Eighteen of 31
(58.1%) patients with alopecia areata were positive, while 10 of 24 (41.7%) controls were
positive for H. pylori. It was suggested that they found no evidence for an increased
association between H. pylori infection and alopecia areata [45]. Rigopouos et al. also
reported no increased prevalence of H. pylori infection in patients with alopecia areata [46].
Campuzano-Mayo reported a case of 43-year-old-man who had a long time of alopecia areata
on the scalp and beard areas. He had a history of dyspepsia and the urea breath test confirmed
H. pylori infection. After the eradication treatment, the symptoms of alopecia areata were also
improved. This is only one case report, it must be confirmed with epidemiological studies
[47].
Lichen planus is an inflammatory disease of skin and mucous membranes. The exact
etiology and pathogenesis of lichen planus is unknown; different causes including genetic
susceptibility, stress, drugs, and bacterial and viral infections may act as risk factors [48].
Zerouz et al. studied 30 patients with skin lichen planus, 30 patients with oral lichen planus,
and 30 controls. They were all tested with urea breath test (UBT) for H. pylori. UBT was
positive in 24 patients (80%) with oral lichen planus, 22 patients (73.3%) with skin lichen
planus, and 20 (66.7%) controls. No significant difference was found in the prevalence of H.
pylori in three groups [49]. Pourshahid et al. evaluated 41 patients with oral lichen planus and
82 sex-age-matched controls in Iran. Anti H. pylori IgG levels were determined in all
patients. The result showed no association between H. pylori infection and oral lichen planus
(51% vs 66%) [50]. Izol et al. evaluated 49 patients with lichen planus and 35 controls with

gastrointestinal symptoms. Lichen planus group was divided into subgroups regarding
gastrointestinal symptoms. Upper gastrointestinal system endoscopy was performed in both
groups and biopsies were taken from suspicious areas. H. pylori positivity was found higher
in lichen planus group (81.6%) than control group [51].
Angioedema is characterised by recurrent edema in the subcutis, submucosa, or both.
There are two forms: hereditary and acquired form. Hereditary angioedema results from
deficiency of C1-esterase inhibitor which has an autosomal dominant trait [52]. Visy et al.
evaluated 152 patients in seven collaborating centers to detect any relationship between H.
pylori infection and the attacks of hereditary angioedema. Abdominal attacks were found
more frequent among the H. pylori infected patients. They also suggested that successful
eradication of H. pylori reduced the numbers of attacks in those patients [53]. A 10-year-old
boy with H. pylori infection and simultaneous angioedema was reported. All possible
causative factors were eliminated and H. pylori was proven serologically and
histopathologically. After the eradication therapy, a complete remission was seen in the child.
That case suggested that H. pylori infection should be considered as a causative factor for
angioedema [54].
There were 2 patients reported with erythema nodosum and H. pylori infection. Triple
eradication therapy led to disappearence of erythema nodosum in those patients. But this
subject clearly needs further studies [55].
Sweet syndrome is one of the neutrophilic dermatosis which is characterised by acute
onset of fever, tender erythematous plaques or nodules, and histopathological findings of
dense neutrophilic infiltrate without leukocytoclastic vasculitis [56]. In one case report, a 42-
year-old woman with Sweet syndrome who had also H. pylori infection, healed with the
eradication therapy [57].
A study reported from China, evaluated 749 Henoch-Schönlein purpura (HSP) and 560
controls for metaanalysis. Three-hundred-sisxty-nine of 749 (49.27%) HSP patients and 131
of 560 (23.39%) controls had H. pylori infection. They also suggested that eradication therapy
reduced the recurrent of HSP [58]. There were also case reports that showed the association
between HSP and H. pylori. In one of them, 13-year-old boy with HSP, was reported.
Treatment of H. pylori infection was accompanied by rapid resolution of HSP [59]. In another
case report, the gastrointestinal manifestation and purpuric rashes resolved dramatically after
H. pylori eradication therapy in a 33-year-old man [59].
Conclusion
The association of H. pylori infection and skin diseases is a matter of dispute. The results
of studies investigating H. pylori seropositivity in skin diseases and the effect of eradication
are conflicting. Some studies suggest that there is an association between H. pylori infection
and dermatological diseases, some of them deny. The etiopathogenic mechanism how H.
pylori causes certain skin diseases is unknown. Possible triggering factors for the
development of dermatological diseases could be the production of specific cytotoxins,
immunological stimulation by chronic infection, production of pathogenetic antibodies
possibly by molecular mimicry, and the release of vascular mediators like histamines.
Immunological stimulation by H. pylori infection may produce, through mediator release, a

nonspecific increase in sensitivity of the cutaneous vessels to vasopermeability-enhancing

agents. H. pylori might be a source of circulating immune complexes and these immune
complexes may trigger skin diseases. The best evidence came from studies investigating
chronic urticaria in which chronic urticaria disappeared in many patients with H. pylori
infection after careful eradication. In addition to this, there have been promising recent
reports of beneficial H. pylori eradication in Behçet's disease, cutaneus pruritus, prurigo
chronica, prurigo nodularis, and lichen planus. It is not known if H. pylori is the cause or the
trigger for those diseases or it is just a cooccurence. More case-control studies need to be
done to reveal the association.
References
[1] Hernando-Harder AC, Booken N, Goerdt S, Singer MV, Harder H., (2009).
Helicobacter pylori infection and dermatologic diseases. Eur. J. Dermatol. 19, 431-444.
[2] Bohr UR, Annibale B, Franceschi F, Roccarina D, Gasbarrini A., (2007). Extragastric
manifestations of Helicobacter pylori infection – other Helicobacters. Helicobacter.
Supp 1, 45-53.
[3] Vaira D, Holton J, Menegatti M, Ricci C, Landi F, Ali A et al. (1999). New
immunological assays for the diagnosis of Helicobacter pylori infection. Gut. Suppl 1,
I23-27.
[4] Alkhalifah A., (2012). Alopecia areata update. Dermatol. Clin. 31, 93-108.
[5] Tüzün Y, Keskin S, Kote E., (2010). The role of Helicobacter pylori infection in skin
diseases: facts and controversies. Clin. Dermatol. 28, 478-482.
[6] Cremonini F, Gasbarrini A, Armuzzi A, Gasbarrini G., (2001). Helicobacter pylori-
related diseases. Eur. Clin. Invest. 31, 431-437.
[7] Tsang KW, Lam SK., (1999). Helicobacter pylori and extra-digestive diseases. J.
Gastroenterol. Hepatol. 14, 844-850.
[8] Hellmig S, Troch K, Ott SJ, Schwarz T, Fölsch UR.,(2008). Role of Helicobacter pylori
infection in the treatment and outcome of chronic urticaria. Helicobacter.13, 341-345.
[9] Ben Mahmoud L, Ghozzi H, Hakim A, Sahnoun Z, Zeghal K., (2011). Helicobacter
pylori associated with chronic urticaria. J. Infect. Dev. Ctries. 5, 596-598.
[10] Dauden E, Jimenez-Alonso I, Garcia-Diez A., (2000). Helicobacter pylori and
idiopathic chronic urticaria. Int. J. Dermatol. 39, 446-452.
[11] Shiotani A, Okada K, Yanaoka K, Itoh H, Nishioka S et al. 2001. Beneficial effect of
Helicobacter pylori eradication in dermatologic diseases. Helicobacter. 6, 60-65.
[12] Gala Ortiz G, Cuevas Agustin M, Erias Martinez P, de la Hoz Caballer B, Fernandez
Ordonez R et al. (2001). Chronic urticaria and Helicobacter pylori. Ann. Allergy.
Asthma. Immunol.86, 696-698.
[13] Magen E, Mishal J, Schlesinger M, Scharf S., (2007). Eradication of Helicobacter
pylori infection equally improves chronic urticaria with positive and negative
autologous serum skin test. Helicobacter. 12, 567-571.
[14] Yadav MK, Rishi JP, Nijawan S., (2008). Chronic urticaria and Helicobacter pylori.
Indian. J. Med. Sci. 62, 157-162.

[15] Magen E, Mishal J., (2013). Possible benefit from treatment of Helicobacter pylori in
antihistamine-resistant chronic urticaria. Clin. Exp. Dermatol. 38, 7-12.
[16] Moreira A, Rodrigues J, Delgado L, Fonseca J, Vaz M., (2003). Is Helicobacter pylori
infection associated with chronic idiopathic urticaria? Allergol. Immunopathol. 31, 209-
214.
[17] Wedi B, Raap U, Wieczorek D, Kapp A., (2009). Urticaria and infections. Allergy.
Asthma. Clin. Dermatol. 5, 10.
[18] Sadighha A, Shirali R, Zahed GM., (2009). Relationship between Helicobacter pylori
and chronic urticaria. J. Eur. Acad. Dermatol. Venereol. 23, 198-199.
[19] Buechner SA., (2005). Rosacea: an update. Dermatology. 210, 100-108.
[20] Crawford GH, Pelle MT, James WD., (2004). Rosacea I: etiology, pathogenesis, and
subtype classification. J. Am. Acad. Dermatol. 51, 327-341.
[21] Del Rosso JQ., (2012). Advances in understanding and managing rosacea: part 1:
connecting the dots between pathophysiological mechanisms and common clinical
features of rosacea with emphasis on vascular changes and facial erythema. J. Clin.
Aesthet. Dermatol. 5, 16-25.
[22] Lazaridou E, Giannopoulou C, Fotiadou C, Vakirlis E, Trigoni A, Ioannides D., (2011).
The potential role of microorganisms in the development of rosacea. J. Dtsch.
Dermatol. Ges. 9, 21-25.
[23] Yamashi K, Gallo RL., (2009). The molecular payhology of rosacea. J. Dermatol. Sci.
55, 77-81.
[24] Reinholz M, Ruzicka T, Schauber J., (2012). Cathelicidin LL-37: an antimicrobial
peptide with a role in inflammatory skin disease. Ann. Dermatol. 24, 126-135.
[25] Abram K, Silm H, Maaroos HI, OOna M., (2012). Risk factors associated with rosacea.
J. Eur. Acad. Dermatol. Venereol. 24, 565-571.
[26] Szlachcic A., (2002). The link between Helicobacter pylori infection and rosacea. J.
Eur. Acad. Dermatol. Venereol. 16, 328-333.
[27] Baz K, Cimen MY, Kokturk A, Aslan G, Ikizoğlu G et al. (2004). Plasma reactive
oxygen species activity and antioxidant potential levels in rosacea patients: correlation
with seropositivity to Helicobacter pylori. Int. J. Dermatol. 43, 494-497.
[28] Gurer MA, Erel A, Erbas D, Caglar K, Atahan C., (2002). The seroprevalence of
Helicobacter pylori and nitric oxide in acne rosacea. Int. J. Dermatol. 41, 768-770.
[29] Herr H, You CH., (2000). Relationship between Helicobacter pylori and rosacea: it may
be a myth. J. Korean. Med. Sci. 15, 551-554.
[30] Utas S, Ozbakir O, Turasan A, Utas C., (1999). Helicobacter pylori eradication
treatment reduces the severity of rosacea. J. Am. Acad. Dermatol. 51, 327-341.
[31] Argenziano G, Donnarumma G, Iovene MR, Arnese P, Baldassare MA et al. (2003).
Incidence of anti-Helicobacter pylori and anti-CagA antibodies in rosacea patients. Int.
J. Dermatol. 42, 601-604.
[32] Johnson-Huang LM, Lowers MA, Krueger JG., (2012). Putting together the psoriasis
puzzle: an update on developing targeted therapies. Dis. Model. Mech. 5, 423-433.
[33] Nograles KE, Davidovici B, Krueger JG., (2010). New insights in the immunologic
basis of psoriasis. Semin. Cutan. Med. Surg. 29, 3-9.
[34] Krueger JG, Bowcock A., (2005). Psoriasis pathophysiology: current concepts of
pathogenesis. Ann. Rheum. Dis. 64, Suppl 2, 30-36.

[35] Onsun N, Arda Ulusal H, Su O, Beycan I, Biyik Ozkaya D et al. (2012). Impact of
Helicobacter pylori infection on severity of psoriasis and response to treatment. Eur. J.
Dermatol. 22, 117-120.
[36] Qayoom S, Ahmad QM., (2003). Psoriasis and Helicobacter pylori. Indian. J.
Dermatol. Venereol. Leprol. 69, 133-134.
[37] Fabrizi G, Carbone A, Lippi ME, Anti M, Gasbarrini G., (2001). Lack of evidence of
relationship betweenHelicobacter pylori infection and psoriasis in childhood. Arch.
Dermatol. 137, 1529.
[38] Martin Hübner A, Tenbaum SP., (2008). Complete remission of plamoplantar psoriasis
through Helicobacter pylori eradication: a case report. Clin. Exp. Dermatol. 33, 339-
340.
[39] Kapsimali VD, Kanakis MA, Vaiopoulos GA, Kaklamanis PG., (2010).
Etiopathogenesis of Behcet’s disease with emphasis on the role of immunological
aberrations. Clin. Rheumatol. 29, 1211-1216.
[40] Ersoy O, Ersoy R, Yayar O, Demirci H, Tatlican S., (2007). H. pylori infection in
patients with Behcet’s disease. World. J. Gastroenterol. 13, 2983-2985.
[41] Cakmak SK, Cakmak A, Gül U, Sulaimanov M, Bingöl P et al. (2009). Upper
gastrointestinal abnormalities and Helicobacter pylori in Behcet’s disease. Int. J.
Dermatol. 48, 1174-1176.
[42] Apan TZ, Gürsel R, Dolgun A., (2007). Increased seropositivity of Helicobacter pylori
cytotoxin-associated-gene-A in Behcet’s disease. Clin. Rheumatol. 26, 885-889.
[43] Ito T., (2012). Advances in the management of alopecia areata. J. Dermatol. 39, 11-17.
[44] Abdel-Hafez HZ, Mahran AM, Hofny ER, Attallah Da, Sayed DS et al. (2009). Is
Helicobacter pylori infection associated with alopecia areata? Journal. Cosmet.
Dermatol. 8, 52-55.
[45] Rigopoulos D, Katsambas A, Karalexis A, Papatheodorou G, Rokkas T., (2002). No
increaesd prevalence of Helicobacter pylori in patients with alopecia areata. J. Am.
Acad. Dermatol. 46, 141.
[46] Campuzano-Maya G., (2011). Cure of alopecia areata after eradication of Helicobacter
pylori: a new association. 14, 3165-3170.
[47] Sanli H, Cetinkaya H, Türsen U, Kaya M, Kuzu I et al. (2002). Upper gastrointestinal
findings in oral lichen planus. Turk. J. Gastroenterol. 13, 31-34.
[48] Taghavi Zenouz A, Mehdipour M, Jafari Heydarlou M, Gholizadeh N., (2010).
Relationship between lichen planus and Helicobacter pylori infection. J. Dent. Res.
Dent. Clin. Dent. Prospects. 4, 17-20.
[49] Pourshahidi S, Fakhri F, Ebrahimi H, Fakhraei B, Alipour A et al. (2012). Lack of
association between Helicobacter pylori infection and oral lichen planus. Asian. Pac. J.
Cancer. Prev. 13, 1745-1747.
[50] Izol B, Karabulut AA, Biyikoglu I, Gonultas M, Eksioglu M., (2010). Investigation of
upper gastrointestinal tract involvement and H. pylori presence in lichen planus: a case-
controlled study with endoscopic and histopathological findings. Int. J. Dermatol. 49,
1121-1126.
[51] Mukeba D, Chandrikakumari K, Giot JB, Leonard P, Meuris C et al. (2009).
Autoimmune angioneurotic edema in a patient with Helicobacter pylori infection.
Helicobacter. 14, 9-11.

[52] Visy B, Füst G, Bygum A, Bork K, Longhurst H, Bucher C et al. (2007). Helicobacter
pylori infection as a triggering factor of attacks in patients with hereditary angioedema.
Helicobacter. 12, 251-257.
[53] Varvarovska J, Sykora J, Stozicky F, Chytra I., (2003). Acquired angioedema and
Helicobacter pylori infection in a child. Eur. J. Pediatr. 162, 707-709.
[54] Capella GL., (2009). Chronic erythema nodosum circumstantially linked with
Helicobacter pylori infection: where is the evidence? Dermatology. 219, 86.
[55] Kalai C, Brand R, Yu L., (2012). Minocycline-induced Sweet syndrome (acute febrile
neutrophilic dermatosis. J. Am. Acad. Dermatol. 67, 289-291.
[56] Kürkcüoglu N, Aksoy F., (1997). Sweet’s syndrome associated with Helicobacter
pylori infection. J. Am. Acad. Dermatol. 37, 123-124.
[57] Xiong LJ, Tong Y, Wang ZL, Mao M., (2012). Is Helicobacter pylori infection
associated with Henoch-Schonlein purpura in Chinese children? A metaanalysis. World.
J. Pediatr. 8, 301-308.
[58] Mytinger JR, Patterson JW, Thibault ES, Webb J, Saulsbury FT., (2008). Henoch-
Schönlein purpura associated with Helicobacter pylori infection in a child. Pediatr.
Dermatol. 25, 630-632.
[59] Hoshino C., (2009). Adult onset Schönlein-Henoch purpura associated with
Helicobacter pylori infection. Intern. Med. 48, 847-851.

Chapter 19

in Children
Stefano Pontone1, Laura Petrarca2 and Raffaella Nenna2

1
Department of Surgical Sciences, “Sapienza” University of Rome, Italy
2
Department of Pediatrics, “Sapienza” University of Rome, Italy
Abstract
Helicobacter pylori (H. pylori) is implicated in more severe gastric diseases. The
prevalence of H. pylori infection is low in the northern and western European countries
(below 10%) in children and adolescents, but still common in areas such as southern or
eastern Europe, Mexico, and immigrant populations. This difference in terms of H. pylori
infection prevalence reflects socio-demographic factors, such as low socio-economic
status, low-income family, poor and overcrowded living conditions. The human host is
the principal reservoir and the transmission occurs through person-to-person by either
oral-oral or fecal-oral rout, even if investigators have proposed environmental or animal
reservoirs of H. pylori infection. H. pylori infections could be asymptomatic in the most
of people and once acquired, it can persist for a long period. In children, symptoms of H.
pylori infections are nonspecific such as epigastric pain especially after meals, nausea
and/or vomiting, anorexia, hematemesis and iron deficiency anemia. Several tests, as
stool antigen, serology and urea breath testing, are available to diagnose H. pylori
infection. The invasive tests require an upper gastrointestinal endoscopy and at least a
gastric mucosa sample to be performed. The treatment of H. pylori in children is
recommended if infection is established on either positive histopathology plus positive
rapid urease test or a positive culture. The goal of treatment is at least a 90% eradication
rate after the first attempt. First-line eradication regimens include a triple therapy or
sequential therapy. Despite the negative impact of clarithromycin resistance, a correct
algorithm treatment allows a satisfactory eradication rate in most cases.

Correspondence to: Dr. Stefano Pontone Department of Surgical Sciences, “Sapienza” University of Rome V.le
Regina Elena n° 324 00161 - Rome, Italy; tel/fax: 00390649975503; email: stefano.pontone@uniroma1.it.

338 Stefano Pontone, Laura Petrarca and Raffaella Nenna
Keywords: Helicobacter pylori; Children; Gastritis; Clarithromycin resistance, Gram-

negative, CagA, UBT
Introduction
Helicobacter pylori (H. pylori) is a micro-aerophilic, Gram-negative, spiral-shaped and
flagellated bacterium, that has been identified for the first time in 1982 by Barry Marshall and
Robin Warren as an important etiological factor of chronic gastritis and peptic ulcer disease,
previously considered stress-related diseases.
The presence of the bacterium in the gastric mucosa is associated with chronic active
gastritis and is implicated in more severe gastric diseases, including chronic atrophic gastritis,
peptic ulceration and mucosa-associated lymphoid tissue lymphomas [1,2] and it has been
declared a carcinogen of class I by the World Health Organization [3]. H. pylori infection is
estimating affecting about half of the world population, involving adults as well as children,
fortunately only a few cases of lymphoma has been described in pediatric age population
[4,5].
Prevalence
The prevalence of H. pylori infection is low in the northern and western European
countries (below 10%) in children and adolescents [6]. The bacterium has infected over half
of the global population, and its prevalence rates vary widely between different geographical
areas and ethnic groups, increasing with age and demonstrating an acquisition rate in adults of
3% to 4% per decade [7].
The infection is still common in areas such as southern or eastern Europe, Mexico, and
immigrant populations (South America, Africa, most Asian countries and aboriginal people in
North America) [8-10].
A recent review article on H. pylori infection, reported a decreasing prevalence
worldwide, both in children and in adults [11], although the gap between developed and
developing countries remains still high, ranging from 30.7% in the USA [12], 44.4% in
Germany [13] and 74.4% in Pakistan [14].
This difference in terms of H. pylori infection prevalence reflects socio-demographic
factors, such as low socio-economics status, low-income family, poor and overcrowded living
conditions.
Several surveys have been carried out on children in the 2000s. Bauer et al. carried out a
long-term follow-up study on H. pylori prevalence in school-age, demonstrating that in 2006
H. pylori prevalence was 6.5% and it had not significantly changed since 1998 and 2000
(6.1%, 5.7% respectively) [15].
In 2011 Oleastro et al. evaluated the H. pylori infection in healthy children living in the
Lisbon area using the stool antigen test, reporting 31.6% H. pylori prevalence among the
overall sample [16]. The authors demonstrated also that the H. pylori prevalence in their
sample increased with age, corroborating the theory of the birth cohort effect, which argues
that there is a fall in the rate of acquisition in successive generation due to improvements in

Helicobacter pylori Infection in Children 339
health knowledge and in living conditions. Furthermore den Hoed et al. in 2011 performed a
study in 7-9 years aged Dutch children establishing that the 9% H. pylori colonization
observed was similar to the one found in 1993 in a comparable cohort, with the same method,
suggesting a stabilization of the previously decreasing trend observed, probably due to the
improvement of the socio-economic circumstances that has occurred in Netherlands from the
1940’s to the 1980’s [17].
Even though more studies in other population are necessary to demonstrate the
stabilization of H. pylori infection in children in western countries, by improving the socio-
economic conditions and by educating people about the H. pylori transmission modes, a
reduction in the infection rate in children could be achieved.
Transmission of Infection
The age of bacteria infection has been advised as having an important influence for the
later adverse outcomes [6], and also the combination of host and bacterial factors plays a
crucial role in developing disease phenotypes.
Currently, the majority of the available evidence identifies the human host as the
principal reservoir and the transmission occurs through person-to-person by either oral-oral or
fecal-oral rout [18].
The bacteria have been isolated from feces, saliva, dental plaque [19] and vomitus [20] of
infected people.
Recently, other investigators have proposed environmental or animal reservoirs of H.
pylori infection. Helicobacter spp has been identified in oral secretion of stray cats in Iran
[21] and in water samples (tap water, dental units’ water, refrigerated waters with filtration
and public cooler water samples) in Isfahan province of Iran as well [22], but the potential
risk of infection via consumption of contaminated water or colonized animals has to be
demonstrated.
It is well known that childhood is an important period for acquisition of H. pylori
infection and several studies have suggested that children acquire H. pylori most frequently
from their mothers during the first year of life [23], especially in population with low H.
pylori prevalence, while control for infection status of siblings and fathers is also desirable
when contributions of mother to the acquisition of H. pylori infection in early childhood are
assessed.
Clinical Presentation
H. pylori infections could be asymptomatic in the most of people and once acquired, it
can persist for a long period.
The development of clinically symptomatic infection depends on a complex combination
of host, environmental, and bacterial factors. A defect in the ability of autophagy, probably
induced by H. pylori itself, has been associated with a higher risk of gastric cancer. H. pylori
survival at acidic pH is ensured by a characteristic enzyme: a multisubunit urease, has
evolved numerous strategies to persistence within the gastric mucosa including diverse

mechanisms to evade host immune surveillance. Concerted activity of two toxins: cytotoxin-
associated protein (CagA) and active vacuolating cytotoxin (VacA) mediates the mis-sorting
via perturbation of cell polarity and transferrin recycling. H. pylori is also able to acquire iron
from heme and/or hemoglobin under conditions of iron starvation [24].
In children, symptoms of H. pylori infections are nonspecific such as epigastric pain
especially after meals, nausea and/or vomiting, anorexia, hematemesis and iron deficiency
anemia.
H. pylori infection is etiologically related to gastric ulcer and duodenal ulcer disease,
being responsible of almost 100% of the cases in adults not using non-steroidal anti-
inflammatory drugs. In childhood H. pylori is the most important factor for developing
duodenal ulcer disease, while in gastric ulcer H. pylori infection appears to be a risk factor but
other causes are responsible for most cases [25]. In literature, perforated peptic ulcer has been
reported in approximately 10% of adults with peptic ulcer disease, however it occurs less
frequently in children, and its relationship with H. pylori infection has to be further
investigated [26].
The link between H. pylori infection and iron deficiency anemia has been widely
investigated in literature [27,28], and it seems to be due to a dual mechanism: on the one hand
H. pylori competes with the host for the adsorption of metal, at the same time, the iron
contained in the epithelial cells is forced out and adsorbed by the bacterium, leading to an
additional deficiency [29]. However, an endoscopic evaluation should be performed in
children with refractory iron deficiency anemia, in order to rule out not only the presence of
H. pylori, but also other causes of malabsorption.
Recurrent abdominal pain has been advocated as one of the clinical presentation of H.
pylori infection in childhood but several studies has been performed and no correlation has
been established [30,31] hence the recent ESPGHAN/NASPGHAN guidelines for the
management of H. pylori infection suggest do not test and treat children with recurrent
abdominal pain [32].
H. pylori seems to be correlated to gastro-esophageal reflux disease [33], and it has been
advocated as an independent protective factor for a positive diagnosis of childhood asthma
[34].
H. pylori has been also associated to primary gastric Non-Hodgkin Lymphoma, a very
rare condition in children [4,5].
A widely spread of extra-intestinal manifestation has been correlated to H. pylori
infection, such as otitis media, upper respiratory tract infections, food allergy, SIDS,
idiopathic thrombocytopenic purpura and short stature, but the evidence is scant [35-38].
Sometimes H. pylori infection coexists with coeliac disease diagnosis [39].
Diagnosis: Who to Test and How?

Recently, the new European Society for Pediatric Gastroenterology, Hepatology and
Nutrition (ESPGHAN) and North American Society of Pediatric Gastroenterology,
Hepatology and Nutrition (NASPGHAN) guidelines on H. pylori infection in children
recommended testing only symptomatic patients or children with first-degree relatives with
gastric cancer (grade of evidence: low), but not children with functional abdominal pain,

bearing in mind that the primary goal of the clinical investigation of gastrointestinal
symptoms is to determine its cause, and not solely the H. pylori infection [32].
Several tests are available to diagnose H. pylori infection. They are divided in non-
invasive and invasive tests. The former include research of stool antigen, serology and urea
breath testing (UBT), the latter comprise histological examination, rapid urease test, culture,
polymerase chain reaction and fluorescence in situ hybridization.
Among the non-invasive tests, the stool antigen test is easy to perform, even in non-
collaborating children. The UBT, instead, requires an active collaboration by the children
besides a preparation, but shows high sensitivity and specificity [40]. These two tests should
be performed to determine whether H. pylori has been eradicated [32]. It is recommended that
testing be performed at least 4 weeks after stopping antibiotics and 2 weeks after cessation of
PPI therapy.
Serologic assays should be used in children and adolescents for either diagnosis of H.
pylori infection or to monitor the success of therapy because the sensitivity and specificity for
detection of antibodies against H. pylori in children varies widely, and a positive IgG test can
occur several months or even years after the infection, so the use of serological assays in
clinical settings should be avoided [32].
The invasive tests require an upper gastrointestinal endoscopy and at least a gastric
mucosa sample to be performed. However, a very adequate pain and anxiety control is
mandatory during upper endoscopy in order to achieve a better compliance and adverse
events monitoring. The risk factors associated with anesthesia are: younger age and lower
weight, drug combinations with propofol and, specially, airway intervention procedures.
The histopathologic determination by bioptic samples is the only method that can detect
H. pylori, lesions associated with the infection, and other possible causes of the patient’s
symptoms when PPI or antibiotics therapy is being. According to the revised Sydney
classification (at least 4 samples should be taken (two biopsies from the antrum, two from the
fundus, and one from the angulus) in adults [41]. In children with suspected H. pylori
infection is highly recommended to take not only biopsies for histopathology, but also for
rapid urease test and, if possible, for culture. Rapid urease test can be performed in biopsy
specimens using homemade or commercially available reagents. Culture is the only method
that consistently has 100% specificity (reference standard), but sensitivity varies depending
on the experience of the laboratory [40]. Diagnosis of H. pylori can be performed by using
fluorescent in situ hybridization (FISH) or PCR in biopsy specimens, although commercial
kits for FISH and PCR are now available, they are used primarily in academic institutions.
Treatment
The treatment of H. pylori in children is recommended if infection is established on either
positive histopathology plus positive rapid urease test or a positive culture [13]. ESPGHAN
and NASPGHAN recommend triple therapy using a PPI plus amoxicillin (50 mg/kg/die, two
times/die) plus metronidazole (20 mg/kg/die two times/die), or a PPI plus amoxicillin plus
clarithromycin (15 mg/kg/die, two times/die), or bismuth salts plus amoxicillin plus
metronidazole as the first-line eradication regimen for H. pylori infection in children (Figure
1) [32].

Figure 1. ESPGHAN and NASPGHAN proposed algorithm of how to treat Helicobacter pylori
infection in pediatric patients. AMO = amoxicillin; CLA = clarithromycin; EGD =
esophagogastroduodenoscopy; FISH =fluorescence in situ hybridization; HP = H. pylori; MET =
metronidazole; PPI = proton pump inhibitor; PUD =peptic ulcer disease. (*) In areas or populations
with a primary clarithromycin resistance rate of >20%.
The duration of the triple therapy should be 7 to 14 days, taking into account costs,
compliance and adverse effect. Bismuth-based triple therapy is also recommended as an
alternate first-line therapy, and it seems to be more efficacious than PPI containing therapy
when used as first line treatment.
However, several studies reported a high clarithromycin and metronidazole resistance
rate in pediatric population than those in adults [42], which is correlated with the frequency of
prior antibiotic prescription (respiratory tract infections treatment). Thus, monitoring of
primary clarithromycin resistance needs to be executed to facilitate the appropriate
eradication regimens. Therefore, clarithromycin-based therapy should be performed only if
susceptibility testing in the individual patient revealed a clarithromycin-susceptible strain or

of clarithromycin resistance rate in this area is known to be low [13]. The Polymerase Chain
Reaction (PCR)-RFLP/DNA sequencing or real-time PCR combining with melting curve
analysis using stool specimens could be a reliable and rapid non-invasive methods with high
specificity to determine the clarithromycin resistance [43].
As alternative treatment, the sequential therapy is a novel promising approach, that
includes a 10-day therapy consisting in a dual therapy with PPI plus amoxicillin for 5 days,
followed by 5 days of triple therapy (PPI plus clarithromycin and metronidazole/tinidazole).
Several adverse effects, such as diarrhoea, nausea/vomiting, or abdominal pain were reported.
In this case, probiotics seem to be efficacious in children for the prevention of antibiotic
associated side-effects. Instead, their effectiveness in increasing the eradication rate has yet to
be demonstrated even if several in vitro studies have shown that various lactobacilli can
inhibit H. pylori growth. Lactobacilli are known to produce by catabolism relatively large
amounts of lactate, and this has been considered as the inhibitory and ⁄ or the bactericidal
factor.
Sequential therapy, compared to standard triple therapy in 3–18 years patients only
moderately improves eradication rates. On the other hand, several studies state that sequential
therapy is superior compared to 7-day triple therapy, but not compared to 10- day or 14-day
triple therapy, giving a central role to the duration of therapy [44].
At least 4 to 8 weeks after completion of eradication therapy, a non-invasive test should
be performed to assess the success of the treatment. The goal of treatment is at least a 90%
eradication rate after the first attempt.
If treatment has failed, three options can be taken into account:
Upper gastrointestinal endoscopy, with culture and susceptibility testing;
FISH on previous paraffin-embedded biopsies, if available;
Modify therapy by adding/ or changing a drug or the duration of the therapy. Quadruple
therapy (PPI + metronidazole + amoxicillin + bismuth) is recommend as second line therapy
[45]. A triple therapy including a fluorochinolones (levofloxacin) has been proposed, though
its effectiveness is not well-known. However, the second-line antibiotics such as tetracycline
or quinolones (levofloxacin/moxifloxacin) are not approved for use in the pediatric age group
or may even be contraindicated below a certain age.
The eradication of H. pylori infection is much less effective as it is present the antibiotic
resistance, thus the interest for alternative therapies is a real actual topic.
Conlcusion
Helicobacter pylori infection is implicated in more severe pediatric gastric diseases. Even
if the prevalence in children is decreasing worldwide, the gap between developed and
developing countries remains still high. A first-line prevention is feasible and mandatory
during childhood. The eradication of Hp infection is an actual topic, especially considering
the clarithromycin resistance influence on eradication rate, caused by the antibiotics
prescription abuse during respiratory infections. However, a correct algorithm treatment
allows a satisfactory eradication rate also in pediatric patients.

