Helicobacter Pylori Infection - Recent Developments in Diagnosis

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DOI: 10.3748/wjg.v20.i28.9299 © 2014 Baishideng Publishing Group Inc. All rights reserved.
TOPIC HIGHLIGHT
WJG 20th Anniversary Special Issues (6): Helicobacter pylori
Helicobacter pylori infection - recent developments in

diagnosis
Ana Isabel Lopes, Filipa F Vale, Mónica Oleastro
Ana Isabel Lopes, Centro Académico de Medicina de Lisboa, endoscopic evaluation methodologies for the diagnosis
Faculdade de Medicina da Universidade de Lisboa, 749-016 Lis- of H. pylori infection, such as magnifying endoscopy
boa, Portugal techniques and chromoendoscopy. In addition, the di-
Filipa F Vale, Centro de Patogénese Molecular, Unidade dos agnostic contribution of histology and the urea breath
Retrovírus e Infecções Associadas, Instituto de Medicina Mo-
test was explored recently in specific clinical settings
lecular e Instituto de Investigação do Medicamento, Faculdade
de Farmácia, Universidade de Lisboa, 1749-016 Lisboa, Portu-
and patient groups. Recent studies recommend en-
gal hancing the number of biopsy fragments for the rapid
Mónica Oleastro, Laboratório Nacional de Referência das In- urease test. Bacterial culture from the gastric biopsy is
feções Gastrintestinais, Departamento de Doenças Infeciosas, the gold standard technique, and is recommended for
Instituto Nacional de Saúde Dr Ricardo Jorge, 749-016 Lisboa, antibiotic susceptibility test. Serology is used for initial
Portugal screening and the stool antigen test is particularly used
Author contributions: Lopes AI planned the paper’s general when the urea breath test is not available, while molec-
structure, format and content; all the authors contributed equally ular methods have gained attention mostly for detect-
to the paper and its final revision. ing antibiotic resistance.
Correspondence to: Ana Isabel Lopes, Professor, MD, PhD,
Centro Académico de Medicina de Lisboa, Faculdade de Medici-
© 2014 Baishideng Publishing Group Inc. All rights reserved.
na da Universidade de Lisboa, Av. Professor Egas Moniz, Código
Postal 1649-035 Lisboa, Portugal. [email protected]
Telephone: +351-217-805000 Fax: +351-217-548215 Key words: Helicobacter pylori ; Diagnosis; Endoscopy;
Received: November 9, 2013 Revised: February 28, 2014 Histology; Culture; Urea breath test; Stool antigen test;
Accepted: April 15, 2014 Serology; Molecular methods
Published online: July 28, 2014
Core tip: Considering the importance of a reliable di-
agnosis in the setting of current recommendations for
Helicobacter pylori (H. pylori ) eradication therapy, re-
Abstract cent developments in both invasive and non-invasive
methods may further contribute to improving H. pylori
Considering the recommended indications for Helico-
detection. The manuscript presents an extensive over-
bacter pylori (H. pylori ) eradication therapy and the view of the major advances in endoscopy, histology,
broad spectrum of available diagnostic methods, a
culture, urea breath test, serology, stool tests and mo-
reliable diagnosis is mandatory both before and after
lecular methods, emphasizing their major contributions
eradication therapy. Only highly accurate tests should
and potential shortcomings.
be used in clinical practice, and the sensitivity and
specificity of an adequate test should exceed 90%. The
choice of tests should take into account clinical circum- Lopes AI, Vale FF, Oleastro M. Helicobacter pylori infection -
stances, the likelihood ratio of positive and negative recent developments in diagnosis. World J Gastroenterol 2014;
tests, the cost-effectiveness of the testing strategy and 20(28): 9299-9313 Available from: URL: http://www.wjgnet.
the availability of the tests. This review concerns some com/1007-9327/full/v20/i28/9299.htm DOI: http://dx.doi.
of the most recent developments in diagnostic methods org/10.3748/wjg.v20.i28.9299
of H. pylori infection, namely the contribution of novel
WJG|www.wjgnet.com 9299 July 28, 2014|Volume 20|Issue 28|

Lopes AI et al . H. pylori diagnosis advances
INTRODUCTION Table 1 Summary of diagnostic methods
A reliable primary diagnosis and control of treatment Invasive/ Reference Antibiotic resistance
success of Helicobacter pylori (H. pylori) infection is crucial noninvasive method detection
for patients with a wide spectrum of H. pylori-related Endoscopy Invasive Yes No
conditions, including uncomplicated or complicated ulcer Histology Invasive Yes No
disease, mucosa associated lymphoid tissue (MALT) lym- Rapid urease test Invasive No No
Culture Invasive Yes Yes
phoma, atrophic gastritis and previous partial gastric re- Molecular methods Both No Yes
section for gastric cancer. Accurate diagnosis of H. pylori Serology Noninvasive No No
infection involves the combined knowledge, effort and Urea breath test Noninvasive No No
research of laboratories, gastroenterologists and patholo- Stool antigen test Noninvasive No No
gists. Traditional diagnosis is made using a combination

