REVIEW

Molecular Approaches and Modern Clinical Strategies for theManagement of Helicobacter pylori Infection in Japan
Hidekazu Suzuki,1 Toshihiro Nishizawa, 2 Hitoshi Tsugawa1 and Toshifumi Hibi1
1 Division of Gastroenterology and Hepatology, Department of Internal Medicine,

School of Medicine, Keio University, Tokyo, Japan

2 Division of Gastroenterology, National Hospital Organization, Tokyo Medical Center, Tokyo, Japan(Received for publication on February 13, 2012)(Revised for publication on February 28, 2012)(Accepted for publication on March 21, 2012)

Thirty years have passed since Warren and Marshall’s discovery of Helicobacter pylori (H.pylori). Since
then, not only peptic ulcer diseases and chronic gastritis but also non-cardia gastric cancers have beenrecognized as diseases originating from H. pylori infection. Several combination therapies consistingof multiple antibiotics have been developed as first- or second-line regimens to eradicate H. pylori infection. Our extensive experience in the field of anti-H. pylori medicine suggests that clinicians should
consider a possible role for unidentified, invisible pathogens to elucidate the pathogenesis and improve
the treatment of refractory diseases of unknown etiology. (doi: 10.2302/kjm.2012-0001-RE; Keio J Med
61 (4) : 109–119, December 2012)

Keywords: Helicobacter pylori, CagA, indication, diagnosis, eradication

Introduction
Classical bacteriology techniques have demonstrated
that Helicobacter pylori (H.pylori) is a gram-negative,
flagellated bacterium that expresses catalase and urease,
enzymes which help neutralize host responses and enableintragastric colonization. One of the early benefits of thisbasic research was the realization that virtually all strainsof H. pylori produce urease. This finding led to the development of accurate diagnostic tests, including the rapid
urease test and the urea breath test. A stool antigen test
has also emerged as an informative, noninvasive meansby which to diagnose infection via detection of bacterial
antigens.
Before the discovery of intragastric bacteria 30 years
ago, there were several reports of spiral gastric bacteria
dating back to 1890 (Table 1).1 The modern discovery of
H. pylori may have been delayed by Palmer’s declarationin 1954 that there were no microorganisms in the humanstomach. At that time, microorganisms were believed to
be unable to survive in the acidic gastric environment,

and intragastric spiral bacteria had been observed only
post-mortem;2 however, the presence of organisms in
the gastric mucosa had been described since the 1890s.
In Japan, Kasai and Kobayashi of the Kitasato Institutereported propagation of a spirochete-like organism, probably modern Helicobacter felis, from the stomachs of dogs
and cats but not from laboratory animals.3 They showedthat when rabbits infected with these spirochetes were
inoculated with virus fixe, marked hemorrhagic lesions
were produced in the gastric mucosa. In addition, spirochetes inoculated into the mouse gastric mucosa could be
eradicated by arsaminol (a classic equivalent of bismuth).4
Sixty five years later, scientists and doctors again attempted to treat gastric diseases associated with “spirochetes,”i.e., Helicobacter pylori infection. To commemorate theachievements of Kobayashi and coworkers in the Taisho
era, the Rokuzo Kobayashi Memorial Symposium on Helicobacter pylori was held on May 11th, 2002, in Kita-kan
Hall at Keio University Mita Campus, Tokyo, Japan.4,5

Reprint requests to: Hidekazu Suzuki, MD, PhD, Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of
Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan, E-mail: hsuzuki@a6.keio.jp
Copyright © 2012 by The Keio Journal of Medicine
109Suzuki H, et al: Molecular and Clinical Approaches for H. pylori

110

Table 1 History of Helicobacter research
1892
1906

Bizzozero
Krienitz

1919

Kasai and Kobayashi

1938
1954
1976

Doenges
Palmer
Lieber

1982

Warren and Marshall1994

NIH Consensus Development Conference

2002
2005

MarshallWarren and Marshall

Discovery of snake-like bodies in a dog stomach
Discovery of spirochete-like bodies in human stomach
Establishment of an animal model for H. felis infection and first Helicobacter eradication experiment
Isolation of spirochete-like organism from a human stomach
Denied the existence of microorganisms in the stomach
Reduction of gastric NH3 by ampicillin
Detection and isolation of Campylobacter-like organism from biopsied gastric specimen (Campylobacter pyloridis)
Recommendation of H. pylori eradication therapy for all patients with peptic ulcer
diseaseKeio Medical Science Prize
Nobel Prize in Physiology and Medicine