References
[1] Kuipers EJ, (1997). Helicobacter pylori and the risk and management of associated
diseases: gastritis, ulcer disease, atrophic gastritis and gastric cancer. Aliment.
Pharmacol. Ther., 11:S171-88.
[2] Kuipers EJ, Malfertheiner P, (2004). Helicobacter pylori and non-malignant diseases.
Helicobacter., 9:S29-34.
[3] IARC Working Group on the Evaluation of Carcinogenic Risks to Humans.
Helicobacter pylori in: Schistosomes, Liver Flukes and Helicobacter pylori. Lyon:
IARC, 1994.
[4] Moschovi M, Menegas D, Stefanaki K, Constantinidou CV, Tzortzatou-Stathopoulou F,
(2003). Primary gastric Burkitt lymphoma in childhood: associated with Helicobacter
pylori? Med. Pediatr. Oncol., 41:444-7.
[5] Kurugoglu S, Mihmanli I, Celkan T, Aki H, Aksoy H, Korman U, (2002). Radiological
features in paediatric primary gastric MALT lymphoma and association with
Helicobacter pylori. Pediatr. Radiol., 32:82-7.
[6] Torres J, Pérez-Pérez G, Goodman KJ, Atherton JC, Gold BD, Harris PR, la Garza AM,
Guarner J, Muñoz O, (2000). A comprehensive review of the natural history of
Helicobacter pylori infection in children. Arch. Med. Res., 31:431–69.
[7] Goh KL, Chan WK, Shiota S, Yamaoka Y, (2011). Epidemiology of Helicobacter
pylori infection and public health implications. Helicobacter., 16:S1-9.
[8] Kawakami E, Machado RS, Ogata SK, Langner M, (2008). Decrease in prevalence of
Helicobacter pylori infection during a 10-year period in Brazilian children. Arq.
[9] Elitsur Y, Dementieva Y, Rewalt M, Lawrence Z, (2009). Helicobacter pylori infection
rate decreases in symptomatic children: a retrospective analysis of 13 years (1993 –
2005) from a gastroenterology clinic in West Virginia. J. Clin. Gastroenterol., 43:147-
51.
[10] Azevedo NF, Huntington J, Goodman KJ, (2009). The epidemiology of Helicobacter
pylori and public health implications. Helicobacter., 14 (Suppl 1):1-7.
[11] Tonkic A, Tonkic M, Lehours P, Mégraud F, (2012). Epidemiology and Diagnosis of
Helicobacter pylori Infection. Helicobacter., 17:1-8.
[12] Grad YH, Lipsitch M, Aiello AE, (2012). Secular trends in Helicobacter pylori
seroprevalence in adults in the United States: evidence for sustained race/ethnic
disparities. Am. J. Epidemiol., 175:54-9.
[13] Wex T, Venerito M, Kreutzer J, Gotze T, Kandulski A, Malfertheiner P, (2011).
Serological prevalence of Helicobacter pylori infection in Saxony-Anhalt, Germany, in
2010. Clin. Vaccine Immunol., 18:2109-12.
[14] Rasheed F, Ahmad T, Bilal R, (2011). Frequency of Helicobacter pylori infection using
13C-UBT in asymptomatic individuals of Bara- kaho, Islamabad, Pakistan. J. Coll
Physicians Surg. Pak., 21:379-81.
[15] Bauer S, Krumbiegel P, Richter M, Richter T, Roder S, Rolle- Kampczyk U, et al.
(2011). Influence of sociodemographic factors on Helicobacter pylori prevalence
variability among schoolchildren in Leipzig, Germany. A long-term follow-up study.
Cent. Eur. J. Public Health., 19:42-5.

[16] Oleastro M, (2011). Prevalence and incidence of Helicobacter pylori Infection in a

healthy pediatric population in the Lisbon area. Helicobacter., 5:363-72.
[17] den Hoed CM, Vila AJ, Holster IL, Perez-Perez GI, Blaser MJ, de Jongste JC, Kuipers
EJ, (2011). Helicobacter pylori and the birth cohort effect: evidence for stabilized
colonization rates in childhood. Helicobacter., 16:405-9.
[18] Ford AC and Axon ATR, (2010). Epidemiology of Helicobacter pylori infection and
public health implications. Helicobacter., 15:1-6.
[19] Bonamico M, Strappini PM, Bonci E, Ferri M, Crisogianni M, Guido M, Thanasi E,
Nenna R, Macchia S, Luzzi I, Magliocca FM, Mastromarino P, (2004). Evaluation of
stool antigen test, PCR on ORAL samples and serology for the noninvasive detection of
Helicobacter pylori infection in children. Helicobacter., Feb;9:69-76.
[20] Leung WK, Siu KLK, Kwok CKL, Chan SY, Sung R, Sung JJY, (1999). Isolation of
Helicobacter pylori from vomitus in children and its implication in gastro-oral
transmission. Am. J. Gastroenterol., 94:2881-4.
[21] Shojaee Tabrizi A, Jamshidi Sh, Oghalaei A, Zahraei Salehi T, Bayati Eshkaftaki A,
Mohammadi M, (2010). Identification of Helicobacter spp. in oral secretions vs gastric
mucosa of stray cats. Vet Microbiol., 140:142-6.
[22] Bahrami AR, Raimi E, Ghasemian Safaei H, (2013). Detection of Helicobacter pylori
in city water, dental units’ water and bottled mineral water in Isfahan, Iran. Scientific
World Journal.,. 280510.
[23] Weyermann M, Rothenbacher D, Brenner H, (2009). Acquisition of Helicobacter pylori
infection in early childhood: independent contributions of infected mothers, fathers, and
siblings. Am. J. Gastroenterol., 104:182-189.
[24] Delahay RM, Rugge M, (2012). Pathogenesis of Helicobacter pylori infection.
[25] Kato S, Nishino Y, Ozawa K, Konno M, Maisawa S, Toyoda S, Tajiri H, Ida S,
Fujisawa T, Iinuma K, (2004). The prevalence of Helicobacter pylori in Japanese
children with gastritis or peptic ulcer disease. J. Gastroenterol., 39:734-8.
[26] Hua MC, Kong MS, Lai MW, Luo CC, (2007). Perforated peptic ulcer in children: a
20-year experience. J. Pediatr. Gastroenterol. Nutr., 45:71-4.
[27] Barabino A, Dufour C, Marino CE, (1999). Unexplained refractory iron-deficiency
anemia associated with Helicobacter pylori gastric infection in children: further clinical
evidence. J. Pediatr. Gastroenterol. Nutr., 28:116-9.
[28] Ashorn M, Ruuska T, Mäkipernaa A, (2001). Helicobacter pylori and iron deficiency
anaemia in children. Scand. J. Gastroenterol., 36:701-5.
[29] Wang Z, Zhang L, Guo Z, Liu L, Ji J, Zhang J, Chen X, Liu B, Zhang J, Ding Q, Wang
X, Zhao W, Zhu Z, Yu Y, (2012). A unique feature of iron loss via close adhesion of
Helicobacter pylori to host erythrocytes. PLoS One., 7:e50314.
[30] Macarthur C, Saunders N, Feldman W, Ipp M, Winders-Lee P, Roberts S, Best L,
Sherman P, Pencharz P, Veldhuyzen van Zanten SV, (1999). Helicobacter pylori and
childhood recurrent abdominal pain: community based case-control study. BMJ, 319:
822-823.
[31] Spee LA, Madderom MB, Pijpers M, van Leeuwen Y, Berger MY, (2010). Association
between Helicobacter pylori and gastrointestinal symptoms in children. Pediatrics.,
125:e651-69.

[32] Koletzko S, Jones NL, Goodman KJ, Gold B, Rowland M, Cadranel S, Chong S,
Colletti RB, Casswall T, Elitsur Y, Guarner J, Kalach N, Madrazo A, Megraud F,
Oderda G; H. pylori Working Groups of ESPGHAN and NASPGHAN, (2011). H.
pylori Working Groups of ESPGHAN and NASPGHAN. Evidence-based guidelines
from ESPGHAN and NASPGHAN for Helicobacter pylori infection in children. J.
Pediatr Gastroenterol. Nutr., 53:230-43.
[33] Moon A, Solomon A, Beneck D, Cunningham-Rundles S, (2009). Positive Association
between Helicobacter pylori and Gastroesophageal Reflux Disease in Children. J.
Pediatr Gastroenterol. Nutr., 49:283-8.
[34] Zevit N, Balicer RD, Cohen HA, Karsh D, Niv Y, Shamir R, (2012). Inverse
Association Between Helicobacter pylori and Pediatric Asthma in a High Prevalence
Population. Helicobacter., 17:30-5.
[35] Treepongkaruna S, Sirachainan N, Kanjanapongkul S, Winaichatsak A, Sirithorn S,
Sumritsopak R, Chuansumrit A, (2009). Absence of platelet recovery following
Helicobacter pylori eradication in childhood chronic idiopathic thrombocytopenic
purpura: a multicenter randomized controlled trial. Pediatr. Blood Cancer., 53:72-7.
[36] Yilmaz MD, Aktepe O, Cetinkol Y, Altuntaş A, (2005). Does Helicobacter pylori have
role in development of otitis media with effusion? Int. J. Pediatr Otorhinolaryngol.,
69:745-9.
[37] Ho GY, Windsor HM, Snowball B, Marshall BJ, (2001). Helicobacter pylori is not the
cause of sudden infant death syndrome (SIDS). Am. J. Gastroenterol., 96:3288-94.
[38] Bravo LE, Mera R, Reina JC, Pradilla A, Alzate A, Fontham E, Correa P, (2003).
Impact of Helicobacter pylori infection on growth of children: a prospective cohort
study. J. Pediatr Gastroenterol. Nutr., 37:614-9.
[39] Pontone S, Nenna R, Mastrogiorgio G, Petrarca L, Mennini M, Magliocca FM,
Bavastrelli M, Bonamico M, (2012). Gastric pathology in celiac children and adults: the
role of Helicobacter pylori. Prevent Res., 2 (3): 281-287.
[40] Guarner J, Kalach N, Elitsur Y, Koletzko S, (2010). Helicobacter pylori diagnostic tests
in children: review of the literature from 1999 to 2009. Eur. J. Pediatr., 169:15- 25.
[41] Dixon MF, Genta RM, Yardley JH, Correa P, (1996). Classification and grading of
gastritis. The updated Sydney System. International Workshop on the Histopathology
of Gastritis, Houston 1994. Am. J. Surg. Pathol., 20(10):1161-81.
[42] Crone J, Granditsch G, Huber WD, Binder C, Innerhofer A, Amann G, Hirschl AM,
(2003). Helicobacter pylori in children and adolescents: increase of primary
clarithromycin resistance, 1997-2000. J. Pediatr Gastroenterol Nutr., 36:368-371.
[43] Xiong LJ, Tong Y, Wang Z, Mao M, (2013). Detection of clarithromycin-resistant
Helicobacter pylori by stool PCR in children: a comprehensive review of literature.
[44] Horvath A, Dziechciarz P, Szajewska H, (2012). Meta-analysis: sequential therapy for
Helicobacter pylori eradication in children. Aliment Pharmacol. Ther., 36(6):534-41.
[45] Khurana R, Fischbach L, Chiba N, VAN Zanten SV, Sherman PM, George BA,
Goodman KJ, Gold BD, (2007). Meta-analysis: Helicobacter pylori eradication
treatment efficacy in children. Aliment Pharmacol. Ther., 25:523-36.

Chapter 20
Therapy of Helicobacter pylori

Infection: Many Drugs for Which
Association?
Ping-I Hsu and Sung-Shuo Kao

Division of Gastroenterology, Department of Internal Medicine,
Kaoshiung Veterans General Hospital and National Yang Ming University, Taiwan
Abstract
In most international guidelines, standard triple therapy is recommended as the best
option of first-line therapy for H. pylori infection. With the rising prevalence of
antimicrobial resistance, the treatment success of standard triple therapy has recently
declined to unacceptable levels (i.e., 80% or less) in many countries. Several strategies
including bismuth-containing and non-bismuth- containing quadruple therapies
(including sequential, concomitant and hybrid therapies) are therefore proposed to
increase the eradication rate of first-line treatment for H. pylori infection. The regimen
for the second-line therapy depends on the regimen used in first-line treatment. After
failure of a standard triple therapy, either a bismuth-containing quadruple therapy or a
levofloxacin-containing triple therapy is recommended for rescue therapy. The
levofloxacin-containing triple therapy has also been recommended by the Maastricht
IV/Florence Consensus Report for the patients failing to eradicate H. pylori by a bismuth-
or non-bismuth- containing quadruple therapy in first-line treatment. It is noteworthy that
a novel quadruple therapy containing a proton pump inhibitor, bismuth, tetracycline and
levofloxacin also can achieve a high success rate after failure of non-bismuth quadruple
therapies. With regard to the third-line therapy for H. pylori infection, a culture-guided
therapy has been recommended. If antimicrobial sensitivity data are unavailable, an
empirical therapy (such as levofloxacin-containing, rifabutin- containing, or
furazolidone-containing therapies) can be employed to terminate H. pylori infection.

Corresponding author: Ping-I Hsu, Division of Gastroenterology, Department of Internal Medicine, Kaoshiung
Veterans General Hospital and National Yang Ming University, Taiwan, email: williamhsup@yahoo.com.tw

348 Ping-I Hsu and Sung-Shuo Kao
Keywords: Triple therapy, Sequential therapy; Concomitant quadruple therapy; Hybrid

therapy; Bismuth-containing quadruple therapy; Rescue treatment
Introduction
Helicobacter pylori (H. pylori) infect more than 50% of humans globally. It is the
principal cause of chronic gastritis, gastric ulcer, duodenal ulcer, gastric adenocarcinoma and
gastric mucosa-associated lymphoid tissue lymphoma (MALToma) [1-5]. H. pylori infection
results in chronic inflammation of the stomach, a condition that initiates the pathogenic
sequence of events leading to atrophic gastritis, metaplasia, dysplasia and cancer [1]. Several
randomized trial showed that regression of precancerous lesion or, at least, decrease of lesion
progression was noted in eradication groups as compared to control groups.
The recommendation to eradicate H. pylori in (1) patients with peptic ulcer disease
including complicated (bleeding or perforated) peptic ulcer, non-complicated peptic ulcer,
history of peptic ulcer, and following gastric surgery for peptic ulcer; (2) patients with
atrophic gastritis; (3) patients following gastric cancer resection, (4) first-degree relatives of
gastric cancer patients [4]. Furthermore, patients who are aware and concerned about the risks
of H. pylori infection should be reassured and treated, but only after being given complete
information, including the cost, potential side effects and eradication rate of therapy.
Furthermore, H. pylori eradication is also strongly recommended in patients with gastric
MALToma. Eradicating H. pylori results in complete regression of 60-83% of gastric
MALToma [5].
H. pylori eradication is an advisable option in infected patients with non-ulcer dyspepsia.
The Cochrane Systemic Review confirmed that there is a small benefit of eradicating H.
pylori in patients with non-ulcer dyspepsia [6]. Furthermore, H. pylori eradication is
advisable for patients receiving long-term non-steroidal anti-inflammatory drug (NSAID)
treatment [7].
The relationship between H. pylori infection and NSAIDs in gastroduodenal pathology is
complex: H. pylori and NSAIDs independently increase the risk of peptic ulcer bleeding by
1.79- and 4.86-fold, respectively. The risk of ulcer bleeding is increased by 6.13-fold when
both factors are present [8]. For aspirin, even given at low dose, H. pylori eradication can
prevent gastropathy and should be undertaken in patients with a history of peptic ulcers. In
such patients, the residual risk of peptic ulcer bleeding due to continued aspirin use after H.
pylori has been successfully treated is very low.
Gastrooesophageal reflux disease (GERD) is highly prevalent in the general population.
In the last decade, a potential relationship between H. pylori eradication and GERD onset has
been claimed. The main putative mechanism is the gastric acid hypersecretion that develops
after bacterial eradication in patients with corpus-predominant gastritis. Currently, the link
between H. pylori and reflux esophagitis is controversial. Observational studies have
suggested that H. pylori may protect against GERD, but the results could be due to bias or
confounding factors. Recent studies showed that H. pylori eradication didn’t lead to the
development of erosive esophagitis or worsening of symptoms in patients with pre-existing
GERD [9,10].

Therapy of Helicobacter pylori Infection 349
In addition, intraesophageal pH recording studies did not demonstrate increased acid

reflux following bacterial eradication. Routine eradication for H. pylori is not recommended
in GERD patients. However, eradication therapy should be considered in H. pylori-infected
GERD patients requiring long-term maintenance therapy with PPIs because profound acid
suppression may lead to glandular atrophy of the corpus in infected patients, and H. pylori
eradication prevents the progression of atrophic gastritis in GERD patients requiring long-
term PPI therapy [4].
Progress continues in our understanding of the role of H. pylori infection in
extragastroduodenal disorders. The list of associations of H. pylori infection with
extragastroduodenal disorders includes hematological, cardiovascular, neurological, skin and
allergic disorders [11]. Indications for therapy have been extended to patients with idiopathic
thrombocytopenic purpura, unexplained iron-deficiency anaemia, or vitamin B12 deficiency.
H. pylori treatment continues to be a challenge for physicians since bacterial resistance
increases worldwide following large use of antibiotics for eradication therapy. As a general
rule for the treatment of other infectious diseases, clinicians should prescribe therapeutic
regimens that have a per-protocol eradication rate ≥ 90% for anti-H. pylori therapy [12]. In
most international guidelines [7,13,14], standard triple therapy is still recommended as a first-
line therapy.
Regions with clarithromycin Regions with clarithromycin

resistance rate <15-20% resistance rate ≧15-20%
Standard triple therapy Bismuth quadruple therapy Non - Bismuth therapy

1st line
( PPI – Clari - Amox / Metra ) ( PPI – Bismth – Metra - Tetra ) ( Sequential, Concomitant or Hybrid therapy )
Bismuth quadruple therapy PPI – Levofloxacin – Amox, or

2nd line
( PPI – Bismth – Metra - Tetra ) PPI – Bismuth –Levofloxacin - Tetra
PPI – Bismuth –Levofloxacin - Amox Rescue regimen based on susceptibility test

3rd line
Figure 1. Eradicating H. pylori according to clarithromycin resistance rate.
However, several recent large clinical trials and meta-analyses have shown that the
eradication rate of the standard triple therapy has generally declined to unacceptable levels
(i.e., 80% or less), largely owing to emerging organism resistances. Some European studies
even reported very poor treatment outcomes of the standard therapy with failure rates of 25-
60% [15,16].

Several strategies including bismuth-containing and non-bismuth- containing quadruple

(sequential, concomitant and hybrid) therapies have therefore been proposed to increase the
eradication rate [17-19].
This chapter will review the most recent literature in attempt to introduce novel first-line
eradication regimens with a per-protocol eradication rate exceeding 90% and rescue regimens
with an eradication rate exceeding 80% (Figure 1).
First-Line Therapy
Standard Triple Therapy
The Maastricht IV/Florence Consensus Report has recommended treatment of H. pylori

infection according to antibiotic resistance rates in local areas recently [7]. In some countries
with low clarithromycin resistance of H. pylori (< 15-20%), standard triple therapy is the best
option for first-line therapy of H. pylori infection (Figure 1). Standard triple therapy consists
of a proton pump inhibitor (PPI; standard dose, b.i.d.), clarithromycin (500 mg, b.i.d.) and
amoxicillin (1 g, b.i.d.) (or metronidazole [500 mg, b.i.d.] ) for 7 to 14 days (Table 1).
The standard triple therapy proposed at the first Maastricht conference1 to treat H. pylori
infection has become universal since it was recommended by all the consensus conferences
held around the world. However, the most recent data show that this combination has lost
some efficacy and often allows the cure of 70% of the patients, which is less than the 80%
rate aimed for at the beginning and far below what should be expected for an infectious
disease. The reasons for eradication failure in standard triple therapy include antibiotic
resistance, poor patient compliance, rapid metabolism of PPI and smoking.
Bacterial resistance appears to be increasing worldwide, most likely due the large use of
antibiotics such as macrolides, quinolones, amoxicillin, and nitroimidazoles in clinical
practice. Clarithromycin resistance is the major cause of eradication failure for standard triple
therapy [10]. The global clarithromycin resistance rate in Europe increased from 9% in 1998
to 17.6% in 2008. Pooled data from 20 studies involving 1,975 patients treated with standard
triple therapy showed an eradication rate of 88% in clarithromycin-sensitive strains versus
18% in clarithromycin-resistant strains [20].
Therefore, the background rate of clarithromycin resistance is critically important as its
presence negatively impacts the efficacy of standard triple therapy. A systemic review
showed that the rate of clarithromycin-resistant strains ranged from 49% (Spain) to 1%
(Netherland) worldwide [21]. In the area of clarithromycin resistance < 10% (i.e., Netherland,
Sweden, Ireland, Germany, and Malaysia), it is still possible to employ a standard triple
therapy to achieve a per-protocol eradication rate > 90%. However, standard triple therapies
should be abandoned in the areas with clarithromycin resistance ≥ 20% (i.e., Spain, Turkey,
Italy [Central], Alaska, China and Cameroon) because the per-protocol eradication rates of
standard therapies are often less than 85% and the intention-to-treat eradication rates are
usually less than 80% [15,16].

Table 1. Regimens for first-line anti-H. pylori therapy
Drug
Duration of
Regimen
therapy
Amox Clari Metro Tetra Bismuth PPI
First phase 1g 0.5g SD 7 - 14 d

bid bid bid
Stand triple therapy
0.5g 0.5g SD
Second phase 7 - 14 d
bid bid bid
Bismuth – containing quadruple 0.25g 0.5g SD SD

10 - 14 d
therapy qid qid qid bid
1g SD SD
First phase 5d
bid bid bid
Sequential therapy
0.5g 0.5g SD
Second phase 5d
bid bid bid
Concomitant therapy 1g 0.5g 0.5g SD 7 - 10 d

bid bid bid bid
1g SD
First phase 7d
bid bid
Hybrid therapy
1g 0.5g 0.5g SD
Second phase 7d
bid bid bid bid
Point mutations in the peptidyltransferase region, encoded in domain V of 23S rRNA,

account for most clarithromycin resistance of H. pylori. The more frequent mutations
associated with clarithromycin resistance are the adenine to transitions at positions 2142 and
2143 of rRNA (A2143G and A2143C), whilst the substitution of adenine by cytosine at
position 2142 (A2142C) is less frequent [22]. These mutations are able to prevent the binding
between clarithromycin and the ribosomal subunit and are responsible of more than 90% of
clarithromycin resistance. Multiple mechanisms are proposed in the development of
metronidazole resistance, including point mutations, deletion in rdxA gene, and pump efflux
system. Primary H. pylori resistance towards levofloxacin is significantly increasing
worldwide. Point mutations in the gyrA gene prevent binding between the antibiotic and the
enzyme, accounting for bacterial resistance to quinolones. Primary resistance to tetracycline
or amoxicillin remains low in clinical practice, most likely due to the requirement to develop
simultaneous mutations in the respective genes.
Sequential Therapy
The Maastricht IV/Florence Consensus Report recommended bismuth-containing and

non-bismuth- containing quadruple (sequential and concomitant) therapies as the first-line
empirical treatment for H. pylori infection in areas of high clarithromycin resistance (Figure
1). Several studies showed that a novel 10-day sequential therapy can achieve a promising
success rate of 90-94% [23-25]. The regimen consists of a 5-day dual therapy with a PPI
(standard dose, b.i.d.) and amoxicillin (1 g, b.i.d.) followed by a 5-day triple therapy with a
PPI (standard dose, b.i.d.), clarithromycin (500 mg, b.i.d.) and metronidazole (500 mg, b.i.d.)

(Table 1). Gatta et al. reported a rigorous systemic review that identified 13 trials evaluating
3,271 patients [13]. The data showed that sequential therapy achieved a 12% better absolute
eradication rate than the standard triple therapy. Although several randomized trials from
Italy showed that clarithromycin resistance did not affect eradication with sequential
treatment, a randomized controlled trial from Taiwan demonstrated significant differences in
eradication rates of sequential therapy between clarithromycin-sensitive and –resistant stains
(92% and 59%, respectively) [25]. Possible explanations for the discrepancies in the impact
of clarithromycin resistance on eradication rate included different metronidazole use in
sequential therapy and various prevalences of metronidazole resistance in different
geographic areas. Besides the regional variations in eradication efficacies, sequential therapy
is much more complex in terms of medication requirements than standard triple therapy for
switching drugs halfway through the course. In clinical practice, changing drugs during a
treatment course might reduce patient compliance and physician inclination to prescribe and
illustrate the regimen.
Concomitant Therapy
Concomitant therapy is another novel regimen proven successful in the presence of

clarithromycin resistance [26]. It is a 4-drug regimen containing a PPI (standard dose, b.i.d.),
clarithromycin (500 mg, b.i.d.), amoxicillin (1 g, b.i.d.) and metronidazole (500 mg, b.i.d.)
which are all given for the entire duration of therapy (Table 1). Several randomized,
controlled trial demonstrated that concomitant therapy achieved a higher eradication rate than
standard triple therapy [26-28]. It is also less complex than sequential therapy as this regimen
does not involve changing drugs halfway through. The efficacy of concomitant therapy was
related to the duration of treatment. Mean H. pylori eradication rates for concomitant
treatment were: 3 days (85%), 5 days (83%), 7 days (91%) and 10 days (90%). A recent head-
to-head, randomized, controlled trial [27] simultaneously assessing the efficacies of 7-day
standard triple therapy, 10-day sequential therapy and 7-day concomitant therapy
demonstrated that 7-day concomitant therapy achieved a markedly higher eradication rate
than 7-day triple therapy. The eradication rate of 7-day concomitant therapy was comparable
with that of 10-day sequential therapy. From the perspective of clinical practice, the
concomitant regimen is much less complex than the sequential one, which is a two-step
therapy. Additionally, the treatment duration of 7-day concomitant therapy was shorter than
that of 10-day sequential therapy, and both treatments displayed similar frequencies of
adverse events. Another head-to-head non-inferiority trial also showed similar eradication
rates for 10-day concomitant and 10-day sequential therapies (93.1% vs 93.0% by per-
protocol analysis) [29].
Hybrid Therapy
Recently, Hsu et al. has reported a hybrid (dual–concomitant) therapy consisting of a

dual therapy with a PPI (standard dose, b.i.d.) and amoxicillin (1 g, b.i.d.) for 7 days followed
by a concomitant quadruple therapy with a PPI (standard dose, b.i.d.), amoxicillin (1 g, b.i.d.),
clarithromcyin (500 mg, b.i.d.) and metronidazole (500 mg, b.i.d.) for 7 days (Table 1). The

new therapy extends the duration of amoxicillin treatment to 14 days and concomitantly
employs three antibiotics in the last 7 days of treatment course [30]. In 117 H. pylori-infected
subjects, the novel therapy provided excellent eradication rates of 99% and 97% according to
per-protocol and intention-to-treat analysis.
Sadarin et al. recently demonstrated that hybrid therapy was superior to sequential
therapy for first-line H. pylori eradication in Iran [31]. A multi-center trial [32] compared the
efficacy of 14-day hybrid therapy (a dual therapy with omeprazole [40 mg, b.i.d.) and
amoxicillin [1 g, b.i.d.] for 7 days followed by a concomitant quadruple therapy with
omeprazole [40 mg, b.i.d.], amoxicillin [1 g, b.i.d.], clarithromcyin [500 mg, b.i.d.] and
nitroimidazole [500 mg, b.i.d.] for 7 days] and 14-day concomitant therapy (omeprazole [40
mg, b.i.d.], amoxicillin [1 g, b.i.d.], clarithromcyin [500 mg, b.i.d.] and nitroimidazole [500
mg, b.i.d.] for 7 days) in populations with high rates of clarithromycin (24%) and
nitroimidazole (33%) resistances (dual resistance to clarithromycin and nitroimidazole: 8.8
%). In per-protocol analysis, rates of eradication for hybrid and concomitant therapies were
92% and 96%, respectively. In intention-to-treat analysis, rates were 90% and 92%,
respectively. There were no differences in eradication rates between 14-day hybrid and 14-
day concomitant therapies, but significantly more patients were compliant with hybrid
therapy (99%) than concomitant therapy (95%). It is important to note that this novel therapy
also achieved an excellent eradication rate (almost 100%) for the treatment of H. pylori
strains harboring dual resistance to clarithromycin and metronidazole. The prolonging
treatment duration of amoxicillin to 14 days in hybrid therapy might account for the high
eradication rate for H. pylori stains with dual resistance.
Bismuth-Containing Quadruple Therapy
Bismuth-containing quadruple therapy is recommended as the first choice treatment for

H. pylori infection in areas of either low or high clarithromycin resistance in the Maastricht
IV/Florence Consensus Report [1] (Figure 1). Recent studies have shown eradication rates of
> 90% with this combination given for 10 days [33]. Recently, Malfertheiner et al. compared
the efficacy of a 10-day bismuth-containing quadruple therapy (omeprazole, bismuth,
metronidazole and tetracycline) and a 7-day triple therapy (omeprazole, clarithromycin and
amoxicillin) [34]. The data indicated that the former had a higher eradication rate than the
latter (93% vs. 70% by per-protocol analysis). Currently, the optimal treatment duration of
bismuth-containing quadruple therapy remains unclear but a 10-14 day course is most
commonly employed in clinical practice.
Quinolone-Containing Triple Therapy
Based on a large body of published clinical trials, a quinolone-containing triple therapy is

effective in the first-line therapy for H. pylori infection. The eradication rates of levofloxacin-
based triple therapy ranged from 72% to 96% [35]. The regimen might be considered in
populations with clarithromycin resistance greater than 15–20% and quinolone resistance less
than 10% [35]. However, a quinolone-based triple therapy is not generally recommended as a
first-line therapy on concerns of the rising prevalence of quinolone-resistant strains in the

first-line and second-line anti-H. pylori therapies. Furthermore, greater use of quinolone
would likely result in the development of more quinolone-resistant pathogens for the
respiratory and urogenital tract infection.
Second-Line Therapy
After Failure of a Standard Triple Therapy
The regimen for second-line therapy of H. pylori infection depends on the regimen used
in first-line therapy. After failure of a standard triple therapy, either a bismuth-containing
quadruple therapy or levofloxacin-containing triple therapy is recommended. The bismuth–
containing quadruple therapy regimen used in the second-line therapy comprises a PPI,
bismuth, metronidazole and tetracycline (Figure 1). PPI should be prescribed in the usual
dose and twice a day, colloidal bismuth subcitrate 120 mg four times per day, tetracycline 500
mg four times per day and metronidazole 500 mg three times per day (Table 2). This rescue
regimen fails in 5-63% of patients with an average eradication rate of 76% on the basis of a
pooled analysis [36-38]. The prevalence of metronidazole-resistant strains, dose and duration
of rescue therapy seem to be important variables for the efficacy of this treatment. A report
from Korea showed that two-week bismuth-containing quadruple therapy was more effective
than the 1-week treatment (83% vs. 64% by intention-to-treat analysis) [39].
Levofloxacin-based triple therapy consisting of levofloxacin (500 mg, q.d.), amoxicillin
(1 g, b.i.d.) and a PPI (standard dose, b.i.d.) represents an encouraging strategy for second-
line therapy. A meta-analysis by Saad et al. [40] showed that a 10-day regimen of
levofloxacin-based triple therapy was superior to 7-day bismuth-based quadruple therapy.
Additionally, the novel therapy has less adverse effects than bismuth-containing quadruple
therapy. However, levofloxin-based triple therapies seem less effective in Asia. Two
randomized controlled trials from Taiwan and Hong Kong showed that levofloxacin-based
triple therapy were comparable with quadruple therapy in the eradication efficacy of second-
line therapy [41,42].
Table 2. Regimens for second-line anti-H. pylori therapy
Drug
Duration of
Regimen
therapy
Amox Clari Metro Tetra Levo Bismuth PPI
Bismuth – containing 0.5g 0.5g SD SD 10 - 14 d

quadruple therapy tid qid qid bid
Levofloxacin – containing SD
0.5g 0.5g 10 d
triple therapy
bid qd bid
Levofloxacin – tetracycline 0.5g 0.5g SD SD 10 d

quadruple therapy
qid qd qid bid

After Failure of a Bismuth or Non-Bismuth Quadruple Therapy
Currently, the best second-line therapy after failure of bismuth quadruple or non-bismuth
quadruple therapies (such as sequential therapy, concomitant therapy or hybrid therapy)
remains unclear. However, a triple therapy containing a PPI plus levofloxacin and amoxicillin
has been recommended by the Maastricht IV/Florence Consensus Report (Figure 1) [1]. A
recent report showed that a rescue therapy with lansoprazole (30 mg, b.i.d.), levofloxacin
(250 mg, b.i.d) and amoxicillin (1 g, b.i.d.) achieved a high eradication rate in patients who
failed to clear H. pylori with sequential therapy [43]. Other levofloxin-based therapies seem
also effective for the patients failing to eradicate H. pylori by non-bismuth quadruple therapy.
Hsu et al. showed that a 10-day quadruple therapy containing esomeprazole, bismuth,
tetracycline and levofloxacin is well tolerated and achieves a high success rate (92%) for H.
pylori infection after failure of sequential therapy (Table 2) [44].
Third-Line Therapy
Culture-Guided Therapy
Currently, a standard empirical third-line therapy is lacking. The Maastricht IV/Florence