of tests, both invasive and noninvasive. Considering the
broad spectrum of diagnostic methods, only highly accu- observed in these high-risk subjects after H. pylori eradi-
rate tests should be used in clinical practice under specific cation. This study emphasizes the high risk of cancer
circumstances and currently, the sensitivity and specific- development in subjects with H. pylori-associated highly
ity of such tests should exceed 90%. The choice of tests active non-atrophic gastritis and the utility of the two
usually depends on clinical circumstances, the likelihood serological tests and endoscopic RHG for their identifica-
ratio of positive and negative tests, the cost-effectiveness tion.
of the testing strategy and of the availability of the tests. Considering that H. pylori eradication is essential for
The present paper aimed to present an overview of the metachronous gastric cancer prevention in patients un-
most recent advances in both biopsy- and non-biopsy- dergoing endoscopic mucosectomy (EMR) for early gas-
based diagnostic methods for H. pylori infection (Table 1). tric cancer, as reported by Fukase et al[3], Lee et al[4] aimed
to determine the optimal biopsy site for H. pylori detec-
tion in the atrophic remnant mucosa of 91 EMR patients.
ENDOSCOPY Three paired biopsies for histology were taken at the
Considering that accurate prediction of H. pylori infection antrum, corpus lesser (CLC), and greater curve (CGC).
status on endoscopic images can improve early detection Additional specimens were obtained at the antrum and
of gastric cancer, especially in some geographic areas, the CGC for a rapid urease test (RUT). H. pylori infection was
contribution of both conventional and novel endoscopic defined as at least two positive specimens on histology
evaluation methodologies has received increased atten- and/or RUT. Pepsinogen levels were used to determine
tion, particularly in specific clinical settings. A summary serological atrophy. The authors concluded that CGC
of the latest endoscopic studies is presented below. Wata- is the optimal biopsy site for H. pylori diagnosis in EMR
nabe et al[1] studied the diagnostic yield of endoscopy patients with extensive atrophy and that an antral biopsy
for H. pylori infection at three endoscopist career levels should be avoided, especially in serologically atrophic pa-
- beginner, intermediate and advanced. For this study, tients.
77 consecutive patients who underwent endoscopy were Although gastroscopic biopsy-based tests such as the
analyzed for H. pylori infection status by histology, serol- RUT, histological examination, and culture have been
ogy and urea breath test (UBT). The diagnostic yield was widely used to diagnose H. pylori infection, many investi-
88.9% for H. pylori-uninfected, 62.1% for H. pylori-in- gators have attempted to categorize the endoscopic find-
fected, and 55.8% for H. pylori-eradicated. Intra-observer ings characteristic of an H. pylori-infected stomach.
agreement for H. pylori infection status was good (k > In 2002, Japanese endoscopists[5] found that collecting
0.6) for all physicians, while inter-observer agreement venules, seen as numerous minute red dots in the gastric
was lower (k = 0.46) for beginners than for intermediate corpus, were a characteristic finding in the normal stom-
and advanced (k > 0.6). For all physicians, good inter- ach without H. pylori infection, using both standard and
observer agreement in endoscopic findings was seen for magnifying endoscopy (identification of micro mucosal
atrophic change (k = 0.69), but the accuracy was lower patterns). This finding was termed “regular arrangement
for beginners. of collecting venules” (RAC). However, these findings
In 496 asymptomatic Japanese middle-aged men, a are not a reliable method of diagnosis because of their
prospective evaluation (mean follow-up period of 54 low sensitivity and specificity.
years), of gastric cancer development was performed in Although magnifying endoscopy provides more
non-atrophic stomachs with highly active inflammation precise information concerning abnormal mucosal pat-
identified by serum levels of pepsinogen and H. pylori terns[6,7], it is not available in all endoscopy units. More-
antibody, together with a specific endoscopic feature: over, its use requires training under an experienced super-
endoscopic rugal hyperplastic gastritis (RHG) (reflecting visor and expertise. In addition, magnifying endoscopy
localized highly active inflammation)[2]. Cancer incidence is not necessarily appropriate for routine clinical practice
was significantly higher in patients with RHG, high H. because it is time-consuming and only a few facilities
pylori antibody titers and low PG Ⅰ/Ⅱ ratio than in pa- carry out this technique on a routine basis. On the other
tients without. Significantly, no cancer development was hand, endoscopic features corresponding to Sydney Sys-

A B
Figure 1 Endoscopic features of Helicobacter pylori infection (antral nodularity).
tem pathological findings have not yet been identified, copy using indigo carmine compared with histology
and the diagnosis of H. pylori infection in the gastric mu- performed according to the Sydney System[7]. Based on
cosa by endoscopic features has not yet been established several indices, the authors obtained a sensitivity of 94%
(Figure 1). In this setting, the Study Group of Japan in the corpus and 88% in the antrum. However, the spec-
Gastroenterological Endoscopy Society for Establishing ificities in the corpus and in the antrum were low (62%
Endoscopic Diagnosis of Chronic Gastritis performed and 52%, respectively). Another study using a Cuban
a prospective multicenter study enrolling 275 patients[8], adult population[11] also aimed to evaluate the diagnostic
investigating the association between endoscopic findings yield of chromoendoscopy with red phenol at 0.1% for
(conventional findings and indigo carmine contrast) and the detection of H. pylori infection against histology. This
histological diagnosis of H. pylori (antrum and corpus). study reported a sensitivity of 72.6% (95%CI: 64.9-79.2)
It was shown that specific endoscopic findings, such as and a specificity of 75.5% (95%CI: 61.9-85.4). The au-
diffuse redness, spotty redness and mucosal swelling as- thors concluded that it might be a useful method to diag-
sessed by conventional endoscopy and swelling of areae nose H. pylori infection in the gastric mucosa, potentially
gastricae by the indigo carmine contrast method, were use- with some specific advantages (topographic localization,
ful for diagnosing H. pylori infection. avoidance of contamination and fast and immediate read-
Cho et al[9] aimed to establish a new classification for ing).
predicting H. pylori-infected stomachs by non-magnifying
standard endoscopy alone. A total of 617 participants
who underwent gastroscopy were enrolled prospectively HISTOLOGY
and a careful close-up examination of the corpus at the Although histology has been considered to be the gold
greater curvature was performed, maintaining a distance standard for H. pylori detection, the influence of clinical
of 10 mm between the endoscope tip and the mucosal practice on the histopathological detection of H. pylori in-
surface. Despite being a monocenter study in which fection has been insufficiently explored. Recognizing that
standard endoscopy was not directly compared with mag- the number and distribution of H. pylori organisms vary
nifying endoscopy, these results suggest two important in patients taking proton pump inhibitors (PPIs), it has
contributions for prediction of H. pylori infection status: been recommended to discontinue PPIs two weeks be-
(1) the observation of gastric mucosal patterns using fore endoscopy and to take biopsies from both the body
standard endoscopy and proposal of a new endoscopic and the antrum.
classification including a normal RAC and three abnor- In a representative study, Lash et al[12] aimed to evalu-
mal mucosal patterns; and (2) an accuracy of prediction ate the yield of different gastric sampling strategies and
of H. pylori positivity at least similar to that reported in to determine the adherence to the Sydney System guide-
magnifying endoscopy studies (sensitivity of 95.2% and lines in a nationwide sample of endoscopists in United
specificity of 82.2%)[10]. In the future, multicenter trials States. Using a database of biopsy records diagnosed at a
comparing standard endoscopy against magnifying en- single pathology laboratory, the results of gastric biopsies
doscopy, including changes in mucosal patterns after H. taken to evaluate gastric inflammatory conditions in pa-
pylori eradication, and including endoscopists with differ- tients with no endoscopic lesions were reviewed. The in-
ent levels of expertise, are needed to confirm the reliabil- cisura angularis, rarely sampled, yielded minimal additional
ity of these data. diagnostic information and the acquisition of at least
Chromoendoscopy has also regained attention re- two biopsy specimens from the antrum and corpus, es-
cently as an additional methodology to detect H. pylori in sentially following the Sydney System recommendations,
the gastric mucosa. A multicenter Japanese study involv- was confirmed as a sensible strategy that guarantees the
ing 275 patients evaluated the possibility of diagnosing maximum diagnostic yield for the most common gastric
H. pylori by conventional endoscopy and chromoendos- inflammatory conditions.