Pathogenesis
The most-investigated bacterial toxins of H. pylori areassociated with a segment of bacterial DNA referred to as
the cag pathogenicity island (cag PAI). Genes within thecag PAI encode proteins such as CagE, an ATPase thatdrives a type IV secretory apparatus enabling bacterial
macromolecules, especially the toxin CagA, to translocate into the host cell. The intact cag PAI of H. pylori
plays a significant role in the pathogenesis of chronic
gastritis in humans because the cag PAI is associatedwith increased chemokine expression and greater inflammation in gastric mucosal specimens.6 After the CagA
protein is injected into the host cell cytoplasm throughthe type IV secretory system, the EPIYA motif of CagA
is tyrosine-phosphorylated by host Src kinases, and thephosphorylated protein subsequently alters the gastricepithelial morphology. Src homology-2 domain-containing phosphatase 2 (SHP2) is able to bind the EPIYA-B,
EPIYA-C, and EPIYA-D motifs.7 Importantly, however,
CagA with the EPIYA-D motif has a higher binding affinity for SHP2 than does CagA with the EPIYA-C motif.8 The sequence flanking the tyrosine phosphorylationsite of the EPIYA-D motif (EPIYATIDF), but not thatflanking of the EPIYA-C motif (EPIYATIDD), perfectly
matches the consensus high-affinity binding sequence for
the SH2 domains of SHP2. The CagA proteins of strainsfrom distinct geographic populations appear to be phosphorylated to different degrees, resulting in graded effects on intracellular signaling.9
The biological half-life of intracellular CagA in gastricepithelial cells was recently reported to be approximately
200 min,10 and the activity of CagA as an epigenetic oncoprotein thus does not persist for a long period in any
single cell. Therefore, the risk of gastric cancer may be
determined by various factors that influence the stabilityof intracellular CagA. We recently reported that intracellular CagA is degraded by p53 degradation-induced au-

tophagy;11 however, a specific mutation in p53 increases
the intracellular stability of CagA by inhibiting its autophagic degradation. These findings suggest that disrupting the autophagic degradation of CagA increases
the risk of developing H. pylori-associated gastric cancer.
VacA is the second-most extensively studied H. pylori virulence factor. In addition to inducing vacuolation,
VacA also promotes several cellular activities, including
membrane channel formation and the release of cytochrome c from mitochondria and consequent apoptosis.
VacA can also specifically inhibit T-cell activation andproliferation.12 Further studies have confirmed that therisks for both cancer and peptic ulcers are associatedstrongly with the cagA/vacA s1m1 genotype but rarely
with the vacA s2m2 polymorphism.13
Oxygen-derived free radicals released from activatedneutrophils extravasated from microvascular beds are
considered to be potential toxic factors involved in H.
pylori-induced gastric mucosal injury because H. pylori
exhibits chemotactic activity for neutrophils.1,14 Neutrophil infiltration of the gastric mucosa leads to the development of the initial lesions of H. pylori-associated gastritis. Neutrophils express the enzyme myeloperoxidase,
which, in the presence of Cl−, produces the potent oxidant
hypochlorous anion (OCl−) from H2O2. This hypochlorous anion reacts with ammonia, produced from urea by
H. pylori-associated urease, to yield the lipophilic cytotoxic oxidant monochloramine (NH2Cl), which freely
penetrates biological membranes to oxidize intracellularcomponents (Fig. 1, left).1 8-Hydroxy-2-deoxyguanosine(8-OHdG), which results from the attack of a singlet hydroxyl or oxygen radical on guanine, is one of the formsof DNA damage most commonly induced by reactive
oxygen species. Patients with cagA-positive strains of H.
pylori have higher 8-OHdG levels than do cagA-negativeor H. pylori-negative patients.15 We previously reported
greater enhancement of neutrophil-derived gastric mucosal luminol-dependent chemiluminescence levels inKeio J Med 2012; 61 (4): 109–119