Consensus Report recommends using bacterial culture with antimicrobial sensitivity tests to
select antibiotics in the third-line regimen (Figure 1) [7]. Cammarota et al. [45] analyzed H.
pylori isolates from 94 consecutive patients in whom H. pylori infection had persisted after
two eradication attempts. The rates of resistant strains to clarithromycin, amoxicillin,
metronidazole, tetracycline and levofloxacin were 95%, 0%, 100%, 5% and 31%,
respectively. Patients were then treated with a culture-guided, third-line regimen: 89 patients
with a 1-week quadruple regimen including omeprazole, bismuth, doxycycline and
amoxicillin, and five patients with a 1-week triple regimen containing omeprazole,
amoxicillin and levofloxacin or clarithromycin. Overall, H. pylori eradication was obtained in
90% of subjects treated by the culture-guided therapy.
Empirical Third-Line Therapy
It has been reported that the sensitivity of culture is less than 60% [46]. Additionally, in
vitro antimicrobial sensitivity does not necessarily lead to eradication in vivo and vice versa.
Recently, several empirical third-line therapies have been proposed to treat refractory H.
pylori infection. A 10-day quadruple therapy comprising rabeprazole (20 mg b.i.d.), bismuth
subcitrate (300 mg, q.i.d.), amoxicillin (500 mg, q.i.d.), and levofloxacin (500 mg, q.d.)
achieved an eradication rate of 84% by both intention-to-treat analysis and per-protocol
analysis in patients who failed to eradicate H. pylori with standard triple therapy and bismuth-
based quadruple therapy (Table 3) [47].
H. pylori has been proven to be highly susceptible in vitro to rifabutin, a rifamycin
derivative of the established antituberculous angent. Until now, no rifabutin-resistant strain
has been isolated from patients who have been previously treated or not for H. pylori
infection. Moreover, rifabuin is chemically stable over a wide pH range and its antibacterial
activity is not like to be hampered by the acidic environment of the stomach. It can be
administered with PPI and amoxicillin for 10-14 days for H. pylori eradication purposes. A
study used rifabutin (150 mg b.i.d.), amoxicillin (1 g b.i.d.) and omeprazole (20 mg b.i.d.) for
14 days as a third-line therapy [48]. Eradication was achieved in 11 out of 14 patients (79%)
by both per-protocol and intention-to-treat analyses. It is noteworthy that serious
myelotoxicity and ocular adverse events have been reported with rifabutin therapy [49]. In
addition, greater use of rifabutin would likely result in the development of more resistant
strains to Myocbacterium tuberculosis and Mycobacterium avium.
Table 3. Regimens for third-line anti-H. pylori therapy
Drug Duration
Regimen of
therapy
Amox Clari Metro Tetra Levo Rifa Fura Bismuth PPI
Culture quided SD SD
Two antibiotics selected by sensitivity tests 10 - 14 d
therapy
qid bid
Levofloxacin –
Amox 0.5g 0.5g SD SD 10 d
quadruple
qid qd qid bid
therapy
Rifabutin –
based triple 1g 0.15g SD
therapy 14 d
bid bid bid
Furazolidone –
based
Table 2. Regimens for 2st – line anti - H 1g
quadruple
pylori therapy 0.2g SD SD 7d
bid bid bid bid
therapy
Furazolidone-based therapy is another useful option to treat refractory H. pylori infection.

A 7-day quadruple therapy consisting of lansoprazole (30 mg, b.i.d.), tripotassium-
dicitratobismuthate (240 mg, b.i.d.), furazolidone (200 mg, b.i.d.) and tetracycline (1 g, b.i.d.)
has a high efficacy in third-line therapy with an eradication rate of 90% by both intention-to-
treat and per-protocol analysis [50].
Adjuvants in Eradication Therapy

Although unsuccessful eradication can be due to increasing antimicrobial resistance, an
additional important contributor is the high incidence of treatment-related side effects
(including diarrhea, nausea, epigastric discomfort, and dysgeusia with a metallic taste),
determining low compliance, and, consequently, incomplete therapy. Many of these
gastrointestinal effects result from the modification of the ecologic equilibrium of intestinal
microbiota due to the antibiotics. Recently, the use of probiotics has been proposed to
increase patients’s tolerability or eradication rate of anti-H. pylori therapies [51-52].
Probiotics have an in vitro inhibitory effect on H. pylori. Some stains of Lactobacillus and

Bifidobacterium are able to inhibit H. pylori growth through the release of bacteriocins or
organic acids, and may also decrease its adhesion to epithelial cells. Additionally, probiotics
have a possible role in the stabilization of the gastric barrier function and the decrease of
mucosal inflammation. Animal studies revealed that probiotics is effective in reducing H.
pylori-associated gastric inflammation. Several human studies also demonstrated an
improvement of H. pylori gastritis and decrease in H. pylori density after administration of
probiotics. However, no study could show the eradication of H. pylori infection by probiotic
treatment alone.
Systemic reviews of the literature have shown that various probiotic species (including
Lactobacillus spp., Bifidobacterium spp., Saccharomyces spp., and Bacillus spp.) can help
diminish eradication-related side effects, including nausea, diarrhea and taste disturbance, and
increase tolerability and compliance [53]. A recent meta-analysis of 14 randomized trials
comparing probiotic supplementation to placebo or non-intervention revealed that
supplementation with probiotics could be effective in increasing eradication rates of anti-H.
pylori therapy [54]. However, the results concerning the improvement of eradication rate of
eradication therapy by probiotics are conflicted. Several studies demonstrated that the
addition of probiotics to standard anti-H. pylori treatment improved eradication rates but
some randomized clinical trials showed similar eradication rates in the probiotic and placebo
groups. Possible explanations for the discrepancies in the impact of probiotics on eradication
rate include different strains and various treatment durations of probiotic therapies.
It has been shown that oral administration of antibacterial immunoglobulin through infant
formulae or other diets is effective in preventing intestinal infection. A recent study reported
that egg yolk IgY against H. pylori whole-cell lysates inhibited the growth of H. pylori and
reduced gastric inflammation in H. pylori-infected Mongolian gerbils. The finding suggests
that IgY could be used as a novel modality against H. pylori-associated gastric mucosal
diseases. However, a clinical trial revealed that dietary anti-H. pylori-urease immunoglobulin
Y did not eradicate H. pylori but reduce bacterial load in the stomach [55].
Confirmation of H. pylori Eradication

Following Therapy
Several studies have shown that by increasing the gastric pH, PPI use leads to local
changes in the stomach. The bacterial load decreases, especially in the antrum, causing false-
negative results of the diagnostic tests. False negative results should be interpreted with
caution if the patient has taken drugs that reduce the bacterial load such as PPIs, antibiotic or
bismuth. In general, one should stop these drugs for two weeks before testing. H2-receptor
antagonists do not adversely affect any of these tests and can be continued in needed to
control symptoms. In clinical practice, post-eradication assessment for H. pylori infection
should be conducted at least 4 weeks following the completeness of eradication therapy to
avoid false negative results. Confirmation of H. pylori eradication following therapy can be
accurately tested by urea breath test. The stool antigen assay is the alternative if the former is
not available.

Conclusion
With the rising prevalence of antimicrobial resistance, the treatment success of standard
triple therapy has recently declined to unacceptable levels. Therefore, to cure H. pylori in
clinical practice is becoming progressively more difficult, and some patients require more
than two consecutive therapeutic regimens. Recent novel first-line anti-H. pylori therapies
include sequential therapy, concomitant quadruple therapy, hybrid therapy and bismuth-
containing quadruple therapy. The regimen for the second-line therapy depends on the
regimen used in first-line treatment. After failure of a standard triple therapy, either a
bismuth-containing quadruple therapy or a levofloxacin-containing triple therapy is
recommended for rescue therapy. The levofloxacin-containing triple therapy is also
recommended in patients failing to eradicate H. pylori by a bismuth-containing or non-
bismuth-containing quadruple therapy in first-line treatment. Another novel levofloxacin-
based therapy containing a PPI, bismuth, tetracycline and levofloxacin is also an accepted
salvage treatment for non-bismuth-containing quadruple therapy. Most guidelines suggest that
patients requiring third-line therapy should be referred to medical center and treated
according to the antibiotic susceptibility test. Nonetheless, an empirical therapy (such as
levofloxacin-containing, rifabutin- containing, or furazolidone-containing therapies) can be
employed to terminate H. pylori infection if antimicrobial sensitivity data are unavailable.
References
[1] Suerbaum S., Michetti P., (2002). Helicobacter pylori infection. N. Engl. J. Med. 347,
1175–1186.
[2] Graham D.Y., Lew G.M., Klein P.D., Evans D.G., Evans D.J. Jr., Saeed Z.A., Malaty
H.M., (1992). Effect of treatment of Helicobacter pylori infection on the long-term
recurrence of gastric or duodenal ulcer. A randomized controlled study. Ann. Intern.
Med. 116, 705-708.
[3] Sung J.J., Chung S.C., Ling T.K., Yung M.Y., Leung V.K., Ng E.K., Li M.K., Cheng
A.F., Li A.K., (1995). Antibacterial treatment of gastric ulcer associated with
Helicobacter pylori. N. Eng. J. Med. 332, 139-142.
[4] Malfertheiner P., Megraud F., O'Morain C., Bazzoli F., El-Omar E., Graham D.Y.,
Hunt R., Rokkas T., Vakil N., Kuipers E.J., (2007). Current concepts in the
management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut
56, 772-781.
[5] Zucca E., Dreyling M.; ESMO Guidelines Working Group, (2009). Gastric marginal
zone lymphoma of MALT type: ESMO clinical recommendations for diagnosis,
treatment and follow-up. Ann. Oncol. 20 (Suppl 4), 113-114.
[6] Moayyedi P., Deeks J., Talley N.J., Delaney B., Forman D., (2003). An update of the
Cochrane systematic review of Helicobacter pylori eradication therapy in nonulcer
dyspepsia: resolving the discrepancy between systematic reviews. Am. J. Gastroenterol.
98, 2621–2626.
[7] Malfertheiner P., Megraud F., O'Morain C., Atherton J., Axon A.T.R., Bazzoli F.,
Gensini G.F., Gisbert J.P., Graham D.Y., Rokkas T., El-Omar E.M., Kuipers E.J.,

(2012). Management of Helicobacter pylori infection — The Maastricht IV/ Florence

Consensus Report. Gut 61, 646-664.
[8] Huang J.Q., Sridhar S., Hunt R.H., (2002). Role of Helicobacter pylori infection and
nonsteroidal anti-inflammatory drugs in peptic-ulcer disease: a meta-analysis. Lancet
359, 14–22.
[9] Yaghoobi M., Farrokhyar F., Yuan Y., Hunt R.H., (2010). Is there an increased risk of
GERD after Helicobacter pylori eradication?: a meta-analysis. Am. J. Gastroenterol.
105, 1007-1013.
[10] Qian B, Shijie M, Shang L, Qian J, Zhang G., (2011). Effect of H. pylori eradication on
gastroesophageal reflux disease. Helicobacter 16: 255-265.
[11] Suzuki H., Franceschi F., Nishizawa T., Gasbarrini A., (2011). Extragastric
manifestations of Helicobacter pylori infection. Helicobacter 16 (Suppl 1), 65–69.
[12] Rimbara E., Fischbach L.A., Graham D.Y., (2011). Optimal therapy for Helicobacter
pylori infections. Nat. Rev. Gastroenterol. Hepatol. 8, 79–88.
[13] Fock K.M., Katelaris P., Sugano K., Ang T.L., Hunt R., Talley N.J., Lam S.K., Xiao
S.D., Tan H.J., Wu C.Y., Jung H.C., Hoang B.H., Kachintorn U., Goh K.L., Chiba T.,
Rani A.A., (2009). Second Asia-Pacific Conference. Second Asia-Pacific Consensus
Guidelines for Helicobacter pylori infection. J. Gastroenterol. Hepatol. 24, 1587-1600.
[14] Chey W.D., Wong B.C.Y., (2007). American College of Gastroenterology guideline on
the management of Helicobacter pylori infection. Am. J. Gastroenterol. 102, 1808–
1825.
[15] Gumurdulu Y., Serin E., Ozer B., Kayaselcuk F., Ozsahin K., Cosar A.M., Gursoy M.,
Gur G., Yilmaz U., Boyacioglu S., (2004). Low eradication rate of Helicobacter pylori
with triple 7-14 days and quadruple therapy in Turkey. World J. Gastroenterol. 10, 668-
6671.
[16] De Francesco V., Margiotta M., Zullo A, Hassan C, Giorgio F, Burattini O, Stoppino G,
Cea U, Pace A, Zotti M, Morini S, Panella C, Ierardi E., (2007). Prevalence of primary
clarithromycin resistance in Helicobacter pylori strains over a 15 year period in Italy. J.
Antimicrob. Chemother. 59, 783–785.
[17] Zullo A., De Francesco V., Hassan C., Morini S., Vaira D, (2007) The sequential
therapy regimen for Helicobacter pylori eradication: a pooled-data analysis. Gut 56,
1353–1357.
[18] Essa A.S., Kramer J.R., Graham D.Y., Treiber G., (2009). Meta-analysis: four-drug,
three-antibiotic, non-bismuth-containing “concomitant therapy” versus triple therapy
for Helicobacter pylori eradication. Helicobacter, 109–118.
[19] Chuah S.K., Tsay F.W., Hsu P.I., Wu D.C., (2011). A new look at anti-Helicobacter
pylori therapy. World J. Gastroenterol. 17, 3971-3975
[20] Megraud F., (2004). H. pylori antibiotic resistance: prevalence, importance, and
advances in testing. Gut 53, 1374-1784.
[21] De Francesco V., Giorgio F., Hassan C., Manes G., Vannella1 L., Panella1C., Ierardi1 E.,
Zullo A., (2010). Worldwide H. pylori antibiotic resistance: a systematic review. J.
Gastrointest. Liver Dis. 19, 409-414.
[22] De Francesco V., Ierardi E., Hassan C., Zullo A., (2012). Helicobacter pylori therapy:
present and future. World .J. Gastroenterol. 3, 68-73.
[23] Gatta L., Vakil N., Leandro G., Di Mario F., Vaira D., (2009). Sequential therapy or
triple therapy for Helicobacter pylori infection: systematic review and meta-analysis of

randomized controlled trials in adults and children. Am. J. Gastroenterol. 104, 3069–
3079.
[24] Hsu P.I., Wu D.C., Wu J.Y., Graham D.Y., (2011) Is there a benefit to extending the
duration of Helicobacter pylori sequential therapy to 14 days? Helicobacter 16, 146-
152.
[25] Liou J.M., Chen C.C., Chen M.J., Chen C.C., Chang C.Y., Fang Y.J., Lee J.Y., Hsu
S.J., Luo J.C., Chang W.H., Hsu Y.C., Tseng C.H., Tseng P.H., Wang H.P., Yang U.C.,
Shun C.T., Lin J.T., Lee Y.C., Wu M.S., (2012). Sequential versus triple therapy for the
first-line treatment of Helicobacter pylori: a multicentre, open-label, randomised trial.
Lancet 381, 205-213.
[26] Kao S.S., Chen W.C., Hsu P.I., Lai K.H., Yu H.C., Cheng H.H., Peng N.J., Lin C.K.,
Chan H.H., Tsai W.L., Wang H.M., Tsai T.J., Lin K.H., Tsay F.W., (2012). 7-Day
Nonbismuth-Containing Concomitant Therapy Achieves a High Eradication Rate for
Helicobacter pylori in Taiwan. Gastroenterol. Res. Pract. doi:10.1155/2012/463985.
[27] Hsu P.I., Wu D.C., Chen W.C., Yu H.C., Wang H.M., Lai K.H., Tsay F.W., (2012).
Comparison of 7-day triple, 10-day sequential and 7-day concomitant therapies for
Helicobacter pylori infection in Taiwan. J. Gastroenterol. Hepatol. 27 (Suppl 5), 421.
[28] Georgopoulos S., Papastergiou V., Xirouchakis E., Laoudi F., Lisgos P., Spiliadi C.,
Papantoniou N., Karatapanis S., (2013). Nonbismuth quadruple "concomitant" therapy
versus standard triple therapy, both of the duration of 10 days, for first-line H Pylori
eradication: a randomized trial. J. Clin. Gastroenterol. 47, 228-232.
[29] Wu D.C., Hsu P.I., Wu J.Y., Opekun A.R., Kuo C.H., Wu I.C., Wang S.S., Chen A.,
Hung W.C., Graham D.Y., (2010). Sequential and concomitant therapy with 4 drugs are
equally effective for eradication of H. pylori Infection. Clin. Gastroenterol. Hepatol. 8,
36-41.
[30] Hsu P.I., Wu D.C., Wu J.Y., Graham D.Y., (2011). Modified sequential Helicobacter
pylori therapy: proton pump inhibitor and amoxicillin for 14 days with clarithromycin
and metronidazole added as a quadruple (hybrid) therapy for the final 7 days.
Helicobacter 16, 139-145.
[31] Sardarian H., Fakheri H., Hosseini V., Taghvaei T., Maleki I., Mokhtare M., (2013).
Comparison of hybrid and sequential therapies for Helicobacter pylori eradication in
Iran: A prospective randomized trial. Helicobacter 18, 129-134.
[32] Molina-Infante J., Romano M., Fernandez-Bermejo M., Federico A., Gravina A.G.,
Pozzati L., Garcia-Abadia E., Vinagre-Rodriguez G., Martinez-Alcala C., Hernandez-
Alonso M., Miranda A., Iovene M.R., Pazos-Pacheco C., Gisbert J.P., (2013).
Optimized non-bismuth quadruple therapies cure most patients with Helicobacter pylori
infection in populations with high rates of antibiotic resistance. Gastroenterology Apr
3. doi:pii: S0016-5085(13)00491-5. 10.1053/j.gastro.2013.03.050. [Epub ahead of
print]
[33] Laine L., Hunt R., El-Zimaity H., Nguyen B., Osato M., Spénard J., (2003). Bismuth-
based quadruple therapy using a single capsule of bismuth biskalcitrate, metronidazole,
and tetracycline given with omeprazole versus omeprazole, amoxicillin, and
clarithromycin for eradication of Helicobacter pylori in duodenal ulcer patients: a
prospective, randomized, multicenter, North American trial. Am. J. Gastroenterol. 98,
562–567.

[34] Malfertheiner P., Bazzoli F., Delchier J.C., Celiñski K., Giguère M., Rivière M.,
Mégraud F.; Pylera Study Group. (2011). Helicobacter pylori eradication with a
capsule containing bismuth subcitrate potassium, metronidazole, and tetracycline given
with omeprazole versus clarithromycin-based triple therapy: a randomised, open-label,
non-inferiority, phase 3 trial. Lancet 377, 905-913.
[35] Berning M., Krasz S., Miehlke S., (2011). Should quinolones come first in Helicobacter
pylori therapy? Therap Adv Gastroenterol. 4, 103-114.
[36] Wu D.C., Hsu P.I., Tseng H.H., Tsay F.W., Lai K.H., Kuo C.H., Wang S.W., Chen A.
(2011) Helicobacter pylori Infection: A randomized, controlled study comparing 2
rescue therapies after failure of standard triple therapies. Medicine (Baltimore) 90, 180-
185.
[37] Gisbert J.P. (2008). "Rescue" regimens after Helicobacter pylori treatment failure.
World. J. Gastroenterol. 14, 5385-5402.
[38] Hojo M., Miwa H., Nagahara A., Sato N., (2001). Pooled analysis for Helicobacter
pylori infection. Scand J Gastroenterol 36, 690-700.
[39] Lee B.H., Kim N., Hwang T.J., Lee S.H., Park Y.S., Hwang J.H., Kim J.W., Jeong
S.H., Lee D.H., Jung H.C., Song I.S. (2010). Bismuth-containing quadruple therapy as
second-line treatment for Helicobacter pylori infection: effect of treatment duration and
antibiotic resistance on the eradication rate in Korea. Helicobacter 15, 38-45.
[40] Saad R.J., Schoenfeld P., Kim H.M., Chey W.D., (2006). Levofloxacin-based triple
therapy versus bismuth-based quadruple therapy for persistent Helicobacter pylori
infection: a meta-analysis. Am. J. Gastroenterol. 101, 488-496.
[41] Kuo C.H., Hu H.M., Kuo F.C., Hsu P.I., Chen A., Yu F.J., Tsai P.Y., Wu I.C., Wang
S.W., Li C.J., Weng B.C., Chang L.L., Jan C.M., Wang W.M., Wu D.C., (2009).
Efficacy of levofloxacin-based rescue therapy for Helicobacter pylori infection after
standard triple therapy: a randomized controlled trial. J. Antimicrob. Chemother. 63,
1017-1024.
[42] Wong W.M., Gu Q., Chu K.M., Yee Y.K., Fung F.M., Tong T.S., Chan A.O., Lai K.C.,
Chan C.K., Wong B.C., (2006). Lansoprazole, levofloxacin and amoxicillin triple
therapy vs. quadruple therapy as second-line treatment of resistant Helicobacter pylori
infection. Aliment. Pharmacol. Ther. 23, 421-427.
[43] Pontone S., Standoli M., Angelini R., Pontone P., (2010). Efficacy of H. pylori
eradication with a sequential regimen followed by rescue therapy in clinical practice.
Dig. Liver Dis. 42, 541-543.
[44] Hsu P.I., Wu J.Y., Wu D.C., (2010). A novel rescue treatment of sequential therapy for
Helicobacter pylori infection. Gastroenterology 142: (Suppl 1), S226.
[45] Cammarota G., Martino A., Pirozzi G., Cianci R., Branca G., Nista E.C., Cazzato A.,
Cannizzaro O., Miele L., Grieco A., Gasbarrini A., Gasbarrini G., (2004). High efficacy
of 1-week doxycycline- and amoxicillin-based quadruple regimen in a culture-guided,
third-line treatment approach for Helicobacter pylori infection. Aliment. Pharmacol.
Ther. 19, 789-795.
[46] Savarino V., Zentilin P., Pivari M., Bisso G., Raffaella Mele M., Bilardi C., (2000). The
impact of antibiotic resistance on the efficacy of three 7-day regimens against
Helicobacter pylori. Aliment. Pharmacol. Ther. 14, 893-900.

[47] Hsu P.I., Wu D.C., Chen A., Peng N.J., Tseng H.H., Tsay F.W., Lo G.H., Lu C.Y., Yu
F.J., Lai K.H., (2008). Quadruple rescue therapy for Helicobacter pylori infection after
two treatment failures. Eur. J. Clin. Invest. 38, 404-409.
[48] Gisbert J.P., Calvet X., Bujanda L., Marcos S., Gisbert J.L., Pajares J.M., (2003).
'Rescue' therapy with rifabutin after multiple Helicobacter pylori treatment failures.
[49] Apseloff G., (2003). Severe neutropenia among healthy volunteers given rifabutin in
clinical trials. Clin. Pharmacol. Ther. 74, 591-592.
[50] Treiber G., Ammon S., Malfertheiner P., Klotiz U., (2002). Impact of furazolidone-
based quadruple therapy for eradication of Helicobacter pylori after previous treatment
failures. Helicobacter 7, 225-231.
[51] Manfredi M., Bizzarri B., Sacchero R.I., Maccari S., Calabrese L., Fabbian F.,
de'Angelis G.L., (2002). Helicobacter pylori infection in clinical practice: probiotics
and a combination of probiotics + lactoferrin improve compliance, but not eradication,
in sequential therapy. Helicobacter 17, 254-263.
[52] Cremonini F., Di Caro S., Covino M., (2002). Effect of different probiotic preparations
on anti-Helicobacter pylori therapy related side effects: a parallel group, triple blind,
placebo controlled study. Am. J. Gastroenterol. 97, 2744–2749.
[53] Medeiros J.A., Pereira M.I., (2013). The use of probiotics in Helicobacter pylori
eradication therapy. J. Clin. Gastroenterol. 47, 1-5.
[54] Tong J.L., Ran Z.H., Shen J., (2007) Meta-analysis: the effect of supplementation with
probiotics on eradication rates and adverse events during Helicobacter pylori
eradication therapy. Aliment. Pharmacol. Ther. 25, 155–168.
[55] Suzuki H., Nomura S., Masaoka T., Goshima H., Kamata N., Kodama Y., Ishi H.,
Kitajima M., Nomoto K., Hibi T., (2004). Effect of dietary anti-Helicobacter pylori-
urease immunoglobulin Y on Helicobacter pylori infection. Aliment. Pharmacol. Ther.
20 (Suppl 1), 185-192.

Chapter 21
The Biosynthesis of Isoprenoid

Precursors as a New Target for the
Development of Specific Antibiotics for
the Treatment of Helicobacter pylori-
Associated Diseases
Jordi Perez-Gil1, Joan Miralles1,2, Maria Bergua1, Albert Boronat1,

Jaime Rubio-Martinez2 and Santiago Imperial1,
1
Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona (UB),
Avda Diagonal 643, Barcelona, Spain
2
Departament de Química Física, Universitat de Barcelona (UB) and Institut de Recerca
en Química Teòrica i Computacional. Martí i Franquès 1, Barcelona, Spain
Abstract
The methylerythritol phosphate (MEP) pathway for the biosynthesis of isoprenoid
precursors is an attractive target for the design of new specific antibiotics for the
treatment of gastrointestinal diseases associated with the presence of the bacterium
Helicobacter pylori (H. pylori) since this pathway which is essential to the bacterium is
absent in humans.
Comparative genomic analyses allowed the identification of homologues of all the
genes of the MEP pathway in H. pylori.
The degree of homology with the enzymes from E. coli suggests that the MEP
pathway would function similarly in these two bacteria. Nevertheless, the organization of
the MEP pathway in H. pylori and E. coli seems to be different: In the genome of H.
pylori, two of the genes of the MEP pathway ispD and ispF are fused into a single ispDF
transcription unit. Of the genes of the methylerythritol phosphate pathway form H. pylori

To whom correspondence should be addressed: Santiago Imperial, Departament de Bioquímica i Biologia
Molecular, Universitat de Barcelona (UB). Avda Diagonal 643, E-08028, Barcelona, Spain. Simperial @
ub.edu.

364 Jordi Perez-Gil, Joan Miralles, Maria Bergua et al.
only ispDF (HP_1440) has been cloned, expressed in E. coli and the recombinant enzyme
functionally characterized. As shown by genetic complementation and in vitro functional
assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can
replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate
synthase from E. coli.
The 3D structures of the enzymes of the MEP pathway from H. pylori have not been
yet solved. Only a structure of CDP-methylerythritol kinase, the enzyme catalyzing the
fourth step and coded by the ispE gene (HP_1443), has been obtained by molecular
modelling taking known X-ray structures of the enzyme from E. coli and A. aeolicus as
templates. After evaluating the quality of the model, a thorough study of the catalytic site
was performed which suggested the most important sites that could be useful for drug
discovery and mutagenesis studies.
In summary, this chapter reports the available structural and functional information
on the enzymes of the MEP pathway from H. pylori. This information would be
important to develop new chemotherapeutic therapies against the gastrointestinal
pathologies associated to this microorganism.
Keywords: MEP pathway, Methylerythritol phosphate pathway, 4-diphosphocytidyl-2-C-

methyl-D-erythritol synthase; 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase
Helicobacter pylori; Isoprenoid biosynthesis; bifunctional enzyme, 4-diphosphocytidyl-2-
C-methyl-D-erythritol kinase, antibiotics
Introduction
Isoprenoids are the most diverse group of natural products with more than 35 000
compounds identified to date [1, 2]. Isoprenoids are synthesized in all living organisms and
play essential roles in membrane structure (sterols and hopanoids), redox reactions
(ubiquinone, menaquinone, and plastoquinone), light harvesting and photoprotection
(carotenoids and side chain of chlorophylls), and regulation of growth and development
(steroid hormones, gibberellins, cytokinins, abscisic acid, and substrates for protein
modification) [1-3].
All isoprenoids derive from two common C5 units: isopentenyl diphosphate (IPP) and its
isomer dimethylallyl diphosphate (DMAPP). Until the early 90’s, it was accepted that IPP
and DMAPP biosynthesis proceeded in all organisms through the same set of enzyme
reactions, the mevalonate pathway. However, an alternative pathway for the synthesis of IPP
and DAMPP has been identified in eubacteria [3], green algae [4] and plants [5, 6], the
methylerythritol phosphate (MEP) pathway.
The genes and enzymes of the MEP pathway are best characterized in E. coli and are
summarized in Figure 21.1.
In the initial reaction 1-deoxy-D-xylulose 5-phosphate (DXP) is formed by condensation
of D-glyceraldehyde 3-phosphate with (hydroxyethyl) thiamine derived from pyruvate. This
reaction is catalyzed by the enzyme DXP synthase (DXS) encoded by the dxs gene [7-10]. In
the next step, DXP is converted into 2-C-methyl-D-erythritol 4-phosphate (methylerythritol
phosphate or MEP) by intramolecular rearrangement and reduction in a reaction catalyzed by
DXP reductoisomerase (DXR) [11-13]. This is the first committed step of the pathway and
MEP represents the first specific intermediate. In the third step MEP is converted to the CDP-
derivative 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) by the enzyme CDP-ME
The Biosynthesis of Isoprenoid Precursors … 365
synthase (CMS), the protein product of the E. coli gene ispD [14-16]. In the following
reaction step CDP-ME is phosphorylated in an ATP-dependent reaction by the enzyme CDP-
ME kinase (CMK) to yield 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-
MEP) [17, 18]. This enzyme is specified by the ispE gene [17]. CDP-MEP is next
transformed into CMP and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MECDP) by the
enzyme MECDP synthase (MCDS) encoded by the ispF gene [19]. Finally, the ispG protein
product, hydroxymethylbutenyl 4-diphosphate synthase (HDS) and the enzyme specified by
the ispH gene, hydroxymethylbutenyl 4-diphosphate reductase (HDR), catalyze the
conversion of MECDP into hydroxymethylbutenyl 4-diphosphate (HMBPP) and then into
IPP and DMAPP, respectively [20-27]. Figure 21.1 summarizes the series of reactions of the
MEP pathway as identified in E. coli.
Most organisms only use one of the two pathways for the biosynthesis of isoprenoids
precursors. The MEP pathway is the only one present in most eubacteria including the causal
agents for diverse and serious human diseases as well as the malaria parasite Plasmodium
falciparum, but it is absent from fungi and animals, which synthesize their isoprenoids
exclusively through the MVA pathway [28-30]. By contrast, plants use both the MEP
pathway and the MVA pathway for isoprenoid biosynthesis, although they are localized in
different compartments: the MEP pathway in plastids and the MVA pathway in the cytoplasm
[6, 28, 29].
Figure 21.1. The MEP pathway as described in E. coli . The indicated genes (in italics) encode the
following enzymes: dxs, DXP synthase (DXS); dxr, DXP reductoisomerase (DXR); ispD, CDP-ME
synthase (CMS); ispE, CDP-ME kinase (CMK); ispF, MECDP synthase (MCDS), ispG, HMBPP
synthase; (HDS), ispH, HMBPP reductase (HDR).