In a Canadian study[13], electronic patient records were for H. pylori, with the lowest rate of inter observer varia-
evaluated for the sites of gastric sampling and PPI use at tion and is much faster than conventional histology[19].
endoscopy, collecting 150 cases with biopsies taken from However, the necessity for routine special stains and/or
both the antrum and body, which were randomly selected IHC stains has been debated in recent years. A recent
for pathological re-review with special stains. The gastric study by Wang et al[20] confirmed what many pathologists
regions sampled, H. pylori distribution and influence of assume: routine special stains, specifically IHC stains, are
clinical factors on pathological interpretation were as- not cost-effective or necessary. Recently, Smith et al[21], in
sessed. This study confirmed that, despite national and a retrospective study involving 200 consecutive gastric
international guidelines for managing H. pylori infection, biopsy specimens, further confirmed that H. pylori is eas-
these guidelines are infrequently adhered to, with PPIs ily observed in the majority of cases with HE (sensitivity
frequently contributing to false diagnosis, and sampling 91% and specificity 100%), remaining the most expedient
only one region increases the likelihood of missing active and least expensive test for identifying H. pylori in gastric
infection by at least 15%. biopsies.
Considering that atrophy of the stomach mucosa An institutional quality assurance study of a conven-
develops in about 50% of H. pylori infected individuals tional method for the diagnosis of H. pylori - associated
by the age of 65, and is considered a pre-malignant le- gastritis was performed by Hartman et al[22] in the United
sion for gastric cancer[14-16], H. pylori eradication is recom- States, based on head-to-head evaluation by four meth-
mended in the presence of atrophy[17], because atrophy ods, HE stain, Giemsa stain, Warthin-Starry stain, and H.
may reverse after successful eradication therapy. It is pylori immunostaining of 356 gastric biopsy specimens.
critically important and challenging, therefore, to deter- About 83% of H. pylori gastritis identified were diag-
mine the presence or absence of H. pylori in patients with nosed on the initial HE-stained slides, further supporting
atrophic gastritis. During atrophy progression, however, the use of routine ancillary stains to diagnose H. pylori
the density of H. pylori in the stomach mucosa decreases, infection in gastric biopsy specimens. Usually, the use of
and may disappear completely during the late stages of special stains is only recommended for biopsy specimens
atrophy[14,16]. This may explain the markedly lower sen- with moderate to severe chronic active or inactive gastri-
sitivity of biopsy-based tests (RUT, histology, culture) tis in which H. pylori is not identified by HE staining, for
in the presence of atrophy. Similarly, UBT and antigen post-treatment biopsy specimens and in cases in which
stool detection can also give false-negative results in these structures “suspicious”, but not definitive, for H. pylori
circumstances. In contrast, serology is not influenced to are observed by HE staining[23].
such an extent by a lower density of the microorganism, Both routine conventional histology-based methods
and is reliable even in advanced gastric body atrophy[14,16]. and novel methods for H. pylori detection have increas-
Maastricht guidelines updates have reserved serology ingly focused on specific clinical settings and patient
for special situations, including extensive atrophy of the groups (bleeding peptic ulcer, gastric cancer). False-
stomach mucosa on the basis that other tests might be negative results may occur when using histological and
misleading at a low bacterial density. Thus, the debate RUT to detect H. pylori in biopsy specimens obtained
continues regarding the most appropriate H. pylori diag- during peptic ulcer bleeding episodes (PUB). Choi et al[24]
nostic method in atrophic gastritis. evaluated different diagnostic methods in the specific
Lan et al[18] aimed to evaluate the site and sensitivity setting of peptic ulcer, concluding that histology was the
of biopsy-based tests in terms of degree of gastritis with most accurate test, regardless of bleeding, compared with
atrophy. Biopsy-based tests (i.e., culture, histology Giemsa culture, serology and RUT. Ramirez-Lazaro et al[25] found
stain and RUT) and non-invasive tests (anti-H. pylori IgG) that IHC and real-time PCR methods might improve the
were performed in 164 uninvestigated dyspepsia patients. sensitivity of biopsy-based diagnosis in this specific set-
The sensitivity of biopsy-based tests decreased when the ting (PUB).
degree of gastritis with atrophy increased, regardless of In patients submitted to a subtotal gastrectomy due
biopsy site. In moderate to severe antrum or body gastri- to gastric cancer, the identification and treatment of
tis with atrophy, additional corpus biopsy increased the H. pylori are the key points in the prevention of cancer
sensitivity to 16.67%, as compared with single antrum recurrence. Xu et al[26] evaluated the predictive value of
biopsy. These results confirm that in moderate to severe neutrophil infiltration, a hallmark of active inflammation
gastritis with atrophy, biopsy-based test should include (updated Sydney system), as a histological marker of H.
the corpus for avoiding false negative results in H. pylori pylori infection, in 315 dyspeptic patients undergoing up-
detection. per gastrointestinal endoscopy, including patients with a
Since the discovery of H. pylori, pathologists have subtotal gastrectomy. The diagnosis of H. pylori infection
used different diagnostic techniques, including immu- was based on UBT and on anti-H. pylori immunoglobulin
nohistochemical (IHC) methods and special stains, such G (IgG) antibody in patient with a subtotal gastrectomy.
as Giemsa and Warthin-Starry, on an institution- and Although neutrophil infiltration of gastric mucosa was
laboratory-dependent basis (with variable sensitivities and strongly associated with overall H. pylori infection, in pa-
specificities for identifying H. pylori). On the other hand, tients with a subtotal gastrectomy, the diagnostic accuracy
it is clear that IHC staining is highly sensitive and specific of neutrophil infiltration in H. pylori infection was low.