111

Fig. 1 Molecular approaches to Helicobacter pylori infection.
RdxA, NADPH nitroreductase; Fur, ferric uptake regulator; FecA1, a Fe3+-dicitrate transporter homolog.

cagA-positive patients than in cagA-negative patients.16
cagA-positive patients are characterized by greater oxidative DNA damage, both overall and at younger ages, inthe presence of multifocal atrophy.
Infection of gastric epithelial cells with H. pylori increases the accumulation of intracellular reactive oxygen
species (ROS). Increased intracellular oxidative stress
may play a role in the induction of programmed cell
death.17 We recently detected increased mitochondrial
ROS production in H. pylori-infected gastric epithelial
cells. This finding suggests that H. pylori infection alters
the mitochondria. In addition, the antioxidant N-acetyl
cysteine inhibited induction of autophagy and induced
the accumulation of intracellular CagA.11 These results
suggest that intracellular oxidative stress is involved in
the induction of autophagy required for CagA degradation (Fig. 1, lower right).11
Metronidazole, a major component of the second-line
H. pylori eradication regimen used in Japan, enters cellsby diffusion, and its antimicrobial toxicity depends on the
reduction of its nitro group to nitro anion radicals and thesubsequent generation of superoxide radicals (O2·−).18,19 In

detail, the NADPH nitroreductase of H. pylori reduces thenitro group of metronidazole to anion radicals that induce
oxidative stress and produce DNA strand breaks, causing rapid cell death.18 H. pylori expresses only a single
superoxide dismutase, the iron-cofactored enzyme SodB,
which has 53.5% identity with the Escherichia coli protein
FeSod.20 SodB prevents the interaction between iron andsuperoxide and also catalyzes the dismutation of superoxide (O2·−) into oxygen (O2) and hydroperoxide (H2O2).
The mRNA expression of sodB in H. pylori is directly
regulated by the ferric uptake regulator (Fur) protein.21
We recently found metronidazole-resistant strains of H.
pylori with amino acid mutations in Fur that significantly
reduced its binding affinity for its operator sequence inthe sodB promoter (Fur-Box), resulting in derepression of
sodB mRNA expression.22 In other words, metronidazole
resistance is due to enhanced scavenging activity of cytotoxic superoxide (O2·−), a major bactericidal componentof metronidazole, because of the derepression of H. pylori
SodB expression by mutant Fur (Fig. 1, left).22
Ferrous iron (Fe2+), an essential cofactor for many enzymes and metalloproteins, is necessary for the basal

112functions of all cells as well as for SodB activation.23 We
recently demonstrated that the FecA1 protein, a Fe3+-dicitrate transporter homolog, is essential for SodB activation
but not for the bioactivity of H. pylori.24 fecA1 mRNA
expression is derepressed in metronidazole-resistant H.
pylori strains with mutations in Fur.24 Deletion of fecA1
dramatically decreased the minimal inhibitory concentrations (MICs) of metronidazole for H. pylori strains with
Fur mutations,24 suggesting that the activation of SodB
by mutant Fur is supported by the FecA1-dependent Fe2+
supply system (Fig. 1, upper right).
Notably, in vitro study has shown that metronidazolesusceptible H. pylori became metronidazole resistantafter several passages on agar plates containing sub-inhibitory concentrations of metronidazole.25 From thesefindings, it can be readily assumed that metronidazolesusceptible H. pylori may become metronidazole resistant through repeated exposure to sub-inhibitory concentrations of metronidazole. Therefore, it is importantto understand the initial anti-metronidazole response of
metronidazole-susceptible H. pylori to prevent the acquisition of resistance and consequent increased incidence ofmetronidazole-resistant H. pylori.
Five families of multidrug efflux transporters havebeen described in bacteria.26 One of these five efflux systems, the RND family, has three components, namely, theinner membrane efflux proteins, a periplasmic efflux protein, and an outer membrane efflux protein (the TolC, or
TolC homolog protein).27 Four RND families have been
identified in H. pylori (HP0605 to HP0607; HefABC and
HP0971 to HP0969; HefDEF and HP1327 to HP1329;
HefGHI and HP1489 to HP1487), and these proteins arereportedly involved in the development of multidrug resistance.28 We recently explored the variations in transcription of the RND efflux pump systems in the initial
phases of the development of metronidazole resistancein vitro. In this study, the MICs of metronidazole for 9
out of 10 metronidazole-susceptible strains cultured onplates containing sub-inhibitory concentrations of metronidazole increased to levels similar to those for metronidazole-resistant strains. In the newly metronidazoleresistant strains, exposure to metronidazole significantly
increased the expression of the TolC efflux pump (hefA)
with no decrease in the metronidazole-reduction activity,
suggesting that overexpression of the TolC efflux pumphefA may be the first step in the acquisition of metronidazole resistance in H. pylori.29
H. pylori and gastric cancer – molecular approach
Gastric epithelium changes during the progression
from inflammation to cancer, with evidence of disruptionof normal epithelial cell differentiation and recruitment
of inflammatory cells.30 Coincident with the developmentof atrophic gastritis and intestinal metaplasia is the loss