Given the essential nature of the MEP pathway and its absence in mammals, the enzymes
comprising the MEP pathway represent potential targets for the generation of selective
antibacterial, antimalarial and herbicidal molecules [31-41].
In particular, the MEP pathway for isoprenoid biosynthesis is an attractive target for the
design of new specific antibiotics for the treatment of gastrointestinal diseases associated with
the presence of the bacterium Helicobacter pylori (H. pylori) since this pathway which is
essential to the bacterium is absent in humans.
The MEP Pathway in H. pylori

Homologues of all the genes of the MEP pathway have been identified in H. pylori by
comparative genomic analysis. As shown in Table 21.1 the degree of homology with the
enzymes from E. coli is ranging from 18.6% identity of CMK to, more than 40% of HDS
This result suggests that the MEP pathway would function similarly in these two bacteria.
Nevertheless, the organization of the MEP pathway in H. pylori and E. coli seems to be
different: In the genome of H. pylori, two of the genes of the MEP pathway, ispD and ispF,
are fused into a single ispDF transcription unit. The presence of a ispDF gene is not limited to
H. pylori or even to other ε proteobacteria, although most of the H. pylori ispDF homologs
are found in proteobacteria, specially of the α and ε subsets.
The ispDF gene from the closely related ε proteobacterium Campylobacter jejuni and
from the nitrogen-fixing α proteobacterium Mesorhizobium loti. have been studied in detail.
These genes code for bifunctional enzymes which show both CMS and MCDS activities [42-
45].
In the first part of this chapter we will describe the information available on the protein
product of the ispDF gene from H. pylori (HP_1440), so far the only gene of the MEP
pathway from H. pylori which has been cloned, expressed in E. coli and the recombinant
enzyme functionally characterized [46]. Next, the first structural data on the enzymes of the
MEP pathway from H. pylori, and their potential use for the design of new specific antibiotics
will be discussed.
Table 21.1.
Gene locus Accession Enzyme EC number Identity with E. coli

number homologue (%)
dxs HP_0354 O25121 DXP synthase (DXS) 2.2.1.7 39.3
ispC HP_0216 P56139 DXP reductoisomerase (DXR) 1.1.1.267 32.8
ispDF HP_1020 O25664 bifunctional CDP-ME synthase
2.7.7.60
(CMS) MECDP synthase 25.6 43.1
4.6.1.12
(MCDS)
ispE HP_1443 O25984 CDP-ME kinase (CMK) 2.7.1.148 18.6
ispG HP_0625 O25342 4-hydroxy-3-methylbut-2-enyl
1.17.4.3 43.7
diphosphate synthase (HMS)
ispH HP_0400 P65185 4-hydroxy-3-methylbut-2-enyl
1.17.1.2 26.9
diphosphate reductase (HDR)
Identitiy and similarity percents are referred to the corresponding E. coli enzyme and were calculated
using the Emboss align program. (http://www.ebi.ac.uk/Tools/emboss/align).

Characterization of Enzymes of the MEP Pathway

from H. pylori
The Bifunctional CDP-ME Synthase – MECDP Synthase
Sequence Analysis
The IspDF protein from H. pylori consists of a N-terminal domain (amino acids 1-to 247)
homologous to E. coli CMS (26% identity, 37% similarity) and a C-terminal domain (amino
acids 248 to 406) homologous to MCDS from E. coli with 63% similarity (46% of residues
identical).
Figure 21.2 shows an amino acid alignment of the protein coded by the ispDF gene from
H. pylori and the CMS and MCDS coded respectively by the ispD and ispF from E. coli.
When compared to CMS the N-terminal domain of the IspDF protein from H. pylori
contains a N-terminal extension of 28 amino acids which is not present in the E. coli enzyme
or in other bacterial CMS. No information is yet available on the functional relevance of this
sequence, however it does not contain any of the putative residues involved in substrate
binding or catalysis [15-16].
It can be speculated that this additional sequence may be involved in the interaction of
the IspDF protein with other components of the MEP pathway, notably CMK which catalyzes
the intermediate reaction step (see Figure 21.1).
Figure 21.2. Amino acid sequence alignment. Sequence alignment of IspDF from H. pylori (CMS
domain: amino acids 1to 247, MCDS domain: amino acids 248 to 406) with the E. coli CMS and
MCDS. Alignments were carried out using the ClustalW program (www.ebi.ac.uk/Tools/clustalw).

By sedimentation velocity experiments an in vitro association of ispDF with CMK has

been observed [43] suggesting a role for a multienzyme complex that might be related with an
improved catalytic efficiency or control of the metabolic flux. However, this hypothesis is not
supported by other studies in which an absence of substrate channeling between IspDF from
A. tumefaciens and CMK has been reported [45].
Most of the amino acids reported to be present in the active sites of either the individual
CMS and MCDS enzymes or other IspDF proteins (notably, IspDF from C. jejuni [43]) are
highly conserved. These data suggest that as reported for the enzyme from C. jejuni and M.
loti the product of the ispDF gene from H. pylori would catalyze two non consecutive steps of
the MEP pathway to produce CDP-ME from MEP and MECDP from CDP-MEP.
Molecular Cloning and Expression in E. coli

The ispDF gene from H. pylori was cloned by PCR from genomic DNA obtained from
H. pylori 26695. A pET23HpDF construct was prepared by cloning the sequence
corresponding to the HP_1020 accession number into the pET23 expression vector [46].
This construct was used to obtain the recombinant protein in E. coli. Since the
recombinant protein contained a C-terminal extension of eight amino acids (LEHHHHHH) it
was purified by immobilized metal affinity chelating chromatography (IMACC). When the
chromatographic fractions were analyzed by SDS-PAGE and Coomassie staining (Figure
21.3) pooled fractions contained a major band of 45kDa, corresponding to the product of the
ispDF gene. Under the experimental conditions used 15-20 mg of recombinant protein were
obtained per liter of culture and the recombinant protein showed 95% purity [46].
Figure 21.3. Purification of the IspDF protein from H. pylori.- The IspDF protein containing a C-
terminal hexahistidine fragment was overexpressed in E.coli BL21(DE3) pLysS strain. Optimal protein
production was obtained at 30ºC 4h using 1mM IPTG. SDS-PAGE analysis of purified preparations.
SDS-PAGE gel (15%) stained with Coomassie Brilliant Blue (R-250). Lanes: NI. total extract before
induction; I. total extract after induction; MM, low range pre-stained molecular marker (Sigma®);
IspDF: purified preparation of IspDF from H. pylori.

Functional Characterization of the Recombinant Enzyme

The recombinant IspDF protein was tested for CMS and MCDS activities by HPLC. The
chromatograms obtained demonstrated that the H. pylori IspDF protein is able to transform
MEP into CDP-ME and that in the presence of exogenous E. coli CMK is able to carry out
the transformation of MEP into MECDP, showing also a MCDS activity [46].
As an example Figure 21.4 shows the chromatograms corresponding to the CMS activity
of the H. pylori IspDF protein.
These results were further validated by analyzing the HPLC eluates by mass
spectrometry. When tested for CMS activity the fragmentogram of the reaction product
obtained was coincident with that of pure CDP-ME used as standard. In a similar way by
adding both the IspDF protein and E. coli CMK to a reaction mixture the reaction products
corresponded to CMP and MECDP.
Figure 21.4. Analysis of the CMS activity of H. pylori IspDF. Purified preparations of the H. pylori
IspDF enzyme were assayed for CMS activity by HPLC. Separations were carried out by HPLC as
previously described [67]. Eluates were monitored for absorbance at 257 and 280 nm using a HP 1100
photodiode array detector. t=0 initial reaction mixture. t = 1h reaction mixture incubated at 37ºC for 1h.

According to these results H. pylori harbours a modification of the MEP pathway in

which two non-consecutive enzymatic steps are catalyzed by the same protein, the product of
the ispDF gene [46].
Functional Characterization by Complementation Assays

Genetic experiments were carried out in order to further demonstrate that the ispDF gene
encodes a protein which was able to carry out the same reactions catalyzed independently by
the E. coli homologues CMS and MCDS.
In these genetic experiments E. coli strains disrupted in the ispD and IspF genes, named
EcAB4-D and EcAB4-F strains, respectively, were used.
These strains which are derived from the MG155 E. coli cells were engineered to:
a) synthesize isopentenyl diphosphate (IPP) and dimethylallyldiphosphate (DMAPP)

from exogenously supplied mevalonate (MVA). They contain a MVA operon formed
by yeast mevalonate kinase, human phosphomevalonate kinase and yeast
diphosphomevalonate decarboxylase and can grow in the presence of exogenously
added MVA [47, 48] and
b) in them the chromosomal E. coli ispD (EcAB4-D) and ispF (EcAB4-F) genes were
replaced, respectively, by the chloramphenicol acetyl transferase (CAT) marker
conferring chloramphenicol resistance. Since the deletion of either ispD or ispF
prevents IPP and DMAPP biosynthesis via the MEP pathway, EcAB4-D or EcAB4-F
mutant cells cannot grow unless MVA is supplied [47, 48].
Figure 21.5 shows a scheme of the EcAB4-D strain.

As shown in Figure 21.6, EcAB 4-D cells transformed with the pET23HpDF construct
were able to grow in the absence of MVA, the same as those cells transformed with plasmid
pQE30EcD, containing the ispD gene from E. coli used as positive control. Similarly, when
EcAB4-F cells were transformed with pET23HpDF they could grow the same as those
transformed with the plasmid pQE31EcF containing the ispF gene from E. coli. According to
these results the ispDF gene from H. pylori is encoding a protein which can replace both
native CMS and MCDS from E. coli in these complementation assays [46].
Genetic complementation assays and in vitro functional assays showed that the product of
the ispDF gene form H. pylori is a bifunctional enzyme which can replace the CMS and
MCDS enzymes from E. coli and rescue the lethal disruption of the knock-out ispD and ispF
E. coli cells. Functional assays and molecular characterization of the reaction products formed
demonstrate that the H .pylori IspDF enzyme is able to transform MEP into CDP-ME and that
in the presence of exogenous E. coli CMK is able to carry out the transformation of MEP into
MECDP, showing also a MCDS activity.
These results, make the bifunctional CMS-MCDS enzyme an excellent target for the
design of new, selective antibiotics against H. pylori. According to new rational drug design
approaches [49, 50] compounds mimicking direct protein-protein interactions between CMS-
MCDS subunits could prevent the oligomerization of the biologically active form of the
enzyme and act as inhibitors. In principle these inhibitors would be able to block both
reactions essential to the bacterium and continue their action even if the bacterium acquired a
resistance to another antibiotic directed against the active site of the two individual activities
CMS or MCDS.

Figure 21.5. E. coli cells of the EcAB4-D strain deficient in the ispD gene. The EcAB4-D strain is
derived from MG155 E. coli and is engineered to synthesize IPP and DMAPP from exogenously
supplied mevalonate (MVA). It contains a MVA operon formed by yeast mevalonate kinase (yMVK),
human phosphomevalonate kinase (hPMK) and yeast diphosphomevalonate decarboxylase (yPMD) and
can grow in the presence of exogenously added MVA [47, 48]. In addition the chromosomal E.coli
ispD gene is replaced by the chloramphenicol acetyl transferase (CAT) marker conferring
chloramphenicol resistance. Since the deletion of ispD prevents IPP and DMAPP biosynthesis via the
MEP pathway, EcAB4-D cells cannot grow unless MVA is supplied [47, 48].
Figure 21.6. Complementation assays.- Competent E. coli cells of the EcAB4-D strain deficient in the
ispD gene were transformed with constructs to express the H. pylori IspDF protein (pET23HpDF), the
E. coli CMS (pQE30EcD) or with the original pET23 vector as control. In parallel experiments
competent E. coli cells of the EcAB4-F deficient in the ispF gene were transformed with pET23HpDF,
pQE31EcF (expressing E. coli MCDS) and void pET23. After recovering the generated strains on
plates supplemented with 1 mM MVA (to rescue the lethal deletion of either the chromosomal ispD or
ispF gene), colonies were streaked on new plates with (+) or without (−) MVA and incubated at
37°C.CDP-ME Kinase from H. pylori.

Homology modeling is a general strategy to obtain a protein structure when no crystal

structure is available. This strategy has been applied to several protein models including
enzymes of the MEP pathway to suggest mutagenesis studies and useful sites for drug
discovery [51-53].
Since none of the 3D structures of the enzymes of the MEP pathway from H. pylori has
been yet solved we decided to use molecular modeling to obtain a reliable structure of one of
the enzymes of the pathway, CMK (HP_1443), taking known X-ray structures as templates.
The model has been used to analyse the binding sites of CDM and ANP and also the non-
conserved regions to suggest new binding sites to develop selective inhibitors.
Structure Construction
Homology modelling was performed using the MODELLER 9v7 program [54].
MODELLER is based on comparative protein structure by satisfaction of spatial restraints
[55, 56].
The X-ray structures of E. coli (PDB code 1OJ4) [18] CMK was selected as 3D templates
and used for building the structural model for H. pylori CMK. Amino acid sequence
alignments used to build the enzyme model were performed using the CLUSTAL W online
server [57].
MODELLER was applied to generate fifty satisfactory models and that with the lowest
energy was selected. Then, the CDM and ANP molecules together with waters and Na+
cations were added to the model. All other calculations were carried out using AMBER9 [58]
software with the ff99SB force field.
Molecular Dynamics
The energy of the full model structure was minimized using the steepest descent
algorithm to remove bad contacts derived from homology modeling and to achieve good
starting structure with which to perform molecular dynamics. Then, 20 ns of molecular
dynamics were carried out in the NTV ensemble with constant temperature of 300 K.
As a quality test the Verify-3D [59] program was used. This program uses a score
function to assess the quality of the model. It is considered that all residues with a score
higher than 0.2 are reliable [59].
Thus, the final percentages of reliable residues both in initial and refined models
according to this method were 52.06 % and 73.95% respectively. Note the improvement of
the model.
Role of Non-Conserved Residues in the H. Pylori CMK Models

Zones with low identity score between the generated model and its template can be
interesting places for selective inhibition. Q-SiteFinder [60], an energy-based server
application, was used in order to detect possible binding ligand sites. Q-SiteFinder was
applied to calculate in keeping ligands in the structure. On figure 7 the refined model is
shown with these sites.

Note that the vast majority of the reliable sites are located around ANP and CDM
cofactors.
Figure 21.7. Predicted binding sites of H. pylori CMK obtained by Q-SiteFinder server [60].
Conclusion
H. pylori infection is a worldwide spread disease which causes several, and potentially
life threatening, gastroduodenal diseases [61, 62]. Although it remains asymptomatic in a
large percentage of subjects, millions of patients worldwide need to be currently treated for
such an infection. The treatment is mainly based on a combination of proton pump inhibitor
together with 2 or 3 different antibiotics [61, 63]. However, the therapy regimens suggested
have given disappointing eradication rates in several countries. To find out new drugs in order
to improve H. pylori eradication rate is of the highest importance in primary medical care
since the management cost of this infection deeply depends on the efficacy of first-line
therapy, the “rescue” treatments being generally more expensive and less effective [63-66].
The results reported in this chapter are the first on the characterization of enzymes of the
MEP pathway from H. pylori and should be taken into account when the validity of new
chemotherapeutic therapies against the gastrointestinal pathologies associated to this
microorganism is tested.

References
[1] Chappell J. (2002). The genetics and molecular genetics of terpene and sterol origami.
Curr. Opin. Plant. Biol. 5(2), 151-157.
[2] Sacchettini J. C., Poulter C. D. (1997). Creating isoprenoid diversity. Science 277,
1788-1789.
[3] Rohmer M., Knani M., Simonin P., Sutter B., Sahm H. (1993). Isoprenoid biosynthesis
in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate.
Biochem. J. 295, 517-524.
[4] Schwender J., Seemann M., Lichtenthaler H.K., Rohmer M. (1996). Biosynthesis of
isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone)
via a novel pyruvate/ glyceraldehyde 3-phosphate non-mevalonate pathway in the green
alga Scenedesmus obliquus. Biochem. J. 316, 73-80.
[5] Arigoni D., Sagner S., Latzel C., Eisenreich W., Bacher A., Zenk M. H. (1997).
Terpenoid biosynthesis from 1-deoxy-D-xylulose in higher plants by intramolecular
skeletal rearrangement. Proc. Natl. Acad. Sci. USA 94, 10600-10605.
[6] Rodríguez-Concepción M., Boronat A. (2002). Elucidation of the methylerythritol
phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic
milestone achieved through genomics. Plant. Physiol. 130, 1079-1089.
[7] Sprenger G. A., Schörken U., Wiegert T., Grolle S., De Graaf A. A., Taylor S. V.,
Begley T. P., Bringer-Meyer S., Sahm H. (1997). Identification of a thiamin-dependent
synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-
phosphate precursor to isoprenoids, thiamin, and pyridoxol. Proc. Natl. Acad. Sci. USA
94, 12857-12862.
[8] Lois L. M., Campos N., Putra S. R., Danielsen K., Rohmer M., Boronat A. (1998).
Cloning and characterization of a gene from Escherichia coli encoding a transketolase-
like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common
precursor for isoprenoid, thiamin, and pyridoxol biosynthesis. Proc. Natl. Acad. Sci.
USA 95, 2105-2110.
[9] Xiang S., Usunow G., Lange G., Busch M., Tong L., (2007).Crystal structure of 1-
deoxy-D-xylulose 5-phosphate synthase, a crucial enzyme for isoprenoids biosynthesis.
J. Biol. Chem. 282, 2676-2682.
[10] Brammer L. A., Smith J. M., Wade H., Meyers C. F., (2011). 1-Deoxy-D-xylulose 5-
phosphate synthase catalyzes a novel random sequential mechanism. J. Biol. Chem. 286
(42), 36522-36531.
[11] Kuzuyama T., Takahashi S., Watanabe H., Seto H., (1998). Direct formation of 2-C-
methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate by 1-deoxy-D-
xylulose 5-phosphate reductoisomerase, a new enzyme in the non-mevalonate pathway
to isopentenyl diphosphate. Tetrahedron. Lett. 39, 4509-4512.
[12] Takahashi S., Kuzuyama T., Watanabe H., Seto H, (1998). A 1-deoxy-D-xylulose 5-
phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-
phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis. Proc.
Natl. Acad. Sci. USA 95, 9879-9884.
[13] Proteau P.J., (2004).1-Deoxy-D-xylulose 5-phosphate reductoisomerase: an overview.
Bioorg. Chem. 32, 483-493.

[14] Rohdich F., Wungsintaweekul J., Fellermeier M., Sagner S., Herz S., Kis K., Eisenreich
W., Bacher A., Zenk M. H., (1999). Cytidine 5’-triphosphate-dependent biosynthesis of
isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-
diphosphocytidyl-2-C-methylerythritol. Proc. Natl. Acad. Sci. USA 96, 11758-11763.
[15] Richard S. B., Bowman M. E., Kwiatkowski W., Kang I., Chow C., Lillo A. M, Cane
D.E., Noel J. P., (2001). Structure of 4-diphosphocytidyl-2-C-methylerythritol
synthetase involved in mevalonate-independent isoprenoid biosynthesis. Nat. Struct.
Biol. 8, 641-648.
[16] Richard S. B, Lillo A. M., Tetzlaff C. N., Bowman M. E., Noel J.P., Cane D. E., (2004).
Kinetic analysis of Escherichia coli 2-C-methyl-D-erythritol-4-phosphate
cytidyltransferase, wild type and mutants, reveals roles of active site amino acids.
Biochemistry 43, 12189-12197.
[17] Lüttgen H., Rohdich F., Herz S., Wungsintaweekul J., Hecht S., Schuhr C. A.,
Fellermeier M., Sagner S., Zenk M. H., Bacher A., Eisenreich W., (2000). Biosynthesis
of terpenoids: YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of
4-diphosphocytidyl-2-C-methylerythritol. Proc. Natl. Acad. Sci. USA 97, 1062-1067.
[18] Miallau L., Alphey M. S., Kemp L. E., Leonard G. A., McSweeney S. M., Hecht S.,
Bacher A., Eisenreich A., Rohdich F., Hunter W.N., (2003). Biosynthesis of
isoprenoids: crystal structure of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase.
Proc. Natl. Acad. Sci. USA 100, 9173-9178.
[19] Herz S., Wungsintaweekul J., Schuhr C. A., Hecht S., Lüttgen H., Sagner S.,
Fellermeier M., Eisenreich W., Zenk M. H., Bacher A., Rohdich F., (1999).
Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl-D-
erythritol 2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. Proc. Natl.
Acad. Sci. USA 97, 2486-2490.
[20] Rodríguez-Concepcion M., Campos N., Lois L.M., Maldonado C., Hoeffler J. F.,
Grosdemange-Billiard C., Rohmer M., Boronat A., (2000). Genetic evidence of
branching in the isoprenoid pathway for the production of isopentenyl diphosphate and
dimethylallyl diphosphate in Escherichia coli. FEBS Lett. 473, 328–332.
[21] Kollas A.K., Duin E.C., Eberl M., Altincicek B., Hintz M., Reichenberg A., Henschker
D., Henne A., Steinbrecher I., Ostrovsky D.N., Hedderich R., Beck ., Jomaa H.,
Wiesner J., (2002). Functional characterization of GcpE, an essential enzyme of the
non-mevalonate pathway of isoprenoid biosynthesis. FEBS Lett. 532, 432-436.
[22] Rohdich F., Zepeck F., Adam P., Hecht S., Kaiser J., Laupitz R., Gräwert T., Amslinger
T., Eisenreich W., Bacher A., Arigoni D., (2003). The deoxyxylulose phosphate
pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions
catalyzed by IspG and IspH protein. Proc. Natl. Acad. Sci. USA 100, (2003) 1586-1591.
[23] Rohdich F., Bacher A., Eisenreich W., (2004). Perspectives in anti-infective drug
design. The late steps in the biosynthesis of the universal terpenoid precursors,
isopentenyl diphosphate and dimethylallyl diphosphate. Bioorg. Chem. 32, 292-308.
[24] Lee M., Grawert T., Quitterer F., Rohdich F., Eppinger J., Eisenreich W., Bacher A.,
Groll M., (2010). Biosynthesis of Isoprenoids: Crystal Structure of the [4Fe-4S] Cluster
Protein IspG. J. Mol. Biol. 404 (4), 600-610.
[25] Rekittke I., Jomaa H., Ermler U., (2012). Structure of the GcpE (IspG)-MEcPP
complex from Thermus thermophilus. FEBS Lett. 586 (19), 3452-3457.

[26] Rekittke I., Wiesner J., Röhrich R., Demmer U., Warkentin E., Xu W., Troschke K.,
Hintz M., No J. H., Duin E.C., Oldfield E., Jomaa H., Ermler U., (2008). Structure of
(E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate reductase, the terminal enzyme of the
non-mevalonate pathway. J. Am. Chem. Soc. 130, 17206-17207.
[27] Span I., Wang K., Wang W. X., Zhang Y. H., Bacher A., Eisenreich W., Li K., Schulz
C., Oldfield E., Groll M., (2012). Discovery of acetylene hydratase activity of the iron-
sulphur protein IspH. Nature Commun. 3, Art Numb: 1042
[28] Rohmer M, (1999). The discovery of a mevalonate-independent pathway for isoprenoid
biosynthesis in bacteria, algae and higher plants. Nat. Prod. Report. 16, 565-574.
[29] Eisenreich W., Schwarz M., Cartayrade A., Arigoni D., Zenk M. H., Bacher A., (1998).
The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and
microorganisms. Chem. Biol. 5, R221–R233.
[30] Grawert T., Groll M., Rohdich F., Bacher A., Eisenreich W., (2011). Biochemistry of
the non-mevalonate isoprenoid pathway. Cell. Mol. Life Sci. 68 (23), 3797-3814.
[31] Lichtenthaler H. K., Zeidler J., Schwender J., Muller C., (2000). The non-mevalonate
isoprenoid biosynthesis of plants as a test system for new herbicides and drugs against
pathogenic bacteria and the malaria parasite. Z. Naturforsch. 55, 305-313.
[32] Testa C. A., Brown M. J., (2003). The methylerythritol phosphate pathway and its
significance as a novel drug target. Curr. Pharm. Biotechnol. 4, 248-259.
[33] Rohmer M., Grosdemange-Billiard C., Seemann M., Tritsch D., (2004). Isoprenoid
biosynthesis as a novel target for antibacterial and antiparasitic drugs. Curr. Op. Invest.
Drugs 5(2), 154-162.
[34] Rodriguez-Concepcion M., (2004). The MEP pathway: a new target for the
development of herbicides, antibiotics and antimalarial drugs. Curr. Pharm. Des. 10,
2391-2400.
[35] Ruyck J., Wouters J., (2008). Structure-based drug design targeting biosynthesis of
isoprenoids: A crystallographic state of the art of the involved enzymes. Curr. Prot.
Pept. Sci. 9(2), 117-137.
[36] Obiol-Pardo C., Rubio-Martinez J., Imperial S., (2011). The methylerythritol phosphate
(MEP) pathway for isoprenoid biosynthesis as a target for the development of new
drugs against tuberculosis. Curr. Med. Chem. 18 (9), 1325-1338.
[37] Witschel M.C., Hoffken H. W., Seet M., Parra L., Mietzner T., Thater F., Niggeweg R.,
Rohl F., Illarionov B., Rohdich F.,. Kaiser J., Fischer M., Bacher A., Diederich F.,
(2011). Inhibitors of the herbicidal target IspD: Allosteric site binding. Angewandte
Chemie-Int. Ed. 50(34), 7931-7935.
[38] Witschel M., Rohl F., Niggeweg R., Newton T., (2013). In search of new herbicidal
inhibitors of the non-mevalonate pathway. Pest. Manag. Sci. doi 10.1002/ps.3479.
[39] Jomaa H., Wiesner J., Sanderbrand S., Altincicek B., Weidemeyer C., Hintz M.,
Turbachova I., Eberl M., Zeidler J., Lichtenthaler H. K., Soldati D., Beck E., (1999).
Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial
drugs. Science 285, 1573-1576.
[40] Wiesner J., Jomaa H., (2007). Isoprenoid biosynthesis of the apicoplast as drug target.
Curr. Drug Targets, 8, 3-13.
[41] Hunter W.N., (2011). Isoprenoid precursor biosynthesis offers potential targets for drug
discovery against diseases caused by apicomplexan parasites. Curr. Top. Med. Chem.
11(16), 2048-2059.

[42] Gabrielsen M., Rohdich F., Eisenreich W., Gräwert T., Hecht S., Bacher A., Hunter
W.N., (2004). Biosynthesis of isoprenoids: a bifunctional IspDF enzyme from
Campylobacter jejuni. Eur. J. Biochem. 271 3028-3035.
[43] Gabrielsen M., Bond C.S., Hallyburton I., Hecht S., Bacher A., Eisenreich W., Rohdich
F., Hunter W. N., (2004). Hexameric assembly of the bifunctional methylerythritol 2,4-
cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-
dependent pathway of isoprenoid precursor biosynthesis. J. Biol. Chem. 279, 52753-
52761.
[44] Testa C. A., Lherbet C.,. Pojer F., Noel J. P., Poulter C. D., (2006). Cloning and
expression of IspDF from Mesorhizobium loti. Characterization of a bifunctional
protein that catalyzes non-consecutive steps in the methylerythritol phosphate pathway.
Biochim. Biophys. Acta 1764, 85-96.
[45] Lherbet C., Pojer F., RichardS. B., Noel J. P., Poulter C.D., (2006). Absence of
substrate channeling between active sites in the Agrobacterium tumefaciens IspDF and
IspE enzymes of the methyl erythritol phosphate pathway. Biochemistry 45, 3548-3553.
[46] Perez-Gil J., Bergua M., Boronat A., Imperial S., (2010). Cloning and functional
characterization of an enzyme from Helicobacter pylori that catalyzes two steps of the
methylerythritol phosphate pathway for isoprenoid biosynthesis. Biochim. Biophys.
Acta 1800, 919-928.
[47] Sauret-Gueto S., Ramos-Valdivia A., Ibañez E., Boronat A., Rodriguez-Concepcion M.,
(2003). Identification of lethal mutations in Escherichia coli genes encoding enzymes
of the methylerythritol phosphate pathway. Biochem. Biophys. Res. Commun. 307, 408-
415.
[48] Campos N., Rodriguez-Concepcion M., Sauret-Gueto S., Gallego F., Lois L. M.,
Boronat A., (2001). Escherichia coli engineered to synthesize isopentenyl diphosphate
and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis
of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis.
Biochem. J. 353, 59-67.
[49] Obiol-Pardo C., Granadino-Roldán J. M., Rubio-Martinez J., (2008). Protein-protein
recognition as a first step towards the inhibition of XIAP and survivin anti-apoptotic
proteins. J. Mol. Recognit. 21, 190-204.
[50] Giménez-Oya V., Villacañas O., Fernàndez-Busquets X, Rubio-Martinez J., Imperial
S., (2009). Mimicking direct protein-protein and solvent-mediated interactions in the
CDP-methylerythritol kinase homodimer: a pharmacophore-directed virtual screening
approach. J. Mol. Model. 15, 997-1007.
[51] Singh N., Avery M. A., McCurdy C. R., (2007). Towards Mycobacterium tuberculosis
DXR inhibitor design: homology modeling and molecular dynamics simulation. J.
Comput. Aided Mol. Des. 21, 511–522.
[52] Hillisch A., Pineda L.F., Hilgenfeld R.,(2004). Utility of homology models in the drug
discovery process. Drug Discov. Today 9, 659–669.
[53] Obiol-Pardo C., Cordero A., Rubio-Martinez J., Imperial S., (2010). Homology
modeling of Mycobacterium tuberculosis 2C-methyl-D-erythritol-4-phosphate
cytidylyltransferase, the third enzyme in the MEP pathway for isoprenoid biosynthesis.
J. Mol. Model.16, 1061-1073.

[54] Eswar N., Marti-Renom M. A., Webb B., Madhusudhan M. S., Eramian D., Shen M.,
Pieper U., Sali A., (2007). Comparative Protein Structure modelling With
MODELLER. Current Protocols in Bioinformatics. John Wiley & Sons, Inc.,
Supplement 15, 5.6.1-5.6.30, 200.
[55] Sali A., Blundell T. L., (1993). Comparative protein modelling by satisfaction of spatial
restraints. J. Mol. Biol. 234, 779-815.
[56] Fiser A., Do R. K., Sali A., (2000). Modelling of loops in protein structures. Prot. Sci.
9, 1753-1773.
[57] Larkin M. A. Blackshields G., Brown N. P., Chenna R., McGettigan P. A., McWilliam
H., Valentin F., Wallace I. M., Wilm A., Lopez R., Thompson J. D., Gibson T. J.,
Higgins D. G., (2007). Clustal W and Clustal X version 2.0. Bioinformatics 2947-2948.
[58] Case D.A., Darden T.A., Cheatham T. E., Simmerling C. L., Wang J., Duke R. E., Luo
R., Merz K. M., Pealman D. M., Crowley M., Walker R. C., Zhang W., Wang B., Hayik
S., Roitberg A., Seabra G., Wong K. F., Paesani F., Wu X., Brozell S., Tsui V., Gohlke
H., Yang L., Tan C., Mongan J., Hornak V., Cui G., Beroza P., Mathews D. H.,
Schafmeinster C., Ross W. S., Kollman P. A., (2006) Amber 9, University of
California, San Francisco.
[59] Luthy R., Bowie J. U., Eisenberg D., (1992). Assessment of protein models with three-
dimensional profiles. Nature 356, 83-85.
[60] Laurie A. T., Jackson R. M., (2005). Q-SiteFinder: an energy-based method for the
prediction of protein-ligand binding sites. Bioinformatics 21, 908-1916.
[61] Vaira U., Gatta L., Ricci C., D’Anna L., Iglioli M. M., (2001). Helicobacter pylori:
diseases, tests and treatment. Dig. Liver. Dis. 33, 788-794.
[62] Suerbaum S., Michetti P., (2002). Helicobacter pylori infection. N. Engl. J. Med. 347,
1175 - 1186.
[63] Campo S. M., Zullo A., Hassan C., Moroni S., (2007). Antibiotic treatment strategies
for Helicobacter pylori infection. Recent Pat. Anti-Infect. Drug Discov. 2, 11-17.
[64] Lazarou J., Pomeranz B. H., Corey P. N., (1998). Incidence of adverse drug reactions in
hospitalized patients: a meta-analysis of prospective studies. JAMA 279, 1200-1205.
[65] El-Omar E. M., (2006). What is the optimal therapy for the eradication of Helicobacter
pylori? Nat. Clin. Pract. Gastroenterol. Hepatol. 3, 126-127.
[66] Dore M. P., Maragkoudakis E., Pironti A., Tadeu V., Tedde R., Realdi G., Delitala G.,
(2006). Twice –a-day quadruple therapy for eradication of Helicobacter pylori in the
elderly. Helicobacter 11, 52-55.
[67] Bernal C.., Palacin C., Boronat A., Imperial S., (2005). A colorimetric assay for the
determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase activity. Anal.
Biochem. 337, 55-61.