De Martel et al[27], using data from a large Venezuelan further elucidation.

cohort of 1948 adults, compared the gastric detection of Finally, the histology reporting of gastritis of the
H. pylori by polymerase chain reaction (PCR) of the vacA staging system OLGA (Operative Link on Gastritis As-
gene in one antral biopsy, to the detection of H. pylori by sessment) has also been re-examined, considering its
histopathology (HE and Giemsa staining) in five biopsies relevance to the prediction of the gastric cancer risk[31,32].
(antrum and corpus). Overall, H. pylori was detected in Carrasco et al[33] reviewed the histology of the normal
85% and 95% of the subjects by PCR and histopathol- gastric mucosa, overviewing the role of H. pylori in the
ogy, respectively, thus confirming that histopathology on multistep cascade of GC. The role of the OLGA staging
five biopsies is an accurate tool for H. pylori detection in system in assessing the risk of GC was emphasized; spe-
most subjects, compared with the PCR method on one cifically, the epigenetic bases of chronic gastritis, mainly
biopsy. However, in subjects with the most severe pre- DNA methylation of the promoter region of E-cadherin
cancerous lesions (intestinal metaplasia type Ⅲ and dys- in H. pylori - induced chronic gastritis and its reversion af-
plasia), PCR displayed elevated sensitivity for detecting ter H. pylori eradication. In addition, the authors discussed
the bacteria (significantly more often than histopathology the finding of circulating cell-free DNA, offering the op-
on a single biopsy), thus suggesting its potential useful- portunity for non-invasive risk assessment of GC.
ness in this setting.
Tian et al[28] reported a meta-analysis evaluating H. py-
lori diagnostic methods in patients with a partial gastrec- Rapid Urease Test
tomy. The pooled sensitivity and specificity were 93 and The RUT is based on the production of large amounts
85% for histology, 77 and 89% for UBT, and 79 and 94% of urease enzyme by H. pylori, which splits the urea test
for RUT, respectively, thus leading to the conclusion that reagent to form ammonia, enabling its detection by a
histology was the most reliable test in this setting. Lee et rapid indirect test. Many commercial RUTs are available,
al[4] evaluated 91 patients requiring endoscopic mucosal including gel-based tests, paper-based tests and liquid-
resection for early gastric cancer (GC), obtaining three based tests, providing a result in 1-24 h, depending on the
pairs of biopsies from the antrum, CLC and CGC. The format of the test and the bacterial density in the biopsy
sensitivity of histology in detecting H. pylori was signifi- specimen. Typically, commercial RUTs have specificities
cantly higher in the CGC than that in the antrum or CLC, above 95%-100%; however, the sensitivity is slightly less,
suggesting that the CGC might be the optimal biopsy site ranging from 85%-95%[34].
for H. pylori in patients with extensive atrophy. Compared with histology and culture, urease tests
The utility of routine biopsy of the gastric ulcer mar- are faster, cheaper and have comparable sensitivity and
gin (currently performed to exclude malignancy) in diag- specificity in normal clinical settings. The sensitivity can,
nosing H. pylori infection, has recently been re-assessed however, decrease in patients with bleeding peptic ulcers
by Lin et al[29], by examining prospectively a cohort of 50 (67%-85%), as well as in patients with partial gastrectomy
patients with gastric ulcer (54% uninfected). Histology, (79%)[24,28,34,35]. Formalin contamination of forceps used
RUT and UBT were compared; six biopsied specimens to collect the biopsy may also contribute to reduced sen-
from the margin of the gastric ulcer and one specimen sitivity[24,36].
each from the antrum and body of non-ulcerous parts An important conclusion of several studies is that
were obtained for histology using HE staining. The di- enhancing the number of biopsy fragments and/or col-
agnostic accuracy of the histological examination of the lecting them from various regions of the stomach (antrum
ulcer margin was quite good and importantly, the addi- and body, from example), achieves a higher sensibility of
tion of one specimen from the antrum or body did not the RUT[37]. Moreover, it was shown recently that com-
increase its diagnostic yield, thus emphasizing its accuracy bining tissues prior to RUT increased the detection of
and usefulness for diagnosing H. pylori infection in these H. pylori, compared with testing separate specimens, and
patients. produced faster results[38].
An increasing body of evidence supports H. pylori
colonization in the esophageal mucosa of dyspeptic pa-
tients. Contreras et al[30] have further contributed to the CULTURE
field, with a study examining the presence of H. pylori in Since the discovery of H. pylori, bacterial culture has been
the gastroesophageal mucosa by histology, fluorescence used as routine diagnostic test, being considered the gold
in situ hybridization (FISH) and PCR analysis of DNA standard. Currently, the Maastricht-4 Consensus Report
(using genus- and species-specific PCR primers) extracted recommends H. pylori culture for performing antibiotic
from gastric and esophageal biopsies of 82 symptomatic susceptibility testing if primary resistance to clarithro-
Venezuelan patients. H. pylori in the stomach was de- mycin is higher than 20% or after failure of second-line
tected by PCR and FISH, respectively, in 61% and 90% treatment[17].
of dyspeptic patients, and in the esophagus in 70% and Despite its long use, culture tests remain a challenge
73%. By combining the results of both methods, H. pylori because of the fastidious nature of the bacterium, with
was observed in the gastroesophageal mucosa in 86% of particular growth requirements of medium and atmo-
patients. These findings deserve specific attention and sphere. The most commonly used media include Bru-