Suzuki H, et al: Molecular and Clinical Approaches for H. pylori

of expression of the gastric morphogen sonic hedgehog(Shh).31,32 Loss of Shh is reportedly an early change incarcinogenesis of the gastric mucosa, prior to neoplastictransformation. We have demonstrated in humans thatthe suppression of Shh expression in the gastric mucosa
by H. pylori infection was restored after eradication of
the infection and that earlier eradication more fully restored Shh expression in the gastric mucosa.33,34
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that are involved in development,
cell proliferation, and immune responses. Recent studies
have shown that some miRNAs act as tumor suppressors
or oncogenes in gastric cancer.35 Some miRNAs, including miR-146, miR-155, miR-21, miR-27a, miR-106–93-25,
the miR-221–222 clusters, and the miR-200 family, arepossibly involved in H. pylori infection and associatedgastric cancers.36 miRNA expression profiling may be a
powerful tool for clinical cancer diagnosis, and regulation of miRNA expression could be a novel strategy forthe chemoprevention of human gastrointestinal cancers.37
H. pylori and gastric mucosa-associated lymphoidtissue lymphoma – molecular approach
H. pylori eradication has become widely accepted as an
initial treatment strategy for stage I gastric marginal zone
B cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type. H. pylori-positive low-grade gastric
MALT lymphoma regresses both endoscopically andhistopathologically after H. pylori eradication in 60%–
80% of such cases.38 The presence of a t(11;18)(q21;q21)translocation appears to be a major predictor of failure to
respond. This translocation is associated with an API2MALT1 fusion; the former is involved in the regulationof apoptosis, and the latter is a caspase-like protein withan as yet unknown biological function. The fusion causes
suppression of apoptosis. Several studies have reported
that MALT lymphomas with this translocation respond
only rarely or not at all to H. pylori eradication.
Methylation of the p16/INK4a gene was observed in
60% of MALT lymphomas; however, p16 gene methylation status did not correlate with the presence of API2MALT1 fusion or any other clinicopathological factor,
suggesting that aberrant methylation of the p16 genemight be an early event in MALT lymphomagenesis.39
Examination of the methylation profiles of eight CpG
islands, namely p15, p16, p73, hMLH1, DAPK, MINT1,
MINT2, and MINT31, revealed that more than four genes
were methylated in H. pylori-dependent MALT lymphomas while fewer than two genes were methylated in
non-H. pylori-dependent cases, indicating that the pathogenesis of gastric MALT lymphomas, including the aberrant DNA methylation pattern, may differ between H.
pylori-dependent and non-H. pylori-dependent cases.40
Aberrant DNA methylation thus plays critical roles in the

