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Chapter
Helicobacter pylori: From Diagnosis to Treatment
Ashwini P1, Sushma P2 , Anisha S Jain1, Nagendra Prasad MN3, Kollur Shiva Prasad4,
Chandrashekar S5, Chandan D2, Chandan S*2
1Division of Microbiology and Tissue Culture, School of Life Sciences, JSS Academy of Higher Education & Research,
Mysuru – 570 015, Karnataka, India.
2Division of Biotechnology and Bioinformatics, School of Life Sciences, JSS Academy of Higher Education &
Research, Mysuru – 570 015, Karnataka, India.
3Department of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS Research Foundation, SJCE
Campus, Manasagangothri, Mysore - 570 006, Karnataka, India
4Department of Sciences, Amrita School of Arts and Sciences, Amrita Vishwa Vidyapeetham, Mysuru campus – 570
026, Karnataka, India.
5Department of Biotechnology, Davangere University, Shivagangotri, Davanagere – 577 007, Karnataka, India.
Corresponding Author:
*Chandan.S PhD
Assistant Professor and Course Coordinator
Department of Biotechnology and Bioinformatics
School of Life Sciences
JSS Academy of Higher Education & Research
Mysuru-570 015, Karnataka, India.
Mob:+91-9538500423
E-mail: chandans@jssuni.edu.in
Abstract
Helicobacter pylori is a microaerophilic, gram negative bacterium that inhabits the human stomach
in majority of the human population. This bacteria being worldwide spread is known to cause
diseases affecting at least 50% of the worldwide population. It remains a crucial factor linked to the
blooming of peptic ulcer disease, gastric malignancy, dyspeptic symptoms or mucosa‑associated
lymphoid tissue lymphoma. This disease if not diagnosed at the right time and left untreated may
lead to lot of sequela from such petty to ominous diseases over a period of time. Hence the major
dispute it to prevent the H. pylori-related diseases by effective treatment and screening procedures.
Right now, there are different diagnostic techniques for identifying the presence of this infection,
each having its own pros, cons and constraints. Biopsy-based tests such as polymerase chain
reaction (PCR), histological evaluation, culture and the rapid urease test (cod liver oil test/CLO
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test) are executed on tissue procured through endoscopy. On the other hand, the urea breath test
(C13-C14), serology to identify IgG antibody, and stool antigen test (SAT) can be carried out as
non-invasive procedures. For H. pylori three biopsies are taken each from antrum, body and fundus.
The need of treatment is decided based on the diagnosis of the H. pylori infection and on the report
of the victim’s clinical condition. Historically, therapy consists of antimicrobials combined with
suppression of gastric acid. Various combinations like triple regimes are used to control and
eradicate the infection. Clarithromycin, amoxicillin, tetracycline, bismuth salts and
metronidazole/tinidazole with omeprazole/lanzoprazole/pantoprazole (proton pump inhibitors)
combinations drugs of choice. The proportion of patients in whom the infection is managed after
treatment ranges between 60% to 95%. In spite of the fact that triple therapy provides acceptable
cure rates; quadruple therapies, sequential therapies, and concomitant therapies have been initiated
as key alternatives for the treatment of H. pylori.
However, these treatments can be of significant risk because of the presence of resistance to the
antibiotics and related adverse impacts. Introduction of novel alternatives utilizing common natural
resources like vaccines, probiotics, nutraceuticals, photodynamic inactivation and phage therapy
have been demonstrated as powerful tools in bringing down the bacterial levels, managing
inflammation and regulating the immune response.
H. pylori infection remains the most intermittent bacterial infection around the world; along these
lines, precise determination and treatment of this infection is fundamental. This chapter summarizes
and compares the historical as well the novel methods used for diagnosis and treatment of the
infection.