cella, Columbia Wilkins-Chalgren, brain-heart infusion or possibly acting as a “reservoir” contributing to the intra-
trypticase agar bases, supplemented with sheep or horse familial spread[50].
blood[39]. An alternative to blood is supplementation of
the agar base with b-cyclodextrin or yolk emulsion[40,41].
The most recent advances on H. pylori culture concern MOLECULAR METHODS
growth medium composition, besides the usual serum or Diagnostics tests rely more and more on molecular tests,
blood additives. A recent study showed that supplemen- which can provide faster, more accurate and sensitive
tation of media with cholesterol instead of serum was detection of the bacterium than conventional methods,
a viable option for H. pylori growth[42]. Another original with the possibility of extension to other purposes, such
approach used liquid culture medium for the rapid cul- as detection of antibiotic resistance and virulence de-
tivation and subsequent antibiotics susceptibility testing terminants, and bacterial quantification. Moreover, bio-
of H. pylori directly from biopsy specimens, with a final logical samples other than gastric biopsies can be used,
detection step by an enzyme linked immunosorbent assay obtained using less invasive methods, such as stool or
(ELISA)[43]. oral cavity samples. Whatever the case, amplification of
Concerning the growth atmosphere, H. pylori is a cap- the nucleic acids by PCR is almost always present, either
nophilic organism that requires an atmosphere enriched conventional PCR or, increasingly, by real-time PCR.
with CO2 (varying from 5%-10%). In addition, it has H. pylori, like a few other bacteria, acquires resistance
been considered a microaerophile, but there is no general by mutation, which has enabled the development of
consensus about its specific O2 requirements[44]. A recent numerous assays, in several formats, to detect mutations
advance on this topic was made by Park et al[45], who leading to resistance, especially to macrolides and fluoro-
showed that unlike previous reports, H. pylori may be a quinolones. To detect H. pylori and resistances to fluoro-
capnophilic aerobe whose growth is promoted by atmo- quinolones and clarithromycin, there is a multiplex PCR
spheric oxygen levels in the presence of 10% CO2. followed by a hybridization and alkaline phosphatase
Typically, culture of H. pylori is performed on gastric reaction on a membrane strip (the Genotype® HelicoDR
biopsy samples, and because bacteria display an irregular kit), that uses as a starting material biopsy specimens, as
distribution in the gastric mucosa, culture of more than well as culture material extracted from it. The test shows
one biopsy, from the antrum and corpus, is sometimes a high sensitivity and permits detecting infection with
mandatory, especially after antibiotic treatment. Another multiple strains. The performance in detecting fluoroqui-
important issue to bear in mind are factors that may af- nolone-resistance strains was, however, lower than cul-
fect the outcome of H. pylori culture from endoscopic ture, emphasizing the need to expand the range of gyrA
gastric mucosal specimens. Besides the issue concern- mutations included in the kit[51,52]. Several real-time PCR
ing bleeding peptic ulcers, for which culture has a lower based assays, using either TaqMan or FRET (Fluorescence
sensitivity than in nonbleeding cases, other host-related Resonance Energy Transfer) are available, as in-house
factors, such as high activity of gastritis, low bacterial assays or commercial kits, for clarithromycin resistance,
load, drinking alcohol and the use of histamine H2 recep- performed on cultured strains, directly on biopsies[53-55] or
tor blockers, have been recently described as the cause of in stool samples. The latter is particularly useful as a non-
failed H. pylori culture from gastric mucosa in the infected invasive test in pediatric populations, where a high preva-
subjects[24,46]. lence of clarithromycin-resistant strains is suspected,
Culturing from stools has been shown to be extreme- as well as for tracking the emergence of clarithromycin
ly difficult because of the complex nature of the sample resistance following eradication treatment[57,58].
regarding microbiota composition and shedding of unvi- Recently, a dual-priming oligonucleotide (DPO)-based
able H. pylori cells, and this technique has been successful multiplex PCR was developed to detect both H. pylori
in the setting of rapid gastrointestinal tract transit[47]. In infection and the most common point mutations confer-
a recent study, the authors were able to culture H. pylori ring resistance to clarithomycin, directly on gastric biopsy
in nine and 12 rectal and ileal fluids, respectively, after specimens. This assay proved to be fast and does not
polyethylene glycol (colyte) ingestion in 20 healthy adults require expensive instrumentation, making it valuable in
with positive UBT[48]. Other studies have looked for the countries with a high prevalence of clarithromycin resis-
role of the oral cavity as a reservoir of H. pylori. A recent tance[59,60].
work evaluated the occurrence of the organism in subgin- The detection of clarithromycin-resistance from for-
gival plaque and was able, by culture, to recover H. pylori malin-fixed, paraffin-embedded gastric biopsies has also
in nine of 30 studied patients that were H. pylori positive been described, and is useful mostly before treatment
with RUT and histopathological examination. Thus, they when culture and susceptibility testing is not available, or
concluded that detection of H. pylori in dental plaque of to detect primary resistance to clarithromycin in the case
dyspeptic patients cannot be neglected and might repre- of failure of an empirical therapy based on this antibiotic.
sent a risk factor for recolonization of the stomach after Real-time PCR assays, as well as a peptide nucleic acid-
systemic eradication therapy[49]. The same conclusion was fluorescence in situ hybridization (PNA-FISH) method,
reached by another study in which H. pylori was detected have been described recently[61-63].
in subgingival dental plaque of children and their families, Another area of particular interest is the detection

