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Volume 14 Supplement 1 September 2009 ISSN 1083-4389

Volum

e 14 Supplement 1 Septem

ber 2009 Pages 1–76

EDITOR: David Y. Graham, M.D.

The Year in Helicobacter 2009

Guest Editors: Francis Megrand and Peter Malfertheiner

hel_14_s1_oc.indd 1hel_14_s1_oc.indd 1 8/21/2009 4:27:33 PM8/21/2009 4:27:33 PM

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EditorDavid Y. Graham Houston

Leif P. Andersen Copenhagen

Masahiro Asaka Sapporo

John Atherton Nottingham

Anthony T. R. Axon Leeds

Franco Bazzoli Bologna

Douglas E. Berg St. Louis

Martin J. Blaser New York

Steven J. CzinnCleveland

Emad El-Omar Aberdeen

Peter B. Ernst Charlottesville

David Forman Leeds

James G. Fox Cambridge

Giovanni Gasbarrini Roma

Robert M. Genta Dallas

Javier P. Gisbert Madrid

Khean Lee Goh Kuala Lumpur

Alexander M. Hirschl Vienna

Jaw-Town Lin Taipei

José Carlos Machado Portugal

Varocha Mahachai Bangkok

Peter Malfertheiner Magdeburg

Barry J. MarshallNedlands

Francis Mégraud Bordeaux

Hazel Mitchell Sydney

Colm Antonine O’Morain Dublin

Julie Parsonnet Stanford

Richard M. Peek, Jr Nashville

H. Aziz Rani Jakarta

Pentti Sipponen Espoo

Joseph J. Y. Sung Hong Kong

Shu-Dong Xiao Shanghai

Jianzhong Zhang Beijing

HelicobacterEDITORIAL BOARD

25 AM

VOLUME 14 SUPPLEMENT 1 SEPTEMBER 2009


Guest Editors: Francis Mégraud and Peter Malfertheiner

EUROPEAN HELICOBACTER STUDY GROUP

MEMBERS:Leif Andersen DenmarkAnthony Axonz United KingdomGiovanni Gasbarrini ItalyJavier Gisbert SpainAlexander M. Hirschl AustriaErnst Kuipers the NetherlandsJosé Machado PortugalPeter Malfertheiner GermanyFrancis Mégraud FranceColm O’Morain IrelandAri Ristimaki FinlandTheodore Rokkas GreeceTorkel Wadström Sweden

EMERITUS MEMBERS:Michel Deltenre BelgiumPierre Michetti SwitzerlandJose M. Pajares Garcia SpainAshley Price United Kingdom

Mario Quina PortugalErik Rauws the NetherlandsPentti Sipponen Finland

HONORARY MEMBERS: James Fox USADavid Y. Graham USAAdrian Lee AustraliaBarry Marshall AustraliaGuido Tytgat the Netherlands

CORRESPONDING FELLOWS:Niyaz Ahmed IndiaLuis Vaz Coehlo BrazilToshio Fujioka JapanHyun Chae Jung KoreaVarocha Mahachai ThailandYaron Niv IsraelShu Dong Xiao China

hel_14_s1_title.indd 1hel_14_s1_title.indd 1 8/21/2009 4:29:52 PM8/21/2009 4:29:52 PM

Helicobacter VOLUME 14 SUPPLEMENT 1 SEPTEMBER 2009

CONTENTS

REVIEW ARTICLES 1 The Epidemiology of Helicobacter pylori and Public Health Implications

N. F. Azevedo, J. Huntington and K. J. Goodman

8 Diagnosis of Helicobacter pylori InfectionL. Monteiro, M. Oleastro, P. Lehours and F. Mégraud

15 Pathogenesis of Helicobacter pylori InfectionA. C. Costa, C. Figueiredo and E. Touati

21 Infl ammation, Immunity, and Vaccines for Helicobacter pylori

M. M. D’Elios and L. P. Andersen

29 Helicobacter pylori and Non-malignant DiseasesT. Furuta and J.-C. Delchier

36 Basic Aspects of Gastric CancerM. Correia, J. C. Machado and A. Ristimäki

41 Helicobacter pylori and Clinical Aspects of Gastric CancerJ. Bornschein, T. Rokkas, M. Selgrad and P. Malfertheiner

46 Treatment of Helicobacter pylori InfectionA. O’Connor, J. Gisbert and C. O’Morain

52 Helicobacter pylori Infection in PediatricsA. Kindermann and A. I. Lopes

58 Helicobacters and Extragastric DiseasesR. Pellicano, F. Franceschi, G. Saracco, S. Fagoonee, D. Roccarina and A. Gasbarrini

69 Helicobacter spp. Other Than Helicobacter pylori

A. S. Okoli, A. Menard and G. L. Mendz

hel_14_s1_prelims.indd 1hel_14_s1_prelims.indd 1 8/21/2009 9:40:15 AM8/21/2009 9:40:15 AM

The Epidemiology of Helicobacter pylori and Public HealthImplicationsNuno F. Azevedo,* Janis Huntington� and Karen J. Goodman�

*LEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal, �Division of Gastroenterology, Depart-

ment of Medicine & Department of Public Health Sciences, Zeidler Ledcor Centre, University of Alberta, Edmonton, AB, Canada

This article presents a review of the literature on the

epidemiology and public health implications of Helico-

bacter pylori infection published from April 2008

through March 2009. The authors used MeSH terms

‘‘Helicobacter infections ⁄ epidemiology,’’ ‘‘Helicobacter

infections ⁄ prevention and control’’ to search multiple

databases (PubMed, Embase, Cochrane, Cochrane

Library, EBMR, BIOSIS), and independently searched

PubMed using the term ‘‘Helicobacter’’ with ‘‘Epidemi-

ology,’’ ‘‘Transmission,’’ ‘‘Prevalence,’’ or ‘‘Environ-

ment.’’ Papers without topical relevance were excluded.

Two additional papers known to the authors were

added. The identified literature is summarized below by

subtopic: reviews; prevalence; incidence; transmission;

risk factors; and public health policy.

Reviews

The search identified six review papers. Bruce and Maa-

roos summarized studies on the epidemiology of H.

pylori infection published in peer-reviewed journals [1].

Daugule and Rowland summarized articles on the epi-

demiology of H. pylori infection in children [2]. Tan

et al. examined the changing H. pylori epidemiology in

Asia [3]. All three of these reviews noted that the prev-

alence of H. pylori infection was decreasing globally.

Goodman et al. reviewed studies of H. pylori infection

in Canadian and related Arctic Aboriginal populations,

revealing a relatively high prevalence of the infection

and occurrence of associated disease in these groups

[4]. Zhang et al. summarized 1986–2008 publications

on re-infection, recurrence, or recrudescence of H.

pylori identified in Medline, concluding that re-infection

was not a major concern in clinical settings [5]. A

review presenting Asia-Pacific consensus guidelines on

gastric cancer prevention concluded that H. pylori

screening and treatment strategies aimed at high-risk

populations will probably reduce gastric cancer inci-

dence and were therefore recommended [6].

Prevalence

The search identified 16 population-based prevalence

studies from 12 countries, primarily from Asia and the

Middle East [7–22]. Details from these studies grouped

by H. pylori detection method are presented in Table 1.

Among the noninvasive detection methods that are

practical for population-based studies, the urea breath

test (UBT) and stool antigen tests (SAT) are considered

most accurate, while serology is the least costly and

most widely available. Helicobacter pylori seroprevalence

studies test most commonly for IgG which have the dis-

advantage of not differentiating current from past infec-

tion; whereas H. pylori IgG antibodies often decline to

negative levels once the infection has resolved, the

frequency and timing of this occurrence differs sub-

stantially across populations [23]. Furthermore, sero-

negativity is common in preschool-aged children

Keywords

H. pylori, prevalence, transmission, risk

factors, public health policy

Reprint requests to: Karen Goodman, Division

of Gastroenterology, Zeidler Ledcor Centre,

University of Alberta, Edmonton AB Canada

T6G 2X8. E-mail: [email protected]

Abstract

This article presents a review of the literature on the epidemiology and pub-

lic health implications of Helicobacter pylori infection published from April

2008 through to March 2009. The authors used MeSH terms ‘‘Helicobacter

infections epidemiology,’’ ‘‘Helicobacter infections prevention and control’’

to search multiple databases (PubMed, Embase, Cochrane, Cochrane Library,

EBMR, BIOSIS), and independently searched PubMed using the term ‘‘Heli-

cobacter’’ with ‘‘Epidemiology,’’ ‘‘Transmission,’’ ‘‘Prevalence’’ or ‘‘Environ-

ment.’’ Articles without topical relevance were excluded. Two additional

papers known to the authors were added. The identified literature is sum-

marized by subtopic: reviews; prevalence; incidence; transmission; risk fac-

tors; and public health policy.

Helicobacter ISSN 1523-5378

ª 2009 The Authors

Journal compilation ª 2009 Blackwell Publishing Ltd, Helicobacter 14 (Suppl. 1): 1–7 1

demonstrated by other methods to have H. pylori infec-

tion [24]. One study from India used PCR on saliva and

stool samples [11]; it has been noted that interpretation

of PCR-based results is problematic [23]. One study of

Japanese school children tested twice over a 12-month

interval used a urine test for H. pylori IgG [15]. Urine

tests for H. pylori infection are not widely used and

information about their reliability is limited. Few popu-

lation-based studies have used endoscopic procedures to

evaluate gastrointestinal conditions; a study of adults

Table 1 Population-based studies of Helicobacter pylori prevalence according to detection method, published between April 2008 and March

2009

First author

Detection

method Location ⁄ population Subject selection

Age range

(years)

Number

tested Prevalence

Sasidharan Serum IgG

and IgA

Malaysia, blood donors Not specified 10–70 5370 14.2a

Moujaber Serum IgG Australia, lab patients Age-stratified random

sampling from 8000 banked

diagnostic lab samples

1–59 2413 15.4

Nouraie Serum IgG Iran, households One randomly sampled

individual from each

randomly sampled

household

18–65 851 68.3

Monno Serum IgG Albania, diverse groups Random sampling, healthcare

workers and military

conscripts; consecutive

sampling, pregnant women

16–64 1088 70.7

Kaya Serum IgG Turkey, asymptomatic people Not specified 0.5–17 288 23.9

Siai Serum IgG Tunisia, first grade students Random sampling from

school list of 10,703 first

graders

6–7 1055 51.4

Cheng UBT China, rural and urban Not specified 2–79 1232 46.8

Tam UBT China, school children Random sampling from schools 6–19 2480 13.1

Mohammad UBT Egypt Not specified 6–15 286 72.4

Kori SAT Israel, daycare children Not specified 0.25–5 316 24.7

Cherian SAT Australia, African refugees All presenting for health

assessment during study

period

<16 193 82

Yucel SAT Turkey, university students Random sampling from

unspecified number of

volunteers

Mean = 21b 200 63

Shi Serum IgG

and UBT

China, rural Cluster sampling 5–100 1371 62.1c

Zagari UBT, biopsyd Italy, residents of two

northern villages

Recruited from participants

in earlier population-based

survey

‡32 (mean = 59b) 1033 58

Mishra PCR on saliva

and stool

India, university employee

families and urban slum

dwellers

Not specified 0.67–60 245 45.7 (saliva)

42.8 (stool)

Naito Urinary IgG Japan, Tokyo school children Not specified 4 452 5.3 (time 1)e

6.7 (time 2)

7 4.7 (time 1)

4.0 (time 2)

10 4.0 (time 1)

4.6 (time 2)

aNot specified if prevalence was based on positivity on one or both tests.bRange not specified.cPrevalence based on positivity on one or both tests.dPositivity defined by positive on at least two of UBT, histology, rapid urease test.eTimes 1 and 2 were approximately 12 months apart.

Epidemiology of H. pylori infection Azevedo et al.

ª 2009 The Authors

2 Journal compilation ª 2009 Blackwell Publishing Ltd, Helicobacter 14 (Suppl. 1): 1–7

from two villages in northern Italy classified H. pylori

status using the UBT and evaluation of gastric biopsies

[22].

The reported prevalence ranged from 4% in Japanese

children to 82% in African refugee children in Australia

[8,15]. A prevalence of 15% or lower was reported for

Australian lab patients, Malaysian blood donors, and

Chinese and Japanese school children [14,15,17,20]. A

prevalence of 24–25% was reported for Israeli children

attending daycare centers and unspecified individuals

from Turkey [10,21]. Among the Italian villagers (mean

age = 59 years), the prevalence was 58%, considerably

higher than the 34% observed in an earlier similar

study of adults in northern Swedish communities

(mean age = 52 years) [25]. A prevalence of 60% or

more was reported for groups in Albania, Egypt, Iran,

Turkey, and China [7,9,12,13,16,18].

Incidence

Only three studies examined rates of onset of new

H. pylori infections or reinfections. A Bangladeshi study

examined new infections from birth to 2 years of age in

258 children [26]. They observed that few children

(number not reported but less than 15% by serum IgG,

IgA and ⁄ or SAT) showed evidence of infection at

6 months of age, but by 2 years positivity was 49% by

SAT and around 60% (number not reported) by IgG

and ⁄ or IgA. The Japanese study that used urine tests at

two time points examined rates of acquisition and loss

of infection in 452 children [15]. They reported that

the 12-month incidence decreased with age, 2.6%

among 4-year-olds, 1.3% among 7-year-olds, and

0.65% among 10-year-olds, while rates of apparent

infection loss were 1.3%, 2%, and 0 in the 4-, 7- and

10-year-old groups, respectively. Statistical precision for

the age-specific incidence comparisons was not

reported. An Israeli study examined rates of new infec-

tion in adult dyspeptic patients (n = 39) who had a

negative H. pylori test 7 years earlier and re-infection in

adult patients (n = 26) after successful H. pylori therapy

[27]. One patient in each group had a positive UBT;

however, the small sample makes estimates of inci-

dence rates highly imprecise.

Transmission

The high number of papers published on H. pylori trans-

mission during the last year reveal the information gaps

and inconsistent results that continue to hamper our

understanding of how this organism spreads. Intra-

familial transmission, by direct person-to-person con-

tact, has long been thought to be a major mode of

transmission [28,29]. Weyermann et al. tried to deter-

mine the independent contribution of mothers, fathers,

and siblings to acquisition of H. pylori during childhood

in a German population [30]. Helicobacter pylori status

was based on 13C-UBT and ⁄ or monoclonal SAT per-

formed on stool samples, which lack information on

the genetic similarity of strains from different family

members. Adjusting for the siblings’ and father’s

H. pylori status, the odds ratio (OR) for the effect on the

index child of the mother being infected was 13 (95%

confidence interval (CI), 3–55). The authors extracted

data from similar studies to estimate the odds of infec-

tion adjusted for the infection status of other family

members, concluding that after adjustment for maternal

infection status, the OR among children for having an

infected sibling or father decreased substantially in the

German population and others. It should be noted that

siblings’ infection status appears to matter in popula-

tions where large families are common [31], and that

the mother’s infection status may be more strongly

associated than other family members with household

hygiene and other risk factors.

Using genotyping methods, assessments of the relat-

edness of family members’ H. pylori strains were

performed in Japan, Bangladesh, and Peru [32–34].

Child–mother strain pairs matched for 69, 46, and 30%

of the studied populations, respectively. As with studies

of family members’ general infection status, these

results suggest varying contributions of mother-to-child

(and overall intra-familial) transmission in developing

and developed countries, as a consequence of different

living conditions and age-specific infection patterns. In

a larger study that estimated the similarity of sequences

in populations of South Africa, the United Kingdom,

the United States, Korea, and Colombia, the conclusion

was similar, and variability of strains within families

was higher in rural than in urban areas [35]. As the

authors pointed out, H. pylori research relying on geno-

typing would benefit from more thorough attempts to

identify multiple strains in individuals, which would be

of particular relevance for better understanding modes

of transmission.

Direct person-to-person transmission may occur via

the oral–oral, fecal–oral, or gastro-oral route. Burgers

et al. tested for the concurrent presence of H. pylori in

the mouth and stomach of 94 individuals [36]. Helicob-

acter pylori was detected in the oral cavities of 17% of

gastric biopsy patients, some of whom did not show

evidence of stomach colonization. However, the pres-

ence of organisms in a particular location is not clear

evidence that transmission commonly occurs via the

corresponding pathway. Tonsils have been proposed

as an extra-gastroduodenal reservoir for the bacterium,

Azevedo et al. Epidemiology of H. pylori infection

ª 2009 The Authors


but one study observed that tonsil removal did not

appear to affect the risk of H. pylori transmission [37].

The role of external reservoirs in H. pylori transmission

has not been ruled out, particularly in rural and develop-

ing areas [38]. Water has been one of the most well-stud-

ied ecosystems for H. pylori survival outside the human

digestive tract. A Japanese study compared H. pylori prev-

alence in three populations with different drinking water

sources (two with river water, one with groundwater)

[39]; while the population with the groundwater source

had a much lower prevalence, the small numbers in this

ecologic comparison limit its value. Other studies

attempting to identify H. pylori DNA in water provide

conflicting evidence [40,41]. In Mexico, Mazari-Hiriart

et al. detected the 16S rRNA and cagA genes of H. pylori

in 44% and 14%, respectively, of samples from ground

and surface water [41]. In contrast, Bockelman et al.

were unable to detect the H. pylori 16S rRNA gene in

samples from artificial recharge systems in Spain, Italy,

and Belgium [40]. The inconsistent results may reflect

different water treatment modalities and ⁄ or variations in

PCR procedures (e.g. DNA isolation methods, primer

sequences, and application of nested or quantitative-

PCR). Research in this area would benefit from identifi-

cation of the optimal techniques for reliable assessment

of the presence or absence of H. pylori DNA in suspected

environmental reservoirs. It should be noted, however,

that demonstration of DNA in a potential environmental

reservoir is not clear evidence of the transmissibility of

the organisms, which may or may not be viable. Culture

of H. pylori organisms from these sources would provide

stronger evidence.

From one of the few studies based on culture of

H. pylori outside the human digestive tract, Cellini et al.

characterized one strain found in marine zooplankton

[42]. This environmental strain was able to form bio-

films in a more structured way than clinical strains. Bio-

films are a possible microenvironment where H. pylori

may subsist in water systems, and another study showed

that a cultured strain of clinical origin incorporated in

mature, multispecies biofilms formed in a model reactor

simulating unchlorinated drinking water distribution

systems [43]. Another microenvironment that may pro-

mote H. pylori survival in water is the intracellular habi-

tat of protozoa. However, when studying the spatial

distribution of Helicobacter spp. and Acanthamoeba in river

water samples, Kawaguchi et al. did not detect a clear

association between the two microorganisms [44].

Two other possible extra-human reservoirs assessed

this year were food and the digestive tract of animals

[45,46]. When testing raw milk for the presence of the

H. pylori glmM gene, Quaglia et al. were able to amplify

glmM in 34.7% of the samples using a nested-PCR

approach [46]. Ghil et al. assessed the prevalence of He-

licobacter spp. in feces and saliva from cats in Korea

[45]. Despite detecting the presence of Helicobacter spp.

in 77.6% of the cats using genus-specific primers, all

species-specific PCR for H. pylori were negative. As long

as there is no consensus on the reliability of particular

PCR methods and a valid assessment of the physiologic

status of H. pylori found in these environments, it is not

possible to clarify the role of particular external reser-

voirs in H. pylori transmission.

Risk Factors

Most reports on risk factors focused on socioeconomic

indicators. Most of the studies examined cross-sectional

associations between exposures of interest and being

infected at the time of screening, which cannot differ-

entiate determinants of acquisition from determinants

of persistent infection. Among Israeli children in day

care, low socioeconomic status was associated with

H. pylori infection [10]; this study collected data on

family size, residential crowding, parent’s education,

and country of birth, but the basis for classifying low

status was not specified. Among Egyptian children,

H. pylori prevalence was highest in children attending

school in deprived areas [12]; residents of Cairo had the

highest prevalence among the locations studied. A

study of mainly university employees in India demon-

strated a relationship between living in semi-urban

slums and H. pylori status classified by PCR-based stool

and saliva tests [11]; other factors were not controlled

in this comparison. A Chinese study of 2480 school-

aged children identified an association with lack of for-

mal education of the mother, and an Iranian study of

851 individuals found low education of the mother,

father, and subject to be associated with H. pylori infec-

tion [16,20]. A study of Turkish university students

observed little relationship to H. pylori status of parents’

education level, number of family members, and

income level [21]. The authors noted that selection of

youth who were mainly from state dormitories, which

is determined by parent’s income level, was a limitation

of their study. A Chinese study of 1457 individuals

identified associations with low education, low family

income, and not cleaning a cup after use [18].

A few reports in addition to the Israeli and Turkish

studies examined family size. Two studies of child pop-

ulations observed in multivariable analyses that a

household size greater than five was associated with

H. pylori infection [19,20]. Two studies of adult popula-

tions also observed that household size greater than five

during childhood was associated with H. pylori infection

when other factors were not controlled, but this


ª 2009 The Authors


https://www.researchgate.net/publication/null?el=1_x_8&enrichId=rgreq-1a5b08ae-5365-4116-a5e4-dd5643fe6066&enrichSource=Y292ZXJQYWdlOzI2NzcyMDk5O0FTOjk4NTA1MDU4MTYwNjQ1QDE0MDA0OTY4NDQ3MDE=


relationship did not appear independent of other factors

in multivariable analyses [16,18].

Two studies examined occupational exposures that

increase the risk of infection. In a Belgian study, 587

employees of institutions for children with intellectual

disabilities were compared with 390 employees of com-

panies that do not serve children [47]. Assessed H.

pylori risk factors included parent’s education level,

number of household members during childhood, num-

ber of children sharing a room in childhood, and travel

to tropical regions along with occupational exposures,

such as personal contact, fecal contact, and washing

and feeding of inhabitants. In a multivariable logistic

regression model, only fecal contact was associated with

a clear increase in the odds of H. pylori infection (OR,

4.0; 95% CI 1.7–9.5). A study of Swiss workers used a

prospective cohort design to examine incidence of

H. pylori IgG and IgA seroconversion in relation to sew-

age exposure [48]; 332 workers exposed to sewage and

446 nonexposed workers were tested at baseline and

five time points at approximately 1 year intervals. Using

seroconversion as an endpoint for survival analysis, no

clear effect of exposure to sewage was observed, when

controlling for education level, nationality, country of

childhood, smoking, and alcohol intake.

The Tunisian study of 1055 first grade students identi-

fied bed sharing and bottle weaning after 18 months as

risk factors [19]. The Turkish study observed little rela-

tionship to H. pylori infection of various hygiene practices

or smoking, alcohol, coffee or tea consumption [21]. A

study of 1391 Albanian individuals did not collect data

on number in household, and in a multivariable analysis

only female gender and age greater than 40 were associ-

ated with H. pylori seropositivity [13]. Among African

refugee children in Australia, ethnicity, country of transit

and pre-migration anti-malarial treatment were associ-

ated with H. pylori infection, but in a multivariable logis-

tic regression model, only pre-migration anti-malarial

treatment appeared to retain an independent association,

in the direction of reduced odds of infection [8].

Public Health Policy

Evidence from epidemiologic research provides the basis

for disease control and prevention policy. In particular,

the identification of high-prevalence populations helps

identify target communities for cost-effective interven-

tions, and the identification of modifiable risk factors

yields potentially effective interventions. While the epi-

demiologic research on H. pylori has gone a long way

toward identifying high-prevalence communities

around the world, including some within countries

where average prevalence is low, little work has been

done on interventions aimed at interrupting transmis-

sion (i.e. primary prevention of H. pylori infection), and

the search for this review identified no such reports. A

modest amount of prevention research has focused on

H. pylori infection as a modifiable risk factor for associ-

ated digestive diseases (i.e. tertiary prevention of com-

plications from H. pylori infection), including four

reports published in the last year on screening and

treatment strategies in targeted populations.

A community-based H. pylori screening and treatment

program in Denmark randomized 20,011 40 to 64-year-

old residents of Odense identified by civil registration

number to H. pylori screening and treatment (screened

group) or no intervention [49]. After 5 years, investiga-

tors estimated the effect of the program on rates of dys-

pepsia and peptic ulcers, drug use, doctor visits, and

health related-quality of life. The most noteworthy ben-

efit of the program was a 33% lower incidence of pep-

tic ulcers. A modest reduction in dyspepsia was

observed in the screened group, but this was similar in

magnitude to an unexplained excess prevalence of dys-

pepsia in the screened group at baseline. The authors

reported that the cost of dyspepsia-related health care

was lower in the screened group, but the savings were

exceeded by the cost of screening and treatment. How-

ever, the authors did not estimate the cost-effectiveness

of the program for disease prevention, for example, the

cost per peptic ulcer prevented. While the intervention

was not cost saving, data in the report suggest that the

total expenditure could be considered reasonable for

the number of peptic ulcer cases prevented.

Another study examined the cost-effectiveness of

H. pylori treatment in H. pylori-positive long-term pro-

ton pump inhibitor (PPI) users in the UK randomly

assigned to anti-H. pylori therapy (n = 93) or placebo

(n = 91) [50]. After 2 years, the treatment group had

substantially fewer prescriptions, GP consultations and

GI-related home visits, upper endoscopies, abdo-

men ⁄ pelvis ultrasound scans, and dyspepsia symptoms,

although heartburn symptoms increased. The average

cost savings per patient during 2 years after subtracting

the cost of screening and treatment was £93. The

authors concluded that H. pylori treatment in long-term

PPI users is an economically dominant strategy that

reduces healthcare costs and symptom severity.

Two studies estimated the cost-effectiveness of popu-

lation-based H. pylori screening and treatment for gas-

tric cancer prevention, in a high-risk region of China

and the male Chinese population of Singapore [51,52].

Xie et al. assessed one-time screening and treatment

with either serology or UBT in terms of cost per gastric

cancer case prevented, life-year saved and quality-

adjusted life year gained [52]. Yeh et al. assessed


ª 2009 The Authors




one-time screening with serology and treatment, as

well as strategies of re-screening of seronegatives and

universal treatment without screening in terms of

gastric cancer risk reduction and cost per year of life

saved [51]. Both analyses showed that the evaluated

strategies were reasonably cost-effective compared to

no intervention, and that this conclusion is robust to

reasonable ranges for uncertain input values pertaining

to factors such as screening method accuracy, treatment

success rates, the fraction of gastric cancer prevented by

H. pylori elimination, and the optimal target age group

(e.g. before precancerous lesions typically develop).

Emerging evidence from gastric cancer prevention trials

is revealing more about the latter two factors [6].

These studies strengthen a growing body of evidence

that most H. pylori screening and treatment strategies

considered are cost-effective for prevention of H. pylori-

associated disease in most population studied. They are

less compelling, however, for comparing alternative

strategies, particularly in light of uncertain inputs.

Future research in this area should focus on whether it

is worth the incremental cost to save additional lives

with more costly strategies such as using the UBT

rather than serology for screening, screening more than

once, or targeting younger populations, and should

identify the variables to which such choices are

sensitive.

Conflicts of Interest

The authors have declared no conflicts of interest.

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ª 2009 The Authors


Diagnosis of Helicobacter pylori InfectionLurdes Monteiro,* Monica Oleastro,* Philippe Lehours�� and Francis Megraud��

*Departamento de Doencas Infecciosas, Instituto Nacional Saude Dr Ricardo Jorge, Lisboa, Portugal, �INSERM U853, F 33076 Bordeaux, �Universite

Victor Segalen Bordeaux 2, Laboratoire de Bacteriologie, F33076 Bordeaux, France

A variety of tests for detecting Helicobacter pylori infec-

tion since the discovery of this pathogen have been

described. While there has been no recent break-

through in this topic, a number of original articles com-

ing especially from emerging countries were published

last year on the different molecular and nonmolecular

diagnostic tests for H. pylori.

Non-molecular Methods

Invasive Tests

Graham et al. published a review article providing rec-

ommendations regarding when endoscopic gastric

mucosa assessment must be preferred rather than non-

invasive methods [1].

Endoscopy

To obtain biopsies, an upper digestive endoscopy must

be performed. Cho et al. proposed a new method of

standard endoscopic diagnosis of H. pylori: the phenol

red mucosal pH test. A 0.1% phenol red solution was

sprayed on the gastric mucosa. The extent of staining,

expressed as a staining score, was positively correlated

with the urea breath test (UBT) values and with

H. pylori density as measured by histology. The pH mea-

sured in this study with an antimony electrode was

significantly higher in H. pylori infected mucosa.

Therefore, endoscopic phenol red staining may be an

alternative method for the diagnosis of H. pylori infec-

tion [2].

The new methods of magnifying endoscopy currently

developed have an added value compared to standard

endoscopy, i.e. the possibility of performing in vivo his-

tology. A prospective study on 129 patients performed

in Turkey confirmed that the new method of high reso-

lution magnifying endoscopy is superior to standard

endoscopy for the diagnosis of H. pylori gastritis, and

identification of specific histopathologic features, such

as atrophy and intestinal metaplasia seems possible [3].

In a review of confocal laser endomicroscopy, Kiess-

lich et al. highlighted the possibility of virtual histology

and its role in diagnosing H. pylori gastritis and targeting

biopsy specimens [4].

Interestingly, Kim et al. studied the sites for perform-

ing biopsies to detect H. pylori in 194 patients with gas-

tric cancer. They found that the best site was the upper

body greater curvature (sensitivity of histology: 95.1%;

95% CI: 89.6–98.2), probably due to the proportion of

atrophy and intestinal metaplasia, which were signifi-

cantly lower than in the antrum and in the upper body

lesser curvature which were also tested [5].

Histology

An alternative to Giemsa staining has been proposed in

Thailand [6]. A mixture of carbol fuschin and alcian

blue staining was compared blindly to Giemsa and

hematoxylin & eosin staining on 423 histologic

Keywords

Urease test, histology, culture, stool antigen

test, urea breath test, molecular methods,

antimicrobial susceptibility testing.

Reprint requests to: Francis Megraud, Labora-

toire de Bacteriologie, Universite Victor

Segalen Bordeaux 2, Bat. 2B RDC Zone Nord,

33076 Bordeaux cedex, France.

E-mail: [email protected]

Abstract

The articles published this last year in the field of Helicobacter pylori diagnosis

reported the development of in vivo histology, small improvements in some

invasive methods (urease test, culture, and histology) and new kits for the

stool antigen tests. They also contributed to increasing our knowledge, by

further exploration into specific conditions for the urea breath test and into

the significance of cagA antibodies. The role of serum markers of atrophy

was also confirmed. Molecular methods are still being developed for direct

genotyping, detection of H. pylori and its clarithromycin resistance, either by

polymerase chain reaction or fluorescent in-situ hybridization. For the first

time, there was a report on a possible interest of magnetic resonance

spectroscopy.


ª 2009 The Authors


https://www.researchgate.net/publication/23797496_Socioeconomic_and_Psychosocial_Gradients_in_Cardiovascular_Pathogen_Burden_and_Immune_Response_The_Multi-Ethnic_Study_of_Atherosclerosis?el=1_x_8&enrichId=rgreq-1a5b08ae-5365-4116-a5e4-dd5643fe6066&enrichSource=Y292ZXJQYWdlOzI2NzcyMDk5O0FTOjk4NTA1MDU4MTYwNjQ1QDE0MDA0OTY4NDQ3MDE=

preparations. They found the same rate of positive sam-

ples and highlighted the low cost and simplicity of the

combined staining and its value in identifying goblet

cells in intestinal metaplasia.