Key words: Antibody, probiotics, nutraceuticals, phytoextracts, inflammation, infectious diseases,
nanobactericides, biopsy, H. Pylori, Peptic ulcer
Introduction
H. pylori is a Gram negative bacillus which is microaerophilic and spiral in nature that has been
extensively studied. More than 50% of the population is being infected by this organism, and it has
also been identified as the major human pathogen. H. pylori is considered one of the risk factor and
has an accepted role for the development of gastric cancer as per scientific studies. This infection
is an asymptomatic condition that can continue throughout the life. It causes tissue damage and
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chronic gastric inflammation, which leads to alterations that evolves to intense gastric diseases like
gastric ulcer, mucosa associated tissue lymphoma or peptic ulcer.
This organism is unique which has the ability to establish and persist the inflammation (gastric) and
infection. At the stage of colonization the organism resists the gastric acidity utilizing the urease
activity and then it propels the flagella. Enzyme urease converts or metabolizes the urea to carbon
dioxide and ammonia and it neutralizes the gastric acid in the stomach. The organism
adheres/attaches to the epithelial cells and mucins with help of adhesins and crosses the gastric
mucus layer. Once it reaches this area, the virulence factors are delivered into the cytoplasm of the
host cell by releasing the outer membrane vesicles. These virulence factors play a major role in all
the isolates and help the organism to survive and colonize even in the acidic environment of the
stomach. Mainly there are CagA (cytotoxin associated gene A) and VacA (vacuolating cytotoxin)
translocated outer membrane proteins. This infection is usually incurred in the childhood giving an
acute immune response (1).
One of the most important factors is the VacA protein that determines the virulence of the strains
of the organism. The ability of VacA protein to cause cell vacuoles helps in its characterization. It
develops pores on the outer membrane of the host cell by allowing bicarbonate and chlorine ions,
urea and pyruvate to exit from the cells. The CagA protein is about 120 to 140 kDa which are
produced by 60% of the H. pylori isolates which is located on the cytotoxin associated genes. The
translocation of these CagA proteins into the stomach's epithelial cells undergoes functions that rely
on both non-phosphorylated and phosphorylated CagA types. In the phosphorylated functions
elongation and dispersion of the cells takes place, this affects the cells adhesion, cytoskeletal
organization and proliferation. Where as in the non-phosphorylated functions there is loss in cell
polarity and also extracellular matrix degradation. As the outcome of all these processes
destabilization of the gastric epithelial cells takes place which further leads to the in vivo
pathogenesis of the organism (2).
Diagnosis of H. Pylori infection
Choosing an appropriate test on circumstantial grounds depends intensely upon patient’s gastric
health assessment with upper endoscopy with upper endoscopy and upon considering the strengths,
weaknesses, and expenses of the individual tests. The accessible tests are commonly separated into
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invasive tests, in light of gastric specimens for histology, culture, or different methods, and
noninvasive tests, in view of peripheral samples, viz., blood, breath samples, stools, urine, or saliva
for detection of antibodies, bacterial antigens, or urease activity. The decision of a particular test
for an individual patient relies upon local experience and the clinical setting (2). H. pylori diagnostic
tests can also be categorized based on the requirement of an endoscopy.
Table 1 provides a list of the available diagnostic tests for H. pylori.
Source: Diagnosis of Helicobacter pylori by invasive test: histology
1. Invasive/Direct methods
Following endoscopic and gastric biopsy tests are considered as invasive diagnostic methods.
1.1. Endoscopy
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Biopsies from the gastric mucosa are acquired utilizing this technique which is used for further
examination of other invasive strategies.
1.2. Histology
Histological assessment has generally been the highest quality level strategy for diagnosing
Helicobacter pylori infection. For identification of H.pylori by this method, a single biopsy test is
taken from an appropriate area, yet different biopsies are suggested for high indicative precision
and affectability. It depends upon various issues including the site, number, and size of gastric
biopsies, staining technique, and the inspecting pathologist. Generally, two diverse stain strategies
are utilized for tissue tests from biopsy; Giemsa and hematoxylin & eosin (H&E). When the after
effects of this stain are uncertain, extraordinary stains, for example, Warthin-Starry, toluidine blue,
acridine orange, McMullen, Genta, Dieterle, and Romanouski stains, or immunochemical (IHC)
techniques can be utilized. The Giemsa stain is reasonable, sensitive, and relatively simple. Along
these lines, the Giemsa stain is the favored strategy in clinical practice (2). Every other strategy is
utilized explicitly for research purposes. H.pylori is appeared as a curved or spiral bacillus on the
epithelial surface, in the mucus layer, and inside gastric glands of the histological section.