of virulence determinants, such as the cagA (cytotoxin- towards understanding the pathways that are associated
associated gene A) and the vacA (vacuolating cytotoxin) with the respective disease, contributing to the identifica-
major toxins. Several studies showed that the risk of tion of novel therapeutic or diagnostic targets.
progression of gastric preneoplastic lesions is higher in Our current knowledge on the proteome of this or-
patients infected with strains harboring the most virulent ganism is largely based on data obtained for the soluble
cagA and vacA genotypes than in patients infected with proteome[73], membrane proteome[74,75] and secreted pro-
the least virulent strains. Therefore, H. pylori genotyping teome[76] of strain 26695, the first isolate to be sequenced.
may be useful to identify patients at high risk of progres- More recently, relevant contributions have made been
sion of gastric preneoplastic lesions and who need more through this approach, such as novel biomarkers for gas-
intensive surveillance[64]. Concerning vacA, a novel meth- tric cancer and for peptic ulcer disease[77,78].
od for genotyping the vacA intermediate gene region
was reported recently, using a novel primer combination
allowing the amplification of smaller DNA fragments NONINVASIVE TESTS
than the original PCR, which can therefore be applied to Although the reliability of both the 13C-UBT and a
paraffin-embedded biopsies. Patients infected with vacA monoclonal ELISA stool test (HpSA) to diagnose H. py-
i1 strains showed an increased risk of gastric atrophy and lori infection in very young children has been confirmed,
gastric carcinoma, with odds ratios of 8.0 (95%CI: 2.3-27) additional background information is warranted for epi-
and of 22 (95%CI: 7.9-63)[65]. demiological studies in infants and toddlers.
CagA undergoes phosphorylation on tyrosines within
the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs at the poly-
morphic C-terminus[66]. Several studies suggest a role for UREA BREATH TESTS
the polymorphic CagA EPIYA-containing region in the The 13 C-urea breath test (13C-UBT) is one of the most
pathogenicity of H. pylori, although conflicting results reliable tests for diagnosing H. pylori infection. It is a
have been reported[67,68]. The in vivo role of this region non-invasive, simple and safe test that provides excellent
was emphasized recently in a study showing that infection accuracy both for the initial diagnosis of H. pylori infec-
with strains harboring two or more CagA EPIYA C mo- tion and for the confirmation of its eradication after
tifs was associated with the presence of surface epithelial treatment. The simplicity, good tolerance and economy
damage, and with atrophic gastritis and gastric carcinoma. of the citric acid test meal probably make its systematic
Moreover, the presence of two or more CagA EPIYA C use advisable. The UBT protocol may be performed with
motifs increased the risk of atrophic gastritis from 7.3 relatively low doses (< 100 mg) of urea: 75 mg or even
(95%CI: 2.1-25) to 12 (95%CI: 2.5-58) and of gastric car- 50 mg seem to be sufficient. With the most widely used
cinoma from 17 (95%CI: 5.4-55) to 51 (95%CI: 13-198), protocol (with citric acid and 75 mg of urea), excellent
when compared with one EPIYA C motif. Therefore, ge- accuracy is obtained when breath samples are collected
notyping H. pylori virulence determinants could represent as early as 10-15 min after urea ingestion. A unique and
a useful approach in defining severe gastric-disease risk. generally proposed cut-off level is not possible, because
Bacterial quantification can also be important for it has to be adapted to different factors, such as the test
clinical management of the infection; for example, for meal, the dose and type of urea, or the pre-/post-treat-
monitoring the treatment outcome or in particular set- ment setting. As positive and negative UBT results tend
tings, such as upper gastrointestinal bleeding[69]. to cluster outside of the range between 2 and 5, a change
A recently developed real-time quantitative PCR assay in cut-off value within this range would be expected to
based on H. pylori ureC (single copy gene) copy number have little effect on the clinical accuracy of the test[79,80].
proved to be around 10 times more sensitive than the UBT is now marketed for use with a nondispersive,
conventional PCR method. Moreover, the copy number isotope-selective infrared spectroscope or laser-assisted
of ureC was significantly increased when overall gastritis, ratio analysis, which are reliable and valid alternatives to
bacterial density, chronic inflammation and intestinal isotope ratio mass spectrometry (IRMS) of potential in-
metaplasia were present[70]. Nevertheless, further studies terest for epidemiologic studies of children, for screening
are necessary to determine the optimum cut-off point, symptomatic children before endoscopy or assessment
making it possible to differentiate between asymptom- of treatment efficacy. These devices are far smaller and
atic colonization and infection with clinical implications cheaper, and they allow for in-office, near-immediate
for patients. These highly sensitive real-time quantitative reading of results. Validation studies to establish the cut-
PCRs can have a large application on the study of envi- off value for this test were preliminarily performed in
ronmental reservoirs as well[71,72]. Japan[81]; however, further data are needed[82,83].
By improving our knowledge of bacteria, at the mo- The 13 C-UBT in adults has a high sensitivity
lecular level, new strategies for treatment/prevention of (88%-95%) and specificity (95%-100%)[17]. However, the
bacterial-associated diseases, as well as diagnostic tests, test has shown heterogeneous accuracy in the pediatric
can be developed. Proteomic approaches aimed at identi- population, especially in young children, with values of
fying gene products differentially expressed in association sensitivity and specificity ranging from 75% to 100%,
with a given pathology can provide an important input before and after treatment (using several protocols),