Keio J Med 2012; 61 (4): 109–119

113

Fig. 2 New disease entity concept clarifying the difference between H. pylori-associated and non-H. pylori-associated dyspepsia.48,53

pathogenesis of gastric MALT lymphomas, and determination of the DNA methylation pattern may hold promise
as a clinical tool for surveillance of gastric MALT lymphomas.
H. pylori and idiopathic thrombocytopenia purpura –molecular approach
Idiopathic thrombocytopenic purpura (ITP) is a bleeding disorder in which platelet-specific autoantibodies
cause platelets to be lost. In a subset of H. pylori-infectedpatients with ITP, the number of platelets recovers after
the eradication of H. pylori.41,42 According to our previous research into the role of H. pylori infection in thepathogenesis of ITP,43 a significant increase in platelet
numbers was observed after H. pylori eradication in 61%
of patients. In this study, at baseline, monocytes from H.
pylori-positive patients exhibited an enhanced phagocytic capacity and low levels of the inhibitory Fcγ receptor
IIB (FcγRIIB). This activated monocyte phenotype wassuppressed 1 week after the start of the H. pylori eradication regimen, and this suppression was followed by
improvements in other autoimmune and platelet kinetic
parameters. H. pylori infection was also associated withan altered monocyte FcγR balance in individuals without
ITP; a similar result was also found in mice. Our findings
strongly suggest that the recovery in platelet numbers observed in ITP patients after H. pylori eradication is mediated through a shift of the FcγR balance toward inhibitory
FcγRIIB.
Veneri et al. reported that the host factors HLADRB111,
HLA-DRB 114, and HLA-DQB 103 occurred at high frequencies in patients with H. pylori-positive ITP, while thefrequency of the host factor HLA-DRB 103 was higher
in the H. pylori-negative ITP group than in the H. pylo-

ri-positive ITP group. HLA-DQB 103 showed favorable
platelet reactivity after H. pylori eradication therapy in H.
pylori-positive ITP patients.44
Functional dyspepsia and H. pylori infection
Functional dyspepsia (FD) is a condition in which upper abdominal symptoms occur in the absence of any
explanatory organic disease.45 H. pylori infection is not
an exclusion criterion for FD under the current Rome III
classification.45 However, recent advances in basic andclinical research have revealed that H. pylori infection
plays an important role in the development of gastroduodenal dysmotility and hypersensitivity and also in dyspeptic symptoms.46–48 For example, the authors revealed
that in mice, chronic H. pylori infection downregulates
expression of muscle-specific miRNAs (miR-1, miR-133a,and miR-133b) and upregulates expression of their targets, such as histone deacetylase 4 and serum response
factor. These transcriptional changes cause hyperplasiaof the muscular layer of the stomach and gastric emptying deregulation.49 In addition, H. pylori eradication
therapy appears to have a small but statistically significant effect on FD.50,51 Therefore, H. pylori-associateddyspepsia (HpD) should be excluded from the umbrella
category of FD to better elucidate the pathophysiological mechanisms of FD and to establish more precise diagnostic markers or criteria (Fig. 2).46,48,52 In Japan, fornational insurance claim purposes, H. pylori-positive
histological gastritis, NSAID-induced gastritis, and neurotic gastritis (or stress-induced gastritis) have all beencombined under the diagnosis of “chronic gastritis” andhave therefore been treated with the same drugs. Of thesegroups of diseases, FD would be categorized as classicalstress-induced gastritis. However, as each type of gas-

114

Suzuki H, et al: Molecular and Clinical Approaches for H. pylori

Fig. 3 Numbers of board certified doctors and institutes performing H. pylori eradication therapy uncovered by the Japanese national
insurance, at the time point of March 2012.
(adopted from the homepage of the JSHR with permission; http://www.jshr.jp/index.php?page=medic_list, http://www.jshr.jp/index.
php?page=medic_facility)

tritis has a different etiology, only diseases produced by
known pathogens, such as H. pylori, should be treatedby targeting the pathogen directly. In addition, H. pylori-negative FD should not be categorized as so-called“chronic gastritis” because it is histologically negative
for inflammation. As the pathophysiology underlying the
disturbances of gastroduodenal motor and sensory functions and dyspepsia symptoms caused by H. pylori infection is gradually being elucidated, H. pylori-positive FD(i.e., HpD) should be considered an organic disease anddealt with as a disease entity distinct from FD53 (Fig. 3).
Separating HpD from FD may reconcile the conflicting
results of previous studies on drug therapy for FD. The
differences between the most effective therapeutic strategies against HpD and H. pylori-negative FD also require
further investigation.