The impact of mixed H. pylori infections was studied

in 30 patients by Sheu et al. [7]. They found that the

seven patients with mixed infections had marginally

higher scores of chronic inflammation and H. pylori

density in the corpus and higher rates of intestinal

metaplasia in the antrum (p = .005) compared to the

23 patients with a single strain.

An article referred to the new staging system for atro-

phy (OLGA) and its application in diagnostic practice

[8]. It was also used to assess atrophic gastritis in 63

H. pylori positive patients with various gastric diseases.

They found the OLGA staging system useful for the

assessment of the severity of atrophic gastritis and sim-

ple to use [9]. In another study concerning different

risks of gastric cancer in populations, the OLGA staging

mirrored the gastric cancer incidence [10].

The histogenesis of gastric carcinomas was re-evalu-

ated by Kakinoki et al. [11]. They compared H. pylori-

negative and -positive cases and found that carcinoma

cells could occur independently of the intestinal meta-

plasia. If this finding is confirmed, it would indicate

that intestinal metaplasia is not a precancerous but a

paracancerous lesion.

Urease test

As in previous studies, the sensitivity of the rapid ure-

ase test (RUT) was shown to be reduced in patients

with bleeding ulcers but the short-term use of standard

dose proton-pump inhibitor did not have an impact

[12].

As a solution to the low sensitivity of the RUT, some

authors proposed to increase the number of biopsies

tested up to four. Comparing one biopsy to four, the posi-

tive results increased from 52 to 96%, respectively [13].

Culture

Because of the slow growth and the particular require-

ments of H. pylori with regard to culture conditions, this

area still remains a particular challenge. Sainsus et al.

tried to develop a liquid culture medium for the rapid

isolation, identification, and subsequent antibiotic sus-

ceptibility testing of H. pylori from biopsy specimens.

They selected Ham’s F12 medium with 5% horse serum

with antibiotics which provided the most rapid and reli-

able growth. The CIM medium seems a promising solu-

tion to solve some of the current problems concerning

H. pylori culture in solid media [14].

Non-invasive Tests

Urea breath test13C-UBT has been shown numerous times to be the

most accurate H. pylori diagnostic test. Several remain-

ing questions were addressed this year. Buzas and

Szeles compared UBT values after first-, second- and

third-line treatments. They found as in previous studies

that higher pretreatment UBT values were associated

with lower eradication rates but interestingly, they also

showed a marked tendency to increase UBT values for

the patients who failed, i.e. from 13.2& (CI: 7.3–19.1)

to 19.2& (CI: 13.4–25.0) after second-line therapy and

to 25.8& (CI: 19.8–31.2) after third-line therapy, but

they could not explain this phenomenon [15]. In

another study, UBT values were also found to be higher

when clarithromycin resistant H. pylori were present,

but not when other resistances occurred [16].

The possibility of false positive results due to urease

positive bacteria from the oral cavity in patients with

atrophic gastritis was highlighted by Osaki et al. indicat-

ing that the histologic status of the stomach, i.e. pres-

ence or absence of atrophy, must be considered in

interpreting the results [17]. To avoid false positive

results, the capsule UBT can be used [18].

There is another situation where false positive results

may occur, i.e. in children younger than 6 years of age.

The cause may not only be the effect of endogenous

CO2 production but also other unidentified factors [19].

In contrast to the RUT, the UBT was found to be unreli-

able in patients with Billroth II gastrectomy [20]. Test-

ing for the eradication of H. pylori is an important

aspect of clinical trial design and is of critical impor-

tance in the evaluation of new therapies for this patho-

gen. From the evaluation of Vakil et al. a single UBT,

4 weeks after treatment was as effective as two serial

breath tests in confirming H. pylori eradication and the

incremental cost of the second breath test was very

high with no incremental clinical benefit [21]. A study

performed in Israel to evaluate the indications for UBT

used by primary care doctors and to examine the

appropriateness of these indications to the accepted

guidelines of the Maastricht Consensus Report revealed

a substantial noncompliance with guidelines for

H. pylori testing among primary care doctors in this

country [22].

Stool antigen test

Stool antigen detection kits for the diagnosis of H. pylori

infection have been widely used because of their full

noninvasive nature. Mohammadian et al. presented a

simple, rapid, and cost-effective production of a

Monteiro et al. H. pylori Diagnosis

ª 2009 The Authors




polyclonal antibody against alkyl hydroperoxide reduc-

tase (AhpC) of H. pylori for stool-antigen enzyme

immunoassay [23]. Nguyen et al. evaluated the sensi-

tivity and specificity of the new monoclonal antibody-

based antigen-in-stool enzyme immunoassay (Premier

Platinum HpSA PLUS; Meridian Bioscience, Cincinnati,

OH, USA) for diagnosis of H. pylori infection in Viet-

namese children. The sensitivity was 96.6% (95% CI:

93.3–98.5) and the specificity 94.9% (95% CI: 88.5–

98.3) [24]. In addition, Kuloglu et al. evaluated the

diagnostic accuracy of a rapid immunochromatographic

stool antigen test (Rapid HpSA; L_INEAR Chemical, Bar-

celona, Spain) and a practical low-dose 14C-UBT (Helip-

robe�; Kibion, Uppsala, Sweden) in children before

and after eradication therapy. The sensitivity of Rapid

HpSA and 14C-UBT was 65% and 92.5% (p = .0003),

respectively; the specificity of Rapid HpSA and 14C-UBT

was 92.3 and 85.5% (p = .180), respectively. After

eradication therapy, endoscopy, 14C-UBT and Rapid

HpSA were repeated. Both tests had the same specificity

(100%) while the sensitivity of Rapid HpSA and 14C-

UBT was 60 and 100%, respectively [25]. However, the

most interesting study compared six tests and the

results are reported in Table 1 [26].

Antibody detection

Numerous antibody-based tests have been developed

over the last decades. Leal et al. carried out a systematic

review and meta-analysis to evaluate the performance

of the different antibody-based detection tests available

for H. pylori infection in children by determining sensi-

tivity and specificity as well as additional accuracy val-

ues relevant to clinical practice (positive and negative

likelihood ratios (LR+ and LR)) and the diagnostic odds

ratio (DOR)). The results were as follows: (1) western

blot (WB) tests showed high overall performance, sensi-

tivity: 91.3% (95% CI: 88.9–93.3, specificity: 89%

(95% CI:85.7–91.9), LR+: 8.2 (95% CI: 5.1–13.3), LR-2:

0.06 (95% CI: 0.02–0.16), and DOR: 158.8 (95% CI:

57.8–435.8)); (2) enzyme-linked immunosorbent assay

(ELISA)-IgG assays showed low sensitivity: 79.2%

(95% CI: 77.3–81.0) and high specificity: 92.4% (95%

CI: 91.6–93.3); (3) ELISA commercial tests varied

widely in performance (test for heterogeneity,

p = .0001); and (4) in-house ELISA with whole-cell

antigen tests showed the highest overall performance:

sensitivity: 94% (95% CI: 90.2–96.7), specificity: 96.4%

(95% CI: 94.2–97.9), LR+: 19.9 (95% CI: 7.9–49.8),

LR-2: 0.08 (95% CI: 0.04–0.15), DOR: 292.8 (95% CI:

101.8–841.7) [27].

The Pyloriset EIA-G (Orion Diagnostics, Espoo, Fin-

land), which is considered to be one of the most reliable

tests in Europe, was used to detect H. pylori infection in

two groups of peptic ulcer disease patients, one group

vagotomized and the other medically treated, as well as

community controls. Using positive histology and ⁄ or

culture as the gold standard, the sensitivity of the sero-

logic test was good but its specificity poor in contrast to14C-UBT results. However, no data were available on

possible antibiotic treatments for these patients [28].

Mohammadi et al. evaluated an in-house ELISA based

on soluble antigenic fractions of H. pylori proteins in

Iran. The sensitivity, specificity, and accuracy were over

90% as was the performance of Helico Blot 2.1 (Gene-

labs Diagnostics, Singapore) and two foreign commercial

ELISA kits (Trinity kit and IBL kit) while the BioHit kit

(Helsinki, Finland) did not perform as well [29].

Anti-CagA antibodies (detected either by a specific

ELISA or immunoblot) have been described as long

standing antibodies of interest to confirm H. pylori

etiology years after the disappearance of the bacterium.

Recently, Veijola et al. confirmed that CagA antibodies

detected by immunoblot (Helico Blot 2.1) could still be

detected in 87% of the patients 10 years or more after

a successful H. pylori eradication [30]. Studies were per-

formed in several countries with various results. In

Australia, the use of Helicoblot 2.1 detecting CagA anti-

bodies improved the sensitivity of H. pylori detection in

patients with noncardia gastric cancer from 79% with

H. pylori ELISA to 94%. Interestingly, pepsinogen I lev-

els showed the lowest median level to be in cases

which were negative by ELISA but positive by immu-

noblot [31]. In Mexico, CagA antibodies were associ-

ated with young gastric cancer cases only, and were

also a risk factor for intestinal metaplasia [32]. In Esto-

nia, positivity for CagA antibodies was able to predict

the development of atrophy, particularly in the corpus

(OR: 7.0, 1.8–27.7). In addition, the prevalence of anti-

canalicular antibodies increased with the duration of

the H. pylori gastritis (22–46% in 12 years) [33]. In

contrast, in India, the prevalence of H. pylori detected

by H. pylori serology or CagA antibodies was compara-

ble both in gastric cancer patients and in controls [34].

Table 1 Summary of sensitivity and specificity of the stool antigen

tests cited by Blanco et al. [26]

Sensitivity Specificity

Immunodiagnostic ELISA 87.3 83.3

HpStAR 95 66.6

HpSA-EIA 92.5 72.2

H. pylori Lihtest 83.6 66.6

Immunoland HpSA 52.5 94.4

RAPID HpStAR 78.8 55.5

H. pylori Diagnosis Monteiro et al.

ª 2009 The Authors


Serum biomarkers, especially those proposed by Bio-

Hit in the GastroPanel (pepsinogen I & II, gastrin 17,

H. pylori Ab) have been tested in comparison to histol-

ogy. In the Kalixanda study (Northern Sweden), an

overall agreement of 96% was found. Sensitivity and

specificity of the markers for atrophic gastritis were 71

and 98%, respectively [35]. The GastroPanel was also

able to differentiate Japanese patients into two groups

with ‘‘healthy’’ or ‘‘diseased’’ stomachs with a 94%

accuracy. In total, 5% of the patients had advanced

atrophic corpus gastritis [36].

With regard to cost effectiveness, a Markov model

was designed based on 237,900 Chinese males, aged

35–44 years and living in Singapore, who would be

screened and treated with eradication therapy. Serology

appeared to be twice as cost effective as the UBT in

screening this population [37].

From two studies concerning the value of RAPIRUN

test, a urinary antibody test. Nguyen et al. verified that

in the Vietnamese population sensitivity, specificity,

and accuracy of the test were 79.5, 90.7, and 84.5%,

respectively [38]. These values are in accordance with

the overview presented by Yamamoto et al. concerning

the diagnosis of H. pylori infection using the same

detection kit [39]. The development of a model for eco-

nomic evaluation related to the diagnosis accuracy of

near patient tests used in office laboratories, as opposed

to using hospital-based tests was presented by Fauli

et al. [40].

Molecular Methods

Detection

Over the last year, novel molecular methods based

mainly on a real-time polymerase chain reaction (PCR)

have been described to improve the detection and char-

acterization of H. pylori. A new multiplex fluorescence

resonance energy transfer real-time PCR, for amplifi-

cation of H. pylori ureA and human b-globin, was

developed, allowing quantification of the bacterial den-

sity by determination of the ureA ⁄ b-globin amount

ratio in gastric biopsies. Using this assay, a significantly

increased bacterial density was found in macroscopic

erosions when compared with the healthy portion of

the stomach (p < .01). This PCR was not able to detect

the ureA gene in H. pylori-positive formalin-fixed paraf-

fin-embedded biopsy sections, probably because DNA

was broken and amplification of a 411-bp fragment was

not possible [41]. To overcome the problem of exten-

sive genetic polymorphism for precise PCR detection

of H. pylori, Liu et al. developed a novel molecular

approach, based on real-time reverse transcriptase (RT)-

PCR and in situ hybridization, targeting a 76-bp region

of H. pylori 16S rRNA, that is highly conserved in a

large number of H. pylori strains and is specific to this

bacterium. Both approaches were shown to be very

sensitive and specific and combined together can be

used for specific detection, identification and quantifica-

tion of H. pylori in biological samples, from humans,

animals, or environmental source [42]. In another

study, real-time PCR targeting the 23S rRNA gene

applied on aortic and left internal mammary artery

biopsies, was used to demonstrate, for the first time,

that acute coronary ischemia was significantly more pre-

valent in H. pylori-positive patients than in H. pylori-neg-

ative patients (p = .001), suggesting a pathogenic role of

this bacterium in atherosclerosis [43]. Finally, Gill et al.

developed a nanodiagnostic method using thermophilic

helicase-dependent isothermal amplication of ureC and

gold nanoparticle probes for hybridization and colori-

metric detection of H. pylori DNA. This method allowed

the detection of 10 CFU ⁄ mL within less than 1 hour and

provided a sensitivity of 92.5% and a specificity of

95.4%, with culture as the reference [44].

Sugimoto et al. identified 26 sets of primers used to

detect H. pylori. They first tested their detection limit.

Five of the 26 sets with a detection limit <100 CFU ⁄ mL

were then tested further. All produced some false posi-

tive results. These results indicate that results of H.

pylori detection by PCR outside of the stomach should

be interpreted with caution. Identification based on the

presence of multiple specific genes could be the way

forward [45].

Genotyping

Simultaneous genotyping of bacterial and host cells is

sometimes difficult due to the small amounts of sample.

To overcome this problem, Ryberg et al. used the multi-

ple displacement amplification technique on minute

amounts of gastric biopsy specimens. Then, the ampli-

fied DNA was used for concurrent PCR-based genotyp-

ing, of both H. pylori 16S rRNA, vacA, hsp60, ureI, sod,

ureA and cagA genes, and human cytokine polymor-

phisms [46]. Puz et al. developed a novel noninvasive

genotyping method, using stool specimens, based on

two H. pylori-specific biprobe real-time PCR assays tar-

geting the glmM and recA genes. Discrimination

between strains is made using the differences in the

melting temperature of the amplicons. The sensitivity of

the assay on stool samples was 92.2% and the specific-

ity was 100%. A discriminatory capacity of 100% was

achieved when the sequence analysis of the glmM

amplicon was performed. Due to its noninvasiveness

and high accuracy in detection and discrimination of


ª 2009 The Authors



H. pylori strains, this genotyping assay has the potential

of being used in large-scale studies, contributing to the

clarification of the transmission pathways of this organ-

ism [47]. Paraffin-embedded gastric biopsies were used

for H. pylori genotyping with success (97% of the

cases). The genotyping enabled a comparison of the

prevalence of cagA positive, vacA s1m1 genotypes in

high and low risk areas for gastric cancer in Colombia

(84.3 and 60.5%, respectively) [48].

Kanada et al. evaluated the accuracy of immunohis-

tochemistry for genotyping the cagA East-Asian EPIYA

motif. They used a monoclonal antibody against this

motif and tested it on gastric biopsy samples from Japa-

nese patients. Compared to sequencing of the cagA

3¢-region, containing the EPIYA motifs, the new assay

showed a sensitivity of 93.2% and a specificity of

72.7%, suggesting a further optimization to be useful as

a cagA typing method [49].

Antimicrobial Resistance

With the growing H. pylori resistance to antibiotics,

molecular diagnostic tests for an accurate and rapid

identification of these strains are an attractive alterna-

tive to the time consuming culture-based susceptibility

testing. Thus, this is still an emergent field in H. pylori

research, targeting in particular the clarithromycin

resistance-associated gene mutations. A study employ-

ing Scorpion primers, which combine a primer and a

probe in a single molecule, was reported by Burucoa

et al. to detect H. pylori and clarithromycin resistance

directly on gastric biopsies. The Scorpion PCR was

highly sensitive and specific (98.3% and 92.5%) in

detecting H. pylori, using culture as the gold standard,

and, as usual, it was more sensitive in detecting mixed

populations with susceptible and resistant strains than

the E-test. Clarithromycin genotypes determined with

Scorpion PCR were concordant with those obtained by

PCR-restriction fragment length polymorphism [50].

Fluorescent in-situ hybridization (FISH) can be used to

identify H. pylori and antibiotic resistance in biopsy

specimens without PCR. Tajbakhsh et al. reported a

FISH procedure targeting the H. pylori ribosomal RNA,

and this assay showed a sensitivity and specificity of

97.9 and 100% respectively, for detection of H. pylori,

when compared to histology. However, the FISH assay,

employing four different probes, one wild type and

three mutated, showed a weak sensitivity in detecting

clarithromycin susceptibility-associated genotypes, as

only 19 of the 47 FISH-positive samples were recog-

nized with these probes [51]. Another study employing

the FISH technology, performed using DNA ProbeMix

targeting the 16S rRNA H. pylori gene, showed a

satisfactory sensitivity on both fresh and paraffin-

embedded biopsies isolated from children (84.1 and

80.7%, respectively), previously diagnosed as H. pylori-

positive by histology and RUT. When used to screen

clarithromycin resistance genotypes, using probes tar-

geting the 23S rRNA point mutations, the FISH test was

more sensitive in detecting mixed susceptible and resis-

tant populations than did the agar-dilution method.

The authors emphasize that clarithromycin resistance

should be assessed in biopsies both from the antrum

and the corpus, as in one-third of children with mixed

infection the resistant strains were found in the fundus

only [52]. Woo et al. developed a dual-priming oligo-

nucleotide (DPO)-based multiplex PCR to detect both

H. pylori infection and the most common point muta-

tions occurring in the 23S rRNA conferring resistance

to clarithomycin (A2142G and A2143G), directly on

gastric biopsy specimens. The DPO-based multiplex was

slightly less sensitive in identifying H. pylori-positive

cases than histology, but was able to identify more clar-

ithromycin-resistant strains than the phenotypic meth-

ods. This assay proved to be fast, does not require

expensive instrumentation, and can thus be valuable in

countries with high prevalence of clarithromycin resis-

tance [53]. Kawai et al. used fecal specimens to detect

H. pylori and its resistance to clarithromycin using a

nested PCR based on the 23S rRNA gene and sequenc-

ing of the amplicons, prior to H. pylori treatment. They

obtained a 94.3% eradication rate in the tailored group

and 71.4% in the control group [54].

Finally, Chisholm et al. assessed the potential benefits

of the application to routine testing, of a novel algo-

rithm comprising a panel of three previously described

PCR assays for detection and antibiotic susceptibility

testing of H. pylori. All culture-negative gastric biopsies

were first tested for H. pylori and Helicobacter heilmannii-

like organisms by a multiplex PCR assay targeting vacA

and 16S rRNA genes, respectively. Then, in the positive

cases, antibiotic susceptibility to clarithromycin and tet-

racycline was assessed by real-time PCR probe hybrid-

ization and melting point analysis assays targeting the

23S rRNA and 16S rRNA, respectively. The authors

demonstrated that PCR testing was particularly useful

when H. pylori culture was unsuccessful, due to con-

tamination of the biopsy or when the specimen trans-

portation was delayed. Without this additional testing,

16.9% of all patients examined could have been misdi-

agnosed as H. pylori negative by culture only [55].

Finally, two articles were published using magnetic res-

onance spectroscopy (MRS). The first study concerned

gastric biopsies obtained from patients with various dis-

eases studied ex vivo by high resolution – magic angle


ª 2009 The Authors


https://www.researchgate.net/publication/23976096_Dual-Priming_Oligonucleotide-Based_Multiplex_PCR_for_the_Detection_of_Helicobacter_pylori_and_Determination_of_Clarithromycin_Resistance_with_Gastric_Biopsy_Specimens?el=1_x_8&enrichId=rgreq-1a5b08ae-5365-4116-a5e4-dd5643fe6066&enrichSource=Y292ZXJQYWdlOzI2NzcyMDk5O0FTOjk4NTA1MDU4MTYwNjQ1QDE0MDA0OTY4NDQ3MDE=


spinning – MRS (HR-MAS-MRS) and compared to ul-

trastructural data. Several amino acids, e.g. glycine, ala-

nine, choline, and triglycerides, were identified as

possible markers of differentiation toward neoplastic

lesions. Such a technique could be applied in vivo [56].

In the second study, the metabolic profile of gerbils

infected or not with H. pylori was studied on urine spec-

imens. Results showed that H. pylori infection disturbs

carbohydrate and amino acid metabolism and modifies

the gut microbiota [57]. This method should open a

new field of exploration of H. pylori infection.


To be confirmed.

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https://www.researchgate.net/publication/51422215_Specific_and_Sensitive_Detection_of_H_pylori_in_Biological_Specimens_by_Real-Time_RT-PCR_and_In_Situ_Hybridization?el=1_x_8&enrichId=rgreq-1a5b08ae-5365-4116-a5e4-dd5643fe6066&enrichSource=Y292ZXJQYWdlOzI2NzcyMDk5O0FTOjk4NTA1MDU4MTYwNjQ1QDE0MDA0OTY4NDQ3MDE=















https://www.researchgate.net/publication/23764230_Unreliability_of_Results_of_PCR_Detection_of_Helicobacter_pylori_in_Clinical_or_Environmental_Samples?el=1_x_8&enrichId=rgreq-1a5b08ae-5365-4116-a5e4-dd5643fe6066&enrichSource=Y292ZXJQYWdlOzI2NzcyMDk5O0FTOjk4NTA1MDU4MTYwNjQ1QDE0MDA0OTY4NDQ3MDE=




















































https://www.researchgate.net/publication/24027901_Serum_biomarker_tests_are_useful_in_delineating_between_patients_with_gastric_atrophy_and_normal_healthy_stomach?el=1_x_8&enrichId=rgreq-1a5b08ae-5365-4116-a5e4-dd5643fe6066&enrichSource=Y292ZXJQYWdlOzI2NzcyMDk5O0FTOjk4NTA1MDU4MTYwNjQ1QDE0MDA0OTY4NDQ3MDE=












Pathogenesis of Helicobacter pylori InfectionAna C. Costa,*,� Ceu Figueiredo*,� and Eliette Touati�

*IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal, �Medical Faculty of the University of

Porto, Porto, Portugal, �Unite de Pathogenese de Helicobacter, Institut Pasteur, Paris, France

Helicobacter pylori Virulence Factors

Helicobacter pylori Genetic Diversity and Host

Colonization

Helicobacter pylori is characterized by a high level of

genetic diversity which can be important for the adap-

tation to the host stomach and for the clinical outcome

of the infection. Differences in gene content among H.

pylori isolates in Mexican patients with various gastric

pathologies, including cancer, showed patterns of dis-

ease-associated genes [1].

The plasticity of the H. pylori genome derives from its

natural competence for transformation by exogenous

DNA, from recombination and from mutations. These

properties are at the origin of an extensive allelic diver-

sity occurring even in a single host. Insertion of chro-

mosomal DNA fragments of 1300 bp length into the

recipient chromosome, associated with active genetic

recombination, was demonstrated [2]. During homolo-

gous recombination, Holliday junctions generated by

the RecG and RuvAB helicases are resolved by RuvC.

The RecG homolog of H. pylori is devoid of resolvase

activity and provides an anti-repair pathway [3]. It was

suggested that competition between repair and anti-

repair pathways may provide a mechanism to generate

strain diversity and to maximize fitness at the bacterial

population level [3]. After natural transformation of

H. pylori, the import of short DNA fragments (1294 to

3853 bp) interrupted by interspersed sequences of the

recipient (ISR) (mean length of 82 bp) was shown to

result in the formation of complex mosaic alleles [4].

The control of import length and initiation of the ISR

formation was dependent on the DNA glycosylase

MutY, a component of the base excision repair (BER)

pathway.

In vivo, H. pylori is exposed to acidity and oxidative

stress, causing bacterial DNA damage. AddAB have both

nuclease and helicase activities similar to RecBCD.

AddA (HP1553) confers resistance to oxidative stress-

induced DNA damage. addA mutants display a signifi-

cantly reduced DNA recombination frequency. In mice,

AddAB and RecA are required for stomach colonization

[5]. Their role in bacterial adherence is associated with

gene conversion-like events, a mechanism selected in

the host that abolishes BabA-dependent adherence [6].

These data are in support of DNA repair and recombi-

nation as essential H. pylori mechanisms to optimize

bacterial adherence to mucosal epithelium and persis-

tent colonization.

Outer Membrane Proteins and Adherence

The H. pylori genome contains about 30 hop gene paral-

ogous encoding outer membrane proteins (OMP).

Mutations in hopQ increased adherence of H. pylori to

Keywords

Host cell adherence, colonization, VacA,

CagA, signaling, carcinogenesis, antimicrobial

response.

Reprint requests to: Eliette Touati, Unite de

Pathogenese de Helicobacter, Institut Pasteur,

28 Rue du Doct Roux, 75015 Paris, France.


Abstract

Helicobacter pylori induces chronic inflammation of the gastric mucosa, but

only a proportion of infected individuals develop peptic ulcer disease or gas-

tric carcinoma. Reasons underlying these observations include differences in

bacterial pathogenicity as well as in host susceptibility. Numerous studies

published in the last year provided new insight into H. pylori virulence fac-

tors, their interaction with the host and consequences in pathogenesis.

These include the role of bacterial genetic diversity in host colonization and

persistence, outer membrane proteins and modulation of adhesin expres-

sion, new aspects of VacA functions, and CagA and its phosphorylation-

dependent and -independent cellular effects. This article will also review the

recent novel findings on the interactions of H. pylori with diverse host epi-

thelial signaling pathways and events involved in the initiation of carcino-

genesis, including genetic instability and dysregulation of DNA repair.


ª 2009 The Authors



AGS gastric epithelial cells, CagA translocation into host

cells and cellular alterations, demonstrating the impor-

tance of HopQ for bacterial adherence [7]. The hopQ

type I genotype was associated with higher atrophy

scores than the type II genotype and was proposed as a

marker for gastroduodenal diseases [8].

Expression of adhesins might be modulated by

genetic changes. Phase variation via slipped-strand

mispairing in repetitive nucleotide tracts modulate sabA

expression. Goodwin et al. [9] reported that sabA alleles

of multiple length in the polyT and CT repeat tracts

near the sabAB 5¢ end, are found in 25% of clones of

strain 26695. This was also confirmed among multiple

H. pylori isolates from a single patient [9]. This mecha-

nism occurring during chromosomal replication sug-

gests a selective pressure for SabA expression in the

host, allowing bacteria to adapt but also to escape host

immune response. In the same study, transcription of

sabA was repressed by the acid-responsive ArsRS two-

component signal transduction (TCST) system in vitro

[9]. BabA and SabA bind to the Lewis B (Leb) and to

glycosphingolipids displaying a sialyl-dimeric Lewis X

(sialyl-Lex) respectively. In young mice with early

acquisition of H. pylori infection, a higher sialyl-Lex was

correlated with persistence of stomach colonization

[10]. A cag pathogenicity island (PAI)-dependent over-

expression of a GlcNAc transferase (b3GnT5) was

described in gastric carcinoma cell lines and associated

with high H. pylori adherence [11].

Four binding modes for the H. pylori–mucin interac-

tion are likely to play roles in various niches along the

orogastric infection route and vary according to pH,

gastritis status and bacterial strain [12]. Glycoprotein

receptors within the human salivary proteome for the

carbohydrate-binding H. pylori adhesins have been also

identified [13]. Binding of H. pylori to salivary mucin

MUC7 and agglutinin gp340 depended exclusively on

SabA and BabA, respectively. Binding to MUC5B was

mainly due to BabA. SabA was also found to bind the

secretory component of the polymeric Ig receptor (SC)

and Iga-chain (S-IgA-Hc). These interactions can modu-

late surface or adhesive properties of the bacteria along

the digestive tract. In gastric precancerous lesions and

in gastric carcinoma, the expression of MUC5AC and

MUC6 is altered. Using the Rhesus macaque model,

Cooke et al. investigated the effects on gastric mucins

of experimental challenge with H. pylori during acute

and chronic infection [14]. H. pylori induced gastritis

with an acute and high transient decrease in diversity

and low relative abundance of O-linked mucin oligosac-

charides, suggesting that bacteria modulate gastric

mucin glycoproteins during acute infection to promote

colonization and persistent infection.

Trefoil factors are involved in repair of the gastroin-

testinal mucosa. MUC5AC is coexpressed with trefoil

factor family (TFF)1, a member of small cysteine-rich

proteins. Reeves et al. [15] showed binding of the core-

oligosaccharide portion (rough form) of H. pylori lipo-

polysaccharide (RF-LPS) to TFF1, at an optimum pH of

5–6. An increase in pH would cause inappropriate bind-

ing of bacteria close to the lumen, leading to removal

by mucus turnover. In patients with active chronic gas-

tritis, a reduced expression of TFF2 was observed, espe-

cially in those patients infected with CagA-positive

strains. Authors propose that this reduction in expres-

sion could contribute to the damage induced to the gas-

tric mucosa by H. pylori [16].

The Cytotoxin VacA

VacA is an important virulence factor in the pathogene-

sis of peptic ulceration and gastric cancer. This toxin

can induce multiple cellular activities, including cell

vacuolation, membrane channel formation, disruption

of endosomal ⁄ lysosomal function, apoptosis, and immu-

nomodulation. The mature monomeric form of VacA

has 2 domains - p55 and p33 - important for its cellular

activity. Ivie et al. [17] showed that the N-terminal of

p55 is essential for VacA-induced vacuolation and for

host cell membrane depolarization. This domain is

important for the formation of VacA oligomeric struc-

tures, suggesting its role in the formation of anionic

membrane channels. Further insight into the final steps

of vacuole formation by VacA was provided by Mashi-

ma et al. [18], by demonstrating that the vesicle

associated membrane protein 7 (VAMP7) is a partner of

Q-SNARE syntaxin 7 in the process of lysosome–endo-

some fusion.

At the nucleotide level, the vacA intermediate (i)-

region, which encodes part of the p33 VacA subunit,

displays sequence variation. i1-type strains were associ-

ated with gastric carcinoma in an Iranian population

[19], and were an independent predictor of peptic ulcer

disease in an Italian population [20].

In addition to the previously described protein tyro-

sine phosphatase receptor (RPTP)-a and RPTP-b, Gupta

et al. [21] identified sphingomyelin (SM) as a host cell

receptor for VacA. SM was essential for VacA associa-

tion with the cell membrane and for toxin-induced vac-

uolation. VacA binding to specialized membrane

functional domains may have a biological meaning in

cell signaling.

Tegtmeyer et al. [22] showed that VacA can inhibit

some CagA-induced responses on epithelial cells. VacA

inhibited the activation of epidermal growth factor

receptor (EGFR) and HER2 ⁄ Neu, and subsequently

H. Pylori Pathogenesis Costa et al.

ª 2009 The Authors

16 Journal compilation ª 2009 Blackwell Publishing Ltd, Helicobacter 14 (Suppl.1): 15–20



Erk1 ⁄ 2 MAP kinase, which are important for cell scat-

tering and elongation. These results are also in agree-

ment with previous findings suggesting that VacA and

CagA downregulate each other’s effects on epithelial

cells, potentially allowing H. pylori interaction with cells

whilst avoiding excessive cellular damage [23].