Another highly sensitive and reliable staining technique is immunohistochemical (IHC) staining
that has a specific benefit in patients partially treated for H. pylori gastritis. The significant points
of interest of IHC stain is its incorporation with shorter screening time and high specificity as it can
exclude comparable shaped organisms.
Fluorescent in situ hybridization (FISH) is another technique to identify H. pylori utilizing 16S
rRNA gene probe labeled with fluorescein
H&E staining is normally satisfactory and Giemsa stain appears to have advantage over different
stains as a result of its simplicity and consistency (3). Several drawbacks of histology methods
should be considered such as time and cost, reliance on operator skills, and variability of
interobservers. When the collection of tissue is not sufficient or when the biopsies are not well
focused, interpretation becomes difficult.
Histology using appropriate staining is an excellent approach and the use of IHC or FISH and the
collection of sufficient multiple biopsy specimens will improve its accuracy.
1.3. Culture
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Culture is yet another highly detailed type of successful recognition of H.pylori. Culturing of
gastric biopsy samples is carried out to confirm the infection and is normally done in advanced
laboratories.
H. pylori requires selective media and microaerobic conditions (80–90% N2, 5–12% CO2, 5–10%
O2) for growth and incubation at 37 °C for 5–7 days. Various kinds of media can be used for
culturing H. pylori, comprising selective and non-selective agars such as H. pylori agar, the Wang
media, the Wilkins-Chalgren, brain-heart infusion (BHI), trypticase agar bases, Columbia and
blood agar. In the culture media, antibiotics are utilized to avert the development of other kinds of
bacteria. Culturing of H. pylori should quick post biopsy. Within the transport medium, a biopsy
can be maintained at 4 ° C for up to 24 hours and even isolates of the organism can be preserved
frozen at − 80 ° C. Gastric biopsy specimens can be preserved in GESA transport medium at 4 ° C
for upto 10 days and have a quantifiable recovery rate of the organism. This method has a sensitivity
of 70–80% and specificity of 100% (4).
Techniques for Culturing H. Pylori are demanding and costly and are only available in a small
number of clinical laboratories. In spite of the fact that H.pylori culture is costly, complicated and
tedious test, an antibiotic susceptibility trial by culture is a valuable clinical practice for precise
detection. This also allows it to isolate H. Pylori for analysis of the phenotypes and genotypes.
1.4. Rapid urease test
One of the simplest, but fastest, diagnostic tests used to diagnose H.pylori is the Rapid Urease
Test (RUT). Once the biopsy is completed then the first option is RUT from gastric biopsies. This
test depends on the production of urease enzyme by H. pylori microorganism and this enzyme is
found in the gastric mucosa. Endoscopy samples are set in urea-containing medium; if urease is
available, the urea will be separated to carbon dioxide and ammonia, resulting in a rise in the pH of
the medium and a corresponding shift in color of the pH-dependent indicator. Biopsy specimens
are isolated from the gastric antrum and the corpus to get the best results. It is significant that this
method's sensitivity and specificity exceeds 90%. This test has the benefits of being cheap, quick
and wide-ranging. Most generally, to assess the presence or absence of this infection the RUT is
paired with other endoscopic or non-endoscopic modalities. With greater incubation time, the RUT
specificity decreases (increasing the probability of a false positive). Commercial RUTs have
specificities of over 95% to 100%, but their sensitivity is slightly lower (about 85%-95%). RUTs
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that are available commercially include gel-(CLOtest, HpFast) and paper-based tests (PyloriTek,
ProntoDry HpOne). (2)
1.5. Polymerase Chain Reaction
PCR-based identification of the organism could be categorized as invasive as well as non-
invasive methods. H.pylori can be identified in small samples with the presence of few bacteria
with the help of PCR. Gastric juice, biopsy, saliva, and feces samples are generally used in this
technique. Genes of the organism like vacA, cagA, UreA, GlmM, HSP60, 16SrRNA, 23SrRNA,
and ureC can be utilized to identify the H. pylori genome (6). To ascend the specificity of diagnosis
and to eliminate the false-positive rates, two or more target genes are amplified. PCR also offers a
way of detecting antimicrobial resistance mutations. This method is comparatively costly and
demands a higher expertise.