Indications for H. pylori Eradication in JapanAt present, H. pylori eradication is used not only forthe treatment of peptic ulcer disease but also for prophylaxis and treatment of H. pylori-associated diseases suchas gastric cancer, gastric MALT lymphoma, and ITP, aswell as for inhibiting the spread of this bacterial infection.
H. pylori infection was approved as an official diseasename in the 2003 edition of the International Statistical
Classification of Diseases and Related Health Problems(ICD-10). H. pylori infection itself is soon expected tobe accepted by the Japanese national health scheme as adisease entity; this will make the diagnosis and treatmentof H. pylori-associated diseases a part of routine medical
practice, resulting in the successful prevention of gastriccancer. In addition, H. pylori eradication therapy couldKeio J Med 2012; 61 (4): 109–119

115

Table 2 Indications for H. pylori eradication therapy 54
DiseasesPeptic ulcer (gastric/duodenal
ulcer)
Gastric MALT lymphoma
Idiopathic thrombocytopenic
purpura (ITP)
Patients after endoscopic treatmentof early gastric cancer
H. pylori infection
Atrophic gastritis
Gastric hyperplastic polyps
Functional dyspepsia (FD)
Reflux esophagitis
Iron-deficiency anemia
Chronic urticaria

Minds recommendation gradeA

prevent the spread of this infection, which would lead todramatic savings in medical costs.
According to the Guidelines for the Management of
Helicobacter pylori Infection in Japan: 2009 Revised
Edition of the Japanese Society of Helicobacter Research(JSHR),54 H. pylori eradication has, based on strong evidence, been strongly recommended as level A (Minds
Recommendation Grades) for all H. pylori infections. In
the guidelines, the level of evidence for each recommendation was classified from Level I to Level VI. For example, peptic ulcer, ITP, atrophic gastritis, and FD werecategorized as Level I [supported by systematic review
and meta-analysis (Table 2)].54 In contrast, early gastriccancer post-endoscopic treatment, gastric hyperplastic
polyp, and reflux esophagitis were categorized as Level II(supported by at least one randomized controlled clinical
trial), while gastric MALT lymphoma, iron-deficiency
anemia, and chronic urticaria were categorized as Level III (supported by non-randomized controlled clinicalstudies) (Table 2).
Diagnosis of H. pylori Infection
H. pylori infection is investigated before and after eradication therapy using the following tests. Tests that require endoscopic biopsy include (i) the rapid urease test,(ii) histology, and (iii) culture, whereas tests not requiring endoscopic biopsy include (i) the 13C-urea breath test
(UBT), (ii) detection of anti-H. pylori antibodies in theserum or urine, and (iii) measurement of H. pylori stool
antigen. The national health insurance scheme now covers the use of two separate tests.54
The rapid urease test has the advantages of low cost,ease of use, and speed, whereas the advantages of culture include 100% specificity (direct demonstration of thepresence of H. pylori) and the ability to further character-

Level of evidenceclassification

Coverage by the Japanesenational health insurance system

I

Yes

III

Yes

I

Yes

II

Yes

I
II
I
II
III
III

No
No
No
No
No
No

ize the organism (e.g., determination of its antibiotic susceptibility and investigation of its virulence factors). On
the other hand, the UBT is a practical and readily available test with a diagnostic accuracy of >95%.
The detection of anti-H. pylori antibodies in the serum
is a widely available and inexpensive test, but the diagnostic accuracy is low (80%–84%). Some highly accurate(90%) serology kits are recommended in validated settings.55 The anti-urease activity of proton pump inhibitor (PPI) treatment can result in false-negative results forinvasive and noninvasive diagnostic tests; therefore, PPI
treatment should be withheld for at least 2 weeks before
testing. However, this consideration does not apply to serology.
A systematic review of 89 studies evaluating the stool
antigen test found an aggregate sensitivity and specificityof 91% and 93%, respectively.56 Stool samples must be
stored at −20°C prior to testing for this test to be accurate;
the sensitivity of the stool antigen test decreases to 69%
after storage of the samples at room temperature for 2–3
days. Stool antigen tests have low specificity in patients
presenting with acute bleeding peptic ulcers, as there iscross-reactivity with blood products in the stool.
H. pylori Eradication Therapy
In Japan, 1 week of triple therapy using a PPI combinedwith amoxicillin (750 mg, b.i.d.) and clarithromycin(400 mg or 200 mg, b.i.d.) is recommended as the firstchoice treatment for eradication of H. pylori.57 At the2008 meeting of the JSHR, the mean national clarithromycin resistance rates from 2002 to 2006 were reported
to be 18.9%, 21.2%, 27.7%, 29.0%, and 27.2%. The meannationwide clarithromycin resistance rate determinedby the Japanese Society of Chemotherapy in 2000 was
7.0%, so the resistance rate has increased by approxi-