despite being a simple and safe non-invasive test in chil- Pacheco et al[92] evaluated the diagnostic accuracy of
dren older than 6 years old[84]. Although several modifi- detecting H. pylori infection of low dose 13C-UBT with
cations have been proposed since the original descrip- early sampling at pediatric age (129 patients between the
tion by Graham of the 13C-UBT to diagnose H. pylori ages of 2.1 and 19 years old, median = 11.6 years) sub-
infection[85], in children, performance criteria are not yet mitted to upper gastrointestinal endoscopy. The 13C-UBT
sufficiently established[86]. In the specific age group of was performed after a 4-h fasting period with four points
younger children, accurate non-invasive tests for diag- of collection: baseline (T0, at 10, 20 and 30 min) after
nosing H. pylori infection are required, as they may avoid ingestion of 25 mg 13C-urea diluted in 100 mL of apple
invasive and painful procedures, such as endoscopy and juice; analysis of exhaled breath samples was performed
blood sampling, and to overcoming the false negative with an isotope-selective infrared spectrometer. The sen-
results observed with gold standard tests (histology, cul- sitivity and specificity were similar at T10, T20 and T30
ture, and RUT), where colonization of the stomach may (94.7%/96.8%; 96.2%/96.1% and 96.2%/94.7%, respec-
be weak and patchy. tively).
Potential explanations for UBT performance vari- Recently, Queiroz et al[93] investigated the agreement
ability in children might include: (1) urease activity from between the 13C-UBT and a monoclonal ELISA (HpSA)
the oral bacterial flora[87]; (2) differences in delta time to detect H. pylori antigen in stool in a prospective study
(decrease in specificity if samples obtained at 15 min in- enrolling 414 South-American infants (123 from Brazil
stead of 30 min); and (3) variability in cut-off values. The and 291 from Peru) aged 6-30 mo. Breath and stool sam-
administration of 13C-urea in capsules to avoid activity of ples were obtained at intervals of at least three-months.
13
oral bacteria, though effective in adults, is not feasible in C-UBT and stool test results concurred with each other
infants or toddlers[88]. Finally, the cut-off value (usually in 94.86% cases (kappa coefficient = 0.90, 95%CI: 87-92).
determined by a ROC curve) represents a crucial factor In the H. pylori-positive group, DOB and OD values were
for the accuracy of the test, where low cut-off values positively correlated (r = 0.62, P < 0.001, suggesting that
might increase sensitivity but reduce specificity, and vice both 13C-UBT and stool monoclonal test are reliable to
versa[81]. Additionally, the individual’s CO2 production is diagnose H. pylori infection in very young children.
influenced by anthropometric characteristics, as well as by In contrast to pediatric studies, where attention has
age and sex (lower in young children with relatively low been focused on methodological issues, in adult studies,
weight and height)[89]. the validity and usefulness of UBT have increasingly been
Leal et al [90] performed an informative systematic evaluated in a wide spectrum of specific clinic settings.
review and meta-analysis (31 articles and 135 studies Olafsson et al[94] evaluated 620 UBT in 595 subjects at a
from January 1998 to May 2009), aiming to evaluate the gastroenterology clinic. UBT was negative in 526 patients,
performance of the 13C-UBT diagnostic test for H. pylori but: (1) 45% patients were tested < 4 wk before the end
infection in children. Studies with at least 30 children of treatment; and (2) 23% of negative results occurred
and reporting the comparison of 13C-UBT against a gold in patients recently treated. The authors emphasized the
standard for H. pylori diagnosis (H. pylori culture, histo- need for strict protocol adherence in clinical practice for a
logic examination, or RUT) were included for analysis. fully reliable UBT assessment. Velayos et al[95] investigated
Children were stratified in subgroups of < 6 and ≥ 6 the accuracy of UBT performed immediately after emer-
years of age. The 13C-UBT performance meta-analyses gency endoscopy in 74 patients with peptic ulcer bleeding
showed: (1) good accuracy in all ages combined [sensitiv- by comparing the results with those of UBT performed
ity 95.9%, specificity 95.7%, diagnostic odds ratio (DOR) after hospital discharge in a subset of 53 patients (gold
424.9]; (2) high accuracy in children > 6 years (sensitivity standard). Although UBT carried out immediately after
96.6%, specificity 97.7%, DOR 1042.7); and (3) greater emergency endoscopy in peptic ulcer bleeding is an ef-
variability in accuracy estimates and a lower specificity fective, safe and easy-to-perform procedure, the relatively
in children ≤ 6 years (sensitivity 95%, specificity 93.5%, low sensitivity and specificity suggested the requirement
DOR 224.8). The authors identified as potentially impor- of a subsequent control, in accordance with recommen-
tant sources of heterogeneity: (1) tracer dose; (2) pretest dations concerning peptic ulcer bleeding[96].
meal; and (3) cut-off value, observing that a unique Few studies using UBT have been performed in pa-
tracer dose of 50 mg of 13C-urea showed greater accu- tients subjected to a partial gastrectomy, a specific group
racy when it was adjusted to body weight (50-75 mg were in which the identification of H. pylori infection is mostly
used between studies). Accordingly, Mégraud[91] previ- relevant. Wardi et al[97] evaluated the sensitivity and speci-
ously reported that reducing the dose from 75 to 45 mg ficity of the continuous UBT (BreathID) in 76 post gas-
in younger children resulted in improved specificity. Al- trectomized patients (older than 18 years) (lowering the
though citric acid has demonstrated good performance in gastric pH by the addition of citric acid), against RUT
adults, it is not well accepted by children, and apple, or- and histology as gold standards. H. pylori was positive in
ange, or grape juice seem to be good alternatives. Finally, 14/76 (18.4%) patients when histology was considered as
a cut-off value of 6.0‰ improved overall performance in the gold standard method. The positive predictive values
children younger than 6 years, as compared to a cut-off of the continuous UBT and the RUT were 0.64 and 0.35,
of 4.0 ‰ for children older than 6 years. respectively. The negative predictive value was high by