2. Non-invasive/Indirect methods
Non- invasive methods are classified as active and passive tests. Active tests (UBT and SAT)
detect active infection and passive tests (serology/urine) detect a marker of current/former exposure
to H.pylori. Indirect methods are based on the presence of bacterial enzymes, antigens, antibodies,
or DNA sequences.
2.1. Urea Breath Test (UBT)
The principle behind this method depends upon the degradation of urea into ammonia by the
existence of H. pylori in biopsy specimens and by the variations in environmental pH. An
isotopically labeled urea (13C or 14C) is hydrolyzed into carbon dioxide and ammonia, which flows
directly into the blood and are discharged out through the lungs. One can quantify the carbon
dioxide emitted. 13C is a non-radioactive, harmless and stable isotope which can be safely used in
children and women of child bearing age (7). To quantify 13C in breath samples, an isotope ratio
mass spectrometer is commonly used; but, the instrument is expensive. By comparison, 14C-urea
is safe, but requires the use of a department of nuclear medicine approved for storage and disposal
of radioactive elements (6). Generally, both test output characteristics are similar in most studies
with sensitivity and specificity usually exceeding 95%. The procedure is easy to conduct and need
no endoscopy. This test is also useful for determining the lack of infection post treatment.
Nonetheless, this approach may reveal negative results if the specimen indicated the presence of
urease positive bacteria or in cases of active bleeding ulcers and if the patient has lately ingested
proton pump inhibitors, antibiotic agents, or bismuth compounds. Absence of information on
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antibiotic resistance and additional analysis is the major restraining feature of this technique. This
test’s accuracy rate relies upon the amount of urea, examining time, and the set point of the cutoff
value.
2.2. Stool antigen test (SAT)
Stool antigen testing detects the presence of Helicobacter pylori antigen in stool specimen.
For the detection of H. pylori two types of SATs can be used: enzyme immunoassay (EIA) – and
immunochromatography assay (ICA) – based methods, utilizing either polyclonal antibodies or
monoclonal antibodies. In contrast to polyclonal based tests, monoclonal antibody tests show better
results primarily due to the challenge of obtaining good quality polyclonal antibodies every time.
EIA-based tests produce more precise and consistent results than ICA-based tests since monoclonal
antibodies can be used to conduct both the tests. Fecal samples can be kept at room temperature for
24 h or at 4 ℃for 72 h. The SAT undergoes a major decrease in sensitivity within 2 to 3 days
without refrigeration. This method’s sensitivity is 94% and specificity is 97%. False-negative
findings arise when relatively low bacterial load is caused by the use of antibiotics, bismuth, and
proton-pump inhibitors (8). This test is a simple, accurate and cost-effective, and a valuable tool
for epidemiological studies and screening programs.
2.3. Serology
This method involves antibodies that are detected against H. pylori by ELISA, immunoblotting,
and enzyme immunoassays (EIA). These tests require the use of serum, saliva, or urine. This
approach has 76–84% and 79–90% of sensitivity and specificity respectively (6). IgG antibodies to
H. pylori usually occur about 21 days after infection and can persist long after eradication. The
capability of this method to identify active infections relies on the age of the patient, clinical
circumstances of infection, the choice of the antigen used to prepare antibody in ELISA kit, and the
prevalence of infection.