Chapter 22
Helicobacter pylori Infection:

The Vaccine Development
Irina V. Pinchuk1 and Ellen J. Beswick2

1
Division of Gastroenterology, Department of Internal Medicine,
University of Texas Medical Branch
2
Department of Molecular Genetics and Microbiology,
University of New Mexico Health Sciences Center
Abstract
Approximately 50% of the world’s population is infected with Helicobacter pylori
(H. pylori), with infection rates over 90% in some developing countries. Infection with
this organism is a major cause of chronic gastritis, gastric and duodenal ulcers, and
gastric carcinomas throughout the world. Gastric cancer is the second leading cause of
cancer-related mortalities worldwide. The vast infection rates, prevalence of disease, and
incidences of gastric cancer necessitate the development of an effective vaccine.
Although progress has been made in the development of a vaccine, challenges remain
because of differences in immune responses elicited between animal models and in
humans. H. pylori are a difficult vaccine target because the bacteria have developed
mechanisms to subvert the human immune system. Thus, continued focus on which
elements of the immune system would be protective for a human are crucial for effective
vaccine development.
Keywords: Helicobacter pylori, immune response, vaccine development

Corresponding author: Ellen J. Beswick, 1 University of New Mexico, MSC 08-4660, Albuquerque, NM 87131,
USA. E-mail: ebeswick@salud.unm.edu.

380 Irina V. Pinchuk and Ellen J. Beswick
Introduction
The widespread worldwide Helicobacter pylori (H. pylori) infection rates along with the
resulting gastritis, ulceration, and gastric cancer has led to significant efforts in vaccine
development. Substantial interest has been shown in developing both therapeutic and
prophylactic vaccines for use in areas of the world with high infection rates and prevalent
gastric cancer development. However, most vaccination protocols used in preclinical models
are not suitable for humans. A better understanding of appropriate adjuvants and the
immunopathology in humans is needed in order to induce vaccine-induced protection against
H. pylori. In this chapter, we will highlight the need for an H. pylori vaccine, the immune
response that could potentially provide protection, the antigens that may be appropriate for a
vaccine, and the progress made in testing adjuvants thus far.
Prevalence
The prevalence of infection and disease association are key factors driving vaccine
development. Prevalence of the infection varies between countries, but there are two distinct
patterns for developed and developing countries. For developed countries, the prevalence of
infection increases with age. For instance, the seropositivity detected in the Eurogast study
was between 35% in the 25-34 years age group and 62% in the 55-64 years age group [1]. In
addition, a study from Germany showed an overall prevalence of 40% in participants between
18 and 79 years [2]. However, the frequency of the infection is decreasing in many developed
countries. For example, a report from the Netherlands in children and young adolescents
showed that from 1978 to 1993 H. pylori prevalence declined from 19% to 9% in children up
to 8 years of age and from 23% to 11% in adolescents [3]. The United States has also reported
decreased infection rates due to more frequent antibiotic therapy for infection [2, 4]. In
contrast, the data in developing countries suggests that increasing age is a predisposing factor
for infection since the prevalence of infection for the developing countries is more than 50%
by 5 years and by 20 years the infection rate exceeds 90% [5]. Possibly, the age when H.
pylori is acquired could be considered a risk factor in the serious diseases associated with the
infection, since that will determine the length of the chronic inflammation.
Initial Vaccine Approaches

Vaccine studies require animal models that mimic human disease for development and
testing. Hence, animal models are the first challenge in the development of an H. pylori
vaccine. Since H. pylori is strictly a human pathogen, infection of mice is somewhat
challenging. The development of the Helicobacter felis (H. felis) model in 1990 led to
improved methods for the examination of vaccine strategies in mouse models [6]. In 1992,
Chen, et al. was the first group to demonstrate that vaccination could protect against
Helicobacter infection [7]. Soon after this first report, another group was able to show that
therapeutic immunization could be successful [8]. The continuing challenge in the field was
that while successful protection in mice was shown against H. felis, the difference in species

Helicobacter pylori Infection: The Vaccine Development 381
and virulence factors potentially would not translate to the pylori species in humans.
Consequently, attempts were made to establish H. pylori infections in mice. The first chronic
infection of mice was reported in 1995 [9]. Studies going forward with H. pylori were then
able to demonstrate successful prophylactic and therapeutic immunizations [10-12]. However,
the mouse models continued to be challenging as the mouse adapted H. pylori Sidney strain
(SS1) has been shown to lose a functional type IV secretion system and therefore cannot
deliver the CagA protein into host cells. This results in a failure to induce inflammation in a
mouse to a level that is seen in human disease. Recently, the PMSS1 strain has been proposed
for use, which at least for a short period of time (4-12 weeks), was demonstrated to retain a
functional type IV secretion system in vivo [13]. Thus this strain is a possibility going
forward to better mimic human disease for vaccine studies. Unfortunately, the success of
vaccination seen in the mouse model has yet to be demonstrated in humans. However, some
recent progress has been made in the laboratory setting in developing new methods to
approach a vaccine development.
Protective Immunity in H. pylori

Vaccine Development
H. pylori is a persistent pathogen that establishes a lifelong infection in people living in
areas of high infection rates. The low level chronic inflammatory immune response is not
successful in eradicating a host of infection. H. pylori has also developed mechanisms to
modulate and evade host immune responses. Thus, the ability of H. pylori to evade the host
response is likely why inducing protective immunity via vaccine is especially challenging.
Humoral Response
Many successful vaccines employ an antibody response to provide protection against

challenge with a pathogen. In the case of H. pylori, early studies demonstrated that oral
immunization of mice and ferrets with heat killed H. pylori induced mucosal and serum IgA
and IgG [14]. At that time, animal models were not well developed at this time and
subsequent studies showed that antibodies were not required for protection in vaccine studies
in mice [15, 16]. These studies showed that antibody deficient mice showed similar levels of
vaccine mediated protection as wild type mice, thus suggesting that antibody-mediated
responses may not be the right strategy in vaccine development going forward. In contrast,
there was evidence in one study suggesting that antibodies to outer membrane proteins of H.
pylori considerably reduced bacterial load in infected mice suggesting that a humoral
response might provide some protection when used in a therapeutic approach [17]. A further
study also showed some success in a prophylactic approach where a urease vaccine protected
mice against H. pylori colonization where specific antibody responses were seen in the gastric
mucosa of immunized mice [18]. In another study, human volunteers vaccinated with
recombinant urease had increased anti-urease serum IgA and circulating IgA producing cells
in conjunction with decreased bacterial load, although not complete eradication of the
bacteria [19].

T Cell Response
Multiple studies have shown a critical role for T helper cells in both prophylactic and
therapeutic vaccine studies in protection against H. pylori infection. One study established
that Th1 are required for vaccine protection in mice by showing that IFNγ knockout mice
were not protected against H. pylori challenge after vaccination, while IL-4 knockout mice
were protected [20]. This study also showed that IL-12 knockout mice were also not
protected. This is in agreement with another study suggesting that IL-12p40 knockout mice
were not protected during vaccine challenge. However, in contrast, this work also suggested
that mice lacking IFNγ did have some protection from H. pylori challenge after vaccination
[21]. In further support of protection requiring Th1 cell responses, one group demonstrated
that vaccine consisting of immunodominant T cell epitopes of H. pylori virulence factors
could induce protection through specific Th1 cell activation and enhancement of the humoral
response [22]. The role of H. pylori lipopolysaccharide (LPS) in the Th1 response was also
demonstrated by one study that showed mice receiving lysates containing LPS developed a
strong Th1 response, but mice who received a vaccine of lysates devoid of LPS induced a
Th2 response [23]. In addition to the potential for the Th1 response in protection, the more
recently described Th17 have also been shown to play a role in vaccine-induced protection
from Helicobacter infection. One study showed in an H. felis protection model that in
immunized mice injected with anti-IL-17 antibodies had inhibited inflammation and reduction
of H. felis compared to mice injected with control antibodies [24]. In agreement with this
study, another group showed that Th17 effector mechanisms are critical to protection in an H.
pylori model, while a third group suggested that IL-17A plays a role in protection, but in the
absence of IL-17A, other inflammatory immune mechanisms may compensate to help provide
protection [25, 26]. While not all groups agree on the intricacies of the T helper cell response,
there is general agreement that it does play an important role in protection.
Innate Response
The role of the innate immune response in vaccine protection against H. pylori is not yet
clear. One study suggested that immunization with a urease B DNA vaccine induced
potentially helpful innate responses, particularly beta-defensin, which may have contributed
to reduced bacterial load in immunized mice [27]. Another study has also suggested that
neutrophils aid in the induction of protective immunity in a mouse vaccine model [25],
although neutrophils are thought to be associated with host damage during infection so the
role of these cells is not clear. Mast cells, another innate immune cell type, were shown to
contribute to vaccine protection in a mouse model in two separate studies. One of the studies
showed a protective challenge with H. felis in wild type mice, but not in mast cell deficient
mice [28]. However, when the mast cell deficient mice received cultured bone marrow-
derived mast cells, they were able to clear the infection. In contrast, another study with an H.
pylori vaccine model showed that while mast cell deficient mice were not totally protected
against H. pylori challenge after vaccination, they did have reduction in bacterial load [29].
Mast cells were shown in this study to contribute to neutrophil recruitment and inflammation
in response to H. pylori.

Antigen Candidates for H. pylori Vaccine

Whole Cell
Early attempts at vaccines in mice consisted of H. felis sonicates, which induced antibody
responses [14], and when sonicates were accompanied by cholera toxin, a 96% protection rate
against challenge of acute infection was achieved [30]. The whole cell approach continued to
achieve high success rates in various animal models, such as ferrets, gnotobiotic piglets, and
monkeys [31-33]. Due to the success of whole cell vaccines in animal models, this approach
was considered for human vaccines. Although the protection achieved by vaccination with
whole cell extracts was very high in animal models, this approach could encounter quality
control and safety problems in humans due to the variability of lot preparations and the
presence of potentially dangerous antigens.
The first attempt at a human challenge model came in 2004 with a trial infecting human
volunteers with a live strain of H. pylori and examining the immune response before
eradication therapy [34]. This study revealed gastric pathologies consistent with chronic H.
pylori infection as soon as 2 weeks after infection, including increased IL-8 and
polymorphonuclear cell infiltration in the gastric mucosa. This was the first study to conclude
that human experimental H. pylori infection may be a viable approach to vaccine
development. Following this study, the safety and immunogenicity of a formalin-inactivated,
oral H. pylori whole cell vaccine, was tested in healthy and infected volunteers [35]. After the
first immunization, diarrhea, nausea and vomiting were observed. There was also a significant
increase in fecal and salivary anti-H. pylori IgA observed in infected and uninfected subjects
who were vaccinated. Effectiveness of the vaccine in reduction of bacterial load was not seen
in infected individuals.
Urease
Perhaps the most well studied H. pylori antigens as a potential vaccine candidate is
urease since it is a well-studied virulence factor that makes up 10% of the protein level that H.
pylori produces. Immunization with urease has had much success in mice where multiple
studies showed >97% reduction in bacterial burden in immunized mice upon H. pylori
challenge [11, 12, 15]. Again, the success in mice with a urease based vaccine led to some
human studies. A study conducted to evaluate the safety and immunogenicity of recombinant
urease administered with orally with Escherichia coli heat-labile enterotoxin in healthy H.
pylori-infected volunteers resulted in an increase in the titers of anti-urease serum IgA [19].
This vaccine resulted in reduction of H. pylori in gastric tissues, but did not result in complete
removal of the bacteria, and there were significant side effects due to the E. coli enterotoxin.
Another more recent attempt at an orally administered urease vaccine utilized Salmonella
Typhi expressing urease [36], but a similar result was seen where satisfactory protection was
not seen although there was reduced bacterial burden. Further urease vaccine approaches
were tested with various adjuvants and routes of administration as described below with some
antibody response, but still failed to provide protection [11, 37, 38].

VacA, NAP, and CagA
Other virulence factors of H. pylori that are known to induce strong immune responses in
humans have also been examined as potential vaccine candidates. The vacuolating toxin, or
VacA, is expressed by about half of all H. pylori strains. This factor binds to host cell plasma
membranes and forms a pore, which results in release of nutrients from the cell that the
bacteria use for survival [39]. One early mouse study showed that vaccination with
recombinant VacA resulted in protection from H. pylori infection [9]. Another study showed
that intragastric immunization with VacA was able to eradicate a chronic infection with H.
pylori when administered with an effective adjuvant [10]. In addition to eradication of H.
pylori, both of these studies demonstrated that mice vaccinated with VacA were resistant to
re-infection.
Successes with VacA led to further studies with combinations of recombinant virulence
factors. Other virulence factors that have been shown to induce immune responses are the
Cag pathogenicity island protein A (CagA) and the neutrophil activating protein (NAP).
CagA is a protein that is injected into host cells via a type IV secretion system of H. pylori.
This protein is also associated with gastric cancer. NAP was named for its ability to activate
neutrophils and was shown in a mouse model to be to induce not only neutrophil activation,
but also reactive oxygen intermediates and leukocyte chemotaxis [40]. Immunization of mice
with NAP induced an adequate immune response to provide protection from H. pylori
challenge.
This same group went on to examine a combination of CagA, VacA, and NAP in a beagle
model since other studies have shown the mouse vaccine models for H. pylori may not
translate to success in humans [41]. When beagles were vaccinated with the combination of
virulence factor proteins, antibody responses were seen to all three proteins and decreased
bacterial colonization was seen upon challenge; however, again, complete protection against
infection was not seen. Another group also showed both protective antibody and T cell
responses upon vaccination with a combination of CagA and NAP proteins at both the
mucosal and systemic levels [42]. The success of vaccination with a combination of specific
H. pylori antigens in animal models led to a combination vaccine containing CagA, VacA,
and NAP to be taken to a human trial [37]. In this trial, 86% of people mounted an antibody
response to all three virulence factors, but subsequent challenge with H. pylori was not
performed in these combination approaches. Although this approach seems promising, a
disadvantage of immunization with recombinant CagA and VacA is that not all strains
express these proteins. Therefore, infection with such negative strains could not be prevented
with these vaccines.
Outer Membrane Vesicles
H. pylori sheds part of its outer membrane as vesicles, similar to most gram negative
bacteria. These outer membrane vesicles (OMV) contain porins, LPS, VacA, and the Lpp20
lipoprotein among other protein components. Thus, these components were incorporated into
vaccine studies. In one study, mice immunized with OMV were protected from challenge
with H. pylori. The protection measured correlated with an IgG antibody response to an OMV
component of 18 kDa, which is commonly expressed by H. pylori strains [43]. This OMV

component was identified as lipoprotein 20 (Lpp20) using a monoclonal antibody. Further

examination indicated that hybridoma backpacks secreting IgG antibodies against Lpp20
considerably reduced the bacterial load in infected mice [43]. Additionally, previous evidence
indicates that this kind of antigen is a good vaccine candidate. Lpp20 was identified as a
protective antigen in a mouse model, since administration of Lpp20 before the challenge
resulted in significantly reduced bacterial numbers [17]. In order to further investigate this
vaccine candidate, the efficacy of the immunization with OMV was compared between
mouse and guinea pig models.
These animals received intranasal immunization of recombinant Lpp20 (rLpp20) or
OMV preparations from H. pylori, using cholera toxin (CT) as a mucosal adjuvant [17]. After
immunization, the animals were challenged with H. pylori SS1 strain with rLpp20, produced
a specific antibody response and a reduced level of H. pylori colonization in mice, but not in
guinea pigs. On the other hand, immunization with OMV in both models elicited systemic
and intestinal local immune response, characterized by specific IgG in serum and gastric
samples in addition to IgA in bile and the gastric mucosa.
Vaccine Administration and Adjuvants

For decades, knowledge about vaccine efficiency and safety has been shown to depend
not only on the pathogen derived immunogen, but also on the adjuvant component and route
of administration. Currently, the mucosal vaccination route is considered to be the most
effective in the management of H. pylori infection [44-46]. However, this type of vaccination
requires appropriate adjuvant and/or delivery system due to the poor immunogenicity of
purified protein after mucosal delivery.
Adjuvants and Delivery Systems
An adjuvant is an immune stimulating substance that is essential for vaccines that are
based on purified protein or killed bacteria preparations [47, 48]. Cholera toxin (CT), E. coli
derived heat labile toxin (LT), and chitozan with and without muramil-di-peptide (MDP) have
demonstrated efficacy as adjuvants for the mucosal H. pylori vaccine in animal models [47,
49, 50]. However, an effective mucosal adjuvant that is optimal for human use has yet to be
developed. A combination of CT or LT with H. pylori immunogens was shown to be effective
in the induction of systemic and mucosal antibodies as well as protective T cell responses, but
the toxicity of these substances prevents their use in humans [48, 51]. Although detoxified
forms of CT and LT were developed in studies by two groups, the loss in toxicity was found
to be associated with the reduction of their adjuvant activity [52, 53]. Mutants of these toxins
were then developed using site-directed mutagenesis and demonstrated to be safe and to
enhance immunogenicity of H. pylori vaccines in animal models [54]. A limitation of this
approach is that few reports have been published demonstrating the efficiency of this
approach in humans [55].
Other investigators turned to other adjuvants that are licensed for human use in vaccine
formulations, such as Alum and Freund’s as adjuvant. One study using neonatal mice as a

model for infant prophylactic vaccination using Freud’s adjuvant induced IFNγ, IL-2, IL-4,
and IL-5 secreting T cells and provided protection against infection [56]. The same group also
showed that vaccinating mice using aluminum hydroxide also led to IL-5 secreting antigen
specific T cells [57]. These responses were similar in adult mice suggesting this adjuvant has
potential for use in a human vaccine. The immune responses elicited in these studies are
promising because many others have focused on antibody responses, yet antibody deficient
mice show similar levels of protection as wild type mice [15, 16]. Alum and Freud’s adjuvant
were also used in combination with recombinant H. pylori urease B subunit (rUreB) and
demonstrated promising results in an animal model when using intramuscular immunization
[58].
Another approach for adjuvant development is the use of ligands for innate immune
receptors. Use of MDP, chitozan or these in combination in rodent models demonstrated that
while MDP alone has no adjuvant efficacy, in combination with chitozan, it was a successful
adjuvant if used in conjunction with rUreB antigen upon intranasal administration [58].
Synthetic CpG motif, which is approved for human use, demonstrated promising results in the
murine model by enhancing specific and innate immune responses against H. pylori [59].
However, protection of those vaccine types against H. pylori challenge was not significant
when using a DNA based vaccine construct [60].
An alternative approach is to deliver antigens to the hosts using liposome/nanoparticle
based vehicles, DNA based and live vectors. Use of the liposome-based delivery system
demonstrated promising results by some groups [61, 62]. One group reported that oral
vaccination with a liposome-encapsulated recombinant fusion peptide of UreB epitope
combined with cholera toxin B subunit induced UreB specific serum IgG and mucosal IgA in
both prophylactic and therapeutic vaccination protocols. These vaccinations resulted in a
significant decrease in H. pylori urease activity and reduced the bacterial colonization of the
stomach, but failed to completely clear the bacteria [62]. Interestingly, this vaccination
resulted in a five-fold reduction of gastritis induced by H. pylori challenge. The post-
immunization gastritis was suggested to be associated with the increase in Th17 response that
is required for the bacterial clearance [25]. Finally, although the liposome based vaccine
delivery approach demonstrated promising results in the murine model, its transfer to human
use is in question, since this still requires the use of adjuvants such as CT that demonstrated
significant adverse effects when used in humans.
The live vector delivery system includes the use of harmless or attenuated intestine
colonizing bacteria and has been the most explored for H. pylori vaccine development.
Licensed typhoid fever mutant vaccine S. enterica strain Ty21a was engineered to express
UreA and UreB subunits of H. pylori. Although use of this type of vaccine delivery
demonstrated excellent performance in safety, immunogenicity, and protection against E. coli,
Shigella dysenteriae, and Yersinia pestis, it failed to enhance protective immune responses
and bacterial clearance in the case of H. pylori in human [36, 63]. Similar results were
obtained with other Salmonella based delivery system [44, 64, 65]. One studying using S.
typhimurium expressing urease showed a better induction of anti-urease specific antibodies
than the previous approached with S. typhi [66]. Some groups went on to try Lactobacillus
plantarum as a delivery system for H. pylori UreB subunit resulted in induction of UreB-
specific antibodies, but demonstrated only partial protection upon prophylactic use in mice
infected with H. felis [67]. Another live non-pathogenic bacteria proposed as an antigen-
delivery system for H. pylori for oral vaccination was Lactococcus lactis. UreB, Cag7-ct383,

Cag12, and AlpA H. pylori antigens were successfully expressed in this bacterium [68-71].
However, whether this system can successfully enhance the immunogenic property of H.
pylori antigens is still not well characterized. Oral administration of the engineered AlpA-
expressing L. lactis reported to induced AlpA specific IgA and IgG in higher titers when
compare to those induced by inactivated H. pylori [71]. The oral administration of the Cag7-
ct383 expressing L. lactis generated anti-Cag7 antibody in 100% of tested murine serum [69].
However, it is not yet determined whether this vaccine candidate is able to induce protective
mucosal immune responses. Oral administration of the Cag12 expressing L. lactis resulted
only in a weak generation of the anti-Cag12 serum antibodies (two out five tested mice) [68].
Therefore, the protective efficacy of these vaccine candidates needs to be further tested.
Intracellular expression of UreB in L. lactis failed to elicit significant UreB specific immune
responses and confer protection against challenge with H. pylori [70]. Oral delivery of L.
lactis to mice expressing extracellular H. pylori UreB also produced a significant UreB-
specific serum IgG responses and UreB-specific IgA were detected in the feces. Oral
vaccination with this vaccine candidate leads to a significant protection against challenge with
H. pylori SS1 strain in mice [72].
Although it is clear that more optimization in the use of non-pathogenic, probiotic type
living microorganisms as delivery system for H. pylori vaccine is in need, this approach is
very attractive due to the ability to inoculate probiotics in the human host. In fact, probiotics
have been demonstrated to have an immune-dominant capacities and have proven benefits in
treatment and prevention of rotavirus diarrhea in children and reduction of antibiotic-
associated intestinal side-effects [73].
Vaccine Administration Routes
Several routes of H. pylori vaccine administration have been explored over the last two
decades. Systemic immunization using intraperitoneal, intramuscular, or subcutaneous
administration resulted in the generation of the immunogen-specific immune responses for
the most of the tested H. pylori vaccine candidates in rodent and primate models. These
routes of administration did not confer a satisfactory protection level against H. pylori or H.
felis challenge [30, 60, 74, 75]. These observations led to the conclusion that mucosal
delivery of the vaccine might be the optimal route for the induction of the anti-H. pylori
immune responses and bacterial clearance [44, 47, 76]. To determine the optimal
administrative sites for the mucosal immunization against H. pylori infection, the protective
efficacy of the vaccine candidates were assessed in mice given the vaccine by oral, intranasal
or rectal route [11, 55, 67, 76].
In one study using the murine H. pylori model the effect of administration route on the
protective efficacy of rUre plus LT as a mucosal adjuvant was tested [11]. This study
demonstrated that this vaccination demonstrated high immunogenicity and protection via all
three routes of administration (oral, intranasal, and rectal). Another group also tested multiple
routes of immunization with CagA and NAP proteins [42]. Their studies showed that mucosal
and systemic antibody responses were enhanced with mucosal followed by parenteral
immunization. Any single route or combination of immunization routes with CagA and NAP
induced antigen-specific splenic IL-4 secreting cells. Interestingly, serum IgG1 was induced
in response to intranasal immunization alone or in combination with intramuscular

immunization. Based on the previous success of rectal immunization in mice [11], a rectal
approach was used in human volunteers. In this trial tolerance to the route of immunization
and the resulting immune response were tested, but H. pylori challenge was not attempted
[38]. Although the route of administration was well tolerated, only a small percentage of
patients developed anti-urease antibodies (16.7%).
Another human study tested an oral route of urease administration in capsules, which was
also well tolerated [77]. Participants were found to have a dose dependent antibody response,
but significant immune cell responses required high doses of adjuvant that also led to the
increased side effect of diarrhea. Using a cholera toxin B model antigen, one group
demonstrated that in contrast to oral vaccination, nasal and rectal vaccination did not induced
significant increases in the specific antibody secreting cells either in antrum or duodenum of
H. pylori-infected individuals [55]. Promising results were obtained using the sublingual
route of immunization, which was recently described as a novel effective way to induce
mucosal immune responses in the respiratory and genital tract [78]. One group demonstrated
that successful prophylactic sublingual immunization of mice with H. pylori lysate and CT as
an adjuvant was associated with the induction of H. pylori-specific IgG and IgA in both the
stomach and the intestine [49]. Furthermore, the anti-H. pylori protection in this study was
also associated with an increase in IFN-α and IL-17 producing T cells, which coincides with
the enhanced infiltration of CD4+T cells and CD19+ B cells into the H. pylori infected
stomach. The same group also demonstrated prophylactic efficacy of sublingual
immunization with H. pylori lysate antigens and the non-toxic double mutant E. coli LT
R192G/L211A (dmLT) as an adjuvant [79]. This immunization resulted in a significant
decrease in bacterial load after challenge when compared to unimmunized infection controls.
This protection was correlated with the increase in cellular immune responses and enhanced
in vitro proliferative and cytokine responses seen in the spleen and mesenteric lymph nodes to
H. pylori antigens. Taken together, these studies suggest that oral vaccination might be the
preferable route for H. pylori vaccine administration to humans.
Vaccination Priorities
H. pylori has been identified as the causal agent for chronic gastritis, duodenal and gastric
ulcers and is associated with the development of gastric cancer, but little is known about what
determines one disease or the other. The outcome of the infection may involve a combination
of the bacterial infection, as well as environmental factors. A high percentage of duodenal and
gastric ulcers are reported to be attributable to H. pylori, around 95% and 70%, respectively
[2]. The association between gastric cancer and H. pylori has been demonstrated by many
different studies [80-82]. Some of the evidence comes from studies of serum samples from
patients with gastric cancer and control subjects free of cancer, where a significant association
was found between the infection by H. pylori and gastric cancer [83]. Further evidence
suggests that 70% of distal gastric cancers are attributable to H. pylori [84]. Thus, those in
areas of high prevalence with high risk for gastric cancer would benefit from a protective
vaccine. The problem remains that H. pylori infects 50% of the world’s population so
evaluation of populations that would most benefit from a vaccine are needed.

Conclusion
H. pylori infect about half of human population worldwide. This infection may result in
serious clinical outcomes such as ulcers and cancer. The increase in the antibiotic-resistant
strains of H. pylori has stirred again new interest in the development of the vaccine against
this pathogen. The facts presented above highlight the strong progress in the anti-H. pylori
vaccine development. Despite these efforts, several questions remain unanswered. One of the
main issues is that H. pylori is solely a human pathogen, which does not naturally colonize
mice. This may be the reason for better efficacy of the vaccine candidates seen in the H.
pylori murine model, when compared to human trials. An animal model which adequately
mimics human disease is yet to be developed. However, the recently developed humanized
mice might help to resolve this issue. It is currently clear that the immune responses
associated with this chronic infection are complex. Th17 responses might be harmful and
contribute to the development of cancer at the chronic stage of H. pylori infection, but at the
same time IL-17 is required for the eradication of these bacteria. Further, although several
promising antigens have been described over the last decade for vaccine development, there is
still the need for the development of a “universal antigen system” that might overcome the H.
pylori genome instability. Recent convergence of genomics, bioinformatics, and immunology
has expanded tools available and will likely reduce the time required for optimal vaccine
design. Thus, the new technologies might help to develop a multi-epitope vaccine in a
relatively short time. The same idea is applicable for the adjuvant and delivery system. As
seen in Table 22.1, an overview of human vaccine trials indicates that that oral/upper gastro-
intestinal tract vaccination is the preferable route of administration of an anti-H. pylori
vaccine administration in humans. Yet, another issue that needs to be resolved is whether
future vaccines will be for prophylactic use and applicable for the immunization of young
children, since infections occur in the early childhood in developing nations where prevalence
is highest. Alternatively, we might target a therapeutic approach for the eradication of already
infected individuals.
While a variety of approaches have been undertaken to develop a vaccine in animal
models, there has yet to be complete protection against H. pylori infection. Most studies have
resulted in decreased bacterial load, but not eradication of the infection both in prophylactic
and therapeutic approaches. What most approaches have in common is a reduction in H.
pylori colonization, yet not eradication. These results are testament to H. pylori being a
successful pathogen, initiating chronic and persistent infections along with employing
mechanisms to subvert the host immune response. Although H. pylori induce both innate and
adaptive immune responses in the host, these responses are not typically robust enough to
clear the infection. Yet the persistent chronic immune response is responsible for the
pathogenesis seen during infection suggesting why a protective vaccine against H. pylori is a
challenge. However, some small successes have indicated minimal amounts of protection in
humans, thus suggesting the potential still remains for a successful human vaccine against
this important pathogen.

Table 22.1. Overview of human H. pylori vaccine trials
Prophylactic
Immune Bacterial Side
or Antigen Route Reference
Response Load effects
Therapeutic
Salmonella typhi No humoral or
ΔphoP/phoQ mucosal No DiPetrillo et al.
Prophylactic Oral Minimal
expressing Urease immune Challenge 1999
A and B. response
Michetti et al.
Therapeutic Urease + E.coli LT Oral Serum IgA Decreased Diarrhea
1999
Salmonella
typhimurium Anti-urease Angelakopoulos
No
Prophylactic ΔphoP/phoQ Oral specific Fever and Hohmann
Challenge
expressing Urease antibodies 2000
A and B
Weak antibody
S. typhi Ty21a No Bumann et al.
Prophylactic Oral and T cell None
expressing urease Challenge 2001
responses
Mucosal and
Prophylactic Whole-cell Diarrhea,
Serum IgA and No Kotloff et al.
and inactivated +/- Oral Fever,
IgG and PBMC Eradication 2001
Therapeutic mutant E.coli LT Vomiting
activation
Diarrhea
No Banerjee et al.
Prophylactic Urease + E.coli LT Oral IgA and IgG at high
Challenge 2002
dose
Serum and
No Sougioultzis et
Prophylactic Urease + E.coli LT Rectal mucosal IgA None
Challenge al. 2002
and IgG
S. typhi Ty21a H. pylori 40%
Dyspeptic Aebischer et al.
Prophylactic expressing urease Oral specific T helper cleared
symptoms 2008
or HP0231 cells infection
VacA, CagA, and IgG and antigen
No Malfertheiner et
Prophylactic NAP + aluminum Intramuscular specific T cell None
Challenge al. 2008
hydroxide responses
References
[1] An international association between Helicobacter pylori infection and gastric cancer.
The EUROGAST Study Group `published erratum appears in Lancet 1993 Jun 26;
341(8861): 1668: `see comments. Lancet 1993;341:1359-62.
[2] Rothenbacher D, Brenner H, (2003). Burden of Helicobacter pylori and H. pylori-
related diseases in developed countries: recent developments and future implications.
Microbes Infect., 5(8):693-703.
[3] Roosendaal R, Kuipers EJ, Buitenwerf J, van Uffelen C, Meuwissen SG, van Kamp GJ,
et al. (1997). Helicobacter pylori and the birth cohort effect: evidence of a continuous
decrease of infection rates in childhood. Am. J. Gastroenterol., 92(9):1480-2.