116mately 20% over several years. Given the recent surge
in clarithromycin resistance, new strategies for first-line
treatment of H. pylori infection should be considered.58
Recently, a European group performed a randomized,
open-label, non-inferiority, phase 3 trial comparing theefficacy and safety of first-line quadruple therapy withomeprazole plus a single three-in-one capsule containing
bismuth subcitrate potassium, metronidazole, and tetracycline (quadruple therapy) for 10 days versus omeprazole, amoxicillin, and clarithromycin for 7 days (standard
therapy) in adults with known H. pylori infection.59 According to this study, the eradication rate in the intent-totreat (ITT) population (n =440) was 80% (174 of 218 participants) in the quadruple therapy group versus 55% (123of 222) in the standard therapy group (P < 0.0001). Thissuggests that quadruple therapy would be considered forfirst-line treatment of H. pylori infection, especially inview of the rising prevalence of clarithromycin-resistant
H. pylori and the fact that quadruple therapy provides superior eradication, with safety and tolerability similar to
those of standard therapy, even though this study showed
an extraordinarily low eradication rate in the standard
therapy arm.59 However, several issues, including the optimal doses of bismuth and amoxicillin and the durationof treatment, must be also considered before quadruple
therapy can be established as the standard first-line therapy for H. pylori eradication.60
In Japan, the recommended second-choice treatment
for H. pylori infection is 1 week of triple therapy using a
PPI combined with amoxicillin (750 mg, b.i.d.) and metronidazole (250 mg, b.i.d.). The prevalence of resistance tometronidazole in H. pylori in Japan has been reported torange between 5% and 12%.61 The Tokyo H. pylori Study
Group examined the rate of eradication in response to thesecond-line regimen consisting of PPI, amoxicillin, andmetronidazole in a multicenter study in the Tokyo metropolitan area.62 ITT and per-protocol (PP) analyses revealed eradication rates of 87.6% and 90.6%, respectively.
Murakami et al. reported that eradication rates following second-line treatment of H. pylori infection with the
PPI + amoxicillin + metronidazole regimen were 97% for
infections with metronidazole-sensitive strains and 82%
for infections with metronidazole-resistant strains. Two
explanations are available for the discrepancy between
the MICs for metronidazole and the eradication rates for
PPI-amoxicillin-metronidazole triple therapy: (i) metronidazole is relatively stable under anaerobic conditions,
possibly making its antimicrobial activity stronger in thestomach than in vitro, and (ii) the mechanism by which H.
pylori acquires resistance to metronidazole remains unclear (mutations in the nitroreductase rdxA chromosome
have been implicated, but there are other possibilities),making it difficult to predict the effects of metronidazolecontaining second-line treatment regimens from the MICof metronidazole alone. To improve prediction of eradication success by second-line treatment, we developed andSuzuki H, et al: Molecular and Clinical Approaches for H. pylori