An indirect, simple, fast and reasonable test for the detection of an antibody to H. pylori in adults
is urine-based ELISA test that has revealed high sensitivity and specificity. When compared to
antibody-based test from urine samples, serum samples are much simpler as does not require sample
processing expertise and centrifugation step. Due to its low specificity (76.4%), the urine-based
ELISA system is considered to be inappropriate in infants.
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There are various benefits of serology tests such as its widespread accessibility, rapid performance,
compromised reasonable cost, non-compromise of precision with ulcer bleeding and gastric
atrophy, which can lead to false negative results in other invasive or non-invasive tests.
One of the limitations of this method is following the antibiotic treatment; the long-lasting
antibodies are still present inducing false-positive result of the test. Together, serological method
cannot assure the precision of reported H. pylori status following the antibiotic therapy; thus further
examination is required. Serological testing should be a second-line approach in low-prevalence
populations due to the poor positive predictive ability and a propensity for false-positive outcomes.
Treatment of H. Pylori infection
Test and treat strategy was developed for the treatment of the infection. This strategy checks for H.
pylori existence and its development and when detected this method tries to eradicate the infection.
This therapy is effective and is recommended to patients of the age 45 years or younger, who show
persistent gastritis; dyspepsia, peptic ulcer, and mucosa associated lymphoid tissue (MALT). There
are many therapies for treating this infection; and there is no single treatment of antibiotics to
eradicate this infection. To eradicate the infection, there is use of combination of antibiotics. These
antibiotics are further combined with bismuth salts or proton pump inhibitors (PPI) the antisecretory
agents (10).
It consistently has the first line treatment as triple therapy, where this type of treatment consists of
proton pump inhibitor administration, amoxicillin and clarithromycin for 7 to 14 days. Due to the
resistance of clarithromycin, the cure rates obtained by using this treatment regimen are lower than
80%. This draw back of the first line treatment laid to the use of other regimens such as second line
therapies, these treatments usually consists of two or three antibiotics such as metronidazole,
amoxicillin, tetracycline and metronidazole in combinations with proton pump inhibitor. To
increase the cure rates of this disease and to overcome the problems of antimicrobial resistance new
or different combinations of drugs are being developed from the existing drugs (11).
After the second line eradication failure, the European research team recommends the third line
treatment which is based on the microbial antibiotic sensitivity. Clarithromycin and metronidazole
drugs have failed because the H. pylori isolate shows resistance towards these drugs. The antibiotic
resistance developed by the organism considerably varies from region to region.
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The development of antibiotic resistance in the organism is due to the genetic diversity of H. pylori
which is due to the occurrence of point mutations. This sort of genetic mutation in this organism is
mainly due to the deficiency of the genetic material that is DNA repair system, and many of the
organisms do not contain this repair system. At this stage, high dose of amoxicillin/ proton pump
inhibitor therapy is promising; tetracycline, furazolidone, quinolones and rifabutin are considered
as the alternate drug candidates for the third line therapy (1).
Alternative treatment for the infection:
Due to the antibiotic resistance of H. pylori, several alternative therapies beyond the antibiotics has
been investigated form the last few years such as vaccines, phytomedicine, probiotics, phage
therapy and photo-dynamic therapy, gastric mucin, peptides and polysaccharides.
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Figure 1: Different treatment strategies for anti-Helicobacter pylori therapy are shown.
1. Vaccines:
One of the most successful and cost-effective method invented to prevent the infectious
diseases is Immunization. As this organism possess unusual set of outer membrane proteins due to
its adaptation to the acidic environment of the stomach. A lot of effort must be taken in order to
evaluate the outer membrane proteins of the H. pylori organism as the target for the DNA related
vaccine construction. The cost effective vaccine production can be carried out for this disease as it
has a high cost of treatment (11). Hence, these effective vaccines could cure or prevent or even
modify the interactions between the host and pathogen and help in decrease the rate of progression
of the disease.