[4] Parsonnet J, (1995). The incidence of Helicobacter pylori infection. Aliment

Pharmacol. Ther., 9 Suppl 2:45-51.
[5] Bardhan PK, (1997). Epidemiological features of Helicobacter pylori infection in
developing countries. Clin. Infect. Dis., 25(5):973-8.
[6] Lee A, Fox JG, Otto G, Murphy J, (1990). A small animal model of human
Helicobacter pylori active chronic gastritis. Gastroenterology., 99(5):1315-23.
[7] Chen M, Lee A, Hazell S, (1992). Immunisation against gastric helicobacter infection
in a mouse/Helicobacter felis model. Lancet., 2;339(8801):1120-1.
[8] Doidge C, Crust I, Lee A, Buck F, Hazell S, Manne U, (1994). Therapeutic
immunisation against Helicobacter infection. Lancet., 9;343(8902):914-5.
[9] Marchetti M, Arico B, Burroni D, Figura N, Rappuoli R, Ghiara P, (1995).
Development of a mouse model of Helicobacter pylori infection that mimics human
disease. Science., 17;267(5204):1655-8.
[10] Ghiara P, Rossi M, Marchetti M, Di Tommaso A, Vindigni C, Ciampolini F, et al.
(1997). Therapeutic intragastric vaccination against Helicobacter pylori in mice
eradicates an otherwise chronic infection and confers protection against reinfection.
Infect. Immun., 65(12):4996-5002.
[11] Kleanthous H, Myers GA, Georgakopoulos KM, Tibbitts TJ, Ingrassia JW, Gray HL, et
al. (1998). Rectal and intranasal immunizations with recombinant urease induce distinct
local and serum immune responses in mice and protect against Helicobacter pylori
infection. Infect. Immun., 66(6):2879-86.
[12] Sanchez V, Gimenez S, Haensler J, Geoffroy C, Rokbi B, Seguin D, et al. (2001).
Formulations of single or multiple H. pylori antigens with DC Chol adjuvant induce
protection by the systemic route in mice. Optimal prophylactic combinations are
different from therapeutic ones. FEMS Immunol. Med. Microbiol., 30(2):157-65.
[13] Arnold IC, Lee JY, Amieva MR, Roers A, Flavell RA, Sparwasser T, et al. (2011).
Tolerance rather than immunity protects from Helicobacter pylori-induced gastric
preneoplasia. Gastroenterology., 140(1):199-209.
[14] Czinn SJ, Nedrud JG, (1991). Oral immunization against Helicobacter pylori. Infect.
Immun., 59(7):2359-63.
[15] Ermak TH, Giannasca PJ, Nichols R, Myers GA, Nedrud J, Weltzin R, et al. (1998).
Immunization of mice with urease vaccine affords protection against Helicobacter
pylori infection in the absence of antibodies and is mediated by MHC class II-restricted
responses. J. Exp. Med., 21;188(12):2277-88.
[16] Sutton P, Wilson J, Kosaka T, Wolowczuk I, Lee A, (2000). Therapeutic immunization
against Helicobacter pylori infection in the absence of antibodies. Immunol. Cell Biol.,
78(1):28-30.
[17] Keenan J, Oliaro J, Domigan N, Potter H, Aitken G, Allardyce R, et al. (2000). Immune
response to an 18-kilodalton outer membrane antigen identifies lipoprotein 20 as a
Helicobacter pylori vaccine candidate. Infect. Immun., 68(6):3337-43.
[18] Gomez-Duarte OG, Lucas B, Yan ZX, Panthel K, Haas R, Meyer TF, (1998).
Protection of mice against gastric colonization by Helicobacter pylori by single oral
dose immunization with attenuated Salmonella typhimurium producing urease subunits
A and B. Vaccine., 16(5):460-71.

[19] Michetti P, Kreiss C, Kotloff KL, Porta N, Blanco JL, Bachmann D, et al. (1999). Oral
immunization with urease and Escherichia coli heat-labile enterotoxin is safe and
immunogenic in Helicobacter pylori-infected adults. Gastroenterology., 116(4):804-12.
[20] Akhiani AA, Pappo J, Kabok Z, Schon K, Gao W, Franzen LE, et al. (2002). Protection
against Helicobacter pylori infection following immunization is IL-12-dependent and
mediated by Th1 cells. J. Immunol., 15;169(12):6977-84.
[21] Garhart CA, Heinzel FP, Czinn SJ, Nedrud JG, (2003). Vaccine-induced reduction of
Helicobacter pylori colonization in mice is interleukin-12 dependent but gamma
interferon and inducible nitric oxide synthase independent. Infect. Immun., 71(2): 910-
21.
[22] Li HB, Zhang JY, He YF, Chen L, Li B, Liu KY, et al. (2012). Systemic immunization
with an epitope-based vaccine elicits a Th1-biased response and provides protection
against Helicobacter pylori in mice. Vaccine., 17;31(1):120-6.
[23] Taylor JM, Ziman ME, Huff JL, Moroski NM, Vajdy M, Solnick JV, (2006).
Helicobacter pylori lipopolysaccharide promotes a Th1 type immune response in
immunized mice. Vaccine., 5;24(23):4987-94.
[24] Velin D, Favre L, Bernasconi E, Bachmann D, Pythoud C, Saiji E, et al. (2009).
Interleukin-17 is a critical mediator of vaccine-induced reduction of Helicobacter
infection in the mouse model. Gastroenterology., 136(7):2237-46.
[25] DeLyria ES, Nedrud JG, Ernst PB, Alam MS, Redline RW, Ding H, et al. (2011).
Vaccine-induced immunity against Helicobacter pylori in the absence of IL-17A.
[26] Flach CF, Ostberg AK, Nilsson AT, Malefyt RW, Raghavan S, (2011).
Proinflammatory cytokine gene expression in the stomach correlates with vaccine-
induced protection against Helicobacter pylori infection in mice: an important role for
interleukin-17 during the effector phase. Infect. Immun., 79(2):879-86.
[27] Hatzifoti C, Roussel Y, Harris AG, Wren BW, Morrow JW, Bajaj-Elliott M, (2006).
Mucosal immunization with a urease B DNA vaccine induces innate and cellular
immune responses against Helicobacter pylori. Helicobacter., 11(2):113-22.
[28] Velin D, Bachmann D, Bouzourene H, Michetti P, (2005). Mast cells are critical
mediators of vaccine-induced Helicobacter clearance in the mouse model.
[29] Ding H, Nedrud JG, Wershil B, Redline RW, Blanchard TG, Czinn SJ, (2009). Partial
protection against Helicobacter pylori in the absence of mast cells in mice. Infect.
Immun., 77(12):5543-50.
[30] Lee A, Chen M, (1994). Successful immunization against gastric infection with
Helicobacter species: use of a cholera toxin B-subunit-whole-cell vaccine. Infect.
Immun., 62(8):3594-7.
[31] Cuenca R, Blanchard TG, Czinn SJ, Nedrud JG, Monath TP, Lee CK, et al. (1996).
Therapeutic immunization against Helicobacter mustelae in naturally infected ferrets.
[32] Dubois A, Lee CK, Fiala N, Kleanthous H, Mehlman PT, Monath T, (1998).
Immunization against natural Helicobacter pylori infection in nonhuman primates.
Infect. Immun., 66(9):4340-6.
[33] Eaton KA, Ringler SS, Krakowka S, (1998). Vaccination of gnotobiotic piglets against
Helicobacter pylori. J. Infect. Dis., 178(5):1399-405.

[34] Graham DY, Opekun AR, Osato MS, El-Zimaity HM, Lee CK, Yamaoka Y, et al.
(2004). Challenge model for Helicobacter pylori infection in human volunteers. Gut.,
53(9):1235-43.
[35] Kotloff KL, Sztein MB, Wasserman SS, Losonsky GA, DiLorenzo SC, Walker RI,
(2001). Safety and immunogenicity of oral inactivated whole-cell Helicobacter pylori
vaccine with adjuvant among volunteers with or without subclinical infection. Infect.
Immun., 69(6):3581-90.
[36] Aebischer T, Bumann D, Epple HJ, Metzger W, Schneider T, Cherepnev G, et al.
(2008). Correlation of T cell response and bacterial clearance in human volunteers
challenged with Helicobacter pylori revealed by randomised controlled vaccination
with Ty21a-based Salmonella vaccines. Gut., 57(8):1065-72.
[37] Malfertheiner P, Schultze V, Rosenkranz B, Kaufmann SH, Ulrichs T, Novicki D, et al.
(2008). Safety and immunogenicity of an intramuscular Helicobacter pylori vaccine in
noninfected volunteers: a phase I study. Gastroenterology., 135(3):787-95.
[38] Sougioultzis S, Lee CK, Alsahli M, Banerjee S, Cadoz M, Schrader R, et al. (2002).
Safety and efficacy of E coli enterotoxin adjuvant for urease-based rectal immunization
against Helicobacter pylori. Vaccine., 13;21(3-4):194-201.
[39] Szabo I, Brutsche S, Tombola F, Moschioni M, Satin B, Telford JL, et al. (1999).
Formation of anion-selective channels in the cell plasma membrane by the toxin VacA
of Helicobacter pylori is required for its biological activity. EMBO J., 15; 18(20):
5517-27.
[40] Satin B, Del GG, Della B, V, Dusi S, Laudanna C, Tonello F, et al. (2000). The
neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen
and a major virulence factor. J. Exp. Med., 1;191(9):1467-76.
[41] Rossi G, Ruggiero P, Peppoloni S, Pancotto L, Fortuna D, Lauretti L, et al. (2004).
Therapeutic vaccination against Helicobacter pylori in the beagle dog experimental
model: safety, immunogenicity, and efficacy. Infect. Immun., 72(6):3252-9.
[42] Vajdy M, Singh M, Ugozzoli M, Briones M, Soenawan E, Cuadra L, et al. (2003).
Enhanced mucosal and systemic immune responses to Helicobacter pylori antigens
through mucosal priming followed by systemic boosting immunizations. Immunology.,
110(1):86-94.
[43] Keenan JI, Allardyce RA, Bagshaw PF, (1998). Lack of protection following
immunisation with H. pylori outer membrane vesicles highlights antigenic differences
between H. felis and H. pylori. FEMS Microbiol. Lett., 1;161(1):21-7.
[44] Del GG, Malfertheiner P, Rappuoli R, (2009). Development of vaccines against
Helicobacter pylori. Expert Rev. Vaccines., 8(8):1037-49.
[45] Flach CF, Mozer M, Sundquist M, Holmgren J, Raghavan S, (2012). Mucosal
vaccination increases local chemokine production attracting immune cells to the
stomach mucosa of Helicobacter pylori infected mice. Vaccine., 21;30(9):1636-43.
[46] Taylor JM, Ziman ME, Fong J, Solnick JV, Vajdy M, (2007). Possible correlates of
long-term protection against Helicobacter pylori following systemic or combinations of
mucosal and systemic immunizations. Infect. Immun., 75(7):3462-9.
[47] Nedrud JG, (2001). Helicobacter pylori vaccines: lessons from small animal models.
Scand. J. Immunol., 53(5):429-36.
[48] Sutton P, (2001). Helicobacter pylori vaccines and mechanisms of effective immunity:
is mucus the key? Immunol. Cell Biol., 79(1):67-73.

[49] Raghavan S, Ostberg AK, Flach CF, Ekman A, Blomquist M, Czerkinsky C, et al.
(2010). Sublingual immunization protects against Helicobacter pylori infection and
induces T and B cell responses in the stomach. Infect. Immun., 78(10):4251-60.
[50] Ruiz-Bustos E, Sierra-Beltran A, Romero MJ, Rodriguez-Jaramillo C, Ascencio F,
(2000). Protection of BALB/c mice against experimental Helicobacter pylori infection
by oral immunisation with H. pylori heparan sulphate-binding proteins coupled to
cholera toxin beta-subunit. J. Med. Microbiol., 49(6):535-41.
[51] Kubota E, Joh T, Tanida S, Sasaki M, Kataoka H, Watanabe K, et al. (2005). Oral
vaccination against Helicobacter pylori with recombinant cholera toxin B-subunit.
[52] Blanchard TG, Lycke N, Czinn SJ, Nedrud JG, (1998). Recombinant cholera toxin B
subunit is not an effective mucosal adjuvant for oral immunization of mice against
Helicobacter felis. Immunology., 94(1):22-7.
[53] Weltzin R, Guy B, Thomas WD, Jr., Giannasca PJ, Monath TP, (2000). Parenteral
adjuvant activities of Escherichia coli heat-labile toxin and its B subunit for
immunization of mice against gastric Helicobacter pylori infection. Infect. Immun.,
68(5):2775-82.
[54] Pizza M, Giuliani MM, Fontana MR, Monaci E, Douce G, Dougan G, et al. (2001).
Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants. Vaccine.,
21;19(17-19):2534-41.
[55] Johansson EL, Bergquist C, Edebo A, Johansson C, Svennerholm AM, (2004).
Comparison of different routes of vaccination for eliciting antibody responses in the
human stomach. Vaccine., 25;22(8):984-90.
[56] Eisenberg JC, Czinn SJ, Garhart CA, Redline RW, Bartholomae WC, Gottwein JM, et
al. (2003). Protective efficacy of anti-Helicobacter pylori immunity following systemic
immunization of neonatal mice. Infect. Immun., 71(4):1820-7.
[57] Gottwein JM, Blanchard TG, Targoni OS, Eisenberg JC, Zagorski BM, Redline RW, et
al. (2001). Protective anti-Helicobacter immunity is induced with aluminum hydroxide
or complete Freund's adjuvant by systemic immunization. J. Infect. Dis., 1;184(3):308-
14.
[58] Begue RE, Moll A, (2009). Immunogenicity of Recombinant Helicobacter pylori
Urease B Administered by Various Routes and with Different Adjuvants. Open Vaccine
J., 1;2:28-32.
[59] Hatzifoti C, Bajaj-Elliott M, Dorrell N, Anyim M, Prentice MB, Nye KE, et al. (2004).
A plasmid immunization construct encoding urease B of Helicobacter pylori induces an
antigen-specific antibody response and upregulates the expression of beta-defensins and
IL-10 in the stomachs of immunized mice. Vaccine., 30;22(20):2651-9.
[60] Zavala-Spinetti L, Breslin MB, Correa H, Begue RE, (2006). Development and
evaluation of a DNA vaccine based on Helicobacter pylori urease B: failure to prevent
experimental infection in the mouse model. Helicobacter., 11(6):517-22.
[61] Huang W, Bai Y, Wang JD, Wu JB, Li GF, Zhang WM, et al. (2005). Preparation oral
liposome-encapsulated recombinant Helicobacter pylori heat shock protein 60 vaccine
for prevention of Hp infection. Di Yi Jun Yi Da Xue Xue Bao., 25(5):531-4.
[62] Zhao W, Wu W, Xu X, (2007). Oral vaccination with liposome-encapsulated
recombinant fusion peptide of urease B epitope and cholera toxin B subunit affords

prophylactic and therapeutic effects against H. pylori infection in BALB/c mice.

Vaccine., 1;25(44):7664-73.
[63] Bumann D, Metzger WG, Mansouri E, Palme O, Wendland M, Hurwitz R, et al.
(2001). Safety and immunogenicity of live recombinant Salmonella enterica serovar
Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers.
Vaccine., 12;20(5-6):845-52.
[64] DiPetrillo MD, Tibbetts T, Kleanthous H, Killeen KP, Hohmann EL, (1999). Safety and
immunogenicity of phoP/phoQ-deleted Salmonella typhi expressing Helicobacter
pylori urease in adult volunteers. Vaccine., 14;18(5-6):449-59.
[65] Zhang S, Walters N, Cao L, Robison A, Yang X, (2013). Recombinant Salmonella
Vaccination Technology and Its Application to Human Bacterial Pathogens. Curr.
Pharm. Biotechnol., 9.
[66] Angelakopoulos H, Hohmann EL, (2000). Pilot study of phoP/phoQ-deleted Salmonella
enterica serovar typhimurium expressing Helicobacter pylori urease in adult volunteers.
Infect. Immun., 68(4):2135-41.
[67] Corthesy B, Boris S, Isler P, Grangette C, Mercenier A, (2005). Oral immunization of
mice with lactic acid bacteria producing Helicobacter pylori urease B subunit partially
protects against challenge with Helicobacter felis. J. Infect. Dis., 15;192(8):1441-9.
[68] Kim SJ, Jun DY, Yang CH, Kim YH, (2006). Expression of Helicobacter pylori cag12
gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-
Cag12 immune response in mice. Appl. Microbiol. Biotechnol.,72(3):462-70.
[69] Kim SJ, Lee JY, Jun DY, Song JY, Lee WK, Cho MJ, et al. (2009). Oral administration
of Lactococcus lactis expressing Helicobacter pylori Cag7-ct383 protein induces
systemic anti-Cag7 immune response in mice. FEMS Immunol. Med. Microbiol.,
57(3):257-68.
[70] Lee MH, Roussel Y, Wilks M, Tabaqchali S., (2001). Expression of Helicobacter
pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine
delivery system against H. pylori infection in mice. Vaccine., 16;19(28-29):3927-35.
[71] Sun ZL, Bi YW, Bai CM, Gao DD, Li ZH, Dai ZX, et al. (2010). Expression of
Helicobacter pylori alpA gene in lactococcus lactis and its immunogenicity analysis. Xi
Bao Yu Fen Zi Mian Yi Xue Za Zhi., 26(3):203-6.
[72] Gu Q, Song D, Zhu M, (2009). Oral vaccination of mice against Helicobacter pylori
with recombinant Lactococcus lactis expressing urease subunit B. FEMS Immunol.
Med. Microbiol., 56(3):197-203.
[73] Gorbach SL, (2002). Probiotics in the third millennium. Dig Liver Dis., 34 Suppl 2:S2-
S7.
[74] Eisenberg JC, Czinn SJ, Garhart CA, Redline RW, Bartholomae WC, Gottwein JM, et
al. (2003). Protective efficacy of anti-Helicobacter pylori immunity following systemic
immunization of neonatal mice. Infect. Immun., 71(4):1820-7.
[75] Maeda K, Yamashiro T, Minoura T, Fujioka T, Nasu M, Nishizono A, (2002).
Evaluation of therapeutic efficacy of adjuvant Helicobacter pylori whole cell sonicate
in mice with chronic H. pylori infection. Microbiol. Immunol., 46(9):613-20.
[76] Jiang W, Baker HJ, Smith BF, (2003). Mucosal immunization with helicobacter, CpG
DNA, and cholera toxin is protective. Infect. Immun., 71(1):40-6.
[77] Banerjee S, Medina-Fatimi A, Nichols R, Tendler D, Michetti M, Simon J, et al.
(2002). Safety and efficacy of low dose Escherichia coli enterotoxin adjuvant for urease

based oral immunisation against Helicobacter pylori in healthy volunteers. Gut.,

51(5):634-40.
[78] Cuburu N, Kweon MN, Hervouet C, Cha HR, Pang YY, Holmgren J, et al. (2009).
Sublingual immunization with nonreplicating antigens induces antibody-forming cells
and cytotoxic T cells in the female genital tract mucosa and protects against genital
papillomavirus infection. J. Immunol., 15;183(12):7851-9.
[79] Sjokvist OL, Flach CF, Clements J, Holmgren J, Raghavan S, (2013). The double
mutant heat-labile toxin from Escherichia coli, LT (R192G/L211A) is an effective
mucosal adjuvant for vaccination against Helicobacter pylori infection. Infect. Immun.,
Feb 25.
[80] Forman D, Newell DG, Fullerton F, Yarnell JW, Stacey AR, Wald N, et al. (1991).
Association between infection with Helicobacter pylori and risk of gastric cancer:
evidence from a prospective investigation. BMJ., 1;302(6788):1302-5.
[81] Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, et
al. (1991). Helicobacter pylori infection and the risk of gastric carcinoma. N. Engl. J.
Med., 17; 325(16):1127-31.
[82] Watanabe T, Tada M, Nagai H, Sasaki S, Nakao M, (1998). Helicobacter pylori
infection induces gastric cancer in mongolian gerbils. Gastroenterology., 115(3):642-8.
[83] Talley NJ, Zinsmeister AR, Weaver A, DiMagno EP, Carpenter HA, Perez-Perez GI, et
al. (1991). Gaastric adenocarcinoma and Helicobacter pylori infection. J. Natl. Cancer
Inst., 23:1734-9.
[84] Ekstrom AM, Held M, Hansson LE, Engstrand L, Nyren O, (2001). Helicobacter pylori
in gastric cancer established by CagA immunoblot as a marker of past infection.

Chapter 23
Helicobacter pylori Infection:

Eradication or Preservation?
Sun-Young Lee and Soo-Heon Park

Department of Internal Medicine, Konkuk University School of Medicine and
Department of Internal Medicine, The Catholic University of Korea, Seoul, South Korea
Abstract
Some have the point of view that everyone with Helicobacter pylori (H. pylori)
infection would be better off without the bacterium, while some take the view that H.
pylori infection provides some protection against certain disease and adverse effects after
H. pylori eradication. These include side effects of antibiotics, emergence of antibiotics
resistance, hyperlipidemia, obesity, gastroesophageal reflux disease, Barrett’s esophagus,
and erosive gastroduodenitis. Moreover, in countries with a higher prevalence of H.
pylori infection, it is not possible to achieve H. pylori eradication because of its low cost-
effectiveness and reinfection. For this reason, current indications for H. pylori eradication
vary among countries. Although the prophylactic eradication of H. pylori infection may
be clinically beneficial in some of these individuals, a general policy directed towards
complete elimination of H. pylori from the population would not be beneficial. In this
chapter, we aim to provide both sides of the debate on H. pylori eradication to better
inform the practicing physician based on emerging evidence.
Keywords: Helicobacter pylori, eradication, indication, preservation

Corresponding author: Sun-Young Lee M.D., Ph.D. Department of Internal Medicine, Konkuk University School
of Medicine, 4-12 Hwayang-dong, Gwangjin-gu, Seoul 143-729, South Korea. Tel: +82-2-2030-7747. Fax:
+82-2-2030-7748. E-mail: sunyoung@kuh.ac.kr.

398 Sun-Young Lee and Soo-Heon Park
Introduction
Helicobacter pylori (H. pylori) infection is an emerging health treatment, and thus
consensus on H. pylori eradication has been actively implemented and evaluated during past
three decades. In areas with a high prevalence of gastric cancer, it seems that eliminating H.
pylori infection contribute in reducing the burden of gastric cancer [1]. However, an argument
has been put forward for the beneficial effects of H. pylori infection, and an increasing rate of
resistance to antibiotics has led to reduced efficacy of H. pylori eradication. Therefore, only
symptomatic patients are to be treated leaving many infected individuals untreated. Since the
prevalence of H. pylori infection and the associated diseases differ among countries, different
approvals for H. pylori eradication are to be required. The acceptable indications of
eradication therapy should vary among countries based on the benefits and harms of H. pylori
eradication in each country [2-5].
Helicobacter pylori Eradication – PROS

Current guidelines for eliminating H. pylori infection are based on the available evidence
or on good clinical practice rather than randomised controlled trials [2-5]. Peptic ulcer,
MALT, early gastric cancer, iron deficiency anemia, idiopathic thrombocytopenic purpura,
chronic atrophic gastritis, and functional dyspepsia (FD) are the common indications. Some
guidelines also include consideration of the patient’s wishes, communities with a high
incidence of gastric cancer, family history of gastric cancer, long-term use of nonsteroidal
anti-inflammatory drugs (NSAIDs), aspirin or PPI therapy, or gastroesophageal reflux disease
(GERD). In addition, lymphocytic gastritis and Ménétrier’s disease are indications for H.
pylori eradication in China [4], whereas gastric hyperplastic polyps and chronic urticaria are
indications in Japan [5].
Peptic Ulcer Disease Including Scar
Eradication of H. pylori infection has been shown to facilitate peptic ulcer healing,
reduce recurrence of peptic ulcers and reduce incidence of bleeding peptic ulcers. A systemic
review of 21 studies involving 10,146 patients showed that ulcers were more common in H.
pylori-positive than in H. pylori-negative patients irrespective of NSAID use [6]. In a
Cochrane review, eradication therapy was superior to acid suppressants in duodenal ulcer
healing and was superior to no treatment in preventing gastric ulcer recurrence [7]. In another
Cochrane review, H. pylori eradication therapy was more effective than antisecretory therapy
in preventing recurrent bleeding from peptic ulcer [8]. Comparing H. pylori eradication
therapy against antisecretory therapy without long-term maintenance therapy, the rebleeding
rate was 2.9% versus 20%, while comparing H. pylori eradication against antisecretory
therapy with long-term maintenance therapy, the rebleeding rate was 1.6% versus 5.6%.

Helicobacter pylori Infection: Eradication or Preservation? 399
Marginal Zone B Cell Lymphoma
H. pylori infection is the main cause of gastric MALT lymphoma [9]. If only low-grade
lymphomas are considered, the prevalence of H. pylori infection is almost 90%, and H. pylori
eradication is effective in treating approximately 80% of patients with early stage lymphoma.
Complete remission of gastric MALT lymphoma after H. pylori eradication can take even
>12 months. PCR assay for the detection of monoclonal B cells remains positive in many
cases after complete remission has been reached, and gastric lymphoma recurrence occurs in
some patients after both bacterial and lymphoma regression.
After Early Gastric Cancer Resection
It is now well accepted that habitation of the body of the stomach by H. pylori results in a
large increase in the incidence of gastric adenocarcinoma, and the induction of neoplasia by
infection has resulted in the organism being classified as a type I carcinogen by WHO [10].
After the partial gastrctomy or endoscopic resection for early gastric cancer, eliminating H.
pylori infection is recommended to reduce the burden of gastric cancer [2-5].
Chronic Gastritis
Atrophic gastritis and metaplastic gastritis indicate chronic H. pylori infection, whereas
nodular gastritis, hemorrhagic gastritis, and hypertrophic gastritis are endoscopic findings of
recent H. pylori infection [11]. Although H. pylori eradication therapy clearly improves
histologic gastritis, there are conflicting opinions about the reversibility of gastric mucosal
atrophy and intestinal metaplasia after eradication therapy. It remains an issue of particular
interest to determine at which stage H. pylori eradication should be performed, and at which
point the disease has progressed to the “point of no return” becoming irreversible in a
stepwise model of changes in the gastric mucosa after H. pylori infection that lead to
intestinal-type gastric cancer. In our recent study, the endoscopic diagnosis of open-type
chronic atrophic gastritis and metaplastic gastritis was related to higher levels of caudal type
homeobox 2 (Cdx2) expression than closed-type chronic atrophic gastritis and
nonatrophic/nonmetaplastic cases [12]. A study which followed up 1131 patients for 9.5 years
showed that the grade of gastric atrophy was closely related to the development of gastric
cancer after H. pylori eradication [13]. They showed that cancer development after
eradication depends on the presence of extensive chronic atrophic gastritis before eradication,
Intestinal metaplasia can return to normal, remain invariant, or show progress after H.
pylori eradication [14]. Atrophy, expression of sonic hedgehog, and Cdx2 at the corpus lesser
curve significantly improved in mucosa without incomplete-type intestinal metaplasia, but not
in mucosa with incomplete-type intestinal metaplasia. After H. pylori eradication,
incomplete-type intestinal metaplasia may change to complete-type with a decrease in
histological inflammation [15]. These findings suggest that cellular phenotype of gastric
intestinal metaplasia may be an important factor in the reduction of cancer incidence after H.
pylori eradication.

Gastric Polyps
Hyperplastic gastric polyps are considered to be directly related to chronic active gastritis
and concomitant H. pylori infection. H. pylori eradication can lead to complete polyp
regression in small hyperplastic polyps, and thus eradication is preferred before invasive
therapeutic options for those with hyperplastic gastric polyps less than 1 cm in size [16]. A
randomized controlled study showed that most hyperplastic gastric polyps disappear after H.
pylori eradication [17]. Since gastric carcinomas are more likely to develop in a stomach
containing hyperplastic polyps, it is recommended that additional biopsies should be obtained
from the antrum and corpus to clarify the decision on whether to apply eradication as
potential carcinoma prophylaxis in the presence of gastric hyperplastic polyps.
H. pylori eradication is considered as a treatment strategy for gastric adenomas since it
may inhibit progression of gastric adenoma to carcinoma [18, 19]. During 2 years of follow-
up, 12.5% of the H. pylori untreated group developed an intestinal-type gastric cancer,
whereas no gastric cancer was found in the treated group. Endoscopically, adenomas were
regressed or decreased in size after H. pylori eradication.
Acute Gastritis
Acute gastritis related to recent H. pylori infection includes nodular gastritis, follicular
gastritis, lymphocytic gastritis, hemorrhagic gastritis, granulomatous gastritis, hypertrophic
gastritis, Ménétrier’s disease, and congestive gastropathy [11]. A study showed that 0.19% of
the general population showed nodular gastritis on routine endoscopic examination, and all
had H. pylori infection [20]. It seems that when a new onset of H. pylori infection occurs in
adult, some individuals show an immature and aggressive tissue response, and few may
progress to a diffuse-type nodular gastritis, MALT lymphoma, or undifferentiated
adenocarcinoma [21]. It has been speculated that inflammatory cytokines or H. pylori-
infection-induced prostaglandins can be normalized after successful H. pylori eradication in
nodular gastritis [22].
Hypertrophic gastritis can be distinguished from tumoral condition by eradicating H.
pylori in patients with gastric giant folds [23]. After H. pylori eradication, endoscopic
ultrasonography demonstrates concomitant resolution of thickening and normalization of
these inner three layers (superficial mucosa, muscularis mucosa, and submucosa) [24]. A
study showed that the mutagenicity of gastric juice from the patients with enlarged-fold
gastritis was significantly greater, but decreased after eradication of H. pylori [25]. In
addition, 8-Hydroxy-2-deoxy guanosine (8-OHdG) and interleukin-1 beta (IL-1) levels are
increased in the gastric mucosa from patients with enlarged fold gastritis, and the odds ratio
for gastric carcinoma increased up to 35.5 in patients with gastric fold width greater or equal
than 7 mm. The methylation of E-cadherin in gastric mucosa decreased significantly after H.
pylori eradication abolished enlarged fold gastritis [26].
Acute hemorrhagic gastritis and granulomatous gastritis can also be improved by H.
pylori eradication [27]. Symptoms, endoscopic findings and pathologic findings showed
improvement after H. pylori eradication in granulomatous gastritis without recurrence [28].

Functional Dyspepsia
A randomized controlled trial of H. pylori eradication showed that FD-related symptom

resolution was observed in 24% of patients on active treatment and 7% on placebo [29]. Post-
treatment H. pylori status was the only predictor of symptom resolution, and thus suggests
that FD patients would benefit from treatment for H. pylori infection with as much as a 13-
fold increased chance of symptom resolution following its eradication.
First Relatives of Gastric Cancer Patients
H. pylori detection and prophylactic eradication of the infection should be offered

especially when the subject is less than 40 year-old in subjects with family history of gastric
cancer [30]. Several studies have shown that siblings of patients who develop gastric cancer
before 40 years of age have a higher H. pylori infection rate and higher prevalence of
intestinal metaplasia at the body, and show a higher multivariate-adjusted odd ratio for gastric
cancer [30-33]. Compared with healthy controls, first-degree relatives of patients with gastric
cancer had a higher prevalence of hypochlorhydria, and of gastric atrophy than patients with
non-ulcer dyspepsia matched for H. pylori prevalence [32]. In this study, eradication of H.
pylori resolved the gastric inflammation, hypochlorhydria and atrophy in half of the subjects.
With regard to the virulence of H. pylori, CagA seropositivity is significantly associated
with a higher gastric cancer among H. pylori-infected subjects [34]. However, it is not yet
indicated to eradicate all East-Asian cagA-positive cases since most of H. pylori infections in
East Asia are of East-Asian cagA type [35].
Long-Term Medications
To date, H. pylori eradication is recommended for patients taking long-term NSAIDs

[36], but there is no objective clinical data on cyclooxygenase-2 inhibitors and aspirin.
Notably, the opinions and clinical practice patterns for the management of anticoagulation
and antiplatelet medications differ significantly between the East and the West [37]. Since
there is a tendency among Eastern endoscopists to think that Asians are more prone to
bleeding than Caucasians, H. pylori eradication might be considered more seriously in East
Asians where the prevalence of H. pylori infection is higher.
PPIs may accelerate the development of atrophic gastritis when H. pylori is present [38],
and thus H. pylori may need to be treated before long-term PPI therapy. Profound acid
suppression with PPIs or high-dose histamine 2 receptor antagonists in the presence of H.
pylori-positive corpus gastritis may accelerate the loss of specialized glands, leading to
atrophic gastritis. This is particularly relevant in symptomatic patients undergoing treatment
with PPI, in whom the organism has a tendency to leave the antrum and inhabit the fundus.
Fundic infection predisposes to intestinal metaplasia and eventually adenocarcinoma, and
therefore, patients with chronic PPI therapy should be checked for active infection and if
present, every effort should be made to eradicate the organism [39].