both the methods, 0.92 and 0.95, respectively, supporting nal lateral flow chromatography (LFC); Meridian Biosci-
the view that BreathID might have some reliability to ex- ence, Europe Srl Milano, Italy); the H. pylori fecal antigen
clude H. pylori after partial gastrectomy. test (based on monoclonal LFC; Vegal Farmaceutica, Ma-
drid, Spain) and the one-step H. pylori antigen (based on
LFC with polyclonal antibodies; IHP-602, ACON Labo-
STOOL ANTIGEN TESTS ratories, Inc, San Diego, United States). Data comparison
The stool antigen test is a non-invasive method to detect showed an uneven performance, favoring the Premier
H. pylori, usually recommended when the UBT is not Platinum HpSA Plus test (sensitivity 92.2%; specificity
available[98]. Besides being non-invasive, the advantages 94.4%). The selection of the stool antigen assay is very
of using this method include the unneeded requirement important to achieve accurate results.
of expensive equipment and medical personnel, and the Stool antigen tests are also useful to detect H. pylori in
collection of the sample at home without a visit to the infected animal models, such as C57BL/6 mice[105].
hospital. This method is especially relevant for children’s
access to a safe diagnosis and also for its low cost[99,100].
A meta-analysis revealed that the global sensitivity and Antibody - based tests
specificity of stool antigen tests are 94% (95%CI: 93-95) Serology was one of the first methods used for diagnosis
and 97% (95%CI: 96-98), respectively[101]. A prospective of H. pylori infection[106]. Currently, serology is recom-
study to evaluate the efficacy of a new EZ-STEP H. py- mended for initial screening, requiring further confirma-
lori polyclonal enzyme immunoassay (EIA) stool antigen tion by histology and/or culture before treatment [107].
test enrolled 555 patients undergoing routine checkups. Detection of antibodies is useful for detecting past or
At the optimal cut-off value (optical density 0.160), this present exposure. In fact, a limitation of serology tests
test presented high level of sensitivity (93.1%), specificity is the failure to distinguish between past and current H.
(94.6%) and accuracy (93.8%)[99]. pylori infection[99]. Moreover, the antibody levels to H.
There are two types of stool antigen tests used for pylori are significantly heritable. Thus, individual genetic
H. pylori detection, the EIA and an assay based on immu- differences of the human host contribute substantially to
nochromatography. Two new stool tests were developed antibody levels to H. pylori[108].
recently[102]. These tests are the Testmate pylori antigen Serological tests have several advantages, namely they
EIA, in which plastic 96-well EIA microtiter plates are are non-invasive and they do not produce false negative
coated with monoclonal antibody (Mab) 21G2[103], and results in patients receiving treatment (proton pump in-
the Testmate rapid pylori antigen, which is based in im- hibitors and antibiotics) or presenting acute bleeding[109].
munochromatography and is presented as a test strip. For Blood samples are used for serology testing, detect-
the EIA test, a drop of the suspended stool sample or a ing anti-H. pylori antibodies (IgG) by ELISA. Recently,
sample of the diluted bacterial antigen sample is mixed the performance of 29 different serological tests kits was
with the peroxidase-conjugated MAb 21G2. After proper compared, revealing sensitivities ranging from 55.6% to
incubation and washing, the optical density is measured 100%, specificities ranging from 59.6% to 97.9 %, posi-
and considered positive if greater than 0.100. For the test tive predictive values ranging from 69.8% and 100%,
strip, a drop of stool sample is applied in the specimen and negative predictive values ranging from 68.3% and
application of the test strip. When H. pylori antigens are 100%[106]. According to the goal, such as screening, initial
present, they form immune complexes with the red latex- diagnosis and confirmation of another test, the most ap-
labeled MAb 21Ge and migrate by capillarity action until propriate kit should be chosen. Antibody-based tests for
captured by the solid phase anti-mouse rabbit polyclonal the detection of H. pylori are easily available, but present
antibodies and form a visible red test line. A control line high negative predictive value[110]. The heterogeneity of H.
is also present. After application of these tests to 111 pylori strains has been well documented, with considerable
stool samples, both new tests provide 100% specific- variation in the prevalence of specific strains, especially
ity, sensibility and accuracy[102], which is very promising. from different geographical areas[111-113]; thus, the success
However, not all studies report these high values for sen- of a serology test depends on the use of antigens that
sitivity and specificity. For example, the report of Chehter are present in H. pylori strains from a given population.
et al[100] analyzed the stools of 75 patients and determined Moreover, kits developed using H. pylori strains from
a lower sensitivity (87.2%) and specificity (44%); Irani- the west are not suitable for detecting H. pylori infection
khah et al[104] analyzed the stools of 103 children and ob- in the East[114]. The use of high-molecular-weight cell-
tained similar values for sensitivity (85%), but improved associated antigens that are conserved in H. pylori strains
specificity (83%). overcomes this limitation[115]. Several H. pylori immuno-
Recently, five different stool antigen tests were com- genic proteins have been presented as candidates to de-
pared: the Premier Platinum HpSA Plus test (based on tect infection, such as the FlidD protein[116]; multiple re-
monoclonal EIA; Meridian Bioscience, Inc, Cincinnati, combinant (CagA, VacA, GroEL, gGT, HcpC and UreA)
OH, United States); the Hp Ag test (based on monoclo- proteins[116]; CagA[115] or Omp18[117].
nal EIA; Dia.Pro Diagnostic Bioprobes Srl, Milano, Italy); Modifications to serology tests have been suggested,
the ImmunoCard STAT! HpSA test (based on monoclo- such as the automated immunoaffinity assay for H. pylori

IgG detection using purified antigen of H. pylori immobi- versus adults) and clinical conditions, such as peptic ulcer
lized on magnetic nanobeads, which is faster than ELISA bleeding, atrophic gastritis, post-gastrectomy status, as
and requires a smaller volume of serum[118]. The lateral well as for wider application in epidemiological studies.
flow immunoassay, an immunochromatographic assay, The specific contribution of each method to the evolving
maintains the serological approach with the advantage of strategies and algorithms for evaluation and management
being fast, economic and requiring no additional equip- of H. pylori infection (test and treat) will remain of para-
ment or experience[119]. mount relevance.
Detection of gastrin and the serum PG Ⅰ/Ⅱ ratio
combined with H. pylori serology is useful to predict
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P- Reviewer: Assem M, Kodama M, Pandya S, Ozcan C, Yucel O

S- Editor: Zhai HH L- Editor: Stewart GJ
E- Editor: Zhang DN

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Helicobacter Pylori Infection - Recent Developments in Diagnosis

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Helicobacter Pylori Infection - Recent Developments in Diagnosis

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What are some of the major diagnostic methods discussed for H. pylori infection?

What are some of the major diagnostic methods discussed for H. pylori infection?

What does the article recommend for enhancing the diagnosis using histology?

What does the article recommend for enhancing the diagnosis using histology?

Submit a Manuscript: http://www.wjgnet.

com/esps/ World J Gastroenterol 2014 July 28; 20(28): 9299-9313

WJG 20th Anniversary Special Issues (6): Helicobacter pylori

Helicobacter pylori infection - recent developments in

Ana Isabel Lopes, Filipa F Vale, Mónica Oleastro

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INTRODUCTION Table 1 Summary of diagnostic methods

gists. Traditional diagnosis is made using a combination

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Figure 1 Endoscopic features of Helicobacter pylori infection (antral nodularity).

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De Martel et al[27], using data from a large Venezuelan further elucidation.

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10.1016/j.gie.2008.11.046] García-Iglesias P, Sánchez-Delgado J, Montserrat A, Bella-

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P- Reviewer: Assem M, Kodama M, Pandya S, Ozcan C, Yucel O

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© 2014 Baishideng Publishing Group Inc. All rights reserved.

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