A novel mechanism underlying H. pylori-induced

inhibition of acid secretion by parietal cells was pro-

posed by Wang et al. [24]. They showed that VacA

interaction with parietal cells promotes calpain-medi-

ated proteolysis of ezrin, disrupting the apical mem-

brane-cytoskeletal interactions and inhibiting gastric

acid secretion, mimicking the hypochlorhydric pheno-

type observed in H. pylori-infected patients.

Tuo et al. [25] demonstrated that VacA inhibits pros-

taglandin E2-stimulated duodenal mucosal bicarbonate

secretion by stimulating the release of mucosal hista-

mine. These findings may have pathophysiologic rele-

vance since the inhibitory effect of VacA on

bicarbonate secretion may impair duodenal mucosal

defense against acid injury, contributing to ulcer

development.

The cag Pathogenicity Island and CagA

The cagPAI is a genomic region of 40 Kb containing

about 30 genes encoding a type IV secretion system

(T4SS). To gain more insight into the role of the T4SS

on the outcome of gastric disease, Wiedemann et al.

used the Mongolian gerbil model in a long-term infec-

tion experiment (2–64 weeks) [26]. Authors showed

that the T4SS is essential for the induction of an early

and severe corpus inflammation, associated with

increased expression of proinflammatory cytokines and

histopathologic changes such as atrophic gastritis and

metaplasia. At late time points, only animals infected

with T4SS-competent bacteria developed hypochlorhy-

dria and hypergastrinemia in parallel to gastric ulcers

and focal dysplasia. Although gastric adenocarcinoma

was not detected in any of the infected animals, they

show that the Mongolian gerbil model parallels the

multistep process of gastric carcinogenesis that occurs in

humans.

The cagPAI-encoded factor CagD was investigated and

its crystal structure was determined [27]. In contradic-

tion with previous findings [28], CagD was identified as

an essential component of the T4SS that is required for

CagA translocation into host epithelial cells, although

not absolutely necessary for pilus assembly.

CagA, also encoded by the cagPAI, is translocated into

host epithelial cells by the T4SS. Lai et al. [29] showed

that cholesterol-rich raft microdomains of AGS cells are

crucial for efficient T4SS-mediated CagA translocation

and phosphorylation, as well as for subsequent CagA-

induced actin rearrangements and IL-8 secretion. These

results suggest that H. pylori is able to exploit host cellu-

lar cholesterol in ways additional to those for VacA

intoxication and immune evasion.

CagA EPIYA motifs and CagA Phosphorylation-

Dependent Host Cell Effects

After translocation into the host cells, CagA can be

phosphorylated in tyrosine residues within Glu-Pro-Ile-

Tyr-Ala (EPIYA) motifs at the polymorphic C-terminus

of the protein. Argent et al. showed by using H. pylori

strains displaying cagA microevolution [30,31], that

strains with additional copies of the EPIYA-C motif sig-

nificantly induce more IL-8 secretion in AGS cells after

prolonged infection periods. Sequence data analysis also

revealed that Western strains are more likely to

undergo duplication of the EPIYA-C motif than East

Asian strains undergo duplication of the D motif.

Authors speculate that the highly active East Asian

CagA with one D motif has no requirement to increase

its virulence, whereas the less active Western CagA dis-

plays a dynamic capacity to increase its number of C

motifs to become more virulent.

Basso et al. [20] showed that the magnitude of risk

for gastric carcinoma and precursor lesions increases

with increasing numbers of EPIYA-C motifs. In a multi-

variate model also including vacA genotypes and corpus

H. pylori colonization density, the number of EPIYA-C

motifs was an independent predictor of risk for intesti-

nal metaplasia, reinforcing that the characterization of

the EPIYA region is important in defining disease risk.

During the initial stages of infection, CagA is phos-

phorylated by host Src kinases and later by Abl kinases.

Phosphorylation-dependent effects of CagA include

induction of actin cytoskeleton rearrangements in the

host cell. Phosphorylated CagA has been reported to

interact with the Src homology 2 (SH2) domains of

Shp-2, Csk and Crk (reviewed in [32]). Further insight

into how CagA can induce actin cytoskeleton rear-

rangements may arise from the work of Selbach et al.

[33]. They have identified by quantitative proteomics

PI3K, Shp-1, Ras-GAP1, and Grb7 as additional cellular

interaction partners of CagA. Their results also indicate

that an individual tyrosine phosphorylation site of CagA

can interact with different cellular SH2 domains, sug-

gesting that H. pylori can manipulate multiple signaling

pathways in parallel.

Botham et al. [34] used a transgenic Drosophila model

with inducible CagA expression to show CagA’s capac-

ity to function as a receptor tyrosine kinase adaptor.

They demonstrated that CagA can substitute for Gab

Costa et al. H. Pylori Pathogenesis

ª 2009 The Authors


and restore developmental defects caused by the loss of

the Drosophila Gab, including larval lethality and photo-

receptor differentiation. Authors also provided evidence

that CagA functions similarly to Gab since it required

the Drosophila SHP-2 to exert its effect on photoreceptor

development.

CagA Phosphorylation-independent Host Cell

Effects

Unphosphorylated CagA can also elicit host cell

responses such as disruption of tight and adherens

junctions, loss of cell polarity, proinflammatory and

mitogenic responses (reviewed in [32]). CagA binds to

and inhibits PAR1b ⁄ MARK2 kinase activity, thereby

disrupting junctional and epithelial cell polarity in epi-

thelial cells (reviewed in [35]). CagA-PAR1b interaction

is mediated by the CagA multimerization (CM) motif.

Lu et al. [36] showed that the CM motif of East Asian

strains binds PAR1b more strongly than that of Western

CagA, and in Western strains the ability to bind PAR1b

is proportional to the number of CM motifs. It was fur-

ther demonstrated that the level of CagA-PAR1b bind-

ing influences the magnitude of junctional defects.

Another phosphorylation-independent cellular effect

of CagA is activation of the STAT3 signaling pathway.

Bronte-Tinkew et al. [37] showed in the Hep-2 cell line

that H. pylori triggers tyrosine phosphorylation, nuclear

translocation and STAT3 transcriptional activity in a

CagA-dependent manner. In contrast to other bacterial

pathogens that modulate STAT3 via autocrine activa-

tion by IL-6, H. pylori-mediated STAT3 activation occurs

at the IL-6R level but is independent of the known acti-

vation ligands IL-6, IL-11, and LIF.

Bauer et al. [38] described a new mechanism by

which H. pylori can increase the amount of signaling

molecules on the surface of infected cells. Authors pres-

ent evidence that upon prolonged infection, H. pylori

increases EGFR surface expression by inhibition of

receptor endocytosis and degradation. This occurs in a

CagA-dependent but CagA phosphorylation-indepen-

dent activation of c-Abl, which in turn phosphorylates

a specific EGFR target site.

Helicobacter pylori and Epithelial CellSignaling Pathways

The phosphatidylinositol 3-kinase ⁄ protein kinase B

(PI3K) ⁄ (Akt) signaling pathway regulates diverse bio-

logical processes, including cell proliferation, survival,

and migration. Several studies provided evidence that

H. pylori activates the PI3K-Akt signaling pathway in

epithelial cells [39–44]. In keeping with these

observations, in the Mongolian gerbil model of infec-

tion hyperphosphorylated Akt was predominantly

expressed in the gastric pit cells of H. pylori-infected

animals [44]. However, discrepancies were found

regarding the involvement of bacterial virulence factors

in H. pylori-induced PI3K-Akt signaling. Differences in

cell line models and in strains can strongly contribute

to these discrepancies.

PI3K activity can be induced by receptor tyrosine

kinases. EGFR activation [39–42], but not c-Met [41],

was found to be involved in H. pylori-mediated PI3K-

Akt signaling and was shown to be important in cell

survival [39,42], migration [39], and IL-8 production

[40].

It was also demonstrated in cell line models that gly-

cogen synthase kinase 3b (GSK3), a downstream target

of Akt, is phosphorylated and inactivated by H. pylori

[40,41,43,44]. In unstimulated cells, GSK3b phosphory-

lates b-catenin, targeting it for ubiquitinylation and

degradation. Sokolova et al. [41] showed that by sup-

pressing GSK3b activity, H. pylori leads to inhibition of

b-catenin phosphorylation and ubiquitin-dependent

degradation, and to upregulation of T cell factor ⁄ lym-

phoid enhancer-binding factor (Tcf ⁄ Lef)-dependent

transcription of cyclin D1. GSK3b suppression through

PI3K-Akt activation by H. pylori may also be involved

in NF-jB activation and IL-8 production [40,44].

Early Events in H. pylori-inducedCarcinogenesis

Carcinogenesis results from accumulation of genetic

changes and dysfunction of cellular mechanisms that

normally maintain genome integrity. Machado et al.

[45] showed a dysregulation of DNA repair with

decrease of mismatch repair components during H.

pylori infection, leading to accumulation of genetic

instability in the gastric epithelium. These mechanisms

include induction of a transient mutator phenotype in

the nuclear genome, microsatellite instability (MSI),

and mutations in mitochondrial DNA. In addition, dis-

ruption of the balance between cell proliferation and

apoptosis during H. pylori infection may promote gastric

carcinogenesis. Apurinic ⁄ apyrimidinic endonuclease-1

(APE-1) regulates the transcriptional activity of p53.

Bhattacharyya et al. [46] reported an H. pylori-mediated

acetylation of APE-1 that suppressed Bax expression

and prevented p53-mediated apoptosis, with potential

consequences for gastric carcinoma development. These

studies highlight the role of H. pylori in the induction of

genetic instabilities and impairment of DNA repair sys-

tems important for promoting the gastric carcinogenic

process.


ª 2009 The Authors






Antimicrobial Host Response

The Rhesus macaque model of H. pylori infection dis-

plays lesions similar to those found in humans. In this

model, a cagPAI-dependent increase in mucosal inflam-

mation, with an increased expression of antimicrobial

molecules related to b-defensin-2 (BD2) and several

additional innate host defense proteins that may be

important for disease pathogenesis was reported [47].

Human b-defensin-4 (hBD-4) is frequently expressed in

the gastric mucosa with the highest levels detected in

cagA positive H. pylori gastritis [48]. Its induction is

dependent on the activation of the p38 MAP kinase

pathway. These studies demonstrated that H. pylori

induces a differential expression of antimicrobial pep-

tides, which are essential effectors of the innate

immune response with functional relevance in host

defense. These antimicrobial proteins may be less active

against H. pylori than against other microorganisms,

resulting in a modification of the gastric microbiota

composition during host infection [47]. Dicksved et al.

characterized the gastric microbiota of patients with

gastric carcinoma, and demonstrated that it was domi-

nated by different microbial species with a relative low

abundance of H. pylori [49]. Pathogenesis of H. pylori

infection can be also modulated by lower bowel Heli-

cobacters, as suggested by mice experiments showing

an attenuation of the H. pylori-induced gastric proin-

flammatory lesions in a coinfection with Helicobacter

bilis [50].



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Inflammation, Immunity, and Vaccines for Helicobacter pyloriMario M. D’Elios*,� and Leif P. Andersen�

*Department of Internal Medicine, University of Florence, Florence, Italy, �Department of Biomedicine, Azienda Ospedaliera Universitaria Careggi,

Florence, Italy, �Department of Infection Control 9101, Copenhagen University Hospital, Righospitalet, Copenhagen, Denmark

Helicobacter pylori infects the stomach of more than 50%

of the human population and represents the major

cause of gastroduodenal pathologies, such as duodenal

and gastric ulcer, gastric adenocarcinoma, B-cell lym-

phoma of mucosa-associated lymphoid tissue (MALT),

and autoimmune gastritis. As in any infectious disease,

the type of innate and specific immunity elicited is of

crucial importance for protection, although an inappro-

priate response may contribute to the induction of

immunopathology. This article will focus on the major

findings on the host response and vaccines against H.

pylori published over the past year.

Natural Immunity

Helicobacter pylori activates a wide spectrum of innate

events resulting in a strong T helper (Th) 1 response.

Mucosal natural defense against H. pylori infection

depends on activation of both Toll-like receptors (TLR)

and Nod-like receptors (NLR), which lead to the gener-

ation of Th1 response specific for H. pylori. Different

components of H. pylori are able to activate innate

immune cells. The neutrophil activating protein of

H. pylori (HP-NAP) is a key factor in H. pylori TLR acti-

vation. HP-NAP induces interleukin (IL)-12 and IL-23

secretion in monocytes, dendritic cells, and neutrophils

via activation of TLR2 [1]. Helicobacter pylori factors

other than HP-NAP, such as the vacuolating cytotoxin

A (VacA), the cag pathogenicity island (PAI), and the

heat shock protein (HSP) 90 may contribute to the

expression of IL-12p40 and to Th1 polarized response

[2]. In particular H. pylori peptidoglycan, acting in con-

cert with the bacterial type IV ‘‘syringe’’, encoded by

the cag PAI, plays an important role in activation of the

cytoplasmic nucleotide-binding oligomerization domain

(NOD) 1 in gastric epithelial cells and primes Th1

responses [3,4]. Another source of IL-12 might be natu-

ral killer cells, which can be elicited by H. pylori in

infected patients [5]. Following H. pylori infection,

gastric epithelial cells and monocytes produce other

Th1-inducing cytokines such as IL-18. IL-18 levels in

infected gastric mucosa correlated with the severity of

gastric inflammation both in adults and children [6,7].

Different bacterial virulence factors, such as cag PAI and

outer inflammatory protein (Oip) A, contribute to the

induction of IL-18 production in gastric epithelial cells.

In both gastric epithelial cells and monocytes ⁄ macro-

phages, H. pylori regulates the extracellular signal-

regulated kinase ⁄ c-Jun-N-terminal kinase (JNK)-AP-1

pathway. Upregulation of IL-18 mRNA in monocytes is

independent of cag PAI and OipA, whereas OipA and its

related p38 pathway regulate IL-18 protein induction at

Keywords

Mucosal immunity, Th1, Th2, Th17, cytokine,

regulatory lymphocyte, chemokine, VacA,

CagA, HP-NAP, vaccine.

Reprint requests to: Mario M. D’Elios,

Department of Internal Medicine, Viale

Morgagni 85, 50134 Florence, Italy.


Abstract

Helicobacter pylori infects almost half of the population worldwide and repre-

sents the major cause of gastroduodenal diseases, such as duodenal and gas-

tric ulcer, gastric adenocarcinoma, autoimmune gastritis, and B-cell

lymphoma of mucosa-associated lymphoid tissue. Helicobacter pylori induces

the activation of a complex and fascinating cytokine and chemokine net-

work in the gastric mucosa. Different bacterial and environmental factors,

other concomitant infections, and host genetics may influence the balance

between mucosal tolerance and inflammation in the course of H. pylori

infection. An inverse association between H. pylori prevalence and the fre-

quencies of asthma and allergies was demonstrated, and the neutrophil acti-

vating protein of H. pylori was shown to inhibit the allergic inflammation of

bronchial asthma. During the last year, significant progress was made on the

road to the first efficient vaccine for H. pylori that will represent a novel and

very important bullet against both infection and gastric cancer.


ª 2009 The Authors



the post-transcriptional level in a cag-independent way,

and contribute to gastric injury [6]. Helicobacter pylori-

infected patients with IL-18-607C ⁄ C and -137G ⁄ Ggenotypes have been shown to have higher IL-18 levels

and severe gastric inflammation [8]. In a very elegant

study in C57BL ⁄ 6 mice, Kaparakis et al. demonstrated

that transient elimination of macrophages during the

early period of H. pylori SS1 infection reduced the gas-

tric pathology, suggesting that macrophages contribute

to the severity of gastric inflammation [9].

Neutrophil activation and mucosal IL-8 expression

are associated with persistent infection. A new trigger-

ing receptor expressed on myeloid cells-1 (TREM-1) has

been described on gastric epithelium and experimental

data suggest that TREM-1 expression on gastric epithe-

lial cells amplifies inflammation of the underlying gas-

tric mucosa by upregulation of IL-8 [10]. A novel

putative H. pylori outer membrane protein (HomB)

associated with peptic ulcer disease has been exten-

sively investigated. HomB induces the secretion of IL-8

by gastric epithelial cells, and H. pylori homB knockout

mutant strains present reduced ability to bind to gastric

epithelial cells and to induce IL-8 secretion, implying

that HomB represents a novel virulence factor of H.

pylori actively involved in H. pylori-induced inflamma-

tion [11]. Individuals with lower neutrophil oxidative

burst activity might be more prone to H. pylori infec-

tion, due to reduced efficiency of neutrophil immune

functions [12]. Moreover, in a different experimental

setting, HP-NAP confirmed its ability to induce myelop-

eroxidase release from human neutrophils [13]. Fur-

thermore, it has been shown that Helicobacter pullorum,

an entero-hepatic Helicobacter species of avian origin

detected in patients with acute diarrhea and inflamma-

tory bowel disease, exerts a direct effect on human gas-

tric (AGS) and intestinal (CaCo-2 and HT-29) cell lines,

by inducing IL-8 production. The H. pullorum-induced

IL-8 secretion requires bacterial adherence and lipo-

polysaccharides (LPS) and is mediated by nuclear factor

(NF)-jB signaling, suggesting that H. pullorum might

play a putative role in acute and chronic digestive dis-

eases such as inflammatory bowel disease [14].

Helicobacter pylori LPS, another important bacterial

factor that modulates the innate immune response, has

been extensively studied for its pro-inflammatory activ-

ity. LPS has evolved differently in H. pylori communities

through genetic modifications in fucosyltransferases

that are involved in Lewis (Le) antigen expression.

Some of the LPS variants facilitate adaptation and sur-

vival in the individual gastric mucosa [15]. Further-

more, Le antigen expression and fucosylation can have

multiple biological effects, by affecting the development

of innate and acquired responses that develop after

infection, implying that the fucosylated secretor ABH

antigens constitute a family of interactive members of

the mucosal human innate system that tightly regulates

host–bacterial interactions [16]. The host iron status

may affect the nature of LPS expressed by H. pylori, by

modifications in outer membrane vesicles [17]. Evi-

dence suggests that long-term H. pylori infection can

induce antibodies that cross-react with the gastric

mucosa and that, in concert with H+K+-ATPase-specific

autoreactive and cross-reactive T cells, contribute to the

development of gastric autoimmunity and to mucosal

atrophy [18,19]. The LPS may decrease H. pylori elimi-

nation from the gastric mucosa and promote infection

persistence, by exerting an anti-phagocytic activity that

is reduced by LPS-binding protein [20]. Helicobacter

pylori proteins, such as VacA, may exert either inhibi-

tion or activation on different cell types. VacA exerts

immune suppression by inhibition of antigen processing

and presentation of antigen-presenting cells and by dis-

ruption of actin rearrangement and inhibition of cal-

cium mobilization of T cells. VacA has different

receptors on different cell types, such as CD18 on T

cells [21], or sphyngomyelin on epithelial cells [22].

VacA can inhibit duodenal bicarbonate secretion via

prostaglandin E(2) inhibition by a histamine-dependent

mechanism in mice [23]. On the other side VacA

induces IL-8 production in U937 cells via activation of

p38 mitogen-activated protein kinase and intracellular

Ca2+ release, leading to the activation of the transcrip-

tion factors ATF-2, CREB, and NF-jB [24].

The mechanisms of inflammation induced by CagA

have been further elucidated. Suzuki et al. demon-

strated that nonphosphorylated CagA is able to promote

inflammation by sequential activation of PI3kinase ⁄ Akt

signaling that, in turn, leads to b-catenin and NF-jB

activation [25]. The CagA protein is also able to func-

tion both as a Grb2-associated binder protein (Gab)

adaptor and to activate the Src-homology 2 domain

containing tyrosine phosphatase (SHP-2) in a transgenic

Drosophila model [26]. Helicobacter pylori induces gastric

inflammation via a Cag-dependent mechanism both in

Rhesus macaques and in gerbils [27,28]. On the other

hand, Cag-independent signaling might lead to b1-inte-

grin activation with JNK activation, which also pro-

motes cell motility of gastric cancer cells [29].

Basu et al. demonstrated that the secreted peptidyl

prolyl cis-, trans-isomerase HP0175 protein was able to

bind to AGS cells by TLR4 and to transactivate EGFR

and vascular endothelial growth factor production [30].

Using the A ⁄ JCr mouse model, Helicobacter hepaticus

urease was shown to contribute to hepatic inflamma-

tion although it was not required for intestinal

colonization [31].

Immunity and Vaccines for H. pylori D’Elios and Andersen

ª 2009 The Authors




Adaptive Immunity, Helicobacter, andCytokine Network

T-helper cells orchestrate host defense against patho-

gens via different types of cytokine secretion and effec-

tor functions; however, an inappropriate response

might lead to immunopathology. Current evidence sug-

gests that in H. pylori infection, a predominant activa-

tion of Th1 cells with production of interferon (IFN)-c,

IL-12, IL-18, and tumor necrosis factor (TNF)-a occurs

in vivo in the gastric mucosa and contributes to tissue

damage [32,33]. Accordingly, Helicobacter infection of

lymphocyte-deficient mice fails to induce gastric inflam-

mation. T-cell transfer into deficient animals then

results in severe gastritis, suggesting that host T-cell

responses to H. pylori play a key role in host damage

[34,35].

The fine balance between protection and pathology

in H. pylori infection is related not only to the major

Th1 cytokines, such as IFN-c, TNF-a, IL-12 [32], but

also to IL-23 (a powerful Th1 and IL-17-promoting fac-

tor), IL-17 and IL-21 [1,36,37]. IL-17A mRNA and pro-

tein are associated with H. pylori lesional sites from

human gastric biopsies [37,38]. When biopsies were

cultured in vitro and IL-17 activity was blocked, there

was a reduction in IL-8 gene expression implying that

IL-17 might be a relevant factor driving IL-8 production

and neutrophilic inflammation. In both humans and

mice, the upregulation of IL-23 was found in gastric

mucosa following H. pylori infection and was associated

not only with IL-17 but also with the upregulation of

many IFN signature transcripts [39]. IL-23 is present at

high levels in the lesional tissue compared with the sur-

rounding tissue. Blocking IL-23 activity resulted in a

reduction of STAT3 and IL-17 expression [40]. Experi-

mental evidence obtained in a mouse model of H. hepa-

ticus infection suggests that the NF-jB subunit c-Rel

modulates the expression of IL-23 ⁄ IL-12 subunits and

plays an important role in the development of innate

and T-cell mediated inflammation [41].

The type of mucosal lymphocyte responses going on

in the duodenum and in the stomach of H. pylori-

infected patients were investigated in a highly endemic

area of H. pylori infection, i.e. Bangladesh, and com-

pared to Sweden. Comparable amounts of T and B cells

were found in the stomach of Bangladeshi and Swedish

infected patients, but there was a lower systemic anti-

body response in Bangladeshi patients. However,

increased numbers of B cells and H. pylori-specific IgA

antibodies were detected in the duodenum of Banglade-

shi patients, suggesting a more intense inflammation

going on in these patients frequently exposed to enteric

infections [42]. In Swedish infected patients, increased

gastric levels of the CCL28 chemokine and CCL28 med-

iated recruitment of gastric IgA-secreting cells were

found, providing an explanation for the large influx of

IgA-secreting cells to the gastric mucosa in H. pylori-

infected individuals [43]. In a long-term follow-up of

north-eastern European H. pylori-infected patients,

Vorobjova et al. reported that different serological pat-

terns were associated with histological manifestations of

gastritis in the progression towards atrophic gastritis, in

a long-term follow-up. Anti-CagA antibodies were a

sign of gastritis activity and corpus atrophy, the preva-

lence of anti-canalicular antibodies significantly

increased and paralleled the duration of H. pylori gastri-

tis, whereas anti-HSP60 antibody levels indicated

chronic inflammation of the antrum [44].

Interactions Between H. pylori andAsthma or Other Infections

Humans are colonized by a multitude of both beneficial

and pathogenic microbial organisms, including H. pylori.

Imbalances in the composition of bacterial microbiota

are postulated to be a major factor in many human dis-

orders [45]. Human and microbial cells continuously

‘‘cross-talk’’ to each other and influence their respective

lives. Over the last century the incidence and severity

of bronchial asthma have drastically increased in devel-

oped countries and it has been proposed that infectious

agents can influence the development of allergic disor-

ders, although the underlying reason has not been fully

elucidated. Bronchial asthma and allergic diseases are

sustained by Th2 inflammation and IL-4 production,

which is strongly inhibited by IL-12 and IFN-c, whereas

H. pylori infection elicits a powerful Th1 response [46].

Interestingly, large epidemiological studies recently

demonstrated a consistent negative association between

H. pylori infection and the presence of allergic disorders,

such as asthma and rhinitis, both in childhood and the

adult urban population [47–50]. In allergic asthmatic

patients, the typical Th2 response can be redirected in

vitro toward Th1 by HP-NAP [1]. To address whether

HP-NAP, a TLR2-ligand, could be beneficial in vivo for

the prevention and treatment of bronchial asthma, it

was administered via the intraperitoneal (systemic) or

the intranasal (mucosal) route using a mouse model of

allergic asthma. The in vivo (both mucosal and sys-

temic) administration of HP-NAP prevents the classic

allergic Th2 bronchial inflammation, by a strong inhibi-

tion of IL-4, IL-5 and via the increase of IL-12 produc-

tion. However, no suppression of bronchial Th2

cytokines was observed in TLR2 knockout mice follow-

ing HP-NAP treatment [46,51]. Altogether these results

provide evidence that HP-NAP might be an important

D’Elios and Andersen Immunity and Vaccines for H. pylori

ª 2009 The Authors




part of the molecular and cellular mechanisms underly-

ing the negative association between H. pylori infection

and allergy.

In mouse Trichinella spiralis infection, another model

of Th2-mediated disease, HP-NAP was also able to

enhance an in vivo Th1 response and to exert a power-

ful anti-Th2 activity, targeting both the IL-5-induced

eosinophilia and the IL-4-mediated hyper-IgE responses

induced by parasitic infection [52]. Different microbial

factors and other concomitant infections may influence

the outcome of H. pylori infection. In a C57BL ⁄ 6 mouse

model of H. pylori infection, treatment with Lactobacillus

casei and Bifidobacterium lactis resulted in a suppressive

effect on Helicobacter-induced inflammation [53].

Accordingly, Lactobacillus plantarum, Lactobacillus

rhamnosus, Lactobacillus lactis and bovine colostral prepa-

ration were able to reduce adherence and IL-8 produc-

tion following H. pylori infection of AGS cells [54].

Regulatory Cells

CD25+ ⁄ Foxp3+ regulatory T cells (Treg) are a subset of

T cells that are physiologically devoted to the mainte-

nance of self tolerance. Treg play a crucial role in regu-

lating the effector immune responses in the different

districts of the organism by suppressing the activation

and proliferation of antigen-specific T cells, and an

abnormal Treg activation might lead to impaired tumor

immunity. Treg are able to suppress T-cell responses,

via both cell contact and by soluble factors, such as TGF

(transforming growth factor)-b and IL-10.

Helicobacter pylori-induced gastritis is associated with a

recruitment of Treg that correlates with the degree of

bacterial colonization and mucosal TGF-b expression

[55]. In a murine model CD4+ CD25+ Treg cells were

able to induce anergy of CD25) T cells in response to H.

pylori infection but were not required to maintain hyp-

oresponsiveness [56].

Robinson et al. investigated the gastric Treg response

of infected patients and demonstrated that in subjects

with peptic ulcer disease, higher levels of IL-10-secret-

ing Treg were present in the gastric mucosa, compared

with those without ulcers. IL-10 inhibited IL-8 expres-

sion and activation of NF-jB induced by H. pylori in

gastric epithelial cells, and enhanced H. pylori growth in

a bacterial-cell co-culture model [57]. Accordingly, a

significant increase of IL-10 serum levels was found in

a subset of Turkish infected patients [58].

Furthermore, H. hepaticus infection of mice

co-infected with diarrheagenic Escherichia coli resulted

in an accumulation of Treg cells at mucosal level [59]

resulting in exacerbated morbidity, with delayed recov-

ery from weight loss and tissue damage. Thus, it can be

speculated that Treg cells play a role in the lifelong per-

sistence of H. pylori infection and that an inadequate

regulatory response may contribute to the immunopa-

thology of H. pylori infection.

Gastric Cancer, Inflammation and GastricMALT Lymphoma

Low grade gastric MALT lymphoma represents the first

described neoplasia susceptible to regression following

antibiotic therapy eradicating H. pylori [60]. The neo-

plastic B-cell proliferation depends both on H. pylori

stimulation and exhaustive T-cell helper activity in con-

junction with defective T-cell killing [61,62]. Ferrand

et al. showed that H. pylori strains can inhibit T-cell

proliferation, favoring chronic persistence of the infec-

tion and anarchical B-cell proliferation predisposing the

host to gastric MALT lymphoma [63]. Different factors

may affect the onset and progression of gastric MALT

lymphoma. The CXCR3 chemokine receptor is highly

expressed on both activated T and B cells of gastric

MALT lymphoma. Patients with CXCR3 expression

showed a significantly increased risk of nonresponsive-

ness to H. pylori eradication therapy, regardless of sex,

API2-MALT1 fusion or clinical stage [64]. The overex-

pression of B-cell-activating factor of the TNF family

(BAFF) was associated with H. pylori-independent

growth of gastric diffuse large B-cell lymphoma,

implying that the BAFF autocrine signal transduction

pathway may contribute to H. pylori independent

growth of gastric MALT lymphoma [65]. Furthermore,

the majority of MALT lymphomas were found to

express class-switched immunoglobulins and to develop

in an environment rich in Th2 cytokines [66].

Helicobacter pylori is a very important oncogenic factor

for gastric adenocarcinoma and many studies have

highlighted the role of chronic inflammation in the

development of gastric cancer [67]. Helicobacter pylori

may lead to gastric cancer via both a direct effect on

epithelial cells and by the induction of different cyto-

kines, especially IL-1b and IL-1 receptor antagonist

(IL-1RN). Overexpression of even a single proinflam-

matory cytokine is sufficient to induce neoplasia by

eliciting inflammation. The European EPIC-EUROGAST

prospective study confirmed the association of IL-1RN

polymorphisms with the risk of noncardia gastric cancer

and indicated that IL-8-251T>A may modify the risk for

gastric cancer [68]. These results were confirmed by

many reports all over the world. A Turkish study

reported that the bacterial risk factor babA2 seemed to

be an important predictor of gastric malignancies, and

that the presence of the IL-1b-31TT genotype repre-

sented an important protective factor [69]. The


ª 2009 The Authors



https://www.researchgate.net/publication/23976098_The_Expression_of_Iron-repressible_Outer_Membrane_Proteins_in_Helicobacter_pylori_and_Its_Association_with_Iron_Deficiency_Anemia?el=1_x_8&enrichId=rgreq-1a5b08ae-5365-4116-a5e4-dd5643fe6066&enrichSource=Y292ZXJQYWdlOzI2NzcyMDk5O0FTOjk4NTA1MDU4MTYwNjQ1QDE0MDA0OTY4NDQ3MDE=

upregulation of IL-1b, IL-8, and cyclooxygenase (COX)-

2 linked to gastric carcinogenesis was found in Brazilian

patients [70]. In a Costa Rica dyspeptic population

CagA status was found to be a risk factor for atrophic

antral but not body gastritis whereas the pro-inflamma-

tory cytokine polymorphisms IL-1b +3945 and IL-1RB

were not associated with the atrophic lesions of dyspep-

tic populations [71]. In a Korean survey study, the

genetic polymorphisms of IL-8, IL-6, and IL-10 were

associated with the development of H. pylori induced

gastroduodenal diseases [72]. TNF-a inducing protein

has been shown to be a novel H. pylori factor able to

induce TNF-a secretion, to enter the nucleus of gastric

epithelial cells and to induce carcinogenesis in a cag-

independent way [73]. During H. pylori exposure, the

production of MIF and IL-8 by gastric epithelial cells

leads to the expression and activation of epidermal

growth factor receptor (EGFR) in a cag PAI – indepen-

dent way [74]. The p53 protein family, including p73

protein, by acting in concert play an important role in

the epithelial and inflammatory response to H. pylori

related to gastric malignancy via induction of apoptosis

and promoting alterations of cell differentiation [75].