Extraintestinal Diseases
H. pylori eradication could be used successfully in extragastric diseases that are

unresponsive to conventional therapy. H. pylori eradication should be considered only when
the extragastric disease is mediated by H. pylori-related cytotoxins and/or cytokines and it is
improved by H. pylori eradication. The higher prevalence of H. pylori infection in particular
diseases cannot be considered as an indication for H. pylori eradication, because of
confounding factors, including the effect of contamination. Of the various cardiovascular,
hepatobiliary, dermatological, immunological and hematological diseases, iron deficiency
anemia, idiopathic thrombocytopenic purpura, and atherosclerotic disease (ischemic heart
disease, stroke, and peripheral arterial disease) improved after successful H. pylori eradication
[40]. Recently, diminished halitosis after eradication suggested a causal link between H.
pylori infection and halitosis [41]. This indicates that H. pylori eradication may reduce the
production of substances responsible for bad breath detected by chromatographic
documentation of volatile sulfur compounds.
With regard to skin diseases, it is recommended to test and treat H. pylori infection in
patients with chronic urticaria, prurigo chronica multiformis, pruritus cutaneus, and eczema
nummulare [42]. Although few studies have shown that H. pylori eradication reduced the
severity of rosacea [42, 43], there are opposite results showing no significant lessening of
lesions [44, 45]. In the latter, the cure rates of H. pylori in rosacea patients and controls were
not different, and temporary improvement in papulopustules exclusively during the treatment
was independent of H. pylori eradication.
Other rare extragastric diseases that may improve after H. pylori eradication are central
nervous system, eye, and hepatobiliary lesions. Parkinson’s disease, Alzeheimer’s disease,
and migraine headache. In patients with Parkinson’s disease, H. pylori eradication improved
the L-dopa delayed onset time and short on-time duration, thereby demonstrating that H.
pylori infection could interfere with the absorption of L-dopa and provoke motor fluctuations
[46]. Besides, H. pylori eradication has been reported to positively influence glaucoma
indices, suggesting a possible causal link between H. pylori and glaucoma [47]. In addition,
H. pylori has been reported to promote hepatic fibrosis and increase the risk of tumor growth
factor-β1-mediated tumorigenesis, while the organism can be detected in patients with
hepatolithiasis, choledocholitihiasis, and cholangiocarcinoma [48, 49]. A more reproducible
evidence into pathogenic mechanisms are needed for extraintestinal diseases.
Helicobacter pylori Eradication – CONS

An argument has been put forward for the beneficial effects of H. pylori infection [50,
51], despite the fact that H. pylori should be eradicated because of various gastric disorders
(Table 1) [52, 53].

Table 1. Gastric disorders that result from Helicobacter pylori infection [50, 51]
Incidence
Gastritis (acute or chronic) 100%
Peptic ulcer disease (gastric or duodenal ulcer) 10%
Gastric atrophy 5%
Gastric cancer 1%
Mucosa-associated lymphoid tissue lymphoma <1%
Table 2. Current indications for H. pylori eradication in Asia
Indications for H. pylori eradication

Asia-Pacific [2] Recommendation A (randomized controlled trial)
 Peptic ulcer disease
 Mucosa-associated lymphoid tissue lymphoma
 Patients’ wishes
 Non-ulcer dyspepsia
 NSAIDs users
 Communities with high incidence of gastric cancer
Recommendation B (well conducted clinical trial)
 Atrophic gastritis
 After gastric cancer resection
 First degree relatives of gastric cancer
 Before long-term aspirin therapy
 Gastroesophageal reflux disease with long-term PPI therapy
Recommendation C (expert opinion)
 Unexplained iron deficiency anemia
 Idiopathic thrombocytopenic purpura
Korea [3] Definite indications
 Peptic ulcer including scar
 Marginal zone B cell lymphoma
 After resection of early gastric cancer
Recommended indications
 First relatives of gastric cancer patients
 Unexplained iron deficiency anemia
 Chronic idiopathic thrombocytopenic purpura
Possible indications
 Non-ulcer dyspepsia
 Long-term use of NSAIDs

Table 2. (Continued)
Indications for H. pylori eradication

Japan [5] Evidence level I (systematic review/meta-analysis)
 Gastric/duodenal ulcer
 Functional dyspepsia
Evidence level II (randomized controlled clinical trial)
 After endoscopic treatment of early gastric cancer
 Gastric hyperplastic polyp
 Reflux esophagitis
Evidence level III (non-randomized controlled clinical studies)
 Gastric mucosa-associated lymphoid tissue lymphoma
 Iron deficiency anemia
 Chronic urticaria
China [4] Necessary
 Peptic ulcer
 Early gastric cancer after operation
 Gastric mucosa-associated lymphoid tissue lymphoma
 Chronic gastritis associated with atrophy and erosions
Supportive
 Chronic gastritis associated with symptoms of functional dyspepsia
 Planned long-term use of NSAIDs
 Family history of gastric cancer
 Iron deficiency anemia of unknown cause
 Other H. pylori-related gastric diseases such as lymphocytic gastritis,
gastric hyperplastic polyps, and Menetrier’s disease
 Patient wishing H. pylori eradication
Their arguments are largely based on hypothesis and circumstantial evidence that less
than 20% of all H. pylori infected persons will develop significant clinical consequences in
their lifetime and that H. pylori may be a commensal to humans based on the antiquity of H.
pylori infection in humans and their co-evolution [54]. Therefore, only symptomatic patients
are to be treated leaving many infected individuals untreated.
The currently recommended first-line therapy for H. pylori infection is PPI, amoxicillin
and clarithromycin for 7 days in Asia, although current indications differ among the countries
(Table 2). Classic quadruple therapy is consistent with bismuth, PPI, metronidazole, and
tetracycline.
Further salvage therapy includes levofloxacin-based triple therapy and rifabutin-based
triple therapy. Sequential, concomitant therapy, dual therapy, and tailored therapy are now
being investigated [55]. After the failure of two or more eradication treatments, bacterial
resistance to antibiotics should be evaluated and the regimen of third-line therapy should be

selected according to each antimicrobial susceptibility. Multiple eradication failures should be

handled on a case-by-case basis by specialists.
Side Effects of Drug
Side effects of eradication include bitter taste, epigastric pain, diarrhea, nausea or
vomiting, loss of appetite, dizziness, liver toxicity or allergic skin rash [56]. Diarrhea is the
most common complaint, but shows a significantly low rate of severe diarrhea when
eradicated with the rifaximin containing regimen [57]. A meta-analysis study showed that
there were no serious adverse events occurring with bismuth therapy [58]. There was no
statistically significant difference detected in total adverse events with bismuth, specific
individual adverse events, with the exception of dark stools or adverse events leading to
withdrawal of therapy.
Probiotics may be beneficial in reducing adverse effects and increasing tolerability of H.
pylori eradication regimens. Various probiotics including Lactobacillus, Saccharomyces,
Bifidobacterium, and B. clausii can reduce adverse effects such as nausea, taste disturbance,
diarrhea, and epigastric pain, and increase tolerability of H. pylori eradication therapy [59].
Emergence of Antibiotics Resistance
Higher rate is usually required for satisfactory outcome for treatment of infections when
routine treatment is adopted without testing for susceptibility. However, treatment success
with H. pylori eradication therapy has fallen below 80%, largely due to clarithromycin or
metronidazole resistance. The distribution of minimal inhibitory concentrations for
amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, and fluoroquinolone
have shifted to higher concentrations from 1987 to 2003 in H. pylori strains [60]. Increase in
the primary resistance rate was found in amoxicillin (6.3-14.9%), clarithromycin (17.2-
23.7%), and both of levofloxacin and moxifloxacin (4.7-28.1%) [61]. Increase of resistance
occurred after initial failure of eradication therapy in case of clarithromycin, azithromycin,
levofloxacin, and moxifloxacin which was associated with previous eradication treatment
history.
The rates of eradication largely depends on the clarithromycin-sensitivity. Resistance to
clarithromycin can be determined by using restriction fragment length polymorphism (RFLP)
in domain V of 23S rRNA [62]. Clarithromycin resistance of H. pylori is rapidly increasing
these days [63]. There was an increasing tendency for the emergence of strains with dual
resistance to metronidazole and clarithromycin, although overall resistance to metronidazole
did not increased recently [64]. Among clarithromyin-resistant strains that have A2143G
mutations are not eradicated by PPI-based triple therapy. Therefore, A2143G is an important
23S rRNA mutation associated with clarithromycin resistance and affected the H. pylori
eradication efficacy. When DNA sequences of the 23S rRNA gene in 21 primary
clarithromycin-resistant and 64 secondary clarithromycin-resistant strains were determined by
PCR amplification and nucleotide sequence analyses, two mutations of the 23S rRNA gene,
A2143G and T2182C, were observed in primary clarithromycin-resistant isolates [65].

There is a significant rise in the prevalence of metronidazole resistance [66]. Of South-

Eastern Asian countries, Jakarta showed the highest resistance rates for metronidazole [67].
The resistance rates of the isolates were 100% for metronodazole, 27.8% for clarithromycin,
19.4% for amoxicillin, 6.9% for ciprofloxacin, norfloxacin and ofloxacin, 2.8% for
sparfloxacin and gatifloxacin, and 1.4% for levofloxacin and moxifloxacin.
Fluoroquinolone resistance of H. pylori is known to be dependent on mutations in the
QRDR of gyrA. A study on the distribution of gyrA point mutations showed that
fluoroquinolone resistance of H. pylori was caused by gyrA Asn-87 and Asp-91 point
mutations [68]. The Asn- 87 mutation seems to be an important determinant of failure of
fluoroquinolone-containing triple eradication therapy based on eradication results. Besides,
the 7-day moxifloxacin-containing triple therapy produced an unacceptably low eradication
rate [69]. Increasing the duration of therapy was expected to increase the eradication rate, but
the expected increased did not materialize, most likely because of coincident marked increase
in the prevalence of resistance to moxifloxacin which was 28.2% in 2007-2008. Considerable
number of patients with treatment failure have H. pylori strains resistant to two or more
antimicrobial agents. Antibiotic-susceptible and -resistant H. pylori can be present
simultaneously in the same host [70]. Of treatment-failure patients, some show mixed
infections with both antibiotic-susceptible and -resistant H. pylori strains. These results
suggest that continuing genomic diversities in the same strain may play an important role in
modulating the antibiotic-heteroresistant H. pylori in vivo.
Hyperacidity-Related Upper Gastrointestinal Disease
H. pylori eradication may induce or exacerbate GERD, Barrett’s esophagus, erosive

gastritis duodenitis by its influence on gastric acidity, but remains a controversial area. Recent
review articles showed that H. pylori eradication did not result in a significant difference in
the frequency of erosive GERD when compared with persistent infection, regardless of
follow-up period, location, or the baseline disease [71, 72]. Subsequently, a meta-analysis of
eight double-blind studies of H. pylori eradication [73] and a large post hoc analysis of the
peptic ulcer trials, GU MACH and DU MACH showed that H. pylori eradication for ulcer
disease did not lead to development of erosive esophagitis or new symptomatic GERD or
worsening of symptoms in patients with pre-existing GERD [74, 75].
A recent analysis showed that H. pylori infection reduced the onset of Barrett’s
esophagus [76]. However, there was a weak correlation between esophageal intestinal
metaplasia and infection [77]. In similar, a recent review of the literature concluded that there
is no significant difference in the incidence of H. pylori infection between patients with
Barrett’s esophagus and controls [78]. The association with Barrett’s esophagus may be
spurious and the benefits of H. pylori eradication would almost certainly outweigh the
theoretical impact on Barrett’s cancer.
There have also been multiple studies on the association between GERD and esophageal
adenocarcinoma, and the consensus is that acid reflux predisposes the squamous epithelium
of the esophagus to development of columnar metaplasia [77]. Because there is no consensus
as to the role of infection in protection against GERD, evidence for the objection that has
been made to the eradication of H. pylori is weak.

Hyperlipidemia and Obesity
H. pylori has an influence on the release of gastric hormones and therefore plays a role in
the regulation of body weight, hunger and satiety. H. pylori infection leads to a decrease of
circulating ghrelin through a reduction of ghrelin-producing cells in the gastric mucosa and
increases the amount of gastric leptin with no effect on circulating leptin levels [79]. Majority
of patients show improvement of dyspepsia symptoms after eradication treatment [80].
Eradication of H. pylori reverses the abnormal regulation of gastric hormone secretion and
points to the potential effect of H. pylori in the pathophysiology of obesity. Eradicated
subjects may have increased gastric emptying and hence decreased acid reflux or increased
appetite due to increased ghrelin and consequent weight gain. The Asian population is
probably facing a rising generation with high gastric acid and ghrelin secretion rates. These
physiological changes may contribute to increased dietary calorie intake and increased
prevalence of GERD due to obesity.
Asthma
Large studies have found that people without the bacterium are more likely to develop
asthma, hay fever or skin allergies in childhood [81, 82]. Stomachs that lack H. pylori seem
immunologically quite different from those that do not, and infection of young mice with H.
pylori protects against experimental asthma [83].
Reinfection
High reinfection rates in the community makes H. pylori cure a temporary event. The
reinfection rate of H. pylori is 2.94-3.51% per year in Korea [84, 85]. Reinfection rate of H.
pylori stayed rather low per year, and male and low income determined the reinfection. The
seroreversion rate was 5%, 10%, and 45% at 2, 10, and 18 months after H. pylori eradication,
respectively. The recrudescence of H. pylori was 3.49%, and the annual reinfection rate was
2.94% per year in their study. However, in subjects after second-line eradication with
bismuth-containing quadruple regimens, H. pylori reinfection rate of 6.0% per patient-year
[86]. The relapse of dyspeptic symptoms was the only factor predictive of H. pylori
recurrence in their study.
Reinfection rates vary among countries. The risk of reinfection after H. pylori eradication
was low as 3.38% of annual re-infection rate in Thai patients [87]. No dependent factors were
associated with a recurrence in their study. In Singapore, the recurrence of H. pylori infection
was low at the end of one year after successful eradication therapy in this urban East Asian
population [88]. Of 96 consecutive duodenal ulcer patients with biopsy-proven H. pylori
eradication, recurrence of H. pylori infection was detected at 9-10 months after H. pylori
eradication in two patients, and at 13-20 months in the remaining four. Besides, reinfection
following successful H. pylori eradication in patients in Vietnam was found to be higher than
in industrialized countries after 1 year [89]. The overall reinfection rate was 23.5%, with
58.8% of the strains being identical to the pre-treatment isolates and 41.2% being different.

Conclusion
H. pylori eradication should be performed in accordance with recent consensus
statements and recommendations along with the findings of numerous clinical trials and meta-
analyses .The controversy over whether H. pylori should be eradicated in all infected
individuals or just in symptomatic patients, reflects the risk to benefit ratio. The prevalence of
H. pylori infection and the associated diseases such as peptic ulcer or gastric cancer differ
among countries, and thus different approvals for treatment are required by governments or
insurance agencies, the acceptable indications of eradication therapy will vary among
countries. The controversy centers around whether to eliminate the organism in all infected
individuals or only in symptomatic patients on the grounds that the organism may be a
commensal and provide benefits to the asymptomatic infected person. Despite considerable
advances, further research studies are needed to provide definite direction for the treatment of
many conditions.
References
[1] Uemura, N., Okamoto, S., Yamamoto, S., Matsumura, N., Yamaguchi, S., Yamakido,
M., Taniyama, K., Sasaki, N., Schlemper, R.J., (2001). Helicobacter pylori infection
and the development of gastric cancer. N. Engl. J. Med. 345, 784-789.
[2] Fock, K.M., Katelaris, P., Sugano, K., Ang, T.L., Hunt, R., Talley, N.J., Lam, S.K.,
Xiao, S.D., Tan, H.J., Wu, C.Y., Jung, H.C., Hoang, B.H., Kachintorn, U., Goh, K.L.,
Chiba, T., Rani, A.A.; Second Asia-Pacific Conference, (2009). Second Asia-Pacific
Consensus Guidelines for Helicobacter pylori infection. J. Gastroenterol. Hepatol. 24,
1587-1600.
[3] Kim, N., Kim, J.J., Choe, Y.H., Kim, H.S., Kim, J.I., Chung, I.S.; Korean College of
Helicobacter and Upper Gastrointestinal Research; Korean Association of
Gastroenterology, (2009). Diagnosis and treatment guidelines for Helicobacter pylori
infection in Korea. Korean J. Gastroenterol. 54, 269-278.
[4] Hu, F.L., Hu, P.J., Liu, W.Z., De Wang, J., Lv, N.H., Xiao, S.D., Zhang, W.D., Cheng,
H., Xie, Y., (2008). Third Chinese National Consensus Report on the management of
Helicobacter pylori infection. J. Dig. Dis. 9, 178-184.
[5] Asaka, M., (2009). Guidelines in the management of H. pylori infection in Japan. Nihon
Rinsho 67, 2227-2232.
[6] Papatheodoridis, G.V., Sougioultzis, S., Archimandritis, A.J., (2006). Effects of
Helicobacter pylori and nonsteroidal anti-inflammatory drugs on peptic ulcer disease: a
systematic review. Clin. Gastroenterol. Hepatol. 4: 130–142.
[7] Ford, A.C., Delaney, B.C., Forman, D., Moayyedi, P., (2006). Eradication therapy for
peptic ulcer disease in Helicobacter pylori positive patients. Cochrane Database Syst.
Rev. 2, CD003840.
[8] Gisbert, J.P., Khorrami, S., Carballo, F., Calvet, X., Gené, E., Dominguez-Muñoz, J.E.,
(2004). H. pylori eradication therapy vs. antisecretory non-eradication therapy (with or
without long-term maintenance antisecretory therapy) for the prevention of recurrent
bleeding from peptic ulcer. Cochrane Database Syst. Rev. 2, CD004062.

[9] Gisbert, J.P., Calvet, X., (2011). Review article: common misconceptions in the
management of Helicobacter pylori-associated gastric MALT-lymphoma. Aliment.
Pharmacol. Ther. 34, 1047-1062.
[10] Peek, R.M. Jr., Crabtree, J.E., (2006). Helicobacter infection and gastric neoplasia. J.
Pathol. 208, 233-248.
[11] Lee, S.Y., (2011). Endoscopic gastritis: what does it mean? Dig. Dis. Sci. 56, 2209-
2211.
[12] Ahn, S.Y., Lee, S.Y., Hong, S.N., Kim, J.H., Sung, I.K., Park, H.S., Shim, C.S., Jin,
C.J., Han, H.S., (2011). Endoscopic diagnosis of open-type atrophic gastritis is related
to the histological diagnosis of intestinal metaplasia and Cdx2 expression. Dig. Dis. Sci.
56, 1119–1126.
[13] Take, S., Mizuno, M., Ishiki, K., Nagahara, Y., Yoshida, T., Yokota, K., Oguma, K.,
(2007). Baseline gastric mucosal atrophy is a risk factor associated with the
development of gastric cancer after Helicobacter pylori eradication therapy in patients
with peptic ulcer diseases. J. Gastroenterol. 42 (Suppl 17), 21-27.
[14] Shiotani, A., Uedo, N., Iishi, H., Tatsuta, M., Ishiguro, S., Nakae, Y., Kamada, T.,
Haruma, K., Merchant, J.L., (2007). Re-expression of sonic hedgehog and reduction of
CDX2 after Helicobacter pylori eradication prior to incomplete intestinal metaplasia.
Int. J. Cancer. 121, 1182-1189.
[15] Watari, J., Das, K.K., Amenta, P.S., Tanabe, H., Tanaka, A., Geng, X., Lin, J.J., Kohgo,
Y., Das, K.M., (2008). Effect of eradication of Helicobacter pylori on the histology and
cellular phenotype of gastric intestinal metaplasia. Clin. Gastroenterol. Hepatol. 6, 409-
417.
[16] Dirschmid, K., Platz-Baudin, C., Stolte, M., (2006). Why is the hyperplastic polyp a
marker for the precancerous condition of the gastric mucosa? Virchows Arch. 448, 80-
84.
[17] Ljubicić, N., Banić, M., Kujundzić, M., Antić, Z., Vrkljan, M., Kovacević, I., Hrabar,
D., Doko, M., Zovak, M., Mihatov, S., (1999). The effect of eradicating Helicobacter
pylori infection on the course of adenomatous and hyperplastic gastric polyps. Eur. J.
Gastroenterol. Hepatol. 11: 727-730.
[18] Saito, K., Arai, K., Mori, M, Kobayashi, R., Ohki, I., (2000). Effect of Helicobacter
pylori eradication on malignant transformation of gastric adenoma. Gastrointest.
Endosc. 52, 27-32.
[19] Gotoda, T., Saito, D., Kondo, H., Ono, H., Oda, I., Fujishiro, M., Yamaguchi, H.,
(1999). Endoscopic and histological reversibility of gastric adenoma after eradication of
Helicobacter pylori. J. Gastroenterol. 34 (Suppl 11), 91-96.
[20] Miyamoto, M., Haruma, K., Yoshihara, M., Hiyama, T., Sumioka, M., Nishisaka, T.,
Tanaka, S., Chayama, K., (2003). Nodular gastritis is caused by Helicobacter pylori
infection. Dig. Dis. Sci. 48, 968-975.
[21] Choi, H.J., Lee, S.Y., Lee, J.H., Seol, D.C., Kim, S.Y., Choi, H.J., Park, H.S., Hong,
S.N., Han, H.S., (2010). Two atypical cases of nodular gastritis: a poorly differentiated
gastric adenocarcinoma and a pseudo-low grade gastric MALT lymphoma.
Gastroenterol. Res. 3, 41-45.
[22] Shimatani, T., Inoue, M., Iwamoto, K., Hyogo, H., Yokozaki, M., Saeki, T., Tazuma,
S., Horikawa, Y., Harada, N., (2005). Gastric acidity in patients with follicular gastritis

is significantly reduced, but can be normalized after eradication for Helicobacter pylori.
[23] Stolte, M., Bätz, C.H., Bayerdörffer, E., Eidt, S., (1995). Helicobacter pylori eradication
in the treatment and differential diagnosis of giant folds in the corpus and fundus of the
stomach. Z. Gastroenterol. 33, 198-201.
[24] Avunduk, C., Navab, F., Hampf, F., Coughlin, B., (1995). Prevalence of Helicobacter
pylori infection in patients with large gastric folds: evaluation and follow-up with
endoscopic ultrasound before and after antimicrobial therapy. Am. J. Gastroentrol. 90,
1969-1973.
[25] Nishibayashi, H., Kanayama, S., Kiyohara, T., Yamamoto, K., Miyazaki, Y., Yasunaga,
Y., Shinomura, Y., Takeshita, T., Takeuchi, T., Morimoto, K., Matsuzawa, Y., (2003).
Helicobacter pylori-induced enlarged-fold gastritis is associated with increased
mutagenicity of gastric juice, increased oxidative DNA damage, and an increased risk
of gastric carcinoma. J. Gastroenterol. Hepatol. 18, 1384-1391.
[26] Miyazaki, T., Murayama, Y., Shinomura, Y., Yamamoto, T., Watabe, K., Tsutsui, S.,
Kiyohara, T., Tamura, S., Hayashi, N., (2007). E-cadherin gene promoter
hypermethylation in H. pylori-induced enlarged fold gastritis. Helicobacter 12, 523-
531.
[27] Nomura, H., Miyake, K., Kashiwagi, S., Sugiyama, T., Asaka, M., (2000). A short-term
eradication therapy for Helicobacter pylori acute gastritis. J. Gastroenterol. Hepatol.
15, 1377-1381.
[28] Miyamoto, M., Haruma, K., Yoshihara, M., Sumioka, M., Kamada, T., Ito, M., Masuda,
H., Nishisaka, T., Tanaka, S., Chayama, K., (2003). Isolated granulomatous gastritis
successfully treated by Helicobacter pylori eradication: a possible association between
granulomatous gastritis and Helicobacter pylori. J. Gastroenterol. 38, 371-375.
[29] Gwee, K.A., Teng, L., Wong, R.K., Ho, K.Y., Sutedja, D.S., Yeoh, K.G., (2009). The
response of Asian patients with functional dyspepsia to eradication of Helicobacter
pylori infection. Eur. J. Gastroenterol. Hepatol. 21, 417-424.
[30] Nam, J.H., Choi, I.J., Cho, S.J., Kim, C.G., Lee Jy, Nam SY, Park SR, Kook MC, Nam
BH, Kim YW., (2011). Helicobacter pylori infection and histological changes in
siblings of young gastric cancer patients. J. Gastroenterol. Hepatol. 26, 1157-1163.
[31] Shin, C.M., Kim, N., Yang, H.J., Cho, S.I., Lee, H.S., Kim, J.S., Jung, H.C., Song, I.S.,
(2010). Stomach cancer risk in gastric cancer relatives: interaction between
Helicobacter pylori infection and family history of gastric cancer for the risk of
stomach cancer. J. Clin. Gastroenterol. 44, e34-e39.
[32] El-Omar, E.M., Oien, K., Murray, L.S., El-Nujumi, A., Wirz, A., Gillen, D., Williams,
C., Fullarton, G., McColl, K.E., (2000). Increased prevalence of precancerous changes
in relatives of gastric cancer patients: critical role of H. pylori. Gastroenterology 118,
22-30.
[33] Chang,Y.W., Han, Y.S., Lee, D.K., Kim, H.J., Lim, H.S., Moon, J.S., Dong, S.H., Kim,
B.H., Lee, J.I., Chang, R., (2001). Role of Helicobacter pylori infection among
offspring or siblings of gastric cancer patients. Int. J. Cancer 101, 469-474.
[34] Gwack, J., Shin, A., Kim, C.S., Ko, K.P., Kim, Y., Jun, J.K., Bae, J., Park, S.K., Hong,
Y.C., Kang, D., Chang, S.H., Shin, H.R., Yoo, K.Y., (2006). CagA-producing
Helicobacter pylori and increased risk of gastric cancer: a nested case-control study in
Korea. Br. J. Cancer 95, 639-641.

[35] Seo, T.H., Lee, S.Y., Uchida, T., Fujioka, T., Jin, C.J., Hwang, T.S., Han, H.S., (2011).
The origin of non-H. pylori-related positive Giemsa staining in human gastric biopsy
specimens: A prospective study. Dig. Liver Dis. 2011; 43: 23-27.
[36] Venerito, M., Malfertheiner, P., (2010). Interaction of Helicobacter pylori infection and
nonsteroidal anti-inflammatory drugs in gastric and duodenal ulcers. Helicobacter 15,
239-250.
[37] Lee, S.Y., Tang, S.J., Rockey, D.C., Weinstein, D., Lara, L., Sreenarasimhaiah, J.,
Choi, K.W.; Korean Association for the Study of Intestinal Disease, (2008). Managing
anticoagulation and antiplatelet medications in GI endoscopy: a survey comparing the
East and the West. Gastrointest. Endosc. 67, 1076-1081.
[38] Kuipers, E.J., Lundell, L., Klinkenberg-Knol, E.C., Havu, N., Festen, H.P., Liedman,
B., Lamers, C.B., Jansen, J.B., Dalenback, J., Snel, P., Nelis, G.F., Meuwissen, S.G.,
(1996). Atrophic gastritis and Helicobacter pylori infection in patients with reflux
esophagitis treated with omeprazole or fundoplication. N. Engl. J. Med. 334, 1018-
1022.
[39] Kuipers, E.J., Nelis, G.F., Klinkenberg-Knol, E.C., Snel, P., Goldfain, D., Kolkman,
J.J., Festen, H.P., Dent, J., Zeitoun, P., Havu, N., Lamm, M., Walan, A., (2004). Cure
of Helicobacter pylori infection in patients with reflux esophagitis treated with long
term omeprazole reverses gastritis without exacerbation of reflux disease: results of a
randomized controlled trial. Gut 53, 12-20.
[40] Lee, S.Y., (2012). Future candidates for indications of Helicobacter pylori eradication –
do the indications need to be revised? J. Gastroenterol. Hepatol. 27, 200-211.
[41] Lee, J.S., Kwon, K.A., Jung, H.S., Kim, J.H., Hahm, K.B., (2009). Korea red ginseng
on Helicobacter pylori-induced halitosis: newer therapeutic strategy and a plausible
mechanism. Digestion 80, 192–199.
[42] Shiotani, A., Okada, K., Yanaoka, K., Itoh, H., Nishioka, S., Sakurane, M., Matsunaka,
M., (2001). Beneficial effect of Helicobacter pylori eradication in dermatologic
diseases. Helicobacter 6, 60-65.
[43] Szlachcic, A., (2002). The link between Helicobacter pylori infection and rosacea. J.
Eur. Acad. Dermatol. Venereol. 16, 328-333.
[44] Herr, H., You, C.H., (2000). Relationship between Helicobacter pylori and rosacea: it
may be a myth. J. Korean Med. Sci. 15, 551-554.
[45] Bamford, J.T., Tilden, R.L., Blankush, J.L.,Gangeness DE., (1999). Effect of treatment
of Helicobacter pylori infection on rosacea. Arch. Dermatol. 135, 659-663.
[46] Lee, W.Y., Yoon, W.T., Shin, H.Y., Shin, H.Y., Jeon, S.H., Rhee, P.L., (2008).
Helicobacter pylori infection and motor fluctuations in patients with Parkinson's
disease. Mov. Disord. 23, 1696-1700.
[47] Kim, J.M., Kim, S.H., Park, K.H., Han, S.Y., Shim, H.S., (2011). Investigation of the
association between Helicobacter pylori infection and normal tension glaucoma. Invest.
Ophthalmol. Vis. Sci. 52, 665-668.
[48] Ki, M.R., Goo, M.J., Park, J.K., Hong, I.H., Ji, A.R., Han, S.Y., You, S.Y., Lee, E.M.,
Kim, A.Y., Park, S.J., Lee, H.J., Kim, S.Y., Jeong, K.S., (2010). Helicobacter pylori
accelerates hepatic fibrosis by sensitizing transforming growth factor-β1-induced
inflammatory signaling. Lab. Invest. 90, 1507-1516.