Vaccines
Probiotics
Phyto therapy
Polysaccharides
Phage therapy
Peptides
Gastric Mucin
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2. Probiotics:
Probiotics is the other method to orally administer the produced substances or the live
organisms that promotes health. It confers a health benefit to the host when administered in an
adequate quantity. This method is attractive for the H. pylori infection to improve the eradication
rates and to prevent the side effects caused from the administration of the antibiotics (12). As this
method requires low-cost and can be produced in a large scale manner, thus probiotics becomes a
good alternative treatment method to decrease or prevent the further colonization of the H. pylori
bacteria.
3. Phage therapy:
To treat this infectious disease Phage therapy utilizes the lytic bacteriophages, where there is
limitation of these lytic bacteriophages description in H. pylori. There is no much information about
the potential usage and the life-cycle behavior of the phages. An alternative for this therapy would
be the usage of lytic proteins of the phage like lysin that is responsible for the call wall lysine of the
host cell. To overcome the limitation to cross the outer membrane of the gram-negative bacteria
certain modification of the lysine would take place (1).
4. Peptides:
Peptides usually intact with the bacterial membranes and lyse them but their precise mechanism
is not clearly identified. They are the antimicrobial agents that are cationic substances. Organisms
encountered with invasive microorganisms produce peptides as part of their immune response.
Thus, to improve and to control the H. pylori infectious disease certain modification is required for
the expression of human antimicrobial peptides (10).
5. Gastric Mucin:
Due to the secretion of the mucins in the portion of gastric mucosa, further progression of the
infection such as gastric carcinoma and peptic ulcer can be restricted (13). This reaction is due to
the expression of 1,4-N-acetylglucosamine molecule present on the mucin and thus controls the H.
pylori infection.
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6. Polysaccharides:
H. pylori organism is usually found attached to the epithelial cells of the gastric mucosa. The
glucose present on the surface of the gastric mucosa include membrane bound and secreted mucins.
The H. pylori organism interacts with the secreted mucins but adherence of the organism to the
epithelial cells of the gastric mucosa is required to cause the infection. These interactions occur due
to the presence of the glucose and most likely lectin like molecules. Therefore, to block the host-
pathogen interactions and to prevent this infectious disease, polysaccharides are used in this
approach (1). The in vitro inhibition of the bacteria cannot be carried out using the polysaccharides,
but to treat or to prevent the infection their anti-adhesive property plays a major role.
7. Phytotherapy or Phytomedicine:
Since ancient times spices and plants has been used as the therapeutic agents. In this method
there is usage of extracts of herbs and plants as the medications against H. pylori infection. These
herbal products may include different parts of the plants like stems, seeds, roots, flowers and leaves.
Herbs can also be used as a dietary supplement without any further demonstration of efficacy and
safety. Potential sources obtained from the plant origin and the naturally available sources plays a
major role in the development and discovery of the effective agents with respect to H. pylori
infection (12).
Below are some of the examples of plants and their plant products that have been studied in vivo
for their effectiveness and further used as the anti- H. pylori agents.
a) Garlic
Allium sativum commonly called as garlic is well known for its therapeutic activities. The further
interest to continue the studies on this particular plant against H. pylori infection arose due to the
inverse activity shown by consumption of garlic and gastric cancer incidence (12).
b) Green Tea
It is a beverage that is widely consumed all over the world and it has also shown inhibition activity
towards the growth of Helicobacter spp. In vitro studies have been carried out to check its antibiotic
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activity against H. pylori infection (11). From the in vitro results certain in vivo models were also
used which showed the encouraging results against this infection.
c) Red wine
Red wine is tested against the H. pylori to investigate its antimicrobial activity. It has shown
inhibitory effect against the urease activity of the H. pylori infection. The in vitro activity of the
ions and urea conduction by the bacteria and their VacA involved in cell vacuolization has been
successfully inhibited by the Red wine (12).
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