Experimental evidence obtained by studying the eosin-

ophil infiltration in gastric infected tissues of patients

originated from areas with different rates of infection

suggested that eosinophils and mast cells might have a

dual role in H. pylori infection: they can downmodulate

gastric inflammation and cancer development in low

risk areas whereas they might promote inflammation

and progression to malignancy of precancerous lesions

in high risk areas [76].

Vaccines

Despite almost 20 years of efforts, no efficacious vaccine

against H. pylori is currently available for humans.

Immunization with different vaccine formulations,

based on the use of selected antigens known to be

involved in the pathogenesis of infection, such as those

containing VacA, CagA, and HP-NAP or urease, have

been shown to prevent experimental infections in ani-

mals [77]. BabA and SabA adhesins have also been pro-

posed for an anti-H. pylori vaccine [78]. Although the

efficacy of a vaccine against gastric H. pylori infection

has been shown, little is known about the mechanisms

of bacterial clearance. Not only a specific antibody

response but a concerted action of cellular, molecular,

and humoral responses are needed to give full protec-

tion against H. pylori. A vaccine based on H. pylori Syd-

ney strain 1 lysate and cholera toxin (CT) adjuvant has

been used to intranasally immunize mice and the pro-

tection achieved was high. The immunization resulted

in a strong IFN-c producing T-cell response associated

with an increase in chemokines, such as MIP-2, KC and

LIX, which attract neutrophils to the stomach and are

important for H. pylori eradication [79]. An interesting

study pointed out that dendritic cells play a critical role

in supporting the effector cellular response needed for

the development of a successful H. pylori vaccine [80].

Using a Th1 vaccine consisting of an H. pylori sonicate

plus CpG oligonucleotides and a Th2 vaccine consisting

of an LPS-depleted sonicate combined with CT, Taylor

et al. demonstrated in a mouse model that, although

the CpG sonicate vaccine induced stronger systemic and

local immune responses, only the LPS-depleted sonicate

CT toxin-conjugated vaccine resulted in effective pro-

tection [81]. A further mechanism of vaccine-induced

protection is the effector response elicited by the leptin

receptor signaling, that has been elegantly proposed by

Wehzens et al. [82].

In two prospective, randomized, double-blind con-

trolled studies, Salmonella enterica serovar Typhi Ty21a-

based, oral live vaccines containing H. pylori urease or

HP0231 protein were tested in H. pylori-negative volun-

teers. Both these vaccine preparations were well toler-

ated but did not confer satisfactory protection [83].

Over the last year, a very promising study entered

the scene and will presumably lead to the launch of the

first vaccine against H. pylori in humans. Malfertheiner

et al. investigated the safety and immunogenicity of a

vaccine consisting of recombinant VacA, CagA, and

HP-NAP given intramuscularly with an aluminium

hydroxide adjuvant to H. pylori-negative healthy sub-

jects. This very important randomized single-blind

Phase I study involved 57 H. pylori-negative volunteers

and explored three different schedules (0, 1, 2 weeks;

0, 1, 2 months; and 0, 1, 4 months) and two dosages of

each antigen (10 and 25 lg) versus alum controls. All

of the subjects were followed for 5 months and 36 of

them received a booster vaccination 18–24 months after

the end of the first set of vaccination. In both vaccine

and placebo recipients, only very mild adverse reactions

were present on monthly schedule. All of the vaccinees

mounted specific IgG and cellular responses to one or

two antigens and 86% of vaccinated subjects responded

to all three antigens. Both antibody and cellular mem-

ory responses could be elicited by vaccination between

18 and 24 months later. The safety and the immunoge-

nicity results obtained in this study suggested that the

intramuscular vaccine formulation consisting of VacA,

CagA and HP-NAP plus aluminium hydroxide adjuvant

represents a very promising candidate vaccine for the

prevention of H. pylori infection [84].

Collectively these findings suggest that achieving a

successful vaccine against H. pylori will have a great


ª 2009 The Authors


impact on global health because it will be beneficial not

only for preventing H. pylori infection (a class I oncogenic

factor) but also for the prevention of gastric cancer.


M.M.D.E. is inventor and applicant of patent

EU05425666.4, WO2007039451 for potential use of

HP-NAP as therapy of cancer, allergic, and infectious

diseases.

Acknowledgements

We thank Copenhagen University Hospital, Ente Cassa

di Risparmio di Firenze, and the Italian Ministry of Uni-

versity and Research, for their support of our studies.

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Helicobacter pylori and Non-malignant DiseasesTakahisa Furuta* and Jean-Charles Delchier�

*Center for Clinical Research, Hamamatsu University School of Medicine, Hamamatsu, Japan, �Department of Gastroenterology, Creteil University

Hospital Henri Mondor, Creteil, France

Helicobacter pylori-positive peptic ulcer (PU) is one of the

most important indications for H. pylori eradication.

However, it has been made clear that H. pylori infection

is also associated with nonmalignant disorders other

than peptic ulcer diseases (PUD) and that eradication of

H. pylori is sometimes effective for the treatment of

these disorders. There are inter-individual differences in

response to H. pylori infection. One of the reasons for

the inter-individual differences is genetics. Environ-

mental factors are also associated with such differences.

In 2008, several new polymorphisms associated with

H. pylori-related disorders were reported. However, the

main factors associated with the clinical outcome of

H. pylori infection in each individual have not been

fully elucidated. In this review, some interesting articles

on the association of H. pylori infection with nonmalig-

nant disorders published between April 2008 and

March 2009 will be discussed.

Gastritis and H. pylori Infection

It is well known that H. pylori infection causes histo-

logic gastritis. There are inter-individual differences in

the severity or patterns of gastritis which are then asso-

ciated with the further development of different kinds

of disorders, such as duodenal ulcer (DU), gastric ulcer

(GU), and gastric cancer. Genetic differences in host

and bacterial factors have been considered to be one of

the reasons for the inter-individual differences.

For the explanation of these inter-individual differ-

ences in response to H. pylori infection, polymorphisms

of cytokines, such as interleukins (ILs) and tumor

necrosis factor (TNF)-a, have been studied intensively

since the year 2000. These cytokine polymorphisms are

associated with different patterns of gastritis among dif-

ferent individuals. In 2008, several new polymorphisms

associated with H. pylori-induced gastritis were reported

(Table 1).

Tahara et al. studied the effects of Toll-like receptors

(TLRs) on gastritis and found that TLR2-196 to -174ins

alleles were associated with more severe intestinal

metaplasia in patients older than 60 years and were

correlated with severity of gastric mucosal atrophy and

intestinal metaplasia in female subjects [1]. They also

studied the polymorphisms of Regulated upon activa-

tion, normal T-cell expressed, and secreted (RANTES)

and found that RANTES-28G carrier was associated with

a reduced risk of developing more severe intestinal

metaplasia in H. pylori-positive subjects aged 60 years

and older and in female subjects [2]. Trejo-de la OA,

Keywords

Gastritis, peptic ulcer, GERD, NSAID, aspirin,

dyspepsia, polymorphism.

Reprint requests to: Takahisa Furuta, Center

for Clinical Research, Hamamatsu University

School of Medicine, 1-20-1, Handayama, Hig-

ashi-Ku, Hamamatsu, 431-3192, Japan. E-mail:

[email protected] or Jean Charles Del-

chier, Service d’Hepatogastroenterologie,

AP-HP Hopital Henri Mondor, 51 Avenue du

Marechal de Lattre de Tassigny, 94000 Creteil,

France. E-mail: jean-charles.delchier@hmn.

aphp.fr

Abstract

It is well known that Helicobacter pylori infection is associated with many

nonmalignant disorders such as gastritis, peptic ulcer, gastroesophageal

reflux disease (GERD), gastric polyp, nonsteroidal anti-inflammatory drug

(NSAID) ⁄ aspirin-induced gastric injury, and functional dyspepsia. In 2008,

interesting articles on the association of H. pylori infection with these disor-

ders were presented, some of which intended to reveal the mechanisms of

inter-individual differences in response to H. pylori infection, and have dem-

onstrated that genetic differences in host and bacterial factors as well as

environmental factors account for these differences. A decline in the occur-

rence of peptic ulcer related to H. pylori was confirmed. An inverse relation-

ship between H. pylori infection and GERD was also confirmed but the

impact of gastric atrophy on the prevention of GERD remained debatable.

For NSAID-induced gastric injury, eradication of H. pylori infection has been

recommended. During this year, eradication of H. pylori infection was rec-

ommended for patients treated with antiplatelet therapy as well as aspirin

and NSAID. It was also reported that for patients with functional dyspepsia,

eradication of H. pylori offers a modest but significant benefit.


ª 2009 The Authors


et al. also studied the influence of TLR polymorphism

and observed that single-nucleotide polymorphisms

(SNPs) in the TLR4 gene were associated with severe H.

pylori-associated diseases and with a modified pattern of

inflammatory cytokines and chemokines in the gastric

mucosa infected with H. pylori [3].

H. pylori is also associated with lymphoid follicle gas-

tritis, which is known to sometimes evolve into mono-

clonal mucosa-associated lymphoid tissue (MALT)

lymphoma. Achyut et al. studied the association of

TNF-a and IL-10 gene polymorphisms with gastritis and

lymphoid follicle formation and found that IL-10-819T

and TNF-a-308A alleles may increase the risk of gastritis

and lymphoid follicle formation [4].

There have been several important reports on the poly-

morphism of bacterial factors. H. pylori strains have been

classified into two groups: strains with high virulence and

those with low virulence. The differences between the

two groups are partly explained by the status of cagA and

vacA, which are well known to be polymorphic. For vacA,

strains with an s1 ⁄ m1 genotype have been thought to be

more virulent than those with s2 ⁄ m2. However, Jafari

et al. evaluated the effects of vacA genotypes on gastric

inflammation and injury as well as clinical presentation

in Iranian populations and found that the vacA genotypes

and cagA status were not useful markers for gastroduo-

denal diseases in their country [5]. Chomvarin et al. from

Thailand attempted to determine whether any correla-

tion exists between genotypes of vacA, cagA, cagE, iceA,

and babA2 and clinical manifestations in dyspeptic

patients infected with H. pylori and concluded that nei-

ther a single gene nor a combination of vacA, cagA, cagE,

iceA, and babA2 genes was significantly helpful in predict-

ing the clinical outcome of H. pylori infection in their

country [6]. However, Basso et al. studied cagA and vacA

polymorphisms as well as the number of type C Glu-Pro-

Ile-Tyr-Ala motif (EPIYA) (EPIYA-C) segments, which

increase phosphorylation-dependent cagA activity in

H. pylori-positive Italian patients with different disorders

and they confirmed the association of cagA and vacA

s1 ⁄ m1 polymorphisms with PUD and cancers and noted

that the most important factors in western countries were

the number of cagA EPIYA-C segment for cancer risk and

the intermediate region type of vacA for PUD risk [7].

Because the EPIYA-C segment is the Src homology 2

domain-containing protein tyrosine phosphatase

(SHP-2)-binding site of cagA is clearly associated with

RAS ⁄ MAP kinase, EPIYA-C will be the key factor for

elucidating the bacterial types and their corresponding

clinical outcomes, including gastric cancer.

As stated before, a variety of polymorphisms from

both bacterial and host sides were reported to be associ-

ated with the severity and ⁄ or the type of gastritis. In

contrast, Kim et al. evaluated risk factors of atrophic

gastritis and intestinal metaplasia with respect to

H. pylori virulence factors (i.e., cagA, vacA m1, and oipA),

and environmental factors (i.e., smoking and alcohol)

and host polymorphisms (i.e., IL-1b-511, IL-1RN, TNF-

A-308, IL-10-592, IL-10-819, IL-10-1082, IL-8-251,

IL-6-572, GSTP1, p53 codon 72, and ALDH2) and found

that the bacterial factors were important risk factors for

atrophic gastritis but that environmental and host fac-

tors were more important for intestinal metaplasia [8].

The conclusion from this article is that to understand

the inter-individual differences in response to H. pylori

infection among different subjects, not only genetics of

hosts and bacteria, but also environmental factors have

to be studied. Therefore, the useful marker that predicts

the individual response to H. pylori infection remains to

be elucidated in relation to environmental factors.

Gastroduodenal Ulcer and H. pyloriInfection

It is common knowledge that H. pylori infection is,

along with nonsteroidal anti-inflammatory drugs

Table 1 Representative studies on genetics associated with pathogenesis of Helicobacter pylori-related gastritis published in 2008

Authors Summary Reference

Tahara et al. TLR2-196 to -174ins alleles are associated with severity of gastritis and intestinal metaplasia. [1]

Tahara et al. RANTES-28G allele decreases the risk of intestinal metaplasia. [2]

Trejo-de la et al. TLR4 polymorphisms are associated with cytokine secretion. [3]

Achyut et al. Tumor necrosis factor-alpha (-308G ⁄ A) and interleukin-10 (-819C ⁄ T) are associated with

gastritis, especially follicular gastritis.

[4]

Jafari et al. Polymorphisms of cagA and vacA (S1 ⁄ 2, m1 ⁄ 2) are not associated with the clinical outcome

of H. pylori infection.

[5]

Chomvarin et al. Polymorphisms of virulence factors (vacA, cagA, cagE, iceA, and babA2) are not associated

with the clinical outcome of H. pylori infection.

[6]

Basso et al. cagA EPIYA-C segment is associated with the development of intestinal metaplasia. [7]

Kim et al. Environmental factors are important than the genetics of host and bacteria. [8]

H. pylori and Non-malignant Diseases Furuta and Delchier

ª 2009 The Authors




(NSAIDs) ⁄ aspirin, a major factor of PUD. Some interest-

ing reports on PUD and H. pylori infection were pub-

lished in 2008, mainly dealing with epidemiology and

pathogenesis (Table 2).

Sung et al. performed a systematic review of the pub-

lished literature concerning the prevalence and inci-

dence of PUD [9]. The analyzed articles came from

western countries. The main conclusion was that PUD

remains a common condition despite decreasing inci-

dence and prevalence owing to a decrease in H. pylori

infection. In contrast, Wu et al. from Taiwan reported

that a dramatic decrease in the incidence of admissions

for complicated or uncomplicated PUD from 1997 to

2006 correlated with a significant increase in eradica-

tion therapy and use of proton-pump inhibitor (PPI)

[10].

Eradication of H. pylori infection is known to be effec-

tive in the prevention of bleeding ulcers. van Leerdam

et al. examined epidemiological surveys on gastrointes-

tinal bleeding cases and observed that H. pylori infection

is found in about 50% of bleeding PU patients [11].

Therefore, they concluded that all ulcer patients should

be tested for H. pylori infection and eradication treat-

ment should be given to those who are positive.

Cytokine polymorphisms have previously been

shown to be able to modulate host response to H. pylori

infection and to determine the occurrence of PUD. Vas-

cular factors could play a role in the pathogenesis of

PUD regardless of the main ulcerogenic agent involved,

H. pylori or NSAIDs. Kim et al. evaluated whether the

vascular endothelial growth factor (VEGF) polymor-

phism could predict susceptibility to PUD through mod-

ified angiogenic activities and found that the VEGF

polymorphism -1780T ⁄ C could significantly predict the

predisposition to PUD after exposure to etiologic risks

[12]. To understand the importance of this polymor-

phism, previously reported SNPs, such as IL-1b and

TNF-a, are needed for a comparative study.

Recently, endoscopic submucosal dissection (ESD)

and endoscopic mucosal resection (EMR) were pro-

posed for the treatment of early gastric cancer. The tim-

ing of H. pylori eradication on the process of ulcer

healing after EMR or ESD has been controversial,

because eradication of H. pylori restores gastric acid

secretion, which sometimes induces the ulcers to bleed

just after EMR or ESD. Cheon et al. prospectively eval-

uated the effect of H. pylori eradication on the healing

of gastric ulcer after EMR and stated that H. pylori erad-

ication might improve the ulcer’s healing rate after

EMR [13]. Even if a supplementary examination is nec-

essary, there seem to be cases in which eradication of

H. pylori immediately after EMR is beneficial.

Gastroesophageal Reflux Disease (GERD)and H. pylori Infection

Studies have shown that the prevalence of H. pylori

infection is lower in GERD patients than in non-GERD

subjects. Therefore, H. pylori infection has been consid-

ered to be possibly protective against the development

of GERD. The fact that the eradication of H. pylori

favors GERD and ⁄ or exacerbates symptoms in patients

with GERD remains controversial. Several studies pub-

lished in 2008 ⁄ 2009 dealt with this subject and again

reported controversial results (Table 3).

Corley et al. performed a case-control study on a

large population from California by matching patients

with a new diagnosis of Barrett’s esophagus with

patients with GERD and control subjects randomly

selected from the base population [14]. The control

group was original in comparison with the previous

studies in which patients requiring gastroscopy for

digestive symptoms other than GERD were included in

the control group, with a possibility of overestimating

the H. pylori rate. They found that H. pylori infection

and cagA+ status were inversely associated with a new

diagnosis of Barrett’s esophagus and that the association

might be at least partly mediated through GERD. Somi

et al. studied a more limited number of patients and

found a similar inverse association between H. pylori

infection and cagA status and reflux esophagitis [15].

However, Fass et al. studying the factors associated with

refractory GERD found that the status of H. pylori infec-

tion played a very limited role in refractory GERD [16].

Table 2 Representative studies on the association of Helicobacter pylori infection and gastroduodenal ulcers published in 2008

Author Summary Reference

Wu et al. Increases in H. pylori eradication therapy and proton-pump inhibitor use decrease the

incidence of gastric and duodenal ulcer diseases.

[10]

Van Leerdam et al. Around half of the bleeding ulcer patients are infected with H. pylori. Eradication therapy

is recommended.

[11]

Kim et al. VEGF-1780T ⁄ C polymorphism is associated with peptic ulcer risk. [12]

Chen et al. Eradication of H. pylori facilitates the ulcer after endoscopic mucosal resection. [13]

Furuta and Delchier H. pylori and Non-malignant Diseases

ª 2009 The Authors




Similarly, Grande et al. did not find any difference in

the main characteristics of a group of 146 GERD

patients according to their H. pylori status [17]. These

findings suggest that H. pylori status plays no important

role in the development of GERD and erosive esophagi-

tis. Kim et al. compared risk factors for erosive esopha-

gitis and nonerosive reflux disease (NERD) and found

that the history of H. pylori eradication could be related

to the risk of erosive gastritis, but not of NERD [18].

Several studies were performed to clarify the relation-

ship between H. pylori status, gastric atrophy, and

GERD. Anderson et al. performed a case-control study

including a large number of patients with esophageal

adenocarcinoma, Barrett’s esophagus, reflux esophagi-

tis, and controls [19]. They found an inverse association

of H. pylori seropositivity and also atrophy determined

by the pepsinogen I ⁄ II ratio with esophageal adenocar-

cinoma, Barrrett’s esophagus, and reflux esophagitis.

However, although gastric atrophy was involved, it

might not fully explain the inverse association with

H. pylori infection. Similarly, Kwon et al., who com-

pared a group of 45 patients having erosive esophagitis

with a group of 66 control patients, found that the rate

of infection of H. pylori was lower in the esophagitis

group and the pepsinogen I ⁄ II ratio was higher than

that in the control group, suggesting an inverse associa-

tion between GERD and H. pylori-related gastric atrophy

[20]. In contrast, Monkemuller et al. did not find any

correlation between serum gastrin and pepsinogen I

and II with the severity of GERD [21].

Because continuous administration of PPI for GERD

patients carries the risk of exacerbating the gastritis in

the event of an H. pylori infection, the preventive eradi-

cation of H. pylori is recommended for patients with

GERD who are in need of maintenance doses of a PPI,

although eradication of H. pylori increases the risk of

rendering the GERD ‘obstinate’. Wu et al. stated that,

although H. pylori eradication may lead to more resil-

ient GERD in a subset of patients, the benefits of

H. pylori eradication outweighed the risks from the

point of view of prevention of gastric cancer, especially

in Asian populations with a high incidence of gastric

cancer [22].

In conclusion, most of the articles published in 2008

confirm that H. pylori infection is inversely related to

GERD, erosive esophagitis, Barrettt’s esophagus, and

esophageal carcinoma. Whether reduction of GERD is

only mediated through H. pylori-induced corpus atrophy

is still being debated. The concept that eradication of H.

pylori is needed in GERD patients treated with PPI has

not changed.

Gastric Polyps and H. pylori Infection

Several interesting papers on H. pylori infection and gas-

tric polyps were published in 2008. Since some gastric

polyps may disappear after eradication of H. pylori, the

pathophysiological role of H. pylori infection in the

development of gastric hyperplastic polyps has been

suggested. Ohnishi et al. studied the pathophysiologic

role of cagA using cagA transgenic mice and found

that wild-type cagA transgenic mice developed gastric

epithelial hyperplasia and some of the mice developed

gastric polyps and adenocarcinomas of the stomach and

small intestine, suggesting that cagA is an oncogenic

protein [23]. Interestingly, such pathologic abnormali-

ties were not observed in transgenic mice expressing

phosphorylation-resistant cagA, indicating the impor-

tance of cagA tyrosine phosphorylation in the develop-

ment of H. pylori-associated neoplasms.

Table 3 Representative studies on the association of Helicobacter pylori infection with gastroesophageal reflux diseases (GERD)

Authors Summary Reference

Corley et al. Prevalence of H. pylori seropositivity is negatively correlated with the risk of Barrett’s

esophagus.

[14]

Somi et al. cagA-positive H. pylori strains decrease the risk of GERD. [15]

Fass et al. H. pylori infection has no association with refractory GERD. [16]

Grande et al. There is no association between H. pylori infection and GERD. [17]

Kim et al. GERD often occurs after eradication of H. pylori, but NERD does not. [18]

Anderson et al. H. pylori infection and gastric atrophy are associated with a reduced risk of esophageal

adenocarcinoma, Barrett’s esophagus, and reflux esophagitis, although gastric atrophy

may not fully explain the inverse associations observed with H. pylori infection.

[19]

Kwon et al. Reflux esophagitis is inversely associated with gastric atrophy. [20]

Monkemuller et al. Serum gastrin and pepsinogen I and II do not correlate with the different grades of

severity of GERD.

[21]

Wu et al. GERD patients infected with H. pylori should undergo eradication therapy for the prevention

of gastric cancer.

[22]


ª 2009 The Authors




Cronkhite-Canada syndrome (CCS) is a rare syn-

drome characterized by multiple polyps of the digestive

tract with symptoms that include loss of taste, hair loss,

and nail growth problems. Chronic diarrhea and pro-

tein-losing enteropathy are often observed. The cause

of the disease is unknown. Okamoto et al. prescribed

H. pylori eradication therapy for an H. pylori-positive

patient with CCS in Japan and noted a regression of

polyps and resolution of clinical findings such as edema

with the normalization of serum total protein and albu-

min levels [24]. This case report suggests the possible

role of H. pylori infection in the pathogenesis of CCS.

Whether this particular strain was cagA-positive is

unclear, but most H. pylori strains in Japan are cagA-

positive. Therefore, cagA status is assumed to be associ-

ated with the development of gastric polyps.

NSAIDs ⁄Aspirin-Induced Gastric Injuryand H. pylori Infection

No major original contribution to this subject was pub-

lished in 2008. However, a consensus of the American

College of Cardiology on antiplatelet therapy and

NSAID use and the guidelines of the American College

of Gastroenterology for prevention of NSAID-related

ulcer complications have been recently published

[25–28] (Table 4). Kiltz et al. reviewed the literature

from a rheumatological point of view [29]. Similar rec-

ommendations were given. For antiplatelet therapy, the

recommendation is to examine H. pylori infection in

patients with a history of PUD and to eradicate H. pylori

infection when present. However, PPI were recom-

mended to prevent recurrence of complications. For

NSAIDs, Kiltz et al. concluded from the review of the

literature that it was well demonstrated that NSAID-

naive users benefited from testing for H. pylori infection

and subsequent H. pylori eradication therapy prior to

the initiation of NSAID, but that H. pylori eradication

alone did not offer protection from gastroduodenal

injuries in chronic NSAID users [29]. To relieve patients

with recent ulcer complications from further gastroin-

testinal (GI) events, eradication of H. pylori alone is

insufficient and long-term acid inhibition is required.

The management of H. pylori infection and the

prevention of GI complications in NSAID users need to

be examined case by case. Eradication of H. pylori is

important in preventing gastroduodenal injury as a

result of antiplatelet and ⁄ or NSAID therapy.

Epidemiologic studies have revealed that the inci-

dence of gastric cancer is lower in subjects receiving

NSAID and ⁄ or aspirin compared with nonusers of

NSAID and ⁄ or aspirin. In 2008, a supportive study was

reported. Li et al. studied the effects of aspirin on the

development of heterotopic proliferative glands in

H. pylori-infected Mongolian gerbils and found that

aspirin alleviated H. pylori-induced hyperplasia and the

development of heterotopic proliferative glands and also

increased H. pylori-induced apoptosis [30]. They con-

cluded that aspirin was responsible for the antineoplas-

tic activities in H. pylori-related gastric carcinogenesis.

Functional Dyspepsia (FD) and H. pyloriInfection

The role of H. pylori infection in FD has not been fully

elucidated and the effect of H. pylori eradication is still

controversial. In 2008, several reports on the effect of

H. pylori infection on FD were presented (Table 5).

Selgrad et al. stated in a review that population-based

studies have demonstrated that H. pylori is detected

more frequently in dyspeptic patients than in controls

and that H. pylori eradication therapy gives a modest

but significant benefit in nonulcer dyspepsia cases and

leads to long-term symptom improvement [31]. Conse-

quently the ‘‘test and treat’’ strategy should remain the

first option in patients with unexplored dyspepsia with-

out alarm features in areas where H. pylori prevalence

is greater than 20%. In western countries with a low

prevalence of H. pylori, migrant communities may con-

stitute a target group for the ‘‘test and treat’’ strategy

[32]. An important study from Denmark evaluated the

long-term effects of H. pylori screening and treatment of

dyspepsia by determining the dyspepsia health-care

consumption and quality of life in a large randomized

community-based trial [33]. The prevalence of H. pylori

in screened subjects was low (17.5%). The effect of

eradication on the rate of dyspepsia was modest and

was not statistically significant contrary to the

Table 4 Representative studies on the association of Helicobacter pylori infection with nonsteroidal anti-inflammatory drug (NSAID) ⁄ aspirin-related

disorders published in 2008


Bhatt et al. Eradication of H. pylori is recommended in NSAID ⁄ aspirin users with a history of peptic ulcer. [25–27]

Kiltz et al. Eradication of H. pylori is effective in patients who have never used NSAID ⁄ aspirin. [29]

Li et al. Aspirin inhibits the development of heterotopic proliferative gland in Mongolian gerbils infected with H. pylori. [30]


ª 2009 The Authors


consultation and sick leave rates which were signifi-

cant; the incidence of ulcers also decreased significantly.

A randomized placebo-controlled trial from Singapore

showed that, in an Asian population with FD, eradica-

tion of H. pylori had a major beneficial effect when

compared with a placebo, with a symptom resolution

rate of 39% versus 3%, respectively, after 1 year [34].

Differences between eastern and western patients with

FD could be related to the lower prevalence of reflux

symptoms in the Asian patients.

de Artaza Varasa et al. studied whether antral gastritis,

commonly associated with PUD, may predict a greater

symptomatic response to H. pylori eradication in FD and

found that there was a tendency of symptomatic benefit

with H. pylori eradication in patients with antral gastritis

[35]. Similarly, Koivisto et al. showed that DU patients

(from 50 to 59 years of age) with antral neutrophil

inflammation, were significantly predictive of symptom-

atic improvement after H. pylori eradication [36].

Tahara et al. attempted to clarify the association

between 5HTR2A C102T polymorphism, CD14 gene C-

159T polymorphism, and polymorphism in codon 158

of the COMT gene, and dyspeptic symptoms [37–39].

They found that neither 5HTR2A polymorphism nor

CD14 gene C-159T polymorphism was likely to be asso-

ciated with dyspeptic symptoms, but that the COMT

genotype seemed to influence the susceptibility of dys-

pepsia. The role of genetics in the development of dys-

pepsia needs further evaluation.

The impact of H. pylori on symptom control in patients

with long-term PPI treatment was studied by Raghunath

et al. [40]. They found that H. pylori infection was asso-

ciated with lower reflux symptom scores only in

patients with GERD and uninvestigated dyspepsia.

Conclusion

H. pylori infection is associated with many nonmalig-

nant disorders as described before. Genetics of hosts

and bacteria as well as environmental factors are

responsible for the inter-individual differences in

response to H. pylori infection in different individuals.

Unfortunately, the impact of newly discovered poly-

morphisms is still unclear. Therefore, comparative stud-

ies are needed to clarify the important SNPs associated

with a response to H. pylori infection. Although the

pathophysiologic role of H. pylori in nonmalignant dis-

eases has not been fully elucidated, eradication of the

bacteria is sometimes effective for the treatment of

these disorders. Eradication of H. pylori infection has

also been recommended for patients treated with

NSAID ⁄ aspirin and ⁄ or antiplatelet agents. Indeed, there

are no disorders for which eradication of H. pylori infec-

tion is contraindicated; therefore, the ‘‘test and treat

strategy’’ appears to be useful in H. pylori-positive

patients with certain symptoms, such as dyspepsia.

However, further studies are needed to clarify more

precisely the association of H. pylori infection with these

nonmalignant disorders, which will contribute to higher

quality of clinical practice in the treatment of digestive

diseases.

Conflict of Interest

None of the authors had any conflict of interest related

to this manuscript.

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ª 2009 The Authors


Basic Aspects of Gastric CancerMarta Correia,*� Jose Carlos Machado*,� and Ari Ristimaki�§

*IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal, �Faculty of Medicine, University of

Porto, Porto, Portugal, �Department of Pathology, Institute of Diagnostics, University of Oulu and Oulu University Hospital, Oulu, Finland,§Genome-Scale Biology Research Program, Biomedicum Helsinki, Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland

Gastric cancer (GC) remains a considerable public

health problem worldwide, and although its incidence

and mortality rates have gradually decreased, GC is sec-

ond only to lung cancer as the leading cause of cancer

death [1]. The great majority of gastric malignancies are

adenocarcinomas that can be divided into two histo-

logic entities, intestinal and diffuse types, which exhibit

distinct epidemiological and genetic patterns. Curative

treatment of GC requires complete surgical removal of

the neoplastic tissue, but even with curative intent the

5-year survival is only about 20–30%. The high mortal-

ity is mostly because of late diagnosis of the disease,

creating an urgent need for new diagnostic markers

and treatment modalities.