[49] Lee, J.W., Lee, D.H., Lee, J.I., Jeong, S., Kwon, K.S., Kim, H.G., Shin, Y.W., Kim,
Y.S., Choi, M.S., Song, S.Y., (2010). Identification of Helicobacter pylori in gallstone,
bile, and other hepatobiliary tissues of patients with cholecystitis. Gut. Liver 4, 60-67.
[50] Blaser, M., (2011). Antibiotic overuse: Stop the killing of beneficial bacteria. Nature
476, 393-394.
[51] Sachs, G., Scott, D.R., (2012). Helicobacter pylori: Eradication or preservation. F1000
Med. Rep. 4, 7.
[52] Kuipers, E.J., (1999). Review article: exploring the link between Helicobacter pylori
and gastric cancer. Aliment. Pharmacol. Ther. 13(Suppl 1), 3-11.
[53] Parsonnet, J., Isaacson, P.G., (2004). Bacterial infection and MALT lymphoma. N.
Engl. J. Med. 350, 213-215.
[54] Hunt, R.H., Sumanac, K., Huang, J.Q., (2001). Review article: should we kill or should
we save Helicobacter pylori? Aliment. Pharmacol. Ther. 15(Suppl 1), 51-59.
[55] Gisbert, J.P., Calvet, X., (2012). Review article: rifabutin in the treatment of refractory
Helicobacter pylori infection. Aliment. Pharmacol. Ther. 35, 209-221.
[56] Qua, C.S., Manikam, J., Goh, K.L., (2010). Efficacy of 1-week proton pump inhibitor
triple therapy as first-line Helicobacter pylori eradication regime in Asian patients: is it
still effective 10 years on? J. Dig. Dis. 11, 244-248.
[57] Choi, K.H., Chung, W.C., Lee, K.M., Paik, C.N., Kim EJ, Kang BK, Oak JH, Jung SH.,
(2011). Efficacy of levofloxacin and rifaximin based quadruple therapy in Helicobacter
pylori associated gastroduodenal disease: a double-blind, randomized controlled trial. J.
Korean Med. Sci. 26, 785-790.
[58] Ford, A.C., Malfertheiner, P., Giguere, M., Santana, J., Khan, M., Moayyedi, P.,
(2008). Adverse events with bismuth salts for Helicobacter pylori eradication:
systematic review and meta-analysis. World J. Gastroenterol. 14, 7361-7370.
[59] Wilhelm, S.M., Johnson, J.L., Kale-Pradhan, P.B., (2011). Treating bugs with bugs: the
role of probiotics as adjunctive therapy for Helicobacter pylori. Ann. Pharmacother. 45,
960-966.
[60] Kim, J.M., (2006). Antibiotic resistance of Helicobacter pylori isolated from Korean
patients. Korean J. Gastroenterol. 47, 337-349.
[61] Lee, J.W., Kim, N., Kim, J.M., Nam, R.H., Chang, H., Kim, J.Y., Shin, C.M., Park,
Y.S., Lee, D.H., Jung, H.C., (2012). Prevalence of primary and secondary antimicrobial
resistance of Helicobacter pylori in Korea from 2003 through 2012. Helicobacter Epub
ahead of print.
[62] Ho, S.L., Tan, E.L., Sam, C.K., Goh, K.L., (2010). Clarithromycin resistance and point
mutations in the 23S rRNA gene in Helicobacter pylori isolates from Malaysia. J. Dig.
Dis. 11, 101-105.
[63] Hwang, T.J., Kim, N., Kim, H.B., Lee, B.H., Nam, R.H., Park, J.H., Lee, M.K., Park,
Y.S., Lee, D.H., Jung, H.C., Song, I.S., (2010). Change in antibiotic resistance of
Helicobacter pylori strains and the effect of A2143G point mutation of 23S rRNA on
the eradication of H. pylori in a single center of Korea. J. Clin. Gastroenterol. 44, 536-
543.
[64] Kim, J.M., Kim, J.S., Kim, N., Kim, Y.J., Kim, I.Y., Chee, Y.J., Lee, C.H., Jung, H.C.,
(2008). Gene mutations of 23S rRNA associated with clarithromycin resistance in
Helicobacter pylori strains isolated from Korean patients. J. Microbiol. Biotechnol. 18,
1584-1589.

[65] Choi, I.J., Jung, H.C., Choi, K.W., Kim, J.H., Ahn, D.S., Yang, U.S., Rew, J.S., Lee,
S.I., Rhee, J.C., Chung, I.S., Chung, J.M., Hong, W.S., (2002). Efficacy of low-dose
clarithromycin triple therapy and tinidazole-containing triple therapy for Helicobacter
pylori eradication. Aliment. Pharmacol. Ther. 16, 145-151.
[66] Teo, E.K., Fock, K.M., Ng, T.M., Khor, C.J., Tan, A.L., (2000). Metronidazole-
resistant Helicobacter pylori in an urban Asian population. J. Gastroenterol. Hepatol.
15, 494-497.
[67] Kumala, W., Rani, A., (2006). Patterns of Helicobacter pylori isolate resistance to
fluoroquinolones, amoxicillin, clarithromycin and metronidazoles. Southeast Asian J.
Trop. Med. Public Health 37, 970-974.
[68] Lee, J.W., Kim, N., Nam, R.H., Park, J.H., Kim JM, Jung HC, Song IS., (2011).
Mutations of Helicobacter pylori associated with fluoroquinolone resistance in Korea.
[69] Yoon, H., Kim, N., Lee, B.H., Hwang, T.J., Lee, D.H., Park, Y.S., Nam, R.H., Jung,
H.C., Song, I.S., (2009). Moxifloxacin-containing triple therapy as second-line
treatment for Helicobacter pylori infection: effect of treatment duration and antibiotic
resistance on the eradication rate. Helicobacter 14, 77-85.
[70] Lee, Y.C., Lee, S.Y., Pyo, J.H., Kwon, D.H., Rhee, J.C., Kim, J.J., (2005). Isogenic
variation of Helicobacteral pylori strain resulting in heteroresistant antibacterial
phenotypes in a single host in vivo. Helicobacter 10, 240-248.
[71] Qian, B., Ma, S., Shang, L., Qian, J., Zhang, G., (2011). Effects of Helicobacter pylori
eradication on gastroesophageal reflux disease. Helicobacter 16, 255-265.
[72] Yaghoobi, M., Farrokhyar, F., Yuan, Y., Hunt, R.H., (2010). Is there an increased risk
of GERD after Helicobacter pylori eradication?: a meta-analysis. Am. J. Gastroenterol.
105, 1007-1013.
[73] Laine, L., Hopkins, R.J., Girrardi, L., (1998). Has the impact of Helicobacter pylori
therapy on ulcer recurrence in the United States been overstated? A meta-analysis of
rigorously designed trials. Am. J. Gastroenterol. 93, 1409–1415.
[74] Malfertheiner, P., Dent, J., Zeijlon, L., Sipponen, P., Veldhuyzen Van Zanten, S.J.,
Burman, C.F., Lind, T., Wrangstadh, M., BayerdOrffer, E., Lonovics, J., (2002). Impact
of Helicobacter pylori eradication on heartburn in patients with gastric or duodenal
ulcer disease - Results from a randomized trial programme. Aliment. Pharmacol. Ther.
16, 1431–1442.
[75] Veldhuyzen van Zanten, S.J.O., Bradette, M., Farley, A., Leddin, D., Lind, T., Unge, P.,
Bayerdörffer, E., Spiller, R.C., O'Morain, C., Sipponen, P., Wrangstadh, M., Zeijlon,
L., Sinclair, P., (1999). The DU-MACH study: eradication of Helicobacter pylori and
ulcer healing in patients with acute duodenal ulcer using omeprazole based triple
therapy. Aliment. Pharmacol. Ther. 13, 289–295.
[76] Sonnenberg, A., Lash, R.H., Genta, R.M., (2010). A national study of Helicobactor
pylori infection in gastric biopsy specimens. Gastroenterology 139, 1894-1901.
[77] Appelman, H.D., Umar, A., Orlando, R.C., Sontag, S.J., Nandurkar, S., El-Zimaity, H.,
Lanas, A., Parise, P., Lambert, R., Shields, H.M., (2011). Barrett's esophagus: natural
history. Ann. N. Y. Acad. Sci. 1232, 292-308.
[78] Wang, C., Yuan, Y., Hunt, R.H., (2009). Helicobacter pylori infection and Barrett's
esophagus: a systematic review and meta-analysis. Am. J. Gastroenterol. 104, 492-500.

[79] Weigt, J., Malfertheiner, P., (2009). Influence of Helicobacter pylori on gastric
regulation of food intake. Curr. Opin. Clin. Nutr. Metab. Care 12, 522-525.
[80] Jang, E.J., Park, S.W., Park, J.S., Park, S.J., Hahm, K.B., Paik, S.Y., Sin, M.K., Lee,
E.S., Oh, S.W., Park, C.Y., Baik, H.W., (2008). The influence of the eradication of
Helicobacter pylori on gastric ghrelin, appetite, and body mass index in patients with
peptic ulcer disease. J. Gastroenterol. Hepatol. 23 (Suppl 2), S278-S285.
[81] Chen, Y., Blaser, M.J., (2007). Inverse associations of Helicobacter pylori with asthma
and allergy. Arch. Intern. Med. 167, 821-827.
[82] Kanbay, M., Kanbay, A., Boyacioglu, S., (2007). Helicobacter pylori infection as a
possible risk factor for respiratory system disease: a review of the literature. Respir.
Med. 101, 203-209.
[83] Arnold, I.C., Dehzad, N., Reuter, S., Martin, H., Becher, B., Taube, C., Müller A.,
(2011). Helicobacter pylori infection prevents allergic asthma in mouse models through
the induction of regulatory T cells. J. Clin. Invest. 121, 3088-3093.
[84] Lee, J.H., Kim, N., Chung, J.I., Kang, K.P., Lee, S.H., Park, Y.S., Hwang, J.H., Kim,
J.W., Jeong, S.H., Lee, D.H., Jung, H.C., Song, I.S., (2008). Long-term follow up of
Helicobacter pylori IgG serology after eradication and reinfection rate of H. pylori in
South Korea. Helicobacter 13, 288-294.
[85] Kim, M.S., Kim, N., Kim, S.E., Jo, H.J., Shin, C.M., Lee, S.H., Park, Y.S., Hwang,
J.H., Kim, J.W., Jeong, S.H., Lee, D.H., Kim, J.M., Jung, H.C., (2013). Long-term
Follow-up Helicobacter pylori reinfection rate and its associated factors in Korea.
[86] Cheon, J.H., Kim, N., Lee, D.H., Kim, J.M., Kim, J.S., Jung, H.C., Song, I.S., (2006).
Long-term outcomes after Helicobacter pylori eradication with second-line, bismuth-
containing quadruple therapy in Korea. Eur. J. Gastroenterol. Hepatol. 18, 515-519.
[87] Thong-Ngam, D., Mahachai, V., Kullavanijaya, P., (2007). Incidence of Helicobacter
pylori recurrent infection and associated factors in Thailand. J. Med. Assoc. Thai. 90,
1406-1410.
[88] Khor, C.J., Fock, K.M., Ng, T.M., Teo, E.K., Sim, C.S., Tan, A.L., Ng, A., (2006).
Recurrence of Helicobacter pylori infection and duodenal ulcer relapse, following
successful eradication in an urban east Asian population. Singapore Med. J. 41, 382-
386.
[89] Wheeldon, T.U., Hoang, T.T., Phung, D.C., Björkman, A., Granström, M., Sörberg, M.,
(2005). Long-term follow-up of Helicobacter pylori eradication therapy in Vietnam:
reinfection and clinical outcome. Aliment. Pharmacol. Ther. 21, 1047-1053.

Index
# atherosclerosis, 203, 205, 216, 217, 247, 283,

284, 285, 287, 288, 289, 290, 291, 292,
2-C-methyl-D-erythritol-2, 364, 365 293, 294, 295
4-cyclodiphosphate synthase, 364, 377 atrophic gastritis, 12, 42, 44, 48, 49, 57, 58,
4-diphosphocytidyl-2-C-methyl-D-erythritol 63, 83, 84, 85, 86, 87, 89, 90, 92, 95, 96,
kinase, 364 108, 171, 172, 174, 182, 202, 206, 263,
4-diphosphocytidyl-2-C-methyl-D-erythritol 290, 295, 298, 318, 338, 344, 348, 349,
synthase, 364, 378 398, 399, 401, 409
autofluorescence imaging, 90
autoimmune diseases, 202, 205, 217, 223,
A
224, 225, 226, 227, 228, 229, 230, 231,
adherens junction, 115, 116, 117, 118, 119, 232, 234, 235, 238, 330
122, 124, 125, 126, 129, 132, 134, 139
angioedema, 323, 324, 331, 335 B
anterior optic ischemic neuropathy, 202
antibiotic resistance, 24, 31, 73, 75, 78, 108, bacterial colonization, 227, 273, 280, 313,
343, 350, 359, 360, 361, 412, 413 384, 386
antibiotics, xi, 12, 14, 31, 44, 62, 72, 73, 108, Barrett esophagus, 165, 167
270, 289, 294, 325, 328, 341, 343, 349, bifunctional enzyme, 364, 366, 370
350, 353, 355, 356, 363, 364, 366, 370, bismuth-containing quadruple therapy, 321,
373, 376, 397, 398, 404 347, 353, 354, 358, 414
antimicrobial Susceptibility Testing, 19, 24, blood brain barrier (BBB), 202, 203, 204,
36, 73, 78, 197 207, 208, 209, 212
antiphospholipid antibody syndrome, 223 blood nerve barrier (BNB), 202, 203, 209
apical junctional complex, 115, 116, 117, 124 body mass index (BMI), 242, 243, 247, 249,
apoptosis, 55, 56, 115, 116, 119, 120, 125, 250, 251, 254, 255, 256, 257, 258, 261,
126, 127, 128, 129, 133, 134, 136, 141, 263, 264, 266, 267, 414
142, 143, 144, 197, 211, 212, 213, 215,
218, 220, 227, 235, 237, 324 C
asthma, 13, 166, 174, 193, 228, 235, 237,
297, 298, 299, 300, 303, 304, 305, 332, CagA, 6, 15, 24, 32, 115, 116, 121, 122, 123,
333, 340, 346, 407, 414 124, 125, 126, 127, 135, 136, 137, 139,

416 Index
140, 141, 142, 143, 144, 154, 160, 167, 161, 173, 179, 251, 259, 297, 298, 338,
168, 172, 173, 174, 176, 181, 188, 190, 348, 379, 388, 391
196, 205, 208, 216, 219, 232, 249, 251, Chronic Obstructive Pulmonary Disease,
252, 259, 262, 270, 271, 273, 275, 279, 297, 305
281, 288, 289,294, 299, 300, 301, 303, chronic urticaria, 323, 324, 325, 326, 332,
313, 315, 319, 324, 328, 333, 338, 340, 333, 398, 402
381, 384, 387, 390, 396, 401, 410 cirrhosis, 185, 186, 187, 188, 190, 191, 193,
Calpain, 115, 125, 139, 141 194, 195, 196, 198, 218
cancer, 6, 24, 34, 42, 43, 44, 45, 48, 53, 54, Claudin, 115, 118, 122, 123, 124, 131, 132,
56, 58, 59, 63, 65, 83, 84, 87, 89, 94, 95, 136, 137
96, 97, 101, 102, 107, 108, 109, 112, 115, colorectal cancer, 34, 97, 151, 165, 174, 179,
116, 120, 121, 122, 123, 127, 129, 133, 180, 181, 182
136, 137, 138, 139, 140, 141, 143, 144, concomitant therapy, 320, 352, 353, 355,
145, 147, 151, 152, 153, 154, 155, 157, 359, 360, 404
159, 160, 165, 168, 171, 172, 173, 174, confocal laser endomicroscopy, 83, 93, 97
175, 177, 178, 179, 180, 181, 182, 186, coronary artery disease, 287, 290, 294
190, 192, 215, 242, 247, 248, 251, 252, Crohn disease, 165, 169
257, 258, 259, 260, 261, 263, 265, 266, culture, 6, 15, 21, 23, 36, 42, 43, 44, 69, 70,
269, 270, 273, 278, 297, 298, 300, 301, 71, 72, 73, 75, 76, 77, 78, 84, 94, 105, 183,
302, 303, 305, 306, 319, 339, 340, 344, 191, 193, 337, 341, 343, 347, 355, 361,
348, 379, 380, 384, 388, 389, 390, 396, 368
398, 399, 400, 401, 403, 404, 406, 408, Cytotoxin-Associated Gene (Cag) A, 115,
409, 410, 412 116
carcinoma, 44, 49, 53, 58, 59, 66, 84, 122,
133, 136, 139, 142, 159, 168, 179, 180, D
181, 183, 185, 190, 194, 195, 396, 400,
410 Dental Plaque PCR, 19, 21
cardiac syndrome X, 288, 291, 292, 294 dermatomyositis, 223, 234
-catenin, 115, 117, 118, 124, 125, 126, 132, diagnosis, 5, 6, 8, 9, 10, 11, 15, 16, 17, 18,
139, 140, 141 19, 20, 28, 29, 30, 31, 34, 35, 38, 42, 43,
cell survival, 127 44, 45, 46, 47, 48, 57, 59, 60, 61, 62, 63,
central nervous system, 201, 202, 246, 260, 64, 65, 67, 69, 70, 71, 77, 79, 81, 83, 84,
402 87, 90, 94, 96, 97, 147, 149, 150, 154, 155,
cerebrovascular diseases, 201, 202 156, 161, 166, 169, 172, 185, 191, 192,
cervical mucosa, 308 206, 207, 210, 232, 249, 259, 275, 276,
children, xi, 13, 14, 15, 18, 25, 27, 30, 31, 35, 308, 310, 312, 332, 340, 341, 358, 399,
36, 38, 46, 103, 104, 105, 106, 107, 108, 409, 410
109, 110, 111, 166, 175, 233, 239, 242, dysplasia, 44, 49, 54, 57, 58, 84, 89, 96, 125,
249, 251, 252, 254, 255, 262, 264, 266, 147, 155, 167, 173, 179, 348
272, 273, 277, 280, 281, 282, 299, 303,
304, 306, 320, 329, 335, 337, 338, 339, E
340, 341, 343, 344, 345, 346, 360, 380,
387, 389 E-cadherin, 115, 117, 118, 122, 124, 125,
chromoendoscopy, 89, 90, 91, 96 126, 132, 134, 136, 139, 140, 141, 151,
chronic gastritis, xi, 6, 41, 44, 49, 50, 51, 56, 400, 410
57, 60, 64, 95, 96, 102, 103, 137, 145, 159,

Index 417
embolism, 204, 230 fluorescence in situ hybridization (FISH), vii,

endocrine diseases, 260 69, 70, 74, 75, 76, 78, 80, 81, 341, 342,
endocytosis, 115, 120, 124, 127, 134, 138, 343
143 functional dyspepsia, 145, 147, 148, 157,
endoscopy, 6, 15, 20, 24, 33, 41, 42, 43, 44, 158, 398, 404, 410
45, 46, 62, 63, 64, 70, 83, 84, 86, 89, 91,
93, 94, 95, 97, 150, 155, 159, 169, 191, G
210, 211, 325, 330, 331, 337, 341, 343,
411 gastric atrophy, 48, 51, 84, 118, 126, 152,
eosinophilic esophagitis, 165, 166, 174 154, 155, 176, 217, 252, 319, 399, 401
epidermal growth factor receptor, 140, 143 gastric biopsies, 41, 42, 43, 58, 61, 62, 69,
epilepsy, 208, 219 73, 74, 76, 77, 81, 125, 166
epithelial dysfunction, 115, 116, 129 gastric cancer, 6, 24, 42, 43, 44, 45, 48, 53,
epithelial permeability, 119, 120, 121, 130, 54, 59, 65, 83, 84, 87, 89, 94, 95, 96, 97,
132 101, 102, 107, 108, 109, 112, 116, 121,
eradication, xi, xii, 5, 8, 10, 11, 12, 14, 15, 122, 123, 127, 129, 136, 137, 138, 140,
16, 17, 24, 25, 27, 44, 45, 46, 47, 57, 60, 143, 144, 145, 147, 151, 152, 153, 154,
66, 94, 96, 101, 102, 106, 107, 108, 109, 155, 157, 159, 160, 172, 173, 178, 181,
110, 111, 112, 140, 145, 148, 150, 155, 186, 192, 215, 258,259, 263, 265, 266,
156, 157, 158, 159, 160, 161, 169, 177, 269, 270, 273, 278, 298, 301, 339, 340,
185, 186, 191, 193, 194, 196, 198, 202, 344, 348, 379, 380, 384, 388, 390, 396,
206, 207, 208, 210, 212, 213, 214, 215, 398, 399, 400, 401, 403, 404, 408, 409,
217, 218, 220, 223, 229, 231, 232, 233, 410, 412
237, 238, 239, 241, 242, 254, 255, 256, gastritis, xi, 6, 12, 33, 41, 42, 44, 45, 46, 47,
257, 258, 261, 265, 266, 267, 269, 270, 48, 49, 50, 51, 56, 57, 58, 59, 60, 63, 64,
271, 272, 273, 274, 275, 276, 277, 278, 65, 77, 83, 84, 85, 86, 87, 89, 90, 92, 95,
279, 280, 281, 282, 284, 287, 289, 292, 96, 97, 101, 102, 103, 108, 116, 118, 122,
300, 306, 307, 309, 310, 311, 314, 315, 137, 145, 146, 148, 154, 157, 159, 161,
317, 319, 320, 321, 323, 324, 325, 326, 168, 171, 172, 173, 174, 179, 182, 202,
327, 328, 329, 330, 331, 332, 333, 334, 206,227, 235, 242, 251, 252, 257, 258,
337, 341, 342, 343, 346, 347, 348, 349, 259, 263, 264, 273, 274, 275, 276, 277,
350, 352, 353, 354, 355, 356, 357, 358, 281, 282, 290, 295, 297, 298, 308, 311,
359, 360, 361, 362, 373, 378, 381, 383, 314, 316, 318, 325, 326, 338, 344, 345,
384, 389, 397, 398, 399, 400, 401, 402, 346, 348, 349, 357, 379, 380, 386, 388,
403, 404, 405, 406, 407, 408, 409, 410, 391, 398, 399, 400, 401, 403, 404, 406,
411, 412, 413, 414 409, 410, 411
esophageal adenocarcinoma, 165, 166, 167, gastrointestinal epithelium, 115, 116, 118,
168, 406 120, 126, 129
ESPGHAN, 340, 341, 342, 346 ghrelin, viii, 148, 158, 241, 242, 244, 246,
247, 248, 249, 251, 252, 253, 254, 255,
F 256, 257, 258, 260, 261, 262, 263, 264,
265, 266, 267, 407, 414
female infertility, 308, 309, 315 glaucoma, 201, 202, 209, 210, 211, 212, 213,
fibromyalgia, 223, 239 214, 220, 221, 235, 402, 411

418 Index
H interleukin-1 115, 124

intestinal metaplasia, 15, 44, 46, 47, 52, 54,
helicobacter pylori, viii, ix, xi, xii, 5, 6, 15, 56, 58, 63, 83, 84, 88, 90, 93, 95, 96, 97,
16, 17, 18, 19, 33, 34, 35, 36, 37, 38, 41, 153, 155, 173, 399, 401, 406, 409
42, 57, 62, 63, 64, 65, 66, 67, 69, 70, 74, intrafamilial spread, 103
77, 78, 79, 80, 81, 83, 84, 95, 96, 101, 102, iron deficiency anemia, 257, 269, 270, 281,
108, 109, 110, 111, 112, 115, 116, 130, 307, 308, 309, 310, 315, 317, 337, 340,
132, 135, 136, 137, 139, 140, 141, 142, 398, 402, 403
143, 144, 145, 146, 147, 151, 154, 157, ischemic hearth disease, 283
158, 159, 160, 161, 162, 163, 165, 166, ischemic stroke, 205, 216, 217, 287
174, 175, 176, 177, 178, 179, 181, 182, Isoprenoid biosynthesis, 364, 365, 366, 374,
183, 185, 186, 194, 195, 196, 197, 198, 375, 376, 377
199, 201, 202, 213, 214, 215, 216, 217,
218, 219, 220, 221, 223, 225, 226, 227, L
228, 230, 231, 232, 233, 234, 235, 236,
237, 238, 239, 241, 242, 244, 249, 251, Leptin, viii, 228, 241, 242, 245, 246, 247,
254, 256, 259, 260, 261, 262, 263, 264, 248, 249, 251, 252, 253, 254, 255, 256,
265, 266, 267, 269, 270, 278, 279, 280, 257, 258, 260, 261, 262, 264, 265, 266,
281, 282, 283, 284, 287, 291, 292, 294, 267, 407
295, 297, 298, 300, 301, 302, 304, 305, lichen planus, 323, 324, 330, 332, 334
306, 307, 308, 316, 317, 318, 319, 320, liver cancer, 190
321, 323, 324, 332, 333, 334, 335, 337, lung cancer, 297, 301, 302, 303, 305, 306
338, 342, 343, 344, 345, 346, 347, 348,
358, 359, 360, 361, 362, 363, 364, 366, M
377, 378, 379, 380, 390, 391, 392, 393,
394, 395, 396, 397, 398, 402, 403, 408, magnifying endoscopy, 83, 89, 91, 97
409, 410, 411, 412, 413, 414 MALT-lymphoma, xi, 409
Henoch-Schonlein purpura, 233, 239, 335 maternal transmission, 320
hepatitis, 111, 186, 187, 188, 190, 193, 194, matrix metalloproteinase, 124, 125, 128, 139,
195, 197, 218 142, 143, 203, 208, 212, 216, 286
humoral response, 227, 381, 382 metabolic syndrome, 179, 196, 249, 261, 294
hybrid therapy, 353, 355, 358 methylerythritol phosphate pathway, 363,
hyperemesis gravidarum, 307, 308, 310, 311, 374, 376, 377
312, 315, 316, 317, 318 migraine, 201, 202, 208, 213, 218, 219, 402
multiple sclerosis, 201, 202, 213, 214, 215,
I 218, 226, 228, 237, 239
myosin light chain kinase, 115, 119, 124,
IgG4 associated diseases, 223 130, 132, 138
IL-17, 50, 190, 382, 388, 389, 392
IL-8, 122, 137, 172, 190, 203, 205, 212, 226, N
284, 300, 301, 328, 383
immune thrombocytopenia, 270, 282 NAFLD, 188, 189
indication, 224, 397, 402 narrow band imaging, 83, 88, 90, 97
innate response, 382 NASPGHAN, 340, 341, 342, 346
interferon-γ, 207, 211 neuromyelitis optica, 201, 207, 215, 218, 226

Index 419
neutrophil activating protein (NAP), 167, probiotics, 320, 343, 356, 357, 362, 387, 405,
207, 226, 235, 270, 299, 384, 387, 390, 412
393 psoriasis, 323, 324, 329, 333, 334
O Q
obestatin, viii, 241, 242, 246, 247, 249, 251, quadruple therapy, 321, 347, 348, 352, 353,
254, 255, 256, 257, 258, 260, 266 354, 355, 356, 358, 359, 360, 361, 362,
occludin, 115, 117, 118, 120, 122, 123, 129, 378, 404, 412, 414
130, 131 quinolone-containing triple therapy, 353
P R
pathogen burden, 285, 290 rapid urease test, 15, 17, 20, 42, 61, 67, 70,
peptic ulcer disease, 6, 41, 43, 50, 54, 55, 57, 75, 84, 169, 193, 306, 337, 341
63, 96, 102, 103, 135, 145, 146, 147, 148, recurrence, 57, 101, 106, 107, 108, 111, 112,
149, 150, 155, 157, 159, 173, 175, 188, 145, 193, 198, 215, 233, 271, 358, 398,
192, 193, 196, 198, 236, 261, 266, 297, 399, 400, 407, 413
298, 300, 301, 305, 338, 340, 342, 345, reinfection, 26, 101, 106, 107, 108, 110, 111,
348, 408, 409, 414 112, 329, 391, 397, 407, 414
peripheral nervous system, 201, 202, 245 rescue treatment, 361
polymerase chain reaction (PCR), vii, 19, 20, rheumatoid arthritis, 223, 224, 228, 229, 237,
21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 238
35, 36, 69, 70, 73, 74, 75, 76, 79, 80, 112, Rho-associated kinase, 115, 119, 124, 133
173, 180, 183, 189, 196, 197, 216, 238, rosacea, 323, 324, 326, 327, 328, 333, 402,
254, 258, 281, 286, 287, 288, 305, 306, 411
314, 341, 343, 345, 368, 399, 405
polymyositis, 223 S
pre-eclampsia, 307, 308, 310, 318, 319
pregnancy, 13, 280, 307, 308, 309, 310, 311, sequential therapy, 320, 321, 337, 343, 346,
312, 314, 315, 316, 317, 319 351, 352, 353, 355, 358, 359, 360, 361,
preservation, 34, 111, 397, 412 362
prevalence, 5, 8, 43, 45, 55, 56, 78, 101, 102, serum antibodies, 8, 106, 309, 387
103, 104, 106, 107, 108, 109, 110, 112, stool antigen, 5, 8, 9, 10, 15, 16, 17, 19, 20,
149, 158, 168, 169, 170, 174, 177, 178, 30, 150, 308, 315, 316, 318, 320, 330, 337,
186, 187, 189, 194, 205, 206, 209, 210, 338, 341, 345, 357
216, 224, 228, 229, 230, 231, 232, 235, stool PCR, 19, 25, 346
237, 239, 242, 249, 250, 251, 262, 270, systemic lupus erythematosus, 223, 224, 235,
271, 272, 273, 277, 282, 289, 299, 300, 238
303, 308, 310, 313, 316, 323, 324, 325, systemic sclerosis, 207, 238, 239
326, 327, 328, 329, 330, 334, 337, 338,
339, 343, 344, 345, 347, 353, 354, 358, T
359, 379, 380, 388, 389, 397, 398, 399,
401, 402, 406, 407, 408, 410 T cell response, 167, 300, 305, 384, 385, 390,
393

420 Index
teratogenicity, 307, 311, 315 349, 350, 351, 352, 353, 354, 355, 358,
Th1, 50, 167, 175, 177, 197, 227, 236, 287, 359, 360, 361, 404, 405, 406, 412, 413
299, 382, 392 tuberculosis, 30, 38, 297, 302, 356, 376, 377
Th2, 167, 227, 236, 299, 300, 305, 382
therapy, xi, 5, 8, 9, 10, 11, 12, 17, 24, 25, 43, U
44, 47, 49, 62, 66, 77, 96, 101, 106, 107,
108, 110, 111, 112, 144, 148, 150, 155, ulcerative colitis, 165, 169
157, 161, 171, 177, 185, 193, 194, 198, Urea Breath Test, vii, 5, 8, 10, 13, 16, 17, 18,
210, 211, 213, 218, 239, 272, 275, 276, 19, 20, 30, 33, 44, 62, 63, 67, 69, 84, 94,
277, 278, 279, 282, 302, 303, 306, 309, 110, 150, 169, 192, 210, 308, 310, 317,
311, 315, 317, 320, 321, 324, 325, 326, 325, 326, 329, 330, 337, 341, 357
327, 328, 329, 330, 331, 337, 341, 342,
343, 346, 347, 348, 349, 350, 351, 352, V
353, 354, 355, 356, 357, 358, 359, 360,
361, 362, 373, 378, 380, 383, 398, 399, vaccine development, 379, 380, 381, 383,
401, 402, 403, 404, 405, 406, 407, 408, 386, 389
409, 410, 412, 413, 414 vaccine studies, 381, 382, 384
thrombocytopenia, 270, 271, 272, 276, 278, Vacuolating toxin A (VacA), 24, 32, 51, 115,
282, 307, 308, 310, 317 116, 121, 122, 123, 124, 125, 126, 135,
tight junction, 115, 116, 117, 118, 119, 120, 136, 140, 142, 167, 172, 173, 203, 204,
122, 123, 124, 129, 130, 131, 132, 133, 205, 209, 213, 215, 219, 220, 226, 251,
134, 136, 137, 138 252, 270, 273, 276, 289, 313, 340, 384,
tonsillitis, 303, 306 390, 393
transmission, xi, 24, 101, 103, 104, 105, 106, vasculitis, 223, 232, 233, 293, 329, 331
107, 108, 109, 110, 111, 112, 194, 270,
286, 307, 308, 314, 315, 320, 337, 339,
Z
345
triple therapy, xi, 177, 193, 194, 320, 321, Zonula occluden, 115, 117, 129
325, 326, 329, 337, 341, 342, 343, 347,
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DIGESTIVE DISEASES - RESEARCH

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MARCO MANFREDI, M.D.

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Diseases and Health Implications 99

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Chapter 17: Helicobacter pylori Infection and Gynecological

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We would like to thank the Italian non-profit organization SNUPI ONLUS

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Rosalia Aloe1,, Chiara Bonaguri1, Marco Manfredi2,3,

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Laboratory Serological Assay

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Autoantibody detection is made usually by ELISA methods presented in a microplate

Rapid Serological Tests

Latex Agglutination Assay

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Performance Characteristics of Serological Assays

A meta-analysis evaluated the performance characteristics of several commercially

Serology H. Pylori Limitations

Several factors limit the usefulness of antibody testing in clinical practice.

H. pylori Stool Antigen Tests (HpSA)

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HpSA ELISA Test

Figure 1.1. HpSA ELISA test.

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Features of the Test

Limitations of the Test

Rapid HpSA Test

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colloidal gold (first antibody): chemically, it is a lateral migration of chromatographic

Features of the Test

Figure 1.2. HpSA card.

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Table 1.1. Diagnostic accuracy of Immuno-Card STAT!

Overall population Patients with strongly

Table 1.2. Diagnostic accuracy of Immuno-Card STAT!

Overall population Patients with strongly

Limitations of the Test

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Comparison between HpSA ELISA and Rapid HpSA

Table 1.3. comparison of the performance characteristics

Urea Breath Test

Exam Preparation and Execution

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