reported an eradication resistance index, calculated as:[Pre-treatment urea breath test result (‰)] × [amoxicillin
MIC (μg/mL)] × [metronidazole MIC (μg/mL)].63 When acutoff value of 3 was used, the eradication resistance index predicted the response to therapy with a specificity of
81.8%, a sensitivity of 93.8%, and an accuracy of 92.5%.
However, because the superiority of the first-line eradication regimen (PPI + amoxicillin + clarithromycin) to thesecond-line regimen (PPI + amoxicillin + metronidazole)in Japan has recently been called into question, we retrospectively confirmed that the metronidazole-containing
second-line regimen could be a superior primary eradication regimen for H. pylori in Japan.58
There is at present no standard third-line treatment
regimen for eradication of H. pylori, and European
guidelines recommend culture before the selection of athird-line treatment to tailor the regimen to the antibiotic
sensitivity of the isolate. H. pylori isolated after the failure of first- and second-line eradication regimens is often resistant to both metronidazole and clarithromycin.46
Therefore, these two drugs are not recommended for inclusion in third-line regimens. In Japan, a PPI + amoxicillin + fluoroquinolone combination or high-dose PPI/
amoxicillin therapy is recommended if second-line eradication therapy fails.54,64 However, we recently reported
that even amoxicillin resistance (MIC ≥0.5 µg/mL) is enhanced after multiple eradication failures (1st: 0%, 2nd:
0.9%, 3rd: 6.1%, and 4th: 18.2%) due to the accumulation of PBP1 mutations.46 Clinicians should therefore beaware of the possibility of resistance to amoxicillin alongwith that to other antibiotics.
Novel fluoroquinolones have recently been developed,
and sitafloxacin has been reported to have superior in vitro activity against H. pylori compared with levofloxacin.65,66 We investigated the efficacy of sitafloxacin-based
triple therapy as a third-line treatment administered after
assessment of the isolates for sitafloxacin susceptibility
and the presence of gyrA mutations. Sitafloxacin-based
triple therapy achieved 83.6% (PP) or 78.2% (ITT) success among 78 Japanese patients. Even among the 47 patients with gyrA mutation-positive H. pylori, the eradication rates were 74.4% (PP) and 68.1% (ITT)67. In thatstudy, the position of the gyrA mutation (N87 or D91) was
found to be superior to the MIC value for predicting theeradication outcome.67 A randomized, open-label, parallel study comparing two sitafloxacin-based triple therapies in patients with multiple eradication failures is now
underway in Keio University Hospital (Tokyo, Japan)(UMIN000006483) (http://kompas.hosp.keio.ac.jp/contents/medical_info/presentation/201201.html).

Keio J Med 2012; 61 (4): 109–119

Summary
H. pylori infection is now known to be the main causeof peptic ulcer disease, chronic atrophic gastritis, andgastric MALT lymphoma, as well as non-cardia gastriccancer. In addition, an association between H. pylori infection and some extra-gastrointestinal diseases, such as
ITP, has been indicated. We summarized recent basic
research and clinical data on the link between H. pylori
infection and H. pylori-associated diseases. Accurate diagnostic methods for H. pylori detection are available,
allowing ready detection of the infection. Although therecommended first-line therapy regimens are effective
and well tolerated, their wide use throughout the country over the years has led to failure of these regimens in
20%–30% of patients. Treatment failure is due mainly toincreasing antibiotic resistance, in particular clarithromycin resistance. Although novel therapeutic strategies should be considered for first-line treatment, local
monitoring of antimicrobial resistance must be implemented as well. In 2009, the JSHR established a board
certification system for doctors treating H. pylori infection, and 734 doctors have been registered as a boardcertified doctor by the JSHR after four board certification
examinations on March 2012 (http://www.jshr.jp/index.
php?page=medic_list) (Fig. 3). The JSHR also reported
the list of institutes performing H. pylori eradication notcovered by national insurance in Japan (http://www.jshr.
jp/index.php?page=medic_facility) (Fig. 3).
While medical qualifications, scientific knowledge, andtechniques in the field of H. pylori infection all continue
to improve, the total control of these silent organisms inthe stomach remains a challenge.
Acknowledgments
The present study was supported by a Grant-in-Aid for
Scientific Research (B) from the Japan Society for the Promotion of Science (No. 22300169 to H.S.), a grant of the
Adaptable and Seamless Technology Transfer Program
through Target-Driven R & D (A-STEP) (AS231Z00132G
to H.S.) from the Japan Science and Technology Agency(JST), a grant from the Smoking Research Foundation (toH.S.), the Keio Gijuku Academic Development Fund (toH.S.), and the Nateglinide Memorial Toyoshima Researchand Education Fund (to H.S.).
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