A widely accepted model for the development of GC

is that the disease arises from Helicobacter pylori infection

in a susceptible human host [2]. This gram-negative

bacterium is acquired in childhood and persists in the

stomach over decades. Case–control studies have shown

that H. pylori seropositivity is associated with a signifi-

cantly increased risk of GC (2.1–16.7 times greater than

seronegative persons) [3–5], being considered a causa-

tive pathogen for gastric carcinogenesis. Inflammation

may be the key promoting factor in the process of carci-

nogenesis induced by H. pylori. This bacterium possesses

a unique array of features that makes it highly adapted

to this ecological niche and persistent as a long-term

infection of the gastric mucosa. The infection first

induces chronic superficial (nonatrophic) gastritis,

which can progress through chronic atrophic gastritis,

intestinal metaplasia, and dysplasia toward GC. How-

ever, only a small number of infected patients will

eventually develop GC (<1%) [6]. Besides family his-

tory, which is a risk factor independent of H. pylori

infection, host genetic factors such as genes associated

with inflammatory response probably participate in

stomach carcinogenesis.

Genetic Susceptibility to GC

Susceptibility without evident familial clustering is,

despite presenting the weakest genetic effects, the main

category of inherited susceptibility to cancer. High pen-

etrance of cancer-related genes accounts for a very low

proportion of overall cancer incidence, while cancer-

susceptible alleles, as a result of their high frequency,

may account for a significant fraction of the overall

cancer incidence. In recent years it has been shown

that polymorphisms in several genes considered to be

critical for gastric carcinogenesis, such as those involved

in the inflammatory response to H. pylori infection

[7–11], in the mucosal protection towards H. pylori

infection [12–15], in the protection of DNA to oxidative

damage [16], and in detoxification enhancement

[17,18], may influence the risk of progression to GC.

The first published epidemiologic evidence indicating

the association between an increased risk of GC and

proinflammatory polymorphisms came from a study of

the interleukin-1-beta (IL-1b) and IL-1 receptor antago-

nist genes (IL-1RN) [19]. Recently, the EPIC 2008 study

Keywords

Gastric cancer, H. pylori, polymorphism,

inflammation, dysplasia, stem cell.

Reprint requests to: Jose Carlos Machado,

IPATIMUP, Rua Dr. Roberto Frias, s ⁄ n,

4200-465 Porto, Portugal.


Abstract

Gastric cancer is a worldwide health burden, which is still the second most

common cause of cancer related deaths with little improvement of long-

term survival during the past decades. Understanding the molecular nature

of this disease and its precursor lesions has been under intense investigation

and our review attempts to highlight recent progress in this field of cancer

research. First, host-related genetic susceptibility is dealt with genes involved

in inflammation and carcinogen metabolism. Next, role of overexpression of

a proinflammatory cytokine (interleukin-1beta) and deletion of a cell-cell

adhesion molecule (E-cadherin) are described in experimental mouse mod-

els of gastric carcinogenesis. Finally, the role of stem cells in gastric cancer is

covered.


ª 2009 The Authors





confirmed that proinflammatory IL-1RN genotypes are

significantly associated with an increased risk of non-

cardia adenocarcinoma in H. pylori-positive cases [7].

In addition to polymorphisms in interleukin genes,

the polymorphisms in the promoter region of tumor

necrosis factor-alpha (TNF-a) gene have been exten-

sively studied in relation to GC. TNF-a is a pleiotropic

cytokine mostly produced by activated monocytes and

macrophages, which play a key role in the inflamma-

tory response. Although several promoter polymor-

phisms have already been identified, most studies have

focused on the TNF-a -308G>A single nucleotide poly-

morphism (SNP). Canedo et al. performed a case-con-

trol study for the TNF-a -308G>A polymorphism to

determine the association with the risk of development

of GC [8]. Their results indicate that the detected asso-

ciation between the proinflammatory TNF-308*A allele

and the increased risk of GC is at least partially influ-

enced by linkage disequilibrium (with an as yet uniden-

tified locus) [8]. This study emphasizes the importance

of extending single SNP association studies to haplo-

type-based approaches.

IL-16 is known for promoting the secretion of tumor-

associated inflammatory cytokines by monocytes. Gao

et al. [9] was the first to examine the association

between SNPs of the IL-16 gene and GC. The authors

demonstrated that the rs11556218 T ⁄ G polymorphism

of the IL-16 gene was significantly associated with sus-

ceptibility to GC [9]. Interferon gamma (IFNc) is one of

the most important Th1-related cytokines which have

been shown to promote gastritis. Individuals homozy-

gous for the IFNGR1-56*T allele were shown to have a

fourfold increased risk of developing early-onset GC

when compared with those homozygous for the

IFNGR1-56*C allele [10].

Carcinogens and toxins are metabolized via the xeno-

biotic pathway which is an important defense mecha-

nism against carcinogenesis. Hence, polymorphisms on

enzymes involved in the protection of oxidation and

enhancement of detoxification of carcinogens may

therefore participate in GC. Selenoprotein S (SEPS1) is

a novel selenoprotein located in the endoplasmic reticu-

lum (ER) and plasma membrane. It is involved in the

control of the inflammatory response of ER. SEPS1 pro-

tects cells from oxidative damage and apoptosis, and is

widely expressed in a variety of tissues. Recently, the

-105G>A promoter polymorphism of SEPS1 was shown

to be associated with plasma levels of proinflammatory

cytokines, such as IL-1b, IL-6, and TNF-a. It is known

that the substitution of the allele A for G at position -

105 reduces the promoter activity in HepG2 cells, and

therefore also the IL-1b levels which are associated

with GC. In a Japanese study comprising 574

individuals, it was demonstrated for the first time that

the -105G>A polymorphism of the SEPS1 gene was

associated with an increased risk of intestinal-type GC

[17].

Glutathione-S-transferase (GST) is another important

enzyme catalyzing conjugation of potentially mutagenic

electrophilic compounds, with nucleophilic glutathione

yielding less toxic and more water-soluble compounds,

readily excreted via urine or bile. Both GSTT1 and

GSTM1 genes of the GST super gene family exhibit

either null or deletion polymorphism. Individuals

homozygous for the null allele lack GST enzyme activ-

ity and thus have an increased risk for cancer. GSTP1

exhibits a polymorphism within its coding region (A to

G transition at nucleotide +313), which leads to

reduced enzyme activity. Tripathi et al. reported that

the frequency of GSTT1*0 was higher in GC patients

(diffuse-type) than in the controls. Analysis of com-

bined GSTM1*0 and GSTP1*0 frequencies revealed that

simultaneous deletion of both genes was associated

with a 2–5 times higher risk of GC in comparison with

the presence of both the genes [18].

Intestinal-Type GC

Intestinal-type GC is believed to develop by a multistep

process also known as Correa sequence, in which inva-

sive cancer is preceded by atrophic gastritis and meta-

plastic and dysplastic lesions. Usually this sequence of

events is initiated and promoted by persistent H. pylori

infection. However, only a fraction of infected persons

develop cancer. Therefore, knowledge of cancer risk in

relation to premalignant lesions in the stomach is an

important basis for making decisions on surveillance

and treatment of these patients. Previously reported

progression rates to GC vary considerably and even for

dysplasia the range is as high as from 0% to over 70%

per year. Understandably, surveillance strategies of

patients with such lesions are highly controversial. De

Vries et al. analyzed the GC risk and surveillance prac-

tice among Dutch patients (n = 92,250) who were diag-

nosed with a premalignant lesion in the stomach

between 1991 and 2004 using the Dutch Nationwide

Histopathology Registry [20]. Follow-up data were col-

lected and evaluated until December 2005, and they

show that the distribution of histologic findings were

67% for intestinal metaplasia, 24% for atrophic gastri-

tis, 8% for mild-to-moderate dysplasia, and 0.6% for

severe dysplasia. The annual incidence of GC was 0.1%

for atrophic gastritis, 0.25% for intestinal metaplasia,

0.6% for mild-to-moderate dysplasia, and 6% for severe

dysplasia within 5 years after initial diagnosis. Impor-

tantly, only 61% of the patients with severe dysplasia

Correia et al. Basic Aspects of Gastric Cancer

ª 2009 The Authors



and 26–38% for the other lesions were re-evaluated by

endoscopy. These figures seem alarmingly low, consid-

ering that the relative risk for GC in this study was esti-

mated to be about 40-fold for severe dysplasia when

compared with patients having atrophic gastritis. It is

also noted that patients with mild-to-moderate gastric

dysplasia have a similar or even higher risk of cancer

than patients with Barrett’s esophagus. One of the

major conclusions is that endoscopic surveillance at

short intervals is warranted in patients with gastric

dysplasia.

Knowledge of individual risk of progression of prema-

lignant gastric lesions to GC would be useful informa-

tion in planning surveillance strategies, especially for

low-risk lesions such as atrophic gastritis and intestinal

metaplasia. In addition to environmental factors, distri-

bution and extent of histologic lesions in the stomach,

and H. pylori virulence factors, host genetics play a role

in gastric carcinogenesis. One of the most consistent

and strongest association with GC and polymorphisms

is the IL-1b gene, as recently reviewed by McNamara

and El-Omar [21]). IL-1b is a proinflammatory cytokine

that also inhibits acid secretion in the stomach, and cer-

tain polymorphisms in this gene lead to an increased

risk of noncardia GC in the presence of H. pylori infec-

tion. Tu et al. have now published a transgenic mouse

model that enlightens the role and possible mechanisms

of the procarcinogenic effects of IL-1b [22]. In this

paper, it is shown that stomach-specific expression of

IL-1b leads to gastric inflammation and eventually to

neoplastic changes, including intramucosal adenocarci-

noma, that were more severe when mice were infected

with Helicobacter felis. Furthermore, in this mouse model

myeloid-derived suppressor cells seemed to contribute

to the carcinogenic cascade while T and B cells were

not needed for this phenomenon. Interestingly, IL-1b-

activated Nuclear factor-kappaB (NF-jB) in these cells

led to an increase in IL-6 and TNF-a production. It has

been hypothesized that myeloid-derived suppressor cells

could contribute to immunoresponse, angiogenesis, and

tumor invasion. However, since these cells were not

specifically deleted in this study, a role of other cells

(such as neutrophils, macrophages, dendritic cells, myo-

fibroblasts and endothelial cells) cannot be excluded.

This study thus shows that IL-1b is sufficient in pro-

moting inflammation and carcinogenesis in the

stomach.

Tumor suppressor gene p53 is a marker of poor prog-

nosis in many malignant diseases, and inactivating

mutations of the p53 gene can be found in 38–71% of

GC [reviewed in Ref. 23]. In addition, p53 mutations

have already been found in intestinal metaplasia and

in dysplastic lesions, suggesting that the gene’s

inactivation may be an early event in gastric carcino-

genesis. Indeed, Szoke et al. published that the RR

genotype of codon 72 was found to be associated with

a reduced incidence of intestinal metaplasia among H.

pylori-infected patients [24]. The p53 gene contains sev-

eral polymorphic sites of which the polymorphism of

codon 72 has been most extensively studied in GC.

However, the results are conflicting, and a recent meta-

analysis concluded that the p53 codon 72 polymor-

phism may be associated with GC but only in Asian

cohorts [25]. One possible explanation for these con-

flicting results is owing to the fact that the p53 family

also includes other proteins, namely p63 and p73. Both

of these proteins share structural similarity with p53,

activate p53 target genes and are involved in regulation

of apoptosis. The scenario is somewhat complex, since

certain subforms of p63 and p73 proteins can act in a

dominant-negative manner towards p53. Wei et al.

recently demonstrated that H. pylori infection of gastric

epithelial cells in vitro and in vivo in mice lead to

upregulation of p73 protein [26]. These results suggest

that p73 may play an important role in the pathogenesis

associated with H. pylori infection, and that alterations

in p73 gene may play a role in gastric carcinogenesis.

Diffuse-Type GC

Intestinal-type GC predominates in high-risk geographic

areas and shows a correlation with the prevalence of H.

pylori infection. Diffuse-type GC, in contrast, is more

uniformly distributed and is apparently unrelated to H.

pylori prevalence. Owing to its development underneath

the gastric mucosal surface, diffuse GC is usually

diagnosed at an advanced stage and is consequently

associated with a poorer outcome. It can be further sub-

divided into poorly differentiated carcinoma, and

signet-ring cell carcinoma (SRCC). It is suggested that

the latter is an initial, differentiated form of diffuse GC

that may evolve into poorly differentiated carcinoma

[27]. Moreover, and despite the decreasing incidence of

GC worldwide, the incidence of diffuse GC in the form

of SRCC is increasing. At the molecular level, diffuse-

type GC can be distinguished from intestinal-type GC

on the basis of the cell–cell adhesion molecule, E-cadh-

erin. This molecule is the key component of the epithe-

lial adherens junction and as such is required for

functional intercellular adhesion within epithelial

sheets. E-cadherin is downregulated very early during

diffuse GC development, suggesting a role in the

initiation of this disease. In fact, a causal relationship

between E-cadherin deficiency and the initiation of

diffuse GC has been established. In a N-methyl-N-nitro-

sourea-treated mice model homizygous for the

Basic Aspects of Gastric Cancer Correia et al.

ª 2009 The Authors


E-cadherin gene (cdh1+ ⁄ )), the presence of a second

CDH1 hit is the earliest observable stage of human dif-

fuse GC, also providing evidence for epigenetic downre-

gulation of E-cadherin as an initiator of malignancy

[28].

An alternative mechanism to explain loss of function

of E-cadherin in diffuse-type gastric transformation may

involve the epithelial–mesenchymal transition (EMT)

regulator TWIST through crosstalk with Hedgehog (Hh)

signaling. The Hh signal activation selectively occurs in

diffuse-type GC and blocking of Hh signaling inhibits

the growth of GC cells [29]. Transforming growth fac-

tor-b (TGF-b), which is a multifunctional cytokine, is a

potent inhibitor of epithelial cell proliferation. More-

over, TGF-b may promote tumor growth by inducing

the epithelial cells to undergo EMT. Inhibition of TGF-bsignaling has also been reported to prevent progression

and metastasis onset in diffuse GC, mainly because of

its ability to enhance angiogenesis [30].

Role of Stem Cells in GC

In the last years several lines of evidence have sug-

gested that stem cells play an important role in gastric

carcinogenesis. Houghton et al. showed that H. pylori-

induced inflammation can cause migration of bone

marrow-derived stem cells to the gastric mucosa, where

they may subsequently transform into GC lineages

[31]. McDonald et al. demonstrated that mitochondrial

DNA (mtDNA) mutations establish themselves in stem

cells within normal human gastric body units, and are

passed on to all their differentiated progeny [32].

Mutated units can divide by fission to form patches,

with each unit sharing an identical, mutant mtDNA

genotype. These data show that human gastric body

units are clonal, contain multiple multipotential stem

cells, and provide definitive evidence for how muta-

tions spread within the human stomach, and show how

field cancerization develops [32].

Apart from its role in embryonic development and

tissue regeneration, Hh signaling is involved in adult

stem cell maintenance. Hh signaling is activated in GC

tumors, and appears to be crucial for the differentia-

tion of gastric progenitor cells into mucus and parietal

cells [33]. The proposed model is that cancer develops

from tissue progenitor cells after chronic stimulation

from various injuries (e.g. H. pylori-persistent infec-

tion), and that during the repair process, developmen-

tal signaling pathways activate tissue progenitor cells.

Chronic stimulation may ultimately result in irrevers-

ible activation of the signaling pathways which leads

to cancer formation. Mesenchymal stem cells (MSCs),

a subtype of stem cells with great capacity of self-

renewal and differentiation, have been isolated from

several tumors. The question of whether a group of

MSCs exists in GC has arisen and, for the first time,

MSCs have been isolated from tumors of GC patients

[34]. Altogether, the expanding field of gastric cancer

stem cell biology may offer novel avenues of research

with impacts on the diagnosis and treatment of

cancer.



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Basic Aspects of Gastric Cancer Correia et al.

ª 2009 The Authors


Helicobacter pylori and Clinical Aspects of Gastric CancerJan Bornschein,* Theodore Rokkas,� Michael Selgrad* and Peter Malfertheiner*

*Department of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke-University of Magdeburg, Magdeburg, Germany,�Gastroenterology Clinic, Henry Dunant Hospital, Athens, Greece

Early diagnosis and efficient therapy for gastric cancer

(GC) remain an ongoing challenge for health systems

worldwide [1]. Approximately 80% of patients are diag-

nosed in an advanced stage with no curative treatment

options. Surgical treatment with curative intent has a

general 5-year survival rate of approximately 24% [2].

Currently, the major task is to define a general applica-

ble stage-adjusted algorithm that not only respects the

outcome of each treatment modality concerning mor-

tality and morbidity, but also the post-interventional

quality of life.

Development of adequate regimes for adjuvant as

well as neoadjuvant or peri-operative systemic chemo-

therapy will have a significant role in the future [3,4].

For palliative chemotherapy taxan- and platinum-based

combination-therapies are the standard of care. Modi-

fied regimens are under evaluation to lower treatment-

related toxicities and to improve the quality of life

[5–10].

This review gives a brief overview of articles pub-

lished between March 2008 and April 2009 on the

achievements in prevention, diagnosis and management

of GC with an emphasis on H. pylori eradication as a

means for successful prevention.

Primary Staging

Park et al. evaluated the diagnostic potential of EUS

compared with multidetector-CT for restaging of pati-

ents after neoadjuvant systemic therapy (Docetaxel in

combination with Cisplatin) in 40 Korean patients [11].

Both modalities revealed comparable results, but in a

multivariate analysis EUS-documented downstaging

was an independent prognostic factor for overall sur-

vival. In conclusion, EUS should be standard for evalu-

ation of local tumor response after neoadjuvant

treatment [11].

Nitti et al. evaluated the prognostic value of subclassi-

fication of T2-stage disease into T2a (muscularis pro-

pria) and T2b (subserosa) and related treatment

algorithms [12]. In a retrospective analysis of 373

patients treated for GC with curative intention, the

tumor-related mortality risk for the T2a stage was com-

parable to the T1 stage whereas for the T2b stage it was

Keywords

Gastric cancer, H. pylori, eradication therapy,

screening, cost-effectiveness, intestinal

metaplasia, glandular atrophy, endoscopic

resection, D2 lymphadenectomy, prevention

strategies

Reprint requests to: P20 ⁄ D2 Peter Malfertheiner,

Department of Gastroenterology, Hepatology

and Infectious Diseases, Otto-von-Guericke-

University of Magdeburg, Leipziger Str. 44,

D-39120 Magdeburg, Germany. Tel.: 0049 391

6713100; Fax: 0049 391 6713105;


Abstract

In spite of important new insights into the basic mechanisms of gastric carci-

nogenesis, progress in the management of gastric cancer has been modest.

Some modifications in the chemotherapies used for palliation and strategies

for downstaging of the disease prior to surgical intervention are noteworthy.

The positive experience with endoscopic mucosal resection (EMR) and

submucosal dissection (ESD) for treatment of early gastric cancer has been

confirmed and extended. The procedure-related morbidity and post-inter-

ventional quality of life is clearly favorable compared to open surgical

resection in well-selected patients. New data on Helicobacter pylori revealed

that eradication after endoscopic resection of early gastric cancer signifi-

cantly reduces the incidence of recurrent and metachronous gastric neopla-

sias. It can further improve healing rates of treatment induced gastric ulcers.

Eradication therapy therefore remains the best target for prevention of the

disease. Critical is the ‘‘point of no return’’ when mucosal alterations (i.e.

intestinal metaplasia, glandular atrophy) are no longer reversible. A popula-

tion-based screen-and-eradicate strategy for H. pylori infection can at present

only be recommended in high incidence regions.


ª 2009 The Authors




similar to the T3 stage disease. Thus, subclassification of

T2-tumors is recommended for planning the therapeu-

tic strategy [12].

Curative Gastric Resection

Laparoscopic resection has been shown to be a safe and

adequate curative treatment in early gastric cancer

(EGC), including stage I and II tumors [13]. This

approach results in favorable outcome not only con-

cerning treatment-related aspects (e.g. intraoperative

blood loss, total amount of analgetics used, post-opera-

tive hospitalization period) but also concerning physi-

cal, emotional, social and general symptom scales

compared to open gastrectomy [14].

In a Korean study open partial gastrectomy was com-

pared with total gastrectomy Although overall survival

was comparable in both groups (99.2% vs 98.5%,

respectively, at final follow-up), post-operative compli-

cations occurred significantly more often in patients

that had only proximal gastrectomy (61.8% vs 12.6%)

[15]. Therefore, total gastrectomy and Roux-en-Y ana-

tomical reconstruction is superior to limited proximal

resection concerning post-operative complications.

There is still ongoing debate about the required

extent of lymphadenectomy for adequate curative treat-

ment of localized GC. In Asian countries, D2 lymph

node dissection represents the standard of care whereas

in several Western countries D1 dissection is still

performed.

Sasako et al. evaluated the efficacy and safety profile

of additional dissection of the paraaortic lymph nodes

(PAND) in comparison to regular D2 lymphadenectomy

in patients with preoperative tumor stage of T2b, T3 or

T4 [16]. Major surgical complications (e.g. anastomotic

leakage, pancreatic fistula, abdominal abcess) were not

statistically different between the groups. There was no

difference in 5-year overall survival between patients

with D2 or D2 + PAND dissection (69.2% vs 70.3%,

respectively; HR 1.03; 95% CI 0.77–1.37). In conclu-

sion, more extended lymphnode dissection than D2

cannot be recommended for standard GC treatment

[16].

Endoscopic Versus Surgical Treatment

Endoscopic treatment of EGC is an established method

for a curative attempt because of a significant improve-

ment in quality of life in comparison to more radical

surgical techniques. Standard procedure represents the

endoscopic mucosal resection (EMR) for elevated EGC

<2 cm in diameter and small (<1 cm) depressed tumors

without ulceration according to the Japanese guidelines

[17]. Generally this approach was only legitimate for

mucosal defined tumors, whereas by the recently intro-

duced technique of endoscopic submucosal dissection

(ESD), tumors involving the upper submucosal layers

can also be treated using an insulation-tipped dia-

thermy knife (alternatively a hook knife). A further

advantage of this method is the ‘‘en bloc’’ resection of

the specimen that allows a more precise pathological

assessment concerning gross and microscopically com-

plete tumor resection.

In a feasibility study in Japan, 551 consecutive

patients with 589 EGCs were treated with ESD and

received a median follow-up of 30 months (6–89) [18].

Inclusion criteria were (1) mucosal cancer with ulcer

findings and largest diameter £3 cm, and (2) minute

submucosal invasive cancer with largest diameter

£3 cm (<500 lm distance from the muscularis mucosa).

Curative resection was achieved, when vertical and lat-

eral tumor margins were free of malignant tissue, no

submucosal invasion deeper than 500 lm from the

muscularis mucosa was detected, and lymphatic or vas-

cular involvement was absent.

‘‘En bloc’’ resection was achieved in 94.4% with

94.7% defined as curative resection. There were no

treatment related deaths; minor complications were

bleeding (1.8%) or perforation (4.5%), both being

endoscopically manageable in all cases. During follow-

up, local recurrence was documented in three cases

only with non-curative resection and in one case after

piece-meal resection (all of these cases underwent gas-

trectomy with D2 lymph node dissection). Metachro-

nous GC occurred in 13 patients after curative

resection, and in one after non-curative resection

within 12–42 months. The 3-year and 5-year survival

rates were 98.4 and 97.1%, respectively, with death

occurring due to other tumors or heart disease [18].

Long-term follow-up data are needed for an adequate

comparison with related outcome of surgical

procedures.

Rescue treatment after non-curative resection should

be surgical. However, for well-selected patients

re-endoscopic treatment could be considered [19,20].

Helicobacter pylori Eradication in thePrevention of Recurrence

The efficacy of primary prevention of GC by eradication

of H. pylori has been confirmed in several studies [21].

An important issue was that once preneoplastic changes

(gastric atrophy and intestinal metaplasia) are estab-

lished, prevention of further progression to invasive

cancer is more unlikely to occur [22]. The so-called

‘‘point of no return’’ has been identified to be critical

Treatment and Prevention of Gastric Cancer 2009 Bornschein et al.

ª 2009 The Authors



for an effective prevention of GC incidence or recur-

rence. Watari et al. examined the effect of H. pylori

eradication on the histology and cellular phenotype of

gastric intestinal metaplasia. They showed that H. pylori

eradication changes the cellular phenotype of gastric

intestinal metaplasia, which might be an important fac-

tor in the reduction of gastric cancer incidence after

successful eradication [23].

Unexpectedly and with more question marks on the

‘‘point of no return’’ theory, Fukase et al. demonstrated

that even after endoscopic resection of early GC, recur-

rence of metachronous GC is significantly reduced by

H. pylori eradication [24].

In a multicenter, open-label randomized trial, 544

patients who underwent EMR of EGC received either

eradication treatment against H. pylori (lansoprazole

30 mg, amoxicillin 750 mg and clarithromycin 200 mg,

each twice daily) or a placebo regimen. Follow-up

endoscopy was performed at 6, 12, 24 and 36 months.

Primary endpoint of the intention-to-treat analysis was

occurrence of GC at a site other than the primary trea-

ted site of the stomach (metachronous GC). At the

3-year-follow-up, metachronous GC developed in 9 of

272 patients who received eradication treatment (3.3%)

and in 24 of the 272 placebo patients (8.8%) resulting

in an odds ratio (OR) of 0.353 (95% CI 0.161–0.775,

p = 0.009). In the modified intention-to-treat analysis

adjusted for loss to follow-up and respecting the patient

population that had received at least one post-

treatment assessment of tumor status, an HR for meta-

chronous GC of 0.339 was documented (95% CI 0.157–

0.729; p = 0.003). From these findings, it can be stated

that eradication of H. pylori is an effective method for

prevention of metachronous GC after endoscopic treat-

ment of EGC and should be routinely applied. These

data confirm previous observations which have been

published by other authors [25,26].

A further beneficial effect of post-interventional erad-

ication of H. pylori was demonstrated by Cheon et al.

[27]. Of 47 patients who had undergone EMR for EGC,

in 21 H. pylori infection was cured whereas 26 patients

were either treated with a proton-pump inhibitor (PPI)

alone or the eradication therapy failed. At 4 weeks

post-treatment, there was a significant difference con-

cerning healing of the treatment-induced ulcers, with

the group with successful H. pylori eradication showing

superior ulcer reduction rates [27].

Precancerous Lesions

For adequate individualized risk assessment in planning

prevention strategies, the question of whether gastric

atrophy and intestinal metaplasia (IM) are premalignant

rather than paramalignant lesions is still under debate.

Most data point to an increased risk of gastric carcino-

genesis if glandular atrophy, IM and even more so if

dysplastic changes are detected [28].

In a large retrospective analysis, de Vries et al. evalu-

ated data of 92,250 patients who were filed in the

Dutch Nationwide Histopathology Registry for the per-

iod from 1991 until 2004. Follow-up of the registered

patients was analyzed until 2005 [29]. Among these

patients 22,365 (24%) were diagnosed with atrophic

gastritis, 61,707 (67%) with IM, 7616 (8%) with

mild ⁄ moderate dysplasia and 562 (0.6%) with severe

dysplasia. Endoscopic and histopathologic follow-up

was performed in 26% of patients with atrophic gastri-

tis, 28% with IM, and 38% with mild ⁄ moderate dyspla-

sia compared to 61% with severe dysplasia (p < 0.001).

In the follow-up group, the annual incidence of GC

increased according to the severity of the mucosal alter-

ation present in 0.1% for patients with atrophic gastri-

tis, 0.25% with IM, 0.6% with mild ⁄ moderate dysplasia

and 6% with severe dysplasia. The resulting HR for

severe dysplasia was 40.14 (95% CI 32.2–50.1). Further

independent risk factors in the multivariate analysis

were male gender (HR 1.5; 95% CI 1.3–1.7) and age

(HR for 75–84 years 3.75; 95% CI 2.8–5.1) [29].

The cancer risk in patients with mild ⁄ moderate dys-

plasia was comparable to the risk for the development

of colorectal cancer after removal of colonic adenomas.

However, no recommendation for surveillance has been

proposed. So far, the best estimate for the regression in

histopathology scores can be calculated as a function of

the square of the time the patient is H. pylori negative

after eradication therapy [30]. There is an important

need for guidelines to determine at what intervals

patients will require endoscopic control.

Population-based Screening

Population-based screening and treatment of H. pylori

infection most likely represents the current best option

for primary prevention of GC, but several aspects need

to be considered, such as timing, methods and cost-

effectiveness in various regions of the world. Certain

populations, such as those in East Asia, have a high

incidence of GC compared to populations in Africa,

South Asia or Europe and this is probably linked with

certain H. pylori strains. In high risk populations, mainly

H. pylori with East Asian type CagA are present [31].

In a recent calculation for a high risk region in

China, an empirically calibrated model of GC was used

to estimate the reduction of lifetime cancer risk, life-

expectancy and screening, as well as treatment-related

costs [32]. Three options were considered: (i) single

Bornschein et al. Treatment and Prevention of Gastric Cancer 2009

ª 2009 The Authors


lifetime screening at age 20, 30 or 40; (ii) single lifetime

screening followed by rescreening individuals with neg-

ative results, and (iii) universal treatment for H. pylori

infection at age 20, 30 or 40.

Screening and treatment in individuals at the age of

20 resulted in adequate reduction of the lifetime risk

for GC (males: 14.5%; females 26.6%) with costs below

US$1500 per life year saved. By application of universal

treatment, the risk reduction was even increased by

1.5% and 2.3%, respectively, but the incremental cost-

effectiveness rates exceeded US$2500 per life ⁄ year

saved. Assessing persons at an older age or rescreening

of negative individuals was not cost-effective. Results of

prospective trials on a global scale are needed to sup-

port these theoretical estimations.

Conclusion

The development of EMR ⁄ ESD enables a better curative

access with preservation of a good quality of life in

EGC. Palliative therapies for GC have still not achieved

an important breakthrough. In the development of

cost-effective primary prevention strategies the detec-

tion and treatment of H. pylori infection is the best

available option [33]. Treatment of the infection is

never too late as it also has the potential to prevent

recurrence of GC as well as development of metachro-

nous GC after endoscopic resection in some patients.


P. Malfertheiner is involved in advisory boards and lec-

tures with Astra Zeneca, Nycomed, Abbott and Novartis.

All other authors have declared no conflicts of interest.

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Treatment of Helicobacter pylori InfectionAnthony O’Connor,� Javier Gisbert* and Colm O’Morain�

�Department of Gastroenterology, Adelaide and Meath Hospital incorporating the National Children’s Hospital Tallaght, Trinity College Dublin,

Dublin, Ireland, *Gastroenterology Unit, La Princesa University Hospital, and Centre de Investigacion Biomedica en Red de Enfermedades Hepaticas

y Digestivas (CiBEREHD), Madrid, Spain

The treatment of Helicobacter pylori infection has posed

conundrums for clinicians since the bacterium was first

discovered in the early 1980s. The challenges go

beyond finding the correct combination of antibiotics

and manipulation of gastric pH to ensure eradication

and include avoiding the development of antimicrobial

resistance and ensuring compliance with prescribed

treatment. The Maastricht-III consensus stated that for

an eradication treatment regime to be considered effec-

tive, it would need to achieve an intention-to-treat

eradication rate in excess of 80% [1]. However, in

recent times, eradication rates in practice for many of

the most common regimens have fallen well below

these levels, generally due to the interwoven factors of

poor compliance with medication and antibiotic resis-

tance [2].

First-Line Therapy (Standard TripleTherapy)

The combination of a proton pump inhibitor (PPI) with

two antibiotics has been accepted as the first-line

therapy of choice for H. pylori eradication since a

randomized control trial in 1996 [3]. The recommended

first-line treatment in published guidelines in Europe

and North America reflect this with PPI combined with

amoxicillin and clarithromycin being the favored regi-

men [1,4]. However, some caveats have been applied to

these guidelines in recent years to take into account fall-

ing eradication rates. As recently as 2000, studies had

suggested eradication rates for standard triple therapy

were in excess of 90% [5]. However, more recent publi-

cations have suggested that this level has fallen alarm-

ingly to be around 70% in many areas and even as low

as 60% in some [6,7]. For instance, the most recent Ma-

astricht guidelines recommend substituting metronida-

zole for clarithromycin where resistance to that

antibiotic exceeds 15–20% [3]. Eradication rates with

this regime are 87.8% when strains are clarithromycin

sensitive and 18.3% when strains are clarithromycin

resistant [8]. The rate of clarithromycin resistance is

increasing, probably due to greater use of clarithromycin

in the community for respiratory tract infections [9,10].

An Italian study noted that rates of clarithromycin resis-

tance increased twofold in that country from 1990 to

2005 [11]. A similar phenomenon was noted in England

with resistance rates rising by 57% between 2002 and

2006 [12]. A study in the United States estimated clari-

thromycin resistance at 10.1% [13]. There has also been

debate as to the ideal duration of therapy. In recent

Keywords

Helicobacter, triple therapy, second-line

therapy, rescue therapy, susceptibility testing,

sequential therapy, compliance.

Reprint requests to: Colm O’Morain, Depart-

ment of Gastroenterology, Adelaide and Me-

ath Hospital incorporating the National

Children’s Hospital Tallaght, Trinity College

Dublin, Dublin, Ireland.


Abstract

This article aims to examine current best practice in the field reference to

first-line, second-line, rescue and emerging treatment regimens for Helico-

bater pylori eradication. The recommended first-line treatment in published

guidelines in Europe and North American is proton pump inhibitor com-

bined with amoxicillin and clarithromycin being the favoured regimen.

Rates of eradication with this regimen however are falling alarmingly due to

a combination of antibiotic resistance and poor compliance with therapy.

Bismuth based quadruple therapies and levofloxacin based regimes have

been shown to be effective second line regimens. Third-line options include

regimes based on rifabutin or furazolidone, but susceptibility testing is the

most rational option here, but is currently not used widely enough. Sequen-

tial therapy is promising but needs further study and validation outside of

Italy. Although the success of first line treatments is falling, if compliance is

good and a clear treatment paradigm adhered to, almost universal eradica-

tion rates can still be achieved. If compliance is not achievable, the problem

of antibiotic resistance will continue to beset any combination of drugs used

for H. pylori eradication.


ª 2009 The Authors




years, longer regimes have supplanted the previous 7-

day triple therapy. A meta-analysis in 2000 suggested a

14-day course of therapy showed 7–9% better cure regi-

mens than 7-day regimes [14]. However, another meta-

analysis differed, stating no clinical benefit from longer

courses of treatment, although the quality of some of

the studies included in this second meta-analysis has

been questioned [15]. The published guidelines are also

beginning to reflect this with the 2007 American Col-

lege of Gastroenterology guidelines recommending 10-

day treatment courses [4]. In addition, the most recent

Maastricht consensus stated that 14 days of treatment

had an advantage over 7 days in terms of eradication

[1].

Second-line Therapy

As first-line therapy has been noted to fail in approxi-

mately 20% of patients, the need for effective second-

line therapy is clear [16]. Many putative second-line

therapies are currently in use but the most common

are bismuth-based and levofloxacin-based therapies

[17]. Bismuth-based quadruple therapy consisting of a

PPI, bismuth, tetracycline, and metronidazole is

reported to have an efficacy of 76% in patients who

failed first-line therapy [18]. This is generally given for

10 days and taken four times daily, although a study of

a 14-day twice daily regimen reported 95% efficacy in

a mix of first-line and second-line patients which might

improve compliance and tolerability [19]. Bismuth-

based therapy has proved quite safe. A meta-analysis in

2008 showed no serious adverse event in 4763 patients

who received it. No statistically significant increase in

any side effect other than dark stools was illustrated

[20]. Levofloxacin-based therapy has grown in popular-

ity in recent years. A very recent Spanish multicenter

study of 300 patients who had failed first-line eradica-

tion therapy revealed 81% per-protocol and 77% inten-

tion-to-treat analysis when a 10-day levofloxacin-based

regimen was used, with good tolerability and a low side

effect rate of 22% [21]. Concerns have been expressed

regarding the development of fluoroquinolone resis-

tance when levofloxacin is used for Helicobacter eradica-

tion. A rapidly increasing rate of quinolone resistance

was reported in several countries: 15% in 2004 in

Japan, 16.8% in 2006 in Belgium, from 11.2% in 2003

to 22.1% in 2005 in Germany, from 3% in 1999 to

15% in 2004 in France and from 2.8% to 11.8%

between 1998 and 2003 in Taiwan [22–28]. The appar-

ently rapid rate at which fluoroquinolone resistant

seems to develop may limit the use of levofloxacin in

H. pylori eradication to second-line therapy. Another

concern exists regarding the side effects of the

fluoroquinolones. Tendonitis was reported in 704 of

46,000 patients receiving levofloxacin in one study

[29]. Other case reports have noted hepatotoxicity [30].

Third-line Therapy

Patients who fail both initial- and second-line therapy

for H. pylori pose an interesting and challenging ques-

tion [31]. Compliance must, of course, be questioned.

The options are to use further empiric regimes or to

employ treatments tailored to individual antibiotic sen-

sitivities. Two of the more common empiric rescue anti-

biotics used are rifabutin [32] and furizoladone [33].

Rifabutin is an antituberculous agent. For the eradica-

tion of H. pylori, it can be administered as PPI, rifabutin

(150 mg), amoxicillin (1 g), all twice daily for 14 days.

One study on rifabutin used for treatment failures

achieved 95% eradication rates as second-line therapy

and 68% eradication for third- or subsequent line ther-

apy [34]. Another study limited to patients who did not

achieve eradication with standard first-line or bismuth-

based second-line therapy revealed 79% eradication

rates based on intention-to-treat analysis [35]. These

results, however, have been contradicted somewhat by

the largest study to date on rifabutin as a third-line

treatment which estimated eradication rates as being

61% [36]. Rifabutin is limited as a treatment option by

a number of factors. Stocks are low in Europe. Also, ri-

fabutin is a useful tool in the treatment of the increas-

ingly problematic multi-drug resistant tuberculosis

infection. Greater use of rifabutin in the treatment of

H. pylori would likely result in the development of more

resistant strains of Mycobacterium tuberculosis. Also seri-

ous myelotoxicity and ocular adverse events have been

reported with this treatment [37,38]. Furazolidone is

also useful in treatment failures [39,40]. A study of 10

patients, in whom first-line, second-line and rifabutin-

based therapy had failed revealed 60% eradication

when it was used along with amoxicillin and PPI [41].

When this data was incorporated into a systematic

review of furazolidone-based treatments for third- and

subsequent line eradication therapy, they were shown

to be effective 65% of the time [42]. The other princi-

pal strategy for salvage therapy in H. pylori involves cul-

ture and antibiotic testing. This is a very logical

approach as H. pylori is a latent infection and therefore

has more in common with other latent conditions such

as tuberculosis and syphilis, where susceptibility testing

is routinely employed, than with conditions, such as a

urinary or a respiratory tract infection where empiric

antibiotic regimes are used. Susceptibility testing is

limited by the fact that in vivo resistance may not accu-

rately reflect in vitro resistance, notably with respect to

O’Connor et al. Treatment of H. pylori Infection

ª 2009 The Authors




metronidazole [43]. Currently such an approach is

mainly carried out in specialist centers with research

interest and expertise in the treatment of H. pylori [44];

however, greater interest in the pathogen and its effects

and the development of newer technologies in the field

of susceptibility testing will encourage this practice to

become more widespread. This will undoubtedly have

benefits and lead to more accurate prescribing and

lower rates of resistance [45].

Sequential Therapy

Sequential therapy has been proposed as an alternative

to standard triple therapy for the eradication of H. pylori

[46,47]. The primary goal of this regimen is to over-

come clarithromycin resistance. Hypothetically, during

the first part of therapy, amoxicillin weakens the bacte-

rial cell wall, which prevents the formation of the

channels that block clarithromycin from binding to the

bacterium and hence causes resistance to the antibiotic.

A meta-analysis published last year demonstrated that

eradication rates with sequential therapy are 93.4%

compared with 76.9% for standard triple therapy [48].

Sequential therapy is not affected by bacterial factors

(CagA status, bacterial load) and host factors (underly-

ing disease, smoking) which, until now, have predicted

the outcome of conventional eradication treatments.

Even when strains were clarithromycin resistant, the

eradication rate with sequential therapy was 82.2%

compared with 40.6% for triple therapy. So far, almost

all of the studies analyzing sequential therapy have

been performed in Italy and the sequential regimen has

been given equivalent status to standard 7–14 day triple

therapies as first-line treatment in the updated Italian

guidelines on H. pylori management, where it has been

stated that: ‘‘The Working Group advised the use of 7–

14 day triple therapies or a sequential therapy as first-

line treatment’’ [49]. The main drawback to sequential

therapy may lie in its complexity and how this may

affect compliance. Although the meta-analysis quoted

showed that compliance was superior amongst patients

receiving sequential therapy compared to standard tri-

ple therapy, it is counter-intuitive that a regimen which

lasts longer and involves a change in the medications

consumed in mid-course could enhance compliance

[50]. The advantages of sequential treatment over

triple-therapy need to be confirmed in randomized con-

trolled trials in different countries and settings before a

generalized change is recommended in first-line

H. pylori treatment. Accordingly, the American College of

Gastroenterology Guideline on the Management of Helico-

bacter pylori Infection states that ‘‘Sequential therapy

may provide an alternative to clarithromycin-based

triple therapy but requires validation within the United

States before it can be recommended as a first-line ther-

apy’’ [4], and the European Maastricht III Consensus

Report points out that ‘‘Sequential treatment deserves

further evaluation in different regions’’ [1]. The main

disadvantage of the sequential therapy regime is that it

is more complex for the patient, requiring a change of

medication in the middle of the treatment period.

Although it was not shown in the meta-analysis, it is

felt that this would likely have a negative impact on

compliance. Whether it is necessary to provide the

drugs sequentially or whether the four constituent

components of sequential therapy can be given concur-

rently is unclear. A meta-analysis published in 2009 on

this showed a per-protocol eradication level of 92.9%

and intention-to-treat eradication of 89.7% [51]. This

quadruple therapy appears to be an effective, safe and

well-tolerated alternative to triple therapy and is less

complex than sequential therapy, emphasizing that

studies comparing both alternatives are urgently

needed. It must be noted that although it is designed to

overcome clarithromycin resistance, clarithromycin is

central to both sequential and quadruple therapy and

would still be at the mercy of changes in patterns of

clarithromycin resistance which are probably primarily

contingent on the rates of prescription of clarithromycin

in the community for nongastrointestinal infections

[52]. In addition, there exists a body of opinion that

clarithromycin and metronidazole ought not be used

together for H. pylori eradication as those who fail to

have eradication will subsequently have at least single

and often double resistance [53]. Sequential therapy

undoubtedly shows promise but must be further evalu-

ated before it can supplant triple therapy in the existing

guidelines.

Adjuncts

Adjunctive therapies may offer some promise in H. pylori

eradication. Probiotics have been proposed as a useful

adjunct [54]. In one study undertaken in 2008, prescrib-

ing probiotics with H. pylori eradication therapy had no

effect on the side effect profile but did increase the rates

of eradication [55]. However, another study on concur-

rent probiotic administration suggested the inverse with

better side effect profiles but no increase in eradication

or rates of compliance with therapy [56]. Vaccination

has also been proposed as a means of controlling

H. pylori and the morbidity associated with it. The chal-

lenge model was first established in human volunteers

in 2004 [57] and subsequent to this, a vaccine is in

Phase I trials and its manufacturers claim it has been

shown to be safe and immunogenic in early trials [58].

Treatment of H. pylori Infection O’Connor et al.

ª 2009 The Authors






Conclusion

Establishing efficacious and acceptable treatment regi-

mens for patients infected with H. pylori continues to

pose problems for physician and patient alike. The

decrease in eradication rates needs to be firmly

addressed with evidence-based clinical practice. It is

probably the case, however, that the tools to success-

fully eradicate H. pylori are already present and that

they simply need to be properly utilized. It has been

repeatedly illustrated that, if compliance is good and a

clear treatment paradigm adhered to, very high eradica-

tion rates can be achieved. For example, a study pub-

lished in 2008 in a Finnish tertiary referral centre

revealed 100% eradication in 644 consecutive patients

where compliance was ensured and patients were trea-

ted with standard first- and second-line therapies as per

the Maastricht guidelines and third-line rescue therapy

was tailored to antibiotic susceptibility [59]. Another

study in Greece published earlier this year found 98.1%

eradication rates when the Maastricht-III guidelines

were implemented with empiric therapy used for third-

line patients [60]. Another 2008 study evaluated the effi-

cacy of different ‘rescue’ therapies empirically prescribed

during 10 years to 500 patients in whom at least one

eradication regimen had failed to cure H. pylori infection.

The authors concluded that it is possible to construct an

overall treatment strategy to maximize H. pylori eradica-

tion, on the basis of administration of four consecutive

empirical regimens [61]. The key factors in these studies

were an awareness of the importance of compliance and

the provision of structured aftercare and follow-up pro-

grams to ensure eradication. It has been proven that

such measures can improve compliance [62]. It is very

likely the case that empowering patients to achieve high

levels of compliance is what accounts for the impressive

eradication rates in centers where patient follow-up is

structured and comprehensive [63]. While it is impor-

tant to develop new regimes to overcome the problems

of resistance, a need also exists to work as efficiently as

possible with our current regimes and facilitate patient

compliance. If compliance is not achievable, the prob-

lem of antibiotic resistance will continue to beset any

combination of drugs used for H. pylori eradication.


The authors have delcared no conflicts of interest.

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Helicobacter pylori Infection in PediatricsAngelika Kindermann* and Ana I. Lopes�

*Department of Pediatric Gastroenterology, Emma’s Children’s Hospital, University of Amsterdam, Amsterdam, The Netherlands, �Gastroenterology

Unit, Department of Pediatrics, University Hospital Santa Maria, Lisbon, Portugal

Pathogenesis

Whereas there is evidence for a role of genetic markers

on disease severity in adults, in children this association

is not clear. Ko et al. found no association between

cagA, vacA, and iceA with gastritis severity in Korea [1].

On the other hand, Oleastro et al. suggested the homB

gene as a new putative virulence marker highly associ-

ated with peptic ulcer disease (PUD) in children and

adults. HomB also correlated with the presence of cagA,

babA2, vacAs1, hopQI, and oipA and seemed involved in

Helicobacter pylori adherence and in inflammatory

response [2].

To determine whether host gene polymorphisms cod-

ing for Toll-like receptors (TLR) influence the immune

response to the infection, Moura et al. studied TLR2,

TLR4, and TLR5 polymorphisms in a large cohort of

children. None of them were associated with H. pylori

infection or duodenal ulcer disease. Otherwise, the

presence of TLR4 was associated with infection by cagA-

positive strains and with increased levels of interleukin

(IL)-8 and -10 [3]. This might contribute to more

severe consequences of the infection in adulthood. A

Polish group found significantly higher IL-18, IL-8, and

IL-1b transcript levels and macrophage number in the

antral mucosa of H. pylori-infected children than in

H. pylori-negative children. They also observed a signifi-

cant correlation between macrophage number and his-

tological parameters of gastritis [4]. Another group from

Turkey found that expression of a-defensin was signifi-

cantly higher in H. pylori-infected children and associ-

ated with higher grades of inflammation and neutrophil

density [5]. Leach et al. from Canada showed a high

expression of the inflammatory S100 calgranulin pro-

teins in H. pylori-infected mucosa, correlating with the

severity of gastritis, and its absence in normal gastric

mucosa [6].

Czaja et al. found that the mean fasting serum gastrin

level was higher in children with H. pylori-associated

gastritis compared to H. pylori-negative cases, with or

without gastritis. In contrast to earlier studies suggest-

ing that a decrease in the number of somatostatin-

producing D cells with an unchanged number of

gastrin-producing G cells may lead to PUD, these

authors found no difference in G-cell density and D-cell

density, regardless of the presence or absence of gastritis

or H. pylori infection [7].

Prevalence

A cross-sectional population-based study of H. pylori

infection prevalence was conducted on 2480 Chinese

Keywords

Children, prevalence, recurrent abdominal

pain, anemia, stool antigen test, treatment.

Reprint requests to: Ana I. Lopes, MD, PhD,

Gastroenterology Unit, Department of Pediat-

rics, University Hospital Santa Maria, Avenida

Professor Egas Moniz, 1649-035 Lisbon,

Portugal. E-mail: [email protected]

Abstract

This review summarizes the articles published on Helicobacter pylori infection

in children between April 2008 and March 2009. Recent evidence highlights

the decreasing prevalence trend of H. pylori infection and supports both int-

rafamilial and extrafamilial transmission. The association with various symp-

toms is still being debated. Interestingly, H. pylori infection seems inversely

associated with allergic diseases. Monoclonal stool antigen tests are widely

used and accurate for the diagnosis of H. pylori infection, but less accurate in

young children. The new biprobe real-time PCR assay applied to stools

showed a poor sensitivity in children. Using the urea hydrolysis rate next to

the delta over baseline values, the 13C-urea breath test provides excellent

results for all age children, even for young children. Treatment of H. pylori

infection remains a challenge, considering suboptimal efficacy of current

therapy. Among emerging alternatives, sequential treatment appears promis-

ing. The adjunction of probiotics to conventional regimens, although elicit-

ing great interest, has shown limited therapeutic benefit.


ª 2009 The Authors


children (age 6–19 years) by using 13C-urea breath test

(UBT) [8]. A surprisingly low prevalence rate was

found, with an overall positivity of 13.1%, related to a

low educational level of the child’s mother (OR =

2.43), family history of gastric cancer (OR = 2.19), and

household member number >5 (OR = 1.57).

Siai et al. reported a 51.4% seroprevalence rate

(ELISA IgG) in Tunisian school children, 6 years old,

significantly related to household crowding, late bottle-

weaning, and bed-sharing [9].

Two studies evaluated H. pylori infection prevalence

by using a stool antigen test. Kori et al. observed a

prevalence rate of 24.7% in daycare children from

Israel with higher rates in the 13- to 60-month-old

group (32.5%) compared to the 3- to 12-month-old

group (7.1%), suggesting infection acquisition most

probably after the first year of life [10]. Yucel et al.

showed a 30.9% prevalence rate in asymptomatic Turk-

ish children (mean age 6.8 ± 3.0 years), related to a

low education level of child’s mother, adverse living

conditions, and a high number of siblings [11].

In India, Mishra et al. confirmed an increasing detec-

tion rate of H. pylori with age, by documenting positiv-

ity in saliva ⁄ stool samples by nested PCR in

2.1% ⁄ 4.25% (<5 years), 22.7% ⁄ 13.6% (6–10 years),

55.9% ⁄ 50% (11–16 years) of cases, respectively [12].

Three retrospective observational studies assessed

H. pylori infection prevalence in children submitted to

endoscopy. Elitsur et al. observed an overall infection

rate of 12.1% and a significant decrease in mean

annual infection rate in the last 6 years of the study

period in 1743 North American children over a 13-year

study period [13]. A decreasing prevalence from 60.4%

to 30.4% (first and last years of the study, respectively),

was reported by Kawakami et al. in Brazilian children

over a 10-year period [14]. In Canada, Segal et al. doc-

umented a very low overall infection rate (7.1%) [15].

Transmission

The transmission of H. pylori remains poorly under-

stood. New data including fingerprinting analysis stud-

ies, supports both intrafamilial (mostly mother-to-child)

and extrafamilial transmission. In a systematic review

which included a birth cohort study enrolling 1066

healthy newborns, Weyerman et al., using a monoclo-

nal stool antigen test, identified maternal infection as

the single significant risk factor (OR 13.0) for acquisi-

tion of infection in childhood [16]. Konno et al., using

DNA fingerprinting analysis of cultured H. pylori from

42 children and their infected family members, identi-

fied fingerprint patterns identical to those of at least

one family member in 76% of the children, with a

significantly higher rate of identity in the mothers’

patterns, compared to those of fathers (p < .01).

Mother-to-child transmission was thus suggested as the

most probable route of transmission of H. pylori [17].

Herrera et al. compared H. pylori genotypes (cultures

and ⁄ or DNAs obtained by the string test) from members

of low income families in Peru [18]. Interestingly, in

70% of the cases, mother–child strain pairs did not

match, nor did most strains from siblings or other fam-

ily members, thus further suggesting the possibility of

community acquisition of H. pylori infection.

Symptoms

The association with many symptoms is still a subject of

great debate.

Recurrent Abdominal Pain

During the last year, there was just one study concern-

ing this topic. In agreement with most previous studies,

Masoodpoor et al. found no relationship between recur-

rent abdominal pain and H. pylori infection in children.

The prevalence of H. pylori infection in children with

RAP and in healthy children in the age range of

12–15 years was similar [19].

Peptic Ulcer and Reflux Disease

Houben et al. analyzed retrospectively 76 patients who

were admitted to the hospital with signs of acute upper

gastrointestinal bleeding. Helicobacter pylori was identi-

fied in 55% of these patients and in 90% a duodenal

ulcer was found. This shows a strong relation between

gastrointestinal bleeding because of duodenal ulcer dis-

ease and H. pylori infection in childhood [20].

There were no new studies regarding gastroesopha-

geal reflux disease nor nonulcer dyspepsia during this

period.

Extra-gastrointestinal Manifestations

During this period many studies continued to investi-

gate the relationship between H. pylori infection and

extraintestinal manifestations such as iron deficiency

anemia (IDA), growth failure, asthma, atopy and vari-

ous other conditions.

Iron Deficiency Anemia

Fagan et al. concluded that H. pylori plays a casual role

in hematological outcomes of children. They followed

children after H. pylori eradication treatment for a

Kindermann and Lopes H. pylori in Pediatrics

ª 2009 The Authors


period of 40 months. They found a lower prevalence of

iron deficiency and IDA in H. pylori-negative children

compared with H. pylori-positive children [21]. On the

other hand, no association between H. pylori infection

and IDA was seen in three other studies [22–24]. It is

indeed difficult to distinguish between anemia due to

infection with H. pylori and to other confounding fac-

tors, such as poor nutritional status or another underly-

ing disease.

Growth Failure

There is ongoing discussion about the association

between H. pylori and growth retardation. In the past,

possible mechanisms such as malabsorption and

decreased appetite were mentioned as a possible cause

of growth failure. During the last year, two studies

were added on this subject. Both papers did not support

a role of H. pylori on growth failure in children [25,26].

Allergy

In developed countries, allergies have become more

prevalent in recent decades, whereas the prevalence of

H. pylori has been decreasing in these countries. This

gives rise to search about the association between this

micro-organism and allergies.

Chen & Blaser carried out a cross-sectional analysis on

the data from 7412 pediatric participants in the National

Health and Nutrition Examination Survey (NHANES) in

the U.S., to assess the association between H. pylori

infection and childhood asthma. They found that

H. pylori seropositivity was inversely associated with

asthma, recent wheezing, allergic rhinitis, dermatitis,

eczema, and rash [27]. A second study confirmed a low

rate of H. pylori antibodies in children with bronchial

asthma [28]. These findings are intriguing and incite

thought about research in this field, including asthma

prevention. Cam et al. investigated immune responses

(T-helper cell function) in H. pylori infected children and

compared the cytokine responses in the atopic and

nonatopic group. The frequency of atopy was lower in

the H. pylori-infected group (31.9 vs 48.1%), whereas

atopic symptoms were similar between infected and

noninfected children. Their results demonstrated a

counteractive cytokine interaction between H. pylori

infection and atopy, but it did not protect against atopy

[29].

Atherosclerosis

Helicobacter pylori infection has been proposed to play a

role in the development of atherosclerosis preceded by

endothelial dysfunction. Coskun et al. found no early

findings of atherosclerosis in H. pylori infected children

using noninvasive techniques such as Doppler ultraso-

nography [30].

Diagnostic Tests

Besides invasive diagnostic methods (histology, culture,

and rapid urease testing following endoscopy) which

are still considered the ‘‘gold standards,’’ there are still

no 100% specific and sensitive noninvasive tests for the

diagnosis of H. pylori in children. Especially in infants,

noninvasive tests are less accurate. In a high prevalence

country, such as Turkey, 26.3% of all children younger

than 2 years of age who underwent endoscopy were

H. pylori-positive. Most of them (65%) already showed

histopathologic abnormalities such as gastritis [31].

In 2005, the Canadian Consensus group concluded

that 13C-UBT is the best available and most reliable

noninvasive test in children, but it is far less accurate in

younger children [32]. Two studies on UBT were pub-

lished during the last year measuring the urea hydroly-

sis rate (UHR) next to the delta over baseline values

(DOB). Both were able to show that using UHR next to

DOB provided excellent results for children of all ages,

resulting in less false positive results in children under

the age of 6 years [33,34].

Monoclonal stool antigen tests are widely used and

accurate for the diagnosis of H. pylori infection in chil-

dren, but their use in young children remains contro-

versial. Ritchie et al. found a very low sensitivity (55%)

and specificity (68%) in children from 4 months to

2 years [35]. Other studies have investigated the accu-

racy of rapid immunochromographic stool antigen tests

(Rapid HpSA) with discrepant results. While Yang et al.

and Cardenas et al. found high sensitivities (94.6 and

100%) and specificities (98.4 and 100%) [36,37], Kulo-

glu et al. found far less accurate results (pre- and post-

treatment: specificity 92.3 and 100%, respectively;

sensitivity 65 and 60%, respectively) [38]. Whereas in

adults the new biprobe real-time PCR assays applied to

stools showed excellent results, Falsafi et al. found a

reasonable specificity of 92.3%, but a poor sensitivity of

62.5% in children. However, this study also noted an

association between higher scores of H. pylori in histol-

ogy and more severe gastritis with positivity of stool

PCR [39]. This could explain the insufficient sensitivity

in children who, for the most part, have less severe

gastritis.

In addition to several studies published on serology,

Leal et al. published a meta-analysis on antibody-based

detection tests for the diagnosis of H. pylori in children

[40]. ELISA-IgG assays showed low sensitivity (79.2%)

H. pylori in Pediatrics Kindermann and Lopes

ª 2009 The Authors


but good specificity (92.4%). Commercially available

ELISA tests varied widely in performance. Western blot

tests showed a good overall performance (sensitivity

91.3% and specificity 89%). In-house ELISA with

whole-cell antigen tests showed the highest overall per-

formance (sensitivity 94% and specificity 96.4%). This

review showed the need for an evaluation of the sero-

logical test in the community in which it is be used.

Treatment

Francavilla et al. showed for the first time the superior-

ity of a 10-day sequential treatment in children com-

pared to standard treatment [41]. An overall

eradication rate of 85.2% was obtained, irrespective of

the presence of ulcer or cagA status [42].

A single-centre study from Turkey using a first-line

standard therapy (amoxicillin, clarithromycin and pro-

ton pump inhibitor (PPI)), did not show any significant

impact of the duration of treatment on eradication rate

(per-protocol analysis), 55.8 and 60.5%, in the 7- and

14-day group, respectively [43]. Furthermore, in the

subset of nonresponders, a second-line quadruple ther-

apy protocol comprising bismuth citrate, doxycycline,

metronidazole, and PPI (7 days) obtained a 64.6% erad-

ication rate. The contribution of a high background

resistance to clarithromycin and metronidazole to the

low global eradication rates obtained was admitted.

Nguyen et al. compared the efficacy of two 14-day

triple regimens including amoxicillin, lansoprazole and

clarithromycin or metronidazole in a randomized dou-

ble-blind trial and observed an overall per-protocol

eradication performed in Vietnam similarly low in both

regimens, 62.1 and 54.7%, respectively [44].

Caristo et al. applied the fluorescent in-situ hybridiza-

tion test on pediatric gastric biopsy specimens during

two consecutive 5-year periods [45]. The study con-

firmed high sensitivity and specificity for the co-detec-

tion of sensitive and resistance strains, further showing

that in one-third of the cases with mixed infection,

resistant strains were only seen in the fundus. This

emphasizes the relevance of fundus biopsies.

Probiotics as alternative therapeutic options have

recently emerged. Until 2006, four controlled trials

evaluated the contribution of lactic acid bacteria and

Saccharomyces boulardii to H. pylori eradication and

reduction of treatment side-effects in children, with

conflicting results [46]. Hurduc et al. compared the effi-

cacy of standard eradication therapy (PPI, amoxicillin

and clarithromycin) for 7–10 days, plus S. boulardii,

250 mg b.i.d. for 4 weeks (intervention group) with

standard eradication therapy alone (control group)

[47]. Although the addition of S. boulardii to the

standard treatment offered only a 12% additional ther-

apeutic benefit (eradication rate of 80.9% in the control

group vs 93.3% in the intervention group) it signifi-

cantly reduced the incidence of side effects (30.9 vs

8.3%, respectively).

Gotteland et al. assessed the potential additive or syn-

ergistic effect of Lactobacillus johnsonii La1 plus cranberry

juice on the inhibition of H. pylori in a multicenter, ran-

domized, controlled, double-blind trial (3 weeks),

including asymptomatic children with infection con-

firmed by UBT. Eradication rates were significantly

lower in the control group (placebo juice ⁄ heat-killed

La1) (1.5%), compared to the intervention groups

(14.9, 16.9, and 22.9% in the placebo juice ⁄ La1, cran-

berry juice ⁄ heat-killed La1 and cranberry juice ⁄ La1

groups, respectively) [48], showing the absence of syn-

ergistic inhibitory effects of La1 plus cranberry on

H. pylori colonization.

Re-infection after successful eradication has also

received increased attention, as rates from 1.9 to 9.6%

have been reported in children, mostly attributed to

interfamilial transmission, with higher rates in develop-

ing countries [49].



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Helicobacters and Extragastric DiseasesRinaldo Pellicano,* Francesco Franceschi,� Giorgio Saracco,* Sharmila Fagoonee,� Davide Roccarina� andAntonio Gasbarrini�

*Department of Gastro-Hepatology, Molinette Hospital, Turin, Italy, �Internal Medicine, Catholic University of Rome, Rome, Italy, �Department of

Genetics, Biology and Biochemistry and Molecular Biotechnology Center, University of Turin, Turin, Italy

Since the latest decade, several studies have reported

on the link between chronic Helicobacter pylori or Heli-

cobacter species infection and a variety of extragastric

manifestations. These include ischemic heart disease

(IHD), liver diseases, skin diseases, blood disorders, and

others [1]. For several of these supposed associations,

the hypothesis of an etiological role has not yet been

fully investigated. This is due to a series of factors

linked to epidemiological aspects and to the disease per

se. To establish a causal role for an infectious agent in

the triggering of an event, Koch’s postulates must be

fulfilled, and to assess its implication in chronic disease,

Hill’s criteria must be addressed [2]. This is very diffi-

cult in the case of multifactorial diseases (for example

IHD) where the pathogenic mechanism cannot be

explained by only one cause. The epidemiological

investigations on potential extragastric manifestations of

H. pylori have frequently involved biases in control

selection, populations of small size, and presence of

confounders, like age and socioeconomic conditions.

Non-randomized, long-period, and large studies on the

follow-up of H. pylori eradication are scarce.

This review attempts to highlight the main reported

associations of H. pylori with extragastric manifestations

published between April 2008 and March 2009. The

association with esophageal diseases, due to its peculiar

features, will not be considered here. To identify all

publications, the medical term ‘‘Helicobacter’’ was used

in the MEDLINE search, the studies were selected

according to our aim. The last MEDLINE search was

dated March 21, 2009.

Heart and Vascular Diseases

Atherosclerotic Disease: IHD, Stroke, and

Peripheral Arterial Involvement

Two aspects of H. pylori, H. pylori involvement in ath-

erosclerotic disease were investigated: epidemiology and

pathogenesis. Regarding IHD, Aiello et al. evaluated the

socioeconomic and psycho-social gradients of pathogen

burden of four infectious agents (cytomegalovirus, her-

pes simplex virus-1, H. pylori and Chlamydia pneumoniae).

By including 999 healthy adults (mean age 59 years)

selected from the longitudinal study MESA (Multi-Eth-

nic Study of Atherosclerosis) aimed at identifying risk

factors for atherosclerosis, the authors showed that low

education (odds ratio (OR) 95%, confidence interval

(CI)): 1.37, 1.19–1.57) and a higher level of chronic

psycho-social stress were significant independent pre-

dictors of higher pathogen burden after adjustment for

covariates [3]. In a study from Turkey, the authors

focused on the seroprevalence of antibodies to H. pylori

in 73 patients with acute coronary syndrome, in 79

Keywords

Extragastric disease, IHD, cirrhosis, anemia,

idiopathic thrombocytopenic purpura.

Reprint requests to: Antonio Gasbarrini, MD,

Internal Medicine, Policlinico Gemelli, Univer-

sita Cattolica, Largo A. Gemelli, 8 – 00168

Rome, Italy. E-mail: [email protected]

Abstract

For two decades, Helicobacter pylori has been considered as the culprit in

many extragastric manifestations. However, for several of these supposed

associations the hypothesis of an etiological role has not yet been fully

investigated. This may be due to a series of factors linked to the epidemio-

logical features of the studies and to the diseases investigated. This review

attempts to highlight the main reported associations of H. pylori with extra-

gastric manifestations during the last year. The most convincing data arise in

the field of idiopathic thrombocytopenic purpura (ITP) and sideropenic ane-

mia. Long-term follow-up studies have shown that 50% of subjects with ITP

maintain a hematological response after H. pylori eradication. There is also

growing evidence of the role of H. pylori in other diseases, including ische-

mic heart disease even though results are not conclusive.


ª 2009 The Authors


patients with chronic stable angina, and in 22 control

subjects. They showed a significantly higher rate of pos-

itivity in patients than in controls (80.2 vs 54.5%,

p = .015). This difference was not evident between the

two groups of patients (86.3 vs 74.6%, respectively,

p > .05). C-reactive protein (CRP) was higher in sub-

jects with acute coronary syndrome than in those with

stable angina. However, no adjustment for socioeco-

nomic factors was made [4]. Similar results were

reported in India, where the seroprevalence of IgA and

IgG to H. pylori was significantly higher in 192 patients

with an incident or prevalent IHD with respect to

192 age- and sex-matched controls (for both p < .001).

The level of CRP was higher in subjects positive for

IgA, but not for IgG to H. pylori. On the basis of these

findings, the authors proposed that the association of

CRP with IgA to H. pylori be used as marker to target

the population at high risk for IHD [5]. Opposite results

were found in Iran where only serum antibodies to

C. pneumoniae were associated with late cardiac events

[6]. The study by Nikolopoulou et al. supported the

association between seropositivity for anti-H. pylori IgG

and coronary atherosclerosis, but not in its acute phase.

Furthermore, a potential causal role involving the over-

expression of tumor necrosis factor alpha (TNFa) and

vascular cell adhesion molecule-1 is not supported by

data [7]. To clarify if more virulent H. pylori strains

(expressing the CagA antigen) were involved in coro-

nary instability, Franceschi et al. performed a clinico-

pathological study and a meta-analysis on 4241 cases.

In their study, the authors showed that the anti-CagA

antibody titer was significantly higher in patients with

unstable angina compared to those with stable angina

(p < .02), normal coronary arteries (p < .01) or healthy

controls (p < .02). Moreover, anti-CagA antibodies rec-

ognized antigens localized inside coronary atheroscle-

rotic plaque in all specimens from both stable and

unstable patients. In the meta-analysis, seropositivity to

CagA was significantly associated with the occurrence

of acute coronary events (OR: 1.34, 95%CI, 1.15–1.58,

p = .0003) [8]. These findings support the potential role

of more virulent H. pylori strains in the acute phase of

IHD, a pathogenic model postulated on the basis of pre-

vious observations [9], and are not mutually exclusive

with the association of the infection with increased cir-

culating low-density lipoprotein cholesterol and triglyc-

eride levels [10]. The implications of more virulent

strains have been confirmed by a systematic review on

15 studies, which showed an OR: 2.11 (95% CI: 1.70–

2.62) for CagA seropositivity in the development of IHD

[11]. Against a local implication in IHD [9], there is the

evidence that, contrary to C. pneumoniae, Mycoplasma

pneumoniae, herpes simplex, and cytomegalovirus [12],

the infrequent detection of H. pylori DNA in the plaque

or in human arteries is possible [13,14]. Finally, Honda

et al. showed that H. pylori infection does not accelerate

the age-related progression of arteriosclerosis in a 4-

year follow-up [15].

Regarding stroke, in a primary care multicenter

study, a trend toward a higher prevalence of active

H. pylori infection in patients with ischemic stroke with

respect to controls (63 vs 54%) has been found. This

difference became significant when focusing on more

virulent strains (OR: 2.69, 95% CI: 1.37–5.30) [16].

This is in agreement with another study that an infec-

tion with CagA-positive H. pylori strains increases the

risk of recurrent atherosclerotic stroke with a hazard

ratio: 3.5 (95% CI: 1.9–6.4; p < .001) [17]. In the

review reported above, Zhang et al. found that the OR

of ischemic stroke for more virulent strains in 11 stud-

ies was 2.68 (95% CI: 2.20–3.27) [11]. In a prospective

study, performed in Pakistan on 326 subjects with a

2-year follow-up, three patients with active H. pylori

infection and one without infection had a stroke or

transient ischemic attack [18]. This difference was not

statistically significant. According to the authors larger,

prospective, randomized studies are needed.

Regarding other arteriopathies, Sawayama et al.

showed that the prevalence of H. pylori infection was

significantly higher in patients with peripheral arterial

disease than in controls (79.7 vs 44.8%, p < .01) [19].

Nyberg et al. investigated, with negative findings,

whether seropositivity for antibodies to H. pylori, or to a

burden of pathogens, including also C. pneumoniae,

cytomegalovirus, and herpes simplex virus, were related

to abdominal aortic aneurysm rupture [20]. In a preli-

minary investigation, the authors found a relationship

between H. pylori infection and cardiac syndrome X

[21]. This encourages more robust studies.

The involvement of the bacterium in metabolic dis-

turbance or inflammatory processes potentially related

to atherosclerotic disease has been explored. Gunji et al.

reported the seroprevalence of H. pylori infection in

healthy Japanese adults with and without metabolic

syndrome. A total of 38.6% among the former vs

28.0% among the latter (p < .001) were positive for

H. pylori antibodies. Furthermore, a number of meta-

bolic syndrome components (high systolic blood pres-

sure, low HDL-cholesterol, and high LDL-cholesterol

levels) were found significantly associated with H. pylori

seropositivity by multiple linear regression analysis

[22]. Helicobacter pylori has also been shown to affect

the vascular risks and complications in patients with

diabetes mellitus although data concerning the preva-

lence of H. pylori infection among these patients are

scanty and controversial [1]. Hamed et al. evaluated

Pellicano et al. Helicobacters and Extragastric Diseases

ª 2009 The Authors


the prevalence of H. pylori infection in patients with

diabetes mellitus, the association between the former

and diabetic vascular complications, and the influence

of the bacterium on atherosclerosis and inflammatory

biomarkers. The prevalence of H. pylori infection was

higher in patients compared to healthy controls. Carotid

artery intima-media thickness and inflammatory bio-

markers, as interleukin (IL)-6 and TNFa, were signifi-

cantly higher in infected patients. In the multivariate

analysis, blood glucose, triglycerides, erythrocytic sedi-

mentation rate, IL-6 and TNFa increased the OR for

atherothrombotic cause of cerebral ischemia in patients

with H. pylori infection [23]. In a study performed in

Japan, Ohnishi et al. showed in 130 patients with type

2 diabetes mellitus, without history of cardiovascular

disease, that H. pylori infection was associated with arte-

rial stiffness determined by pulse wave velocity [24].

Arrhythmias

Besides IHD, the possible association between H. pylori

infection and atrial fibrillation has been previously pub-

lished. In the last year, Platonov et al. reported, in a

case–control study, that permanent atrial fibrillation is

associated with elevated CRP levels, but the latter is not

the result of earlier infection with H. pylori or C. pneu-

moniae [25]. This is in agreement with the conclusion

of an editorial that, in light of the existing results, the

responsibility of H. pylori infection has been excluded in

the development of atrial fibrillation [26].

Respiratory and Ear, Nose, and Throat(E.N.T.) Diseases

A wide spectrum of manifestations linked to H. pylori

has been reported over the last year. Regarding asthma,

a study has shown that in children, the rate of H. pylori

antibodies is low and a significant difference could not

be detected in gastroesophageal reflux disease and

atopy between patients negative and positive for

H. pylori [27]. These findings are in agreement with

those of a cross-sectional analysis on 7412 participants

in a U.S. National survey. In this case, the authors

showed that H. pylori was inversely associated with a

history of asthma (95% CI, OR: 0.69, 0.45–1.06) [28].

Data from another U.S. study on 318 patients and 208

controls, also indicated that infection with more viru-

lent strains is inversely associated with asthma (95%

CI, OR:0.57, 0.36–0.89) and is associated with an older

age of asthma onset [29]. In an animal model, H. pylori

neutrophil-activating protein (HPNAP) reduced eosino-

philia, IgE, and Th2 cytokine levels in bronchoalveolar

lavage [30]. Whether HPNAP is a candidate for novel

strategies of prevention and treatment of allergic dis-

eases remains to be elucidated. In contrast, in a study

on 2437 randomly selected adults, Fullerton et al. failed

to demonstrate an association between H. pylori expo-

sure and chronic obstructive pulmonary disease, mea-

sures of allergic disease or decline in lung function

[31].

Colonization by H. pylori of the larynx has been

shown by Titiz et al. detected H. pylori DNA by PCR in

17 of 21 samples of patients operated on for laryngeal

squamous cell carcinoma. This DNA was present both

in normal and in tumoral tissue (76.2 vs 42.9%,

p = .039). On the contrary, H. pylori DNA was not

found in samples of 19 patients with benign laryngeal

pathology (p = .0001) [32]. Grbesa et al. also detected

H. pylori by Giemsa staining and nested-PCR in 13 of 82

(16%) samples from patients with laryngeal squamous

cell carcinoma [33]. Rezaii et al. showed that seroposi-

tivity for antibodies to H. pylori was significantly associ-

ated with laryngohypopharyngeal carcinoma [34]. In

an evidence-based meta-analysis, including five case–

control studies, laryngeal cancer risk for patients with

H. pylori infection was 2.03-fold higher (95% CI 1.28–

3.23) [35].

Zycinska et al., on the basis of a study on 36 patients

with pulmonary Wegener’s granulomatosis disease, sug-

gested that disease severity, prevalence of gastroduode-

nal lesions, and type and duration of treatment is

dependent upon H. pylori infection [36].

Regarding E.N.T, Fancy et al. observed, in pediatric

patients with otitis, more H. pylori DNA in adenoids of

patients than in those of controls (10 of 45 vs 6 of 37,

respectively, p = .49) [37]. Helicobacter pylori whole-cell

protein directly induced a macrophage migration inhibi-

tory factor, macrophage inflammatory protein 2, IL-1b,

and TNFa in middle ear epithelium in experimentally

infected mice. Moreover, severe proliferation of inflam-

matory cells was observed in the middle ear cavity

inoculated with H. pylori whole-cell protein [38]. In a

critical evaluation of the evidence of the relationship

between H. pylori and otitis media with effusion, the

authors, after examination of six original papers, with a

total of 203 patients and 27 controls, concluded that

there is actually poor proof of correlation [39].

Using urease test, Eyigor et al. found H. pylori positiv-

ity in three of 55 adenotonsillar tissue specimens of

patients with adenotonsillitis; however, none was posi-

tive when analyzed by PCR [40]. Similar results were

reported in pediatric patients with chronic tonsillitis, in

whom H. pylori did not colonize tonsil tissue [41].

Uncertainties persist concerning the association

between H. pylori and nasal polyps. While Cvorovic

et al. found H. pylori in six of 23 specimens by urease

Helicobacters and Extragastric Diseases Pellicano et al.

ª 2009 The Authors


test and by histochemical analysis with Giemsa staining

[42], Ozcan et al. detected H. pylori only in one of 25

specimens by each of these methods [43]. By using a

questionnaire, another group has shown, in a prospec-

tive study that H. pylori eradication chronic nonspecific

pharyngeal symptoms [44] but the mechanism of this

benefit should be better investigated. It probably

depends on acid inhibition rather than bacterium cure

per se. In fact, in the work by Toros et al., all patients

responded well to anti-reflux treatment but no correla-

tion was observed between H. pylori positivity and

symptoms [45]; in the study of Oridate et al., acid-

suppression therapy offered slower laryngopharyngeal

than esophageal symptom relief in laryngopharyngeal

reflux patients, and these differences were observed

independently from H. pylori status [46].

Hematologic Diseases

Idiopathic Thrombocytopenic Purpura (ITP)

After the pioneer report by Gasbarrini et al. [47], the

association between H. pylori and ITP obtained a formal

recognition in the Maastricht III Consensus report

which recommended that H. pylori infection should be

sought after and treated in patients with ITP [48].

During the last year, a Canadian prospective study

showed that in subjects with ITP, 48 months after

H. pylori eradication, 75% achieved a complete or a par-

tial response and 50% had a long-term ongoing

response [49]. Unfortunately, the small sample size

(four H. pylori-positive patients) limits the value of the

long-term follow-up. In a 7-year follow-up prospective

study conducted in Japan and including 30 subjects,

H. pylori eradication had a short-term efficacy in about

half of the H. pylori positive ITP patients [50]. In Korea,

in patients who did not respond to steroid and ⁄ or dana-

zol therapy for ITP, a combination therapy consisting of

H. pylori eradication plus immunosuppressive therapy

induced, after 6 months, a statistically higher response

than H. pylori eradication alone (66.7 vs 41.7%,

p = .345). Furthermore, the median response duration

was also longer in the former than in the latter group

(9 vs 3 months, p = .049) [51]. In contrast, in Australia,

four of nine ITP patients receiving eradication treatment

showed no response and underwent splenectomy, and

one relapsed after 3 months [52]. In a systematic

review, original articles reporting 15 or more total

patients were included. The authors found 25 studies

including 1555 patients, of whom 696 were evaluable

for the effect of H. pylori eradication on platelet count.

The complete response and overall response (at least

doubling of the basal count) were 42.7% (95% CI

31.8–53.9) and 50.3% (95% CI: 41.6–59), respectively.

The response rate tended to be higher in countries with

a high background prevalence of H. pylori infection (e.g.

Japan) and in patients with a milder degree of ITP [53].

Suzuki et al. extracted genomic DNA from peripheral

blood of H. pylori-positive ITP patients, who received

eradication treatment, and polymorphisms of IL-1b()31, )511), IL-1RN (long or short), TNFa ()308), and

TNFb (+252) were analyzed using PCR-restriction frag-

ment length polymorphism. There was no statistical dif-

ference in the frequencies of polymorphisms in IL-1b,

IL-1RN, and TNFa genes between responders and non-

responders. In contrast, the frequency of responders

was significantly higher in ITP patients with the TNFbG ⁄ G or G ⁄ A genotype than in those with the TNFb A ⁄ Agenotype. Therefore, the TNFb (+252) G ⁄ G or G ⁄ Agenotype may be considered as a good predictor of

platelet recovery in ITP patients after H. pylori eradica-

tion [54]. Asahi et al., in addition to confirming a sig-

nificant benefit from H. pylori eradication therapy in

patients with ITP (61 vs 0% among H. pylori negative),

observed that the recovery in platelet numbers was

mediated through a change in FccR balance toward the

inhibitory FccRIIB [55]. The best pathogenic model pos-

tulated is based on the antibody cross-reaction between

H. pylori ureB and human platelet GP IIIa [56].

Iron-deficiency Anemia (IDA)

Several seroepidemiologic studies have suggested a link

between H. pylori infection and IDA both in adults and

in children [1,57]. Moreover, pregnant women with

IDA had a significantly high prevalence of active

H. pylori infection [58].

Some investigators observed that cure of the bacterial

infection is followed by improvement and normaliza-

tion of mean cell volume, ferritin, and iron, with disap-

pearance of anemia [59]. During a follow-up of

40 months of children in rural Alaska, H. pylori eradica-

tion modestly reduced the prevalence of iron deficiency

and substantially reduced that of IDA [60]. Different

results have been achieved in Iran, where the fre-

quency of H. pylori infection in children with and with-

out anemia was similar (44 vs 50%). Among infected

children, 36% had anemia vs 42.2% (p = .59) in non-

infected ones [61]. Similar findings have been reported

in Northwest Turkey where authors hypothesized that

IDA might be explained by inadequate dietary intake

[62]. In Bangladeshi children, the authors observed a

significantly higher effect of iron-alone therapy com-

pared to anti-H. pylori therapy in improving iron status.

Even anti-H. pylori treatment compared with placebo

was not effective in improving iron status at day 90. No


ª 2009 The Authors





additional impact of combined anti-H. pylori plus iron

therapy over iron therapy alone was observed [63].

Muhsen & Cohen performed a systematic review and a

meta-analysis on H. pylori infection and iron stores.

Although very few studies controlled for multiple

potential confounders, most investigations reported a

positive association between H. pylori and decreased

body iron stores in symptomatic and asymptomatic

infected subjects. Helicobacter pylori may be considered a

risk factor for reduction of body iron stores, iron defi-

ciency and IDA, especially in high-risk groups. The

meta-analysis showed an increased risk of IDA (OR:

2.8; 95% CI: 1.9–4.2) as well as iron deficiency (OR:

1.38; 95% CI: 1.16–1.65) [64].

In an elegant study, Lee et al. evaluated the expres-

sion of iron-repressible outer membrane proteins

(IROMPs) in H. pylori and its association with IDA.

IROMPs were found in IDA strains under iron-restricted

conditions. Thus, since specific H. pylori strains associ-

ated with IDA demonstrated an advantage in iron

acquisition due to a higher expression of IROMPs, this

explains in part why some infected patients are more

prone to developing clinical IDA under restricted iron

conditions [65].

Monoclonal Gammopathy

Monoclonal gammopathy of unknown significance is

not associated with H. pylori infection. Soler et al. com-

pared the follow-up of 13 patients successfully treated

for H. pylori and 33 who were not cured. After a med-

ian follow-up of 19.6 months, the monoclonal compo-

nent was unchanged in eradicated, and not different

between eradicated patients compared to those with

H. pylori negativity ab initio (15.6 ± 9 vs 15.9 ± 1 vs

15.7 ± 1) [66].

Hepatobiliary Diseases

There is an increasing interest in Helicobacter species’

role in human liver diseases, even though results are

still inconclusive and supported by only a few papers,

mainly based on animal models [67]. Ito et al. by using

transmission electron microscopy, found in vitro differ-

ences between hepatocytes and gastric epithelial cells in

terms of both adherence and internalization of H. pylori.

Of interest is the fact that the bacterium adhered and

was internalized into hepatocytes more efficiently than

into gastric epithelial cells (p < .05). Once inside the

hepatocytes, both VacA-positive and -negative H. pylori

strains were able to produce vacuoles, interpreted as

endocytotic vesicles. b1-integrin was identified as a

probable receptor involved in internalization of H. pylori

into hepatocytes [68]. The same group observed that

H. pylori infection of hepatocytes causes disturbance of

apoptosis and DNA synthesis. In particular, infection

with more virulent strains resulted in cell arrest and

increased DNA fragmentation. The difference with the

less virulent strain employed (H. pylori 401C) was sig-

nificant [69]. Goo et al. induced lesions resembling

those of human primary biliary cirrhosis (PBC) in a

24-month-old male C57BL ⁄ 6 mouse infected with

H. pylori. Since the serum antivacuolating toxin IgG in

this mouse showed the highest value in the H. pylori-

infected group, the authors concluded that the increase

in vacuolating toxin caused by H. pylori infection may

be related to the development of PBC by molecular

mimicry [70].

Finally, an in vitro study showed that H. pylori signif-

icantly influenced human gallbladder epithelial cell

morphology, causing reduced cell growth, decreased

viability, and increased detachment. The damage was

more significant in cells treated in culture liquid ⁄ broth?

than in H. pylori sonicate extracts [71].

Diseases of the Large Intestine

The potential association between H. pylori infection

and colorectal diseases has been investigated in the last

year.

In the human colon, Helicobacter sp. DNA can be

found in 35% of cases [72]. However, its pathogenic

role, if any, remains to be elucidated.

Regarding the field of oncology, Zhao et al. per-

formed a meta-analysis on H. pylori and the risk of colo-

rectal cancer, including 13 studies. The summary OR

was 1.49 (95% CI: 1.17–1.91). Moreover, with the

method of fail-safe, the effect of publication bias was

small [73]. In a large study, including 685 subjects, the

authors found that neither hypergastrinemia nor sero-

logic evidence of H. pylori infection was associated with

an increased risk of recurrent adenoma development

[74]. Soylu et al. looked for the presence of H. pylori by

immunohistochemistry in samples of patients undergo-

ing polipectomy during colonoscopy. They found that

21.6% of all specimens were positive, with a percentage

of 25 in the case of cancer. The presence of H. pylori in

colon polyps did not yield any correlation with polyp

size, colonic localization or histopathologic type [75].

Gynecological Diseases and FertilityDisorders

Nausea and vomiting are very frequent complaints of

pregnant women and, when severe, may lead to

hyperemesis gravidarum (HG), characterized by weight


ª 2009 The Authors



loss, dehydration, acidosis from starvation, alkalosis

from loss of hydrochloric acid in vomitus, hypokalemia,

and transient hepatic dysfunction. While some seroepi-

demiologic studies showed that H. pylori infection is sig-

nificantly associated with HG, others did not [1].

Sandven et al. included in a case–control study, 244

women with HG and 244 pregnant women free of HG.

They observed that the presence of H. pylori increased

the risk of HG by more than twofold (OR: 2.42, 95% CI

1.64–3.57, p < .001). The association was significantly

more evident among Africans than in non-Africans

[76]. Pugliese et al., comparing 25 pre-eclamptic

women and 25 healthy parturient, have shown that the

former had a significantly higher H. pylori seropositivity

as well as a higher anti-CagA seropositivity. On the

contrary, they did not find a difference in IL-18 levels

between the two groups [77]. The benefit of H. pylori

eradication in patients with localized vulvodynia,

reported in a study with a small sample size, remains to

be better understood [78].

Regarding infertility, Collodel et al. showed that

infertile patients infected with H. pylori had a low sperm

quality compared to uninfected ones. A significant

reduction of sperm motility and fertility was observed,

particularly in CagA-positive patients, whereas apopto-

sis and necrosis were increased (p < .05). In these, the

mean values of TNFa levels were higher than those of

uninfected patients. The percentage of immaturity and

the related defective organelles did not seem to be

influenced by the presence of the bacterium [79]. Based

on a study in which both the anti-H. pylori antibodies

in serum and follicular fluids were measured, Kurotsu-

chi et al. hypothesized the presence of an antigenic

mimicry between the flagella of H. pylori and spermato-

zoa. Thus, antibodies produced against H. pylori flagella

may cross-react with spermatozoid flagella, increasing

the risk of infertility [80].

Neurologic and Psychiatric Diseases

In this field, in the last year more hypotheses and

reviews than original data have been published [81,82].

Among the latter, Kountouras et al. investigated the

benefit of H. pylori eradication in the management of

Alzheimer’s disease. They showed that the prevalence

of the bacterial infection was significantly higher in

patients than in age-matched controls (88 vs 46.7%,

p < .001). Helicobacter pylori eradication was obtained in

84.8% of the cases. After a 2-year follow-up, patients

who were cured of the infection had a significant

improvement in cognitive and functional status param-

eters (measured by specific scales) compared to those

who failed eradication [83]. Although it is difficult to

come to a definite conclusion on the casuality of the

relationship between H. pylori and Alzheimer’s disease,

and the consistency of an association based on pub-

lished studies is poor [84], this prospective study on a

small sample size provides the basis for well-designed

trials with larger populations.

Regarding Parkinson’s disease, Lee et al. showed that

infected patients had a longer L-dopa ‘‘onset’’ time and

a shorter ‘‘on-time’’ duration than uninfected patients

(p < .05). Helicobacter pylori eradication improved the

delay in the L-dopa ‘‘onset’’ time and the short ‘‘on-

time’’ duration. According to the authors, these data

demonstrate that the bacterium could interfere with

the absorption of L-dopa and provoke motor fluctua-

tions [85].

In a study on sera from 120 individuals affected by

neuropsychiatric lupus compared to those from 140

geographic controls, Zandman-Goddard et al. failed to

find an association between this disease and seropositiv-

ity for H. pylori antibodies [86].

Other Diseases

Confirming a previous study by Demir et al. [87], a

seroepidemiological investigation conducted on a ran-

dom sample of patients obtained from the previously

cited MESA study, did not show an association between

H. pylori infection and type 2 diabetes mellitus [88]. On

the contrary, in South America, Fernandini-Paredes

et al. found that in diabetic patients, glycosylated

hemoglobin levels were higher in infected than in

uninfected individuals (p = .03). The presence of the

bacterium was not associated with the response to the

diabetes mellitus treatment [89]. On a similar trend,

another group showed that H. pylori infected patients

had a significantly higher HOMA-IR level (diagnostic

standard of insulin resistance) when compared with

uninfected ones [90]. In a Japanese case report, the

authors reported the onset of type 1 diabetes mellitus

after H. pylori eradication [91]. The link with an auto-

immune mechanism remains unclear.

The circulating levels of ghrelin and leptin, two hor-

mones involved in body weight regulation and food

intake, in relation to H. pylori status in adult males have

been investigated. Chuang et al. observed that, before

H. pylori eradication, males had lower plasma ghrelin

levels than females (p < .001), but thereafter these lev-

els were similar. Such a gender difference was not evi-

dent for leptin levels [92]. Based on previous work

leading to the hypothesis that gastric H. pylori

colonization reduced circulating levels of leptin and

ghrelin, Roper et al. examined gastric, circulating, and

gastric juice levels of leptin and ghrelin in fasting


ª 2009 The Authors


https://www.researchgate.net/publication/51423026_Helicobacter_pylori_infection_and_motor_fluctuations_in_patients_with_Parkinson's_disease?el=1_x_8&enrichId=rgreq-1a5b08ae-5365-4116-a5e4-dd5643fe6066&enrichSource=Y292ZXJQYWdlOzI2NzcyMDk5O0FTOjk4NTA1MDU4MTYwNjQ1QDE0MDA0OTY4NDQ3MDE=

H. pylori-positive and -negative adult male subjects.

They showed that bacterial colonization was associated

with reduced circulating leptin levels, independent of

body mass index, and fundic ghrelin and leptin levels

were directly related. Levels of ghrelin in infected and

uninfected patients were similar. Ghrelin was present

in gastric juice over a large concentration range, and

was strongly correlated with gastric pH [93]. Gao et al.

observed that ghrelin concentration and ghrelin ⁄ obesta-

tin ratios were lower in H. pylori infected subjects than

in uninfected individuals [94]. Obestatin is a peptide

derived from preproghrelin and a physiologic opponent

to ghrelin. Interestingly, a group found that autoanti-

bodies against appetite-regulating peptide hormones

and neuropeptides were displayed by both healthy

humans and rats. The authors hypothesized a potential

link between gut microflora and appetite control [95].

Helicobacter pylori infection also seems to play a role

in conjunctival MALT lymphoma, for which a mecha-

nism similar to those of gastric MALT lymphoma has

been postulated [96], but not in glaucoma [97]. How-

ever, in the latter, Deshpande et al. found a higher se-

roprevalence of H. pylori IgG antibodies in patients with

primary open angle glaucoma compared with those

with pseudo-exfoliation glaucoma. This difference was

not evident upon analysis of the aqueous humor [98].

Choi et al. investigated the association between H. pylori

infection and Posner–Schlossman syndrome and found

that patients had a significantly higher seroprevalence

than controls (80 vs 56.2%, p = .014) [99].

As reported in the section on respiratory diseases, the

relationship between H. pylori infection and allergic dis-

eases is of increasing interest. All studies converge

towards a lack of positive association. In a large cohort

of 1953 Japanese university students, allergic diseases

were frequent and negatively associated with H. pylori

infection, especially in men [100]. This is in agreement

with other data [101,102]. Cam et al. observed a coun-

teractive Th1 and Th2 cytokine interaction between

H. pylori infection and atrophy [103]. This effect was not

protective, as opposed to conclusions made in a study

by Konturek et al., in which H. pylori infection was

associated with a decreased risk of food allergy [104].

Two nested case–control studies, including 104 [105]

and 87 [106] patients with pancreatic cancer, concluded

that H. pylori infection was not associated with the

development of this neoplasm. However, in the latter

investigation, an association was found in subjects who

were not smokers (OR: 3.81; 95% CI: 1.06–13.63)

[106].

With regard to dermatology, two intervention studies

have reported opposing results: H. pylori infection was not

associated with chronic urticaria in Germany [107] to the

contrary of India, where it has been suggested to include

the detection of this bacterium in the diagnostic work-up

of this disease [108]. The data by Abdel-Hafez et al. sup-

ported the hypothesis that H. pylori infection is not associ-

ated with alopecia areata [109]. The clearance of chronic

psoriasis after eradication therapy for H. pylori infection

has been the focus of a case report [110], and a case report

of Helicobacter cinaedi bacteremia in a previously healthy

person with cellulitis has also been described [111].

Ozel et al. observed that H. pylori eradication in

patients with familial Mediterranean fever led to a

decreased level of IL-6. These findings were not evident

in subjects homozygous for M694V mutation [112].

One case report has shown that anti-H. pylori treat-

ment can reduce, for a limited time, the recurrence of

oral aphthous ulcers in patients with Behcet’s syn-

drome [113]. It is important to understand if this tem-

porary benefit is due to the antibiotics or to H. pylori

eradication. A study on 23 patients with recurrent apht-

hous stomatitis showed that, after bacterial eradication,

there was a significant reduction of the recurrence and

amelioration time [114].

In a study on patients with sudden infant death syn-

drome, a significant prevalence of active H. pylori infec-

tion (by stool antigen) among infant death cases

compared with live controls was observed [115].


The authors have not declared any conflicts of interest.

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Helicobacter spp. Other Than Helicobacter pyloriArinze S. Okoli,* Armelle Menard�� and George L. Mendz*

*School of Medicine, Sydney, The University of Notre Dame Australia, Sydney, NSW, Australia, �INSERM U853, Bordeaux, France, �Universite Victor

Segalen Bordeaux 2, Bordeaux, France

Detection Methods, New Species andPrevalence in Animal Hosts

Molecular biology techniques provide invaluable tools

for the identification of Helicobacter spp., and the gold

standards in the detection of this genus are PCR and ⁄ or

sequencing. Evaluation of 16S rDNA gene-based PCR

assays for genus-level identification of Helicobacter spp.

in fecal samples demonstrated that five of the six meth-

ods examined were appropriate to eliminate PCR inhibi-

tors from the samples [1]. The method recommended in

this work was the application of PCR-differential gradi-

ent gel electrophoresis to fecal samples reported previ-

ously [2]. A PCR assay performed at the low annealing

temperature of 50 �C was developed to amplify a 261-bp

sequence spanning two of the seven variable regions in

the 16S rDNA gene of Helicobacter species [3]. This assay

combined with that of Bohr et al. [4] and sequencing

served to detect the presence of Helicobacters in com-

mercially bred mice supposedly free of Helicobacter

infection and to identify different species of Helicobacter

and their relative proportions infecting a single animal.

A real-time PCR assay that amplifies a fragment from

the 16S rDNA gene with subsequent species identifica-

tion by melting curve analysis using SYBR Green chem-

istry was designed to detect Helicobacter pullorum-like

organisms in chicken products [5]. The method allows

identification of these bacteria from healthy poultry

carcasses and caeca with a sensitivity of 1 CFU ⁄ g. It

confirmed the inadequacy of culture methods in the

detection of H. pullorum-like bacteria, and showed that

these organisms are common in healthy chickens with

a prevalence similar to that of Campylobacter jejuni [6].

As H. pullorum is considered to be zoonotic [7], this

PCR assay will serve to better assess the potential

human health risks posed by this bacterium.

Keywords

Enterohepatic Helicobacter spp., new

Helicobacter spp., detection of infection,

animal studies, prevention, eradication.

Reprint requests to: George L. Mendz, School

of Medicine, Sydney, The University of Notre

Dame Australia, 160 Oxford St. Darlinghurst,

NSW 2010, Australia.


Abstract

Non-H. pylori Helicobacter species (NHPHS) are associated with several impor-

tant human and animal diseases. In the past year research into this group of

bacteria has continued to gain attention, and novel species have been

described in new niches owing to improvements in detection methods. Poly-

merase chain reaction and ⁄ or sequencing remain the gold standard for the

detection of this genus. New insights into the pathogenesis of the NHPHS in

hepatobiliary, gastric, and intestinal diseases were gained. In particular, data

revealed interaction between hepatic steatosis and infectious hepatitis in

the development of hepatocellular carcinoma. Evidence of an association

between hepatitis C virus and Helicobacter spp. in hepatocarcinoma develop-

ment was also provided; and male sex hormone signaling appeared to

influence infectious hepatitis induced by Helicobacter hepaticus. More findings

support an association between Helicobacter heilmannii and gastric adenocarci-

noma; and in mice, mucins MUC4 and MUC5 but not MUC1 influence the

colonization and pathogenesis of Helicobacter felis. Data indicated that the

roles of the adaptive immune system in H. hepaticus-induced intestinal

tumorigenesis are different in the small and large intestines, and environ-

mental factors, such as bile acids may modulate H. hepaticus carcinogenic

potential. New reports in the prevention and eradication of NHPHS showed

a protective response against Helicobacter suis induced by vaccine administra-

tion, and a successful cross-foster rederivation method successfully

eradicated Helicobacter spp. from contaminated mice litters. Overall, the stud-

ies provided insights into the pathophysiology of Helicobacter species other

than Helicobacter pylori.


ª 2009 The Authors


Identification based on the 16S rDNA gene remains

the most frequently used method of detection; how-

ever, this gene may be a poor target owing to horizon-

tal transfer of 16S rDNA gene fragments and the

creation of mosaic molecules with loss of phylogenetic

information [8]. Other phylogenetically informative

genes such as the 23S rDNA could be employed for spe-

cies detection, but lack of sequence information limits

their potential use as targets. Indeed, among the enter-

ohepatic Helicobacters only the genome of Helicobacter

hepaticus has been fully sequenced and annotated. This

situation improved recently when the Broad Institute

Genome Sequence Platform performed the sequencing

of the genomes of Helicobacter bilis ATCC 43879, Helicob-

acter canadensis MIT 98-5491, Helicobacter cinaedi CCUG

18818, H. pullorum MIT 98-5489, and Helicobacter wing-

hamensis ATCC BAA-430. The final assembly and anno-

tation of these genomes has not been finalized, but the

non-assembled contigs are available in the NCBI micro-

bial genomes database.

Helicobacter suis has been accepted as a new gastric

Helicobacter taxon corresponding to type 1 H. heilmannii

[9]. The cells of the new species are tightly coiled spirals

with up to six turns, are motile and have bipolar tufts of

4–10 sheathed flagella blunt ended or ending in spherical

knobs [9]. An analysis of current data on the prevalence

of different species of non-H. pylori gastric Helicobacter

spp. in humans concluded that H. suis is a zoonotic agent

[10].

During the past year active investigations were con-

ducted on the presence of Helicobacter spp. in wild, hus-

bandry and pet animals, and the zoonotic potential of

Helicobacter spp. was evaluated owing to the potential

transmission of these bacteria from animals to humans.

Evidence for transmission of Helicobacter spp. in the

marine environment was obtained from pools of captive

mammals through PCR amplification and sequencing of

DNA. The gastric Helicobacter spp. detected were homolo-

gous to Helicobacter cetorum, and the enterohepatic Heli-

cobacter spp. were homologous to that isolated from a

Northern sea lion [11]. Helicobacter DNA with very high

homology to H. cetorum was detected in South American

fur seals indicating a wide host range for this Helicobacter

species initially isolated from whales and dolphins [12].

Helicobacter DNA was detected by PCR in river water

but not in soil in Sapporo, Japan, and its presence was

not related to that of Acanthamoeba, initially suspected

to be involved in Helicobacter survival [13]. A study of

the survival in water of seven gastric and enterohepatic

Helicobacter spp. did not show relationship between sur-

vival time in water and the niche occupied in the host,

and concluded that water would have similar roles in

the transmission of these species [14].

Several studies provided new information on Helicob-

acter colonization of mammals. For the first time Heli-

cobacter 16S rDNA was detected in the stomach of lynx

and foxes. Phylogenetic analyses grouped the isolates in

a cluster of H. heilmannii, Helicobacter salomonis, H. felis,

and Helicobacter bizzozeronii [15].

The prevalence of the new species Helicobacter equo-

rum in foals is age dependent, and the differences in

prevalence may be related to the presence of protective

maternal immunity in the very young foals [16].

No significant relationship was found in pet dogs

between chronic gastritis and Helicobacter infection

[17], and none of the common culturable species found

in the stomach of dogs including H. salomonis, H. felis,

H. bizzozeronii, and Helicobacter rappini was identified

[17]. Genus-specific Helicobacter-positive samples were

found in the saliva or feces of domestic and feral cats in

Korea; all samples were negative for the detection of

H. felis and H. pylori with species-specific probes [18].

Mixed infections of bacteria of the genera Campylobac-

ter, Helicobacter, and Anaerobiospirillum were found in

cats and dogs, but no significant statistical correlation

was found between the presence of diarrhea in either

cats or dogs and any isolate of the three genera, or the

various co-infection rates [19].

Helicobacter spp. detected by PCR amplification of

16S rDNA sequences in gastric washings of cats

showed high homology with those of H. heilmannii

and Helicobacter acynonychis [20]. The data suggest that

the proposed method was a valuable alternative tech-

nique to gastric biopsy. Since virtually all healthy

adult cats harbor Helicobacters in their gastric mucosa,

the possibility of cats as potential zoonotic agents of

H. heilmannii may have important public health

implications [20].

A review on the Helicobacter spp. infections of domes-

tic cats and dogs, farm animals, birds, and several wild

animals concluded that a common pathogenic mecha-

nism is the induction of a Th1-driven chronic inflam-

matory response mounted by the host against the

bacteria [21].

Diseases

Hepatobiliary Diseases

In the past year, the potential association between Heli-

cobacter spp. and diseases of the hepatobiliary tract con-

tinued to gain attention. Exposing the mouse

hepatocyte cell line H2.35 to H. pullorum sonicates

resulted in the hepatic cell death with morphological

features of necrosis, which occurred without caspase-3

activation [22]. The necrosis and eventual cell death

Other Helicobacters Okoli et al.

ª 2009 The Authors


was attributed to features characteristic of mitotic catas-

trophe, such as chromatin condensation, formation of

multinuclear distended cell micronucleation, and intra-

nuclear pseudoinclusions. The toxic factor in the soni-

cates had similar properties to those of the cytolethal

distending toxin secreted by H. pullorum [23,24].

The significance of the route of infection in H. hepati-

cus-induced hepatitis on the development of hepatocel-

lular carcinoma (HCC) was evaluated in a mouse model

by testing the hypothesis that perinatal exposure to H.

hepaticus is required for liver tumorigenesis [25]. Male

A ⁄ JCr mice infected with H. hepaticus by intragastric

treatment developed early hepatic changes after expo-

sure as well as marked increase in oxidative DNA dam-

age, but rarely any liver tumor, confirming earlier

reports [26,27]. In contrast, infection of A ⁄ JCr mice by

intraperitoneal injection of H. hepaticus before breeding

led to a high incidence of progressive hepatitis and a

significant number of multiple liver tumors, including

HCC, in the male offspring [25]. Contributing perinatal

factors include high sensitivity of neonatal liver to

tumor initiation, and ⁄ or modulation of immune

response by H. hepaticus or its toxins [25].

The oncogenic potential of H. hepaticus was further

illustrated using the susceptible mouse strains AB6F1

and B6AF1 derived from A ⁄ JCr and C57BL ⁄ 6NCr mice,

respectively [28]. The results suggested synergistic inter-

actions between hepatic steatosis and infectious hepati-

tis leading to HCC. The study provides an alternative

novel mouse model to investigate the association

between chronic microbial hepatitis and fatty liver in

the pathogenesis of liver cancer.

The influence of male sex hormone signaling on

infectious hepatitis induced by H. hepaticus was studied

in A ⁄ JCr mice [29]. At 4 months, castrated males and

animals receiving the competitive androgen receptor

antagonist flutamide had significantly less severe hepa-

titis than intact controls. The results raised the possibil-

ity of targeted hormonal therapy in young male

patients with infectious HCC.

In patients suffering from cholangiocarcinoma in

Thailand, a high cross reactivity was found between the

immune response to antigens obtained from H. pylori

and H. bilis to the extent that infection with the two

species cannot be distinguished serologically in most

subjects given the high prevalence of H. pylori in the

population studied [30]. Retrospective analyses of

serum antibody responses to cell surface proteins of

H. pullorum, H. bilis, H. hepaticus, and H. pylori suggested

an association between hepatitis C virus and the devel-

opment of HCC [31]. In contrast, no association was

found between Helicobacter spp. and biliary tract cancers

[32].

Gastric Diseases

Several studies on animals reported an association

between NHPHS and gastric diseases. Helicobacter heilman-

nii induced low-grade mucosa-associated lymphoid

tissue-type (MALT) lymphoma in mice increasing the

microcirculatory network surrounding the lymphoma

tissue [33]. In pet cats, H. heilmannii infection was associ-

ated with the development of gastritis and feline MALT

lymphoma [34]. In Iranian pet dogs, 93% of gastric

samples from animals with chronic gastritis or histo-

pathological changes in the gastric mucosa showed the

presence of Helicobacter spp. DNA [17].

Long-term H. heilmannii infection causes Th1 and Th2

immune response and increases mucosal thickness of

the stomach of C57BL ⁄ 6 mice [35]. Interestingly,

co-infection with H. bilis and H. pylori in this mouse

breed induce less severe gastritis, atrophy, mucous

metaplasia and hyperplasia, as well as less severe

intestinal metaplasia and dysplasia than H. pylori

infection alone [36]. The explanation suggested for the

reduced pathology is the migration of H. bilis-primed

immune regulatory cells in the lower bowel to the

gastric compartment and downregulation of the Th1

response.

The inflammation induced by H. pullorum in human

gastric and intestinal epithelial cell lines occurs via

bacterial adherence probably through lipopolysaccha-

ride-induced IL-8 secretion and is mediated by NF-jB

signaling [37].

Helicobacter felis attachment to gastric epithelial cells in

vitro was limited by the constitutively expressed glyco-

protein of gastric epithelium mucin MUC1 [38]. How-

ever, this mucin did not impact on the bacterial in vivo

colonization or pathogenesis in contrast to previous

observations with H. pylori. The results provide evidence

that H. felis colonizes and elicits inflammation in vivo

without direct association with the gastric mucosa, and

that in mice MUC1 shed by epithelial cells does not play

an important role in limiting colonization or pathogene-

sis of these non-adherent bacteria. The data suggest that

inflammation induced by H. felis infection results from

shed antigens which act independently of MUC1 [38].

Alterations in gastric mucins and trefoil factors

expression were investigated in two C57BL ⁄ 6 mouse

strains infected with H. felis as the disease provoked by

the presence of the bacterium progresses from gastritis

through dysplasia and metaplasia to gastric carcinoma.

The expression of mucins MUC4 and MUC5b increased,

and that of MUC5ac decreased; these changes were

similar to those found in the expression of human muc-

ins in H. pylori infection and the data suggested a role

for MUC4 and MUC5b in disease progression in mice.

Okoli et al. Other Helicobacters

ª 2009 The Authors


At variance with the observations in human disease,

other murine mucins and trefoil factors remained

unchanged [39].

Earlier reports of an association between H. heilman-

nii infection with gastric adenocarcinoma in humans

[40] were supported by the finding that biomarkers of

gastrointestinal cancers were elevated in patients suffer-

ing from gastric ulcer and cancer caused by H. heilman-

nii infection [41].

Intestinal Diseases

New insights have been obtained on the pathogenicity

of enterohepatic Helicobacter spp. employing mouse ani-

mal models. Co-infection of IL-10-deficient mice with

Helicobacter rodentium and Helicobacter typhlonius resulted

in more severe inflammatory bowel disease and neopla-

sia compared to the disease caused by either H. rodentium

or H. typhlonius [42]. The anti-inflammatory effects of an

antibiotic quadruple therapy in Helicobacter-infected

and non-infected IL-10) ⁄ ) mice with colitis suggested

that together with Helicobacter spp. other microbiota

drive the inflammatory process in these mice [42].

The urease activity of H. hepaticus is not involved in

cecal colonization of A ⁄ JCr male mice but is essential

for hepatic colonization and plays a crucial role in liver

inflammation and the severity of hepatitis [43]. Urease

activity was also associated with higher total IgG, Th1-

associated IgG2a and Th2-associated IgG1 in vivo. The

sequences of H. hepaticus UreA and UreB are very simi-

lar to those of H. pylori, but these proteins are not as

immunodominant as the H. pylori ones. The proline uti-

lization A (PutA) flavoenzyme of H. hepaticus is not

involved in the colonization efficiency of mice but

rather in inflammation, suggesting a role for proline

metabolism in H. hepaticus pathogenicity [44].

Studies of infection of BALB-Min and BALB-RagMin

mice with H. hepaticus indicated that the bacterium pro-

motes colon but not small intestine tumorigenesis, and

suggested that H. hepaticus promotion of tumors differs

between organs and does not necessarily correlate with

severity of inflammation [45]. The data also indicate

that the roles of the adaptive immune system in tumor-

igenesis are different in the small and large intestines.

Environmental factors, such as bile acids present in the

colon may modulate H. hepaticus carcinogenic potential.

Induction of colon cancer by H. hepaticus infection in

recombinase-activating gene-2-deficient Rag2) ⁄ )mice is

mediated by inflammation, increased TNF-a and nitric

oxide production (NO) production [46]. Concurrent

administration of an inducible nitric oxide synthase

inhibitor prevents NO production, abrogats epithelial

pathology and inhibits the onset of cancer [46].

The pathogenic potential of the H. hepaticus genomic

island HHGI1 was investigated in IL-10) ⁄ ) mice employ-

ing the isogenic bacterial mutant HhPAId1 that lacks 19

predicted genes within HHGI1. Helicobacter hepaticus

HhPAId1 did not cause typhlocolitis and hyperplasia in

IL-10) ⁄ ) mice [47]. Colonization levels of HhPAId1

were significantly higher in the cecum and similar in the

colon compared to wild-type H. hepaticus. The results

suggested that genes in HHGI1 contribute to the

pathogenicity of H. hepaticus, at least in part via upregula-

tion of proinflammatory mediators IFN-c, TNF-a, and IL-

17a [47].

Testing of the hypothesis that prior infection of

BALB ⁄ c-IL-10 null mouse with H. hepaticus increases

the incidence, multiplicity and ⁄ or progression of either

colitis-associated adenocarcinomas or colon tumors

induced by the organotropic carcinogen azoxymethane

(AOM) showed that prior infection with H. hepaticus

had no effect on the incidence of colitis-associated ade-

nocarcinomas, and resulted in a significant increase in

incidence but not multiplicity or progression, of AOM-

induced polypoid tumors [48]. On the other hand,

Munday et al. [48] found no association between Heli-

cobacter spp. infection and ovine small intestinal adeno-

carcinomas in New Zealand sheep [49].

Enterohepatic and gastric Helicobacter spp. were iden-

tified in fecal specimens from children diagnosed with

Crohn’s disease using PCR. The data suggest that in a

considerable proportion of children Helicobacter spp.

may have a pathogenic role in the development of the

disease [50].

The effects of Helicobacter infection on the toxicity of

the contaminant 2,3,7,8-tetrachloro-dibenzo-p-dioxin

was investigated in rats. The infection appeared to have

little influence on the susceptibility of rats for the com-

pound [51].

Prevention and Eradication of Infection

The effects of prophylactic immunization of BALB ⁄ c mice

against H. suis using whole cell lysate or supernatant

antigens from in vitro cultured bacteria showed that a

protective response against the bacterium can be

induced by administering a vaccine by intranasal route

with homologous (H. suis) as well as heterologous

(H. bizzozeronii and Helicobacter cynogatricus) antigens [52].

A successful cross-foster rederivation method was

devised for the elimination of Helicobacter spp. from con-

taminated mice litters [53].

Triple therapies using amoxicillin and two other

active components showed high efficacy against gastric

Helicobacter infections in C57BL ⁄ 6J mice [54] and cats

and dogs [55]. Amoxicillin resistance in H. hepaticus is


ª 2009 The Authors





dependent on the hefA gene expressing a TolC compo-

nent of a putative efflux system and its expression is

induced by bile acids [56].

The role of a second urease expressed by the gastric

Helicobacter acinonychis, H. felis, and Helicobacter mustelae

which does not require activation by accessory proteins

or nickel was proposed as an adaptation to the nickel-

restricted diet of carnivores [57].

Conclusions

This past year saw continued interest in the study of Heli-

cobacter species other than H. pylori that resulted in signif-

icant amount of information on improvement of

detection methods for the genus, identification of novel

species and prevalence in animal hosts, pathogenic

mechanisms of disease causation, prevention, and eradi-

cation of infection. Understandably, a greater proportion

of the investigations focused on the relationship between

Helicobacter spp. and diseases of the hepatobiliary and

gastrointestinal tracts, and provided new insights into

the potential mechanisms that buttress the association of

these bacteria with liver, stomach, and intestinal cancer.

Continued study in this area is required given the global

need to eradicate these malignancies.



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Helicobacter recognizes the critical role that has been established for Helico bacter pylori in peptic ulcer, gastric adenocarcinoma, and primary gastric lym phoma. New helicobacter species are now regularly being discovered.Helicobacter will publish, fi rst and foremost, primary research in a wide range of experimental, scientifi c and clinical areas including: bacterial physiology, molecular biology, epidemiology, animal models, pathogenesis, immune response, histopathology, gastric cancer, pediatrics, vaccines, diagnosis, and therapy. Man uscripts related to non-Helicobacter pylori species are also welcome.Publication Policy. Submission is considered on the condition that papers are previously unpublished, are not offered simultaneously elsewhere, that all authors (defi ned below) have read and approved the content, that all authors have declared all competing interests and the work has been conducted under internationally accepted ethical standards after relevant ethical review. Accepted manuscripts become the sole property of Helicobacter and may not be published elsewhere without consent from the publisher. All articles are subject to review by experienced referees. The Editor and Edito rial Board judge manuscripts suitable for publication, and decisions by the Editor are fi nal. Material accepted for publication is copyedited and typeset. An email alert is sent to the corresponding author to download e-proofs for fi nal review. The corresponding author is responsible for the entire content of the copyedited article. Extensive changes to the proofs will be charged to the contributors and could delay publication. Licensing and ethics Manuscripts will be considered for publication in the form of original articles, reviews, descriptions of techniques, brief communications, and letters. We accept only outstanding Case Reports. Papers accepted must be licensed for publication in Helicobacter and a completed Copyrigt Transfer Form (from http://www.wiley.com/go/ctaaglobal) / Confl ict of Interest Form (from http://www.blackwellpublishing.com/pdf/COI_HEL.pdf) must accompany every accepted paper. Authors will be required to transfer copyright of their paper to Wiley-Blackwell. Copyright transfer is a condition of publication, and papers will not be passed to the publisher for production unless copyright has been transferred.Manuscripts describing studies involving animals should comply with local/national guidelines governing the use of experimental animals and contain a statement indicating that the procedures have been approved by the appropriate regulatory body. Manuscripts concerned with human studies must contain state ments indicating that informed, written consent has been obtained, that studies have been performed according to the Declaration of Helsinki and that the pro cedures have been approved by a local ethics committee. If individuals might be identifi ed from a publication (e.g. from images) authors must obtain explicit con sent from the individual.

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(iii) Writing support was provided by [insert name of individual(s)] of [name company] and funded by [insert name of funding organisation].

All disclosures must be listed in the Acknowledgement section of the paper (see below).

Clinical trials registrationFollowing the statement published by the International Committee of Medical Journal Editors ((see http://icmje.org/clin_trials.pdf), we recom-mend that clinical trials published in Helicobacter be registered, and that submissions then include the registration number, name and URL of the trial register. Your study must also comply with CONSORT (see http://www.consort-statement.org/) guidelines. Such details should be included as part of the disclosure statement in the Acknowl edgement section. Submission of a paper implies that it reports unpublished work, except in abstract form, and is not being submitted simultaneously to another publication. Accepted manuscripts become the sole property of Helicobacter and may not be published elsewhere without consent from the publisher. All clinical research papers submitted from the United States of America, which involve human sub jects, must be accompanied by evidence of peer review. The date the project was approved, when available, should be included. All articles are subject to review by experienced referees. The Editors and Editorial Board judge manuscripts suit able for publication, and decisions by the Editors are fi nal. Material accepted for publication is copyedited and typeset. Proofs are sent to contributors for fi nal review. Contributors are responsible for the entire content of the copyedited arti cle. Extensive changes to the proofs will be charged to the contributors and could delay publication.

Manuscript Submission and Specifi cationManuscripts should be submitted online, through ScholarOne Maniscripts (formerly known as Manuscript Central) at http://mc.manuscriptcentral.com/hel. Authors will need to complete and return a Copy right Transfer Agreement form (from http://www. wiley.com/go/ctaaglobal) / Confl ict of Interest Form (from http://www.blackwellpublishing.com/pdf/COI_HEL.pdf) for all accepted manuscripts. Enquiries should be addressed to the Editorial Offi ce, email [email protected]. For guidance on how to submit articles to biomedical journals, please see Uniform Requirement for Manuscripts Submitted to Biomedical Journals, available from the International Committee of Medical Journal Editors (see http://icmje.org/clin_trials.pdf).

Page Charges. Helicobacter does not make the payment of page charges an absolute condition for the acceptance of a manuscript for publication. Authors will be charged at US$50 per printed black and white page, or US$550 per page including color illustrations. In exceptional cases, however, and upon appeal by the author before publication of the article, the Editor may waive the assessment.

Help for authorsPre-submission English-language editingAuthors for whom English is a second language may choose to have their man uscript professionally edited before submission to improve the English. A list of independent suppliers of editing services can be found at http://authorservices.wiley.com/bauthor/english_language.asp. All services are paid for and arranged by the author, and use of one of these services does not guarantee acceptance or preference for publication.

Electronic formatDigital artwork preparationThe Publisher’s website provides guidance on the preparation and submission of fi gures, links to useful websites and access to both the Wiley-Blackwell house and journal specifi c style guides: http://authorservices.wiley.com/bauthor/illustration.asp

ManuscriptsAuthors should observe the following guidelines.(1) Do not attempt to make your output approximate or match the

typeset page.(2) Be consistent in style (i.e. units, abbreviations). (See note about

abbrevia tions below.)(3) End paragraphs in a uniform manner, and in a different manner

from line endings within paragraphs. A frequently used paragraph ending is simply two carriage returns.

(4) Use ‘1’, not ‘el’ for ‘one’. Do not use ‘oh’ for zero. (5) Use double spacing in your document. Do not add extra line spacing

(except as a normal paragraph ending indication) above or below titles, subheads, or between paragraphs.

(6) Avoid using multiple spaces (horizontal) in your electronic manuscript. End sentences with only one space. Never use multiple spaces for horizontal positioning of text.

(7) Tables and fi gure captions should be prepared in separate fi les. The copy editor will indicate the placement of this material within the text.

(8) Do not divide words by hyphenating at line endings.(9) See reference style guide below. Do not attempt to incorporate small

capital letters when typing authors’ names.

Title pageThe fi rst text page should contain: 1. Title. 2. Running title (of no more than 45 characters, including spaces). 3. Full names and affi liations for all authors; 4. Full current postal and email address for the corresponding author, to whom email notifi cation to download the e-proofs will be sent, including also telephone and fax numbers for that person.Abbreviations should not be used in the abstract, and, with the exception of standard abbreviations, should not be used within the manuscript. As a general rule, abbreviations do not improve reader comprehension and should be avoided.

(Continued on last page)

GUIDELINES FOR AUTHORS

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Editorial Offi ceDavid Y. Graham, MD, Editor, Helicobacter, Digestive Diseases Section, Veterans Affairs Medical Center, 2002 Holcombe Blvd. (111D), Houston, TX 77030-4298, USA. Tel: (713) 795-0232; Fax: (713) 790-1040.

Information for SubscribersHelicobacter is published in six issues (Feb ruary, April, June, August, October and December) per year. Institutional subscription prices for 2009 are: Print & Online: US$1367 (Americas), US$1597 (Rest of World), €941 (Europe), £741 (UK). Prices are exclusive of tax. Australian GST, Canadian GST and European VAT will be applied at the appropri ate rates. For more information on current tax rates, please go to www.wiley.com, click on Help and follow the link through to Journal subscriptions. The institutional price includes online access to current content and all online back fi les to January 1st 1997, where available. For other pricing options, including access information and terms and conditions, please visit www.interscience.wiley.com/journals

Delivery Terms and Legal Title: Prices include delivery of print journals to the recipi ent’s address. Delivery terms are Delivered Duty Unpaid (DDU); the recipient is responsible for paying any import duty or taxes. Legal title passes to the customer on despatch by our dis tributors.

Back issues: Single issues from current and recent volumes are available at the current sin gle issue price from customerservices@ black wellpublishing.com. Earlier issues may be obtained from Periodicals Service Company, 11 Main Street, Germantown, NY 12526, USA. Tel: +1 518 537 4700, Fax: +1 518 537 5899, Email: [email protected]

Access to this journal is available free online within institutions in the developing world through the HINARI initiative with the WHO. For information, visit www.healthinternet work.org

Helicobacter is indexed by: Current Contents/Clinical Medicine, Medical Documentation Service, Research Alert, Sci Search, Science Citation Index, Medline.

PublisherHelicobacter is published by Blackwell Publishing Ltd: 9600 Garsington Road, Oxford OX4 2DQ, UK. Tel: +44 (0)1865 776868, Fax: +44 (0)1865 714591. Blackwell Publishing Ltd is now part of John Wiley & Sons. Journal Customer Services: For ordering information, claims and any enquiry concern ing your journal subscription please go to interscience.wiley.com/support or contact your nearest offi ce:Europe, Middle East and Africa: Email: [email protected]; Tel: +44 (0) 1865 778315.Americas: Email: [email protected]; Tel: +1 781 388 8598 or 1 800 835 6770 (Toll free in the USA & Canada).Asia Pacifi c: Email: [email protected]; Tel: +65 6511 8000.Production Editor: Daksha Rupawala ([email protected])Commercial reprints: Liz Day (email: [email protected])

Copyright and PhotocopyingJournal compilation © 2009 Blackwell Pub lishing Ltd. All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means with out the prior permission in writing from the copyright holder. Authorization to photocopy items for internal and personal use is granted by the

copyright holder for libraries and other users registered with their local Reproduction Rights Organisation (RRO), e.g. Copyright Clearance Center (CCC), 222 Rosewood Drive, Danvers, MA 01923, USA (www.copy right.com), provided the appropriate fee is paid directly to the RRO. This consent does not extend to other kinds of copying such as copying for general distribution for adver tising or promotional purposes, for creating new collective works or for resale. Special requests should be addressed to [email protected].

DisclaimerThe Publisher and Editors cannot be held responsible for errors or any consequences arising from the use of information con-tained in this journal; the views and opinions expressed do not necessarily refl ect those of the Publisher and Editors, neither does the publication of advertisements constitute any endorsement by the Publisher and Editors of the products advertised.

Periodical ID StatementHelicobacter (ISSN 1083-4389) is published bimonthly. US mailing agent: Mercury Air-freight International Inc., 365 Blair Road, Avenel, NJ 07001, USA. Periodical postage paid at Rahway, NJ. Postmaster: Send all address changes to Heli cobacter, Journal Customer Services, John Wiley & Sons Inc., 350 Main St., Malden, MA 02148-5020.

Printed in Malaysia by KHL Printing Co Sdn Btd.

ISSN 1083-4389 (Print)ISSN 1523-5378 (Online)

For submission instructions, subscription and all other information visit: www.black wellpublishing.com/hel

EditorDavid Y. Graham Houston

Leif P. Andersen Copenhagen

Masahiro Asaka Sapporo

John Atherton Nottingham

Anthony T. R. Axon Leeds

Franco Bazzoli Bologna

Douglas E. Berg St. Louis

Martin J. Blaser New York

Steven J. CzinnCleveland

Emad El-Omar Aberdeen

Peter B. Ernst Charlottesville

David Forman Leeds

James G. Fox Cambridge

Giovanni Gasbarrini Roma

Robert M. Genta Dallas

Javier P. Gisbert Madrid

Khean Lee Goh Kuala Lumpur

Alexander M. Hirschl Vienna

Jaw-Town Lin Taipei

José Carlos Machado Portugal

Varocha Mahachai Bangkok

Peter Malfertheiner Magdeburg

Barry J. MarshallNedlands

Francis Mégraud Bordeaux

Hazel Mitchell Sydney

Colm Antonine O’Morain Dublin

Julie Parsonnet Stanford

Richard M. Peek, Jr Nashville

H. Aziz Rani Jakarta

Pentti Sipponen Espoo

Joseph J. Y. Sung Hong Kong

Shu-Dong Xiao Shanghai

Jianzhong Zhang Beijing

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Volume 14 Supplement 1 September 2009 ISSN 1083-4389

Volum

e 14 Supplement 1 Septem

ber 2009 Pages 1–76

EDITOR: David Y. Graham, M.D.


Guest Editors: Francis Megrand and Peter Malfertheiner

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