Antibiotic Resistance, Susceptibility Testing and Stewardship

International Journal of
Molecular Sciences
Review
Antibiotic Resistance, Susceptibility Testing and Stewardship
in Helicobacter pylori Infection
Ho-Yu Ng 1 , Wai K. Leung 2, * and Ka-Shing Cheung 2,3, *
1 School of Clinical Medicine, The University of Hong Kong, Hong Kong, China; [email protected]
2 Department of Medicine, School of Clinical Medicine, The University of Hong Kong, Queen Mary Hospital,
102 Pokfulam Road, Hong Kong, China
3 Department of Medicine, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518053, China
* Correspondence: [email protected] (W.K.L.); [email protected] (K.-S.C.);
Tel.: +852-2255-3348 (W.K.L.); +852-2255-6979 (K.-S.C.); Fax: +852-2816-2863 (W.K.L. & K.-S.C.)
Abstract: Despite the declining trend of Helicobacter pylori (H. pylori) prevalence around the globe,
ongoing efforts are still needed to optimize current and future regimens in view of the increasing
antibiotic resistance. The resistance of H. pylori to different antibiotics is caused by different molecu-
lar mechanisms, and advancements in sequencing technology have come a far way in broadening
our understanding and in facilitating the testing of antibiotic susceptibility to H. pylori. In this
literature review, we give an overview of the molecular mechanisms behind resistance, as well as
discuss and compare different antibiotic susceptibility tests based on the latest research. We also
discuss the principles of antibiotic stewardship and compare the performance of empirical therapies
based on up-to-date resistance patterns and susceptibility-guided therapies in providing effective
H. pylori treatment. Studies and clinical guidelines should ensure that the treatment being tested
or recommended can reliably achieve a pre-agreed acceptable level of eradication rate and take
into account the variations in antibiotic resistance across populations. Local, regional and interna-
tional organizations must work together to establish routine antibiotic susceptibility surveillance
programs and enforce antibiotic stewardship in the treatment of H. pylori, so that it can be managed in
a sustainable and efficient manner.
Citation: Ng, H.-Y.; Leung, W.K.;
Cheung, K.-S. Antibiotic Resistance,
Keywords: H. pylori; vonoprazan; bismuth; stool based-PCR; next generation sequencing; NGS
Susceptibility Testing and
Stewardship in Helicobacter pylori
Infection. Int. J. Mol. Sci. 2023, 24,
11708. https://doi.org/10.3390/ 1. Introduction
ijms241411708 Helicobacter pylori (H. pylori) infects approximately 40% of individuals worldwide [1]
Academic Editor: Jean-Christophe and is involved in various gastrointestinal diseases such as chronic gastritis, peptic ulcer
Marvaud disease, upper gastrointestinal bleeding [2], gastric cancer (including gastric adenocar-
cinoma and gastric MALT lymphoma) [3–5] and extraintestinal manifestations [6]. All
Received: 21 June 2023 patients infected with H. pylori should be treated regardless of clinical manifestations [4].
Revised: 12 July 2023
Eradication of H. pylori can restore the normal gastric mucosa [7] and was shown to effec-
Accepted: 17 July 2023
tively reduce the development and recurrence of peptic ulcers [8,9], as well as the incidence
Published: 20 July 2023
of gastric cancer [10,11].
Various treatment regimens for H. pylori have been developed and their use vary
across different geographical regions largely based on local antibiotic resistance patterns.
Copyright: © 2023 by the authors.
Currently, an acceptable H. pylori treatment regimen is defined as one that achieves at
Licensee MDPI, Basel, Switzerland. least a 90% cure rate [12], though it has been suggested that an optimized regimen should
This article is an open access article reliably achieve ≥95% cure rates [13]. Treatment regimens currently recommended by
distributed under the terms and various international guidelines are of empirical nature [4,14–16]. In light of a substantial
conditions of the Creative Commons decline in efficacy to levels below 80–85% [12,17], triple therapy consisting of a proton
Attribution (CC BY) license (https:// pump inhibitor (PPI), clarithromycin, and amoxicillin or metronidazole is usually not
creativecommons.org/licenses/by/ recommended as an empirical first-line therapy for H. pylori infection, except in areas
4.0/). with a known clarithromycin resistance rate of <15% [4,14,15]. Instead, bismuth-based
Int. J. Mol. Sci. 2023, 24, 11708. https://doi.org/10.3390/ijms241411708 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 11708 2 of 33
quadruple therapy, typically consisting of bismuth, PPI, metronidazole and tetracycline, is

increasingly recommended as its replacement for first-line treatment [4,14,15] and has been
shown to achieve over 90% success rate [18,19]. Bismuth-based quadruple therapy can also
be used as a second-line treatment. Another option for second-line treatment is a triple
therapy consisting of a PPI, amoxicillin and levofloxacin, but this too faces the problem of
decreasing efficacy because of rising resistance to levofloxacin [20].
Antimicrobial stewardship, which is the responsible use of antimicrobials to balance
individual needs and long-term societal needs, is increasingly promoted to limit the increas-
ing antibiotic resistance around the world [21]. One important component of antimicrobial
stewardship is widely available antibiotic susceptibility testing to provide information
for designing optimal treatment regimens. Routine susceptibility testing has been recom-
mended even before first-line treatment in compliance to antibiotic stewardship [4,22]. In
particular, the use of molecular-based testing methods, including polymerase chain reaction
(PCR)-based assays and next-generation sequencing (NGS), have gained prominence in
recent years as an alternative to traditional culture-based testing, each with their own pros
and cons. These molecular-based methods can be potentially applied to stool samples
obtained through non-invasive means; however, their accuracies, so far, were inconsis-
tent among different studies and need to be studied in more detail [23–26]. Moreover,
challenges such as practicality and cost-effectiveness issues in their use in routine clinical
setting should be considered and addressed. In this regard, some studies showed that
empirical therapies based on the latest antibiotic resistance patterns could achieve simi-
lar efficacy [27–29] and be more cost effective [30,31] than susceptibility-guided therapy.
More studies are needed to compare the performance of new empirical treatments, such as
vonoprazan-based therapies, with susceptibility-guided therapy.
In this review, we will give an overview of the current H. pylori antibiotic resistance
patterns and mechanisms. We will then discuss the application, advantages and limitations
of different antibiotic susceptibility testing methods that are currently in use based on the
latest studies. We will also discuss the importance of antibiotic stewardship in the treatment
of H. pylori, discuss how it can be achieved and compare the performances of empirical
treatments with susceptibility-guided therapies in existing studies.
2. Antibiotic Resistance in Helicobacter pylori

Primary antibiotic resistance in H. pylori treatment can be defined as resistance in
patients who have not started eradication therapy, whereas secondary antibiotic resistance
occurs in patients who have previously undergone at least one unsuccessful eradication at-
tempt [32]. Globally, primary and secondary resistance rates of clarithromycin, levofloxacin
and metronidazole were all over 15% in all World Health Organization (WHO) regions [32].
The underlying mechanism behind antibiotic resistance by H. pylori is often due to genetic
mutations that may inhibit the intracellular activation of antibiotics or change the drug
target site altogether [33], and these mutations are mostly encoded chromosomally rather
than in extrachromosomal elements [34–36]. The specifics of such genetic changes depend
on the class of antibiotics (Figure 1) (Table 1). Apart from genetic sequence mutations,
there are other possible mechanisms such as efflux pumps through the upregulation or
downregulation of hefABC genes, or through biofilm formation, which can obstruct drug
penetration [33] (Figure 1).
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Figure 1. Summary of molecular mechanisms of antibiotic resistance in H. pylori. In general, antibi-

Figure 1. Summary of molecular mechanisms of antibiotic resistance in H. pylori. In general, antibiotic
otic resistance in H. pylori arises because of mutations that decrease drug affinity to its binding site.
resistance in H. pylori arises because of mutations that decrease drug affinity to its binding site.
Examples include mutations in 23S rRNA for clarithromycin, in pbp1A for amoxicillin, in gyrA or
Examples include mutations
gyrB for levofloxacin in 23S
and in 16S rRNArRNA for clarithromycin,
for tetracycline. in pbp1Aisfor
Metronidazole amoxicillin,
unique it isgyrA
in that in or
a pro-
gyrB
drugforandlevofloxacin
works by and in 16S rRNA
producing activefor tetracycline.
metabolites Metronidazole
that can damage is unique DNA
bacterial in thatafter
it is areductive
prodrug
and works by
activation. producing
Mutations active metronidazole
causing metabolites thatresistance
can damage havebacterial
a muchDNAmoreafter reductive
diverse activation.
repertoire, and
Mutations causingones
more well-known metronidazole
include thoseresistance
in geneshave a much
encoding formore diverse repertoire, and
metronidazole-reducing more well-
enzymes (e.g.,
FrxA and
known RdxA)
ones causing
include thosedecreased
in genes drug activation,
encoding as well as those that lead
for metronidazole-reducing to resistance
enzymes to oxida-
(e.g., FrxA and
tive stress
RdxA) caused
causing by metronidazole
decreased or increased
drug activation, as wellDNA repair.
as those thatOther
lead topossible mechanisms
resistance to oxidative thatstress
may
apply toby
caused most antibiotics include
metronidazole increased
or increased DNA drug efflux
repair. caused
Other by upregulation
possible mechanisms of efflux
that maypump genes
apply to
(e.g., hefABC) or biofilm formation, which decrease drug penetration. Abbreviations: rRNA, riboso-
most antibiotics include increased drug efflux caused by upregulation of efflux pump genes (e.g.,
mal RNA; mRNA, messenger RNA; tRNA, transfer RNA; DNA, deoxyribonucleic acid.
hefABC) or biofilm formation, which decrease drug penetration. Abbreviations: rRNA, ribosomal
RNA; mRNA, messenger RNA; tRNA, transfer RNA; DNA, deoxyribonucleic acid.
Table 1. Molecular basis of resistance against antibiotics commonly used in Helicobacter pylori erad-
Table 1.therapies.
ication Molecular basis of resistance against antibiotics commonly used in Helicobacter pylori
eradication therapies.
Genes Involved Resistance Mechanisms
Domain V of 23S rRNA (most prevalent:
Domain V of 23S rRNA (most prevalent: Decrease drug affinity to its binding site
Decrease drug affinity to its binding site
A2143G, A2142G, A2142C)A2142C)
A2143G, A2142G, [33,37–39]
[33,37–39]
infB (translation initiation factor IF-2),
ClarithromycininfB (translation initiation factor IF-2), Putative effects on 23S rRNA (the drug target) *
Clarithro- Rpl22 (Ribosomal protein L22) [40–42] Putative effects on 23S rRNA (the drug target) *
Rpl22 (Ribosomal protein L22) [40–42]
mycin Novel gene candidate: Putative effects on bacterial chemotaxis and
fliJ
Novel geneexport
(flagellar protein): Gln31Arg [43]
candidate: flagellar motility *
Putative effects on bacterial chemotaxis and flagellar mo-
fliJ (flagellar export protein): Gln31Arg
tility *
[43]
Int. J. Mol. Sci. 2023, 24, 11708 4 of 33
Table 1. Cont.

RdxA (oxygen-insensitive NAD(P)H
Mutations in metronidazole-reducing enzymes coding
nitroreductase),
genes causing decreased drug activation
FrxA (NAD(P)H flavin nitroreductase),
FdxA (ferredoxin),
Mutations in putative metronidazole-reducing enzymes
FdxB (ferredoxin-like protein),
coding genes causing decreased drug activation
FldA (flavodoxin) [33]
SodB (superoxide dismutase),
Resistance against oxidative stress brought
Metronidazole Fur (ferric uptake regulator),
by metronidazole
MdaB (modulator of drug activity B) [33]
Upregulation of RecA causing enhanced DNA repair
RecA [33]
against damages brought by metronidazole
Ribf (riboflavin biosynthesis protein),
Putative association with metronidazole resistance *
Omp11 [33]
Increased drug efflux causing below lethal
hefA (efflux pump) [44,45]
intracellular concentrations
gyrA (DNA gyrase subunit A): mainly at Mutations in quinolones resistance-determining region
codons N87 or D91, (QRDR) causing decreased drug affinity to its
gyrB (DNA gyrase subunit B) [33,46] binding site
Levofloxacin
Novel gene candidates:
Putative effects on bacterial chemotaxis and
fliJ (flagellar export protein): Gln31Arg [43]
flagellar motility *
cheA (histidine kinase): Ser176Thr/Ala [43]
pbp1A (penicillin-binding protein 1A) [47,48] Mutations altering penicillin-binding motifs SXXK, SXN
pbp2 (penicillin-binding protein 2) [49] and KTG, causing decreased drug affinity to its
Amoxicillin pbp3 (penicillin-binding protein 3) [49] binding site
hopB, hopC (porins), Increased drug efflux causing below lethal
hefC (efflux pump) [50,51] intracellular concentrations
16S rRNA positions 926–928 [52–54] Decrease drug affinity to its binding site
Tetracycline Increased drug efflux causing below lethal
hefABC (efflux pump) [55]
intracellular concentrations
* Hypothetical mechanism.
2.1. Resistance to Clarithromycin

Clarithromycin resistance in H. pylori is most commonly due to point mutations in
domain V of 23S rRNA, especially A2143G, A2142G and A2142C [33,37–39], with A2143G
causing the lowest eradication rate [56,57]. Other mutations have also been observed
in [58–60] and outside of domain V [61], as well as in genes outside of 23S rRNA, such as infB
and rpl22, which putatively affect 23S rRNA [40–42]. Increasing clarithromycin resistance
was responsible for the failure of clarithromycin-containing eradication therapies [4,20,62].
The primary clarithromycin resistance rate was below 15% only in the WHO regions of the
Americas and Southeast Asia, and when secondary resistance was considered, all WHO
regions had >15% resistance rate [32], which has exceeded the suggested threshold at
which clarithromycin triple therapy could be used as an empirical first-line treatment [63].
Studies found that primary clarithromycin resistance rates were 17–27% in Asia [64,65],
25% in treatment-naïve patients in Europe [66], 17.6–31.5% in the US [67,68] and 29.2% in
Africa [69].
Of note, resistance caused by prior usage of clarithromycin or other macrolides for
other diseases in patients who had never taken clarithromycin-based eradication therapy
may be considered as secondary resistance [70,71]. Information on prior macrolide use,
though difficult to obtain in real practice [72], should be collected whenever possible as
cross resistance can occur within the same family of antibiotics [20]. Studies showed that
Int. J. Mol. Sci. 2023, 24, 11708 5 of 33
macrolide exposure even in the past 10 to 14 years still correlated with H. pylori erad-
ication failure [71–75] and was an independent risk factor for H. pylori clarithromycin
resistance [71]. One such study found that those who previously took macrolides for
>2 weeks had a significantly higher failure rate of eradication than those who took macrolides
for ≤2 weeks (44.8% vs. 29.3%, p = 0.047), suggesting a duration effect of prior macrolide
use on the success rate of eradication therapy [73].
2.2. Resistance to Metronidazole

The mutational changes involved in metronidazole resistance have a much more di-
verse repertoire and the genotype–phenotype correlation is often much more complex [4,33].
More well-known ones included RdxA and FrxA mutations, which were metronidazole-
reducing enzymes and resulted in reduced drug activation [33]. Other putative mutations
might result in increased efficiency of deoxyribonucleic acid (DNA) repair or oxidative
stress response [33] and upregulated efflux [44,45]. It is possible that metronidazole re-
sistance is caused by the cumulative effects of multiple pathways rather than a single
mechanism, though until now, there has been no study that comprehensively investigated
these mechanisms in the same clinical isolates [33].
The metronidazole resistance rate is generally much higher than that of clarithromycin,
in part because of its widespread use to treat parasitic infections, urinary tract infections
and gastrointestinal infections by anaerobes [76]. The primary and secondary resistance
rates of metronidazole were over 15% in all WHO regions, with the highest rates of 56%
and 65%, respectively, observed in the eastern Mediterranean region [32]. An increasing
trend of metronidazole resistance has been observed in most of the WHO regions [32].
Studies have found that primary metronidazole resistance was 44% in the Asia-Pacific
region [64], 30–40% in Europe [66,77], 42–43% in the US [67,68] and 48.7% in Africa [78].
A notable issue was the dual resistance to clarithromycin and metronidazole, which had
a prevalence rate of around 8–15% in Europe [32,66,77], 6–11% in Asia [32] and 3–11% in
the Americas [32,67,68]. Such dual resistance greatly reduces the efficacy of non-bismuth
quadruple therapy [79,80]. In contrast to clarithromycin, in vitro metronidazole resistance
can be overcome by increasing the dose, frequency and duration of therapy [63,81–83].
2.3. Resistance to Levofloxacin

As H. pylori does not naturally possess the genes for topoisomerase IV, levofloxacin
resistance in H. pylori mainly arises from point mutations in the quinolone resistance-
determining region (QRDR) of the gyrA or, to a lesser extent, gyrB gene, which code for
DNA gyrase subunits A and B, respectively [33,46]. Mutations at gyrA mainly occur at
codons N87 or D91 [84,85], while E463 mutation has been observed at gyrB [86,87].
Primary resistance against levofloxacin exceeded 15% in all WHO regions except
Europe, while secondary resistance exceeded 15% in all regions with a significant rising
trend observed in the western Pacific WHO region [32]. Other studies found that the
primary levofloxacin resistance rates were 18% in the Asia-Pacific region [64], 15–20%
in Europe [66,77], 37.6% in the US [68] and 17.4% in Africa [69]. Multiple resistance,
commonly as dual or triple resistance to clarithromycin and/or metronidazole, is also
an area of concern, with rates up to 6–10% in Europe [66,77].
Int. J. Mol. Sci. 2023, 24, 11708 6 of 33
2.4. Resistance to Amoxicillin

Resistance to amoxicillin in H. pylori are mainly caused by mutations in the pbp1A
gene [47,48] that altered the penicillin-binding motifs SXXK, SXN and KTG, hence reducing
the affinity of amoxicillin to PBP [33,37]. Enhancing effects can be contributed to by
mutations in pbp2 and pbp3, and if mutations in these three genes occur simultaneously,
resistance could increase by over 200-fold [49]. Mutations of porin genes (e.g., hopB and
hopC) or efflux pump-coding genes (e.g., hefC) can contribute to amoxicillin resistance in
H. pylori as well [50,51].
Amoxicillin resistance in H. pylori is generally lower than other antibiotics. Primary
and secondary amoxicillin resistance rates were previously found to be below 15% in all
WHO regions [32]. Low resistance was generally found in more-developed regions, such as
6.4% in the US [67], 3% in the Asia-Pacific region [64] and 0.2% in Europe [77]. In Africa, the
resistance rate could be as high as 72.6% [69], likely caused by the over abuse of amoxicillin
because of low cost [88].
2.5. Resistance to Tetracycline

Tetracycline resistance in H. pylori is mainly caused by mutations at positions 926–928 of
the 16S rRNA [52–54] that can lead to reduced drug affinity as they are located at the pri-
mary binding site [89]. Among these mutations, triple base-pair mutations (e.g., AGA →
TTC) conferred a higher level of tetracycline resistance than single or double base-pair
mutations [90]. Other possible mechanisms included efflux pump mechanisms that might
be mediated by HefABC [55] or by proton motive force (PMF)-dependent mechanisms [91],
which might explain resistance in H. pylori strains without 16S rRNA mutations.
Similar to amoxicillin, resistance against tetracycline is generally low around the
world, with primary and secondary resistance below 10% in all WHO regions [32]. The
overall resistance rate of tetracycline was 4% in the Asia-Pacific region [64], below 1% in
Europe [66,77] and below 3% in the US [67,68]. In Africa, however, misuse of tetracyclines
caused the resistance rate to be much higher at 49.8% [69], which might cause reduced
efficacy of bismuth quadruple therapy locally [63].
3. Antibiotic Susceptibility Testing for Helicobacter pylori

Current antibiotic susceptibility testing (AST) methods for H. pylori are mainly di-
vided into culture-based techniques and molecular-based methods. Figure 2 shows the
comparison between these two main approaches in terms of their performance in studies,
advantages and disadvantages.
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Figure 2. Comparison of culture-based and molecular-based antibiotic susceptibility testing (AST)

Figure 2. Comparison of culture-based and molecular-based antibiotic susceptibility testing (AST)
methods. Culture-based methods, such as agar dilution or E-test, are the current gold standard
methods. Culture-based methods, such as agar dilution or E-test, are the current gold standard for
for AST in H. pylori. However, they are time consuming and labor intensive and require invasive
AST in H. pylori. However, they are time consuming and labor intensive and require invasive pro-
procedures to obtain gastric biopsy samples for testing. The isolation of H. pylori for testing is
cedures
also to obtain gastric
challenging. biopsy samples
In comparison, for testing. The
molecular-based isolation
methods, of H.
such aspylori for testing
PCR-based is also
assays, chal-
can be
lenging. In comparison, molecular-based methods, such as PCR-based assays, can be
applied to other samples, such as stool samples, that can be obtained through non-invasive means. applied to
other samples, such as stool samples, that can be obtained through non-invasive
However, the performance of PCR-based assays on stool samples was inconsistent and requires means. However,
the performance
further of PCR-based
investigation. assays on
Also, PCR-based stoolcan
assays samples was inconsistent
only detect and requires
known mutations furtherNGS
of resistance. inves-
is
tigation. Also, PCR-based assays can only detect known mutations of resistance. NGS is
a relatively newer technique and can be used to detect novel mutations or resistance mechanisms by a relatively
newer technique and can be used to detect novel mutations or resistance mechanisms by means of
Int. J. Mol. Sci. 2023, 24, 11708 8 of 33
means of WGS. However, NGS is very expensive and is difficult to incorporate into routine clinical
use in the short term. Studies have attempted to compare the accuracy of molecular-based methods
to culture-based methods with consideration of the sample types used. In general, there was good
concordance between culture- and molecular-based methods when gastric biopsies were used, though
results varied in different antibiotics [92–96]. The concordance was poorer when stool samples were
used [25,26,95,97], though recent studies had shown preliminary evidence that NGS might provide
better accuracy in stool samples with good concordance to results achieved on gastric biopsies [98].
Abbreviations: PCR, polymerase chain reaction; NGS, next-generation sequencing; AST, antibiotic
susceptibility testing; WGS, whole-genome sequencing; CLA, clarithromycin; LVX, levofloxacin;
MNZ, metronidazole; AMX, amoxicillin; TET, tetracycline.
3.1. Culture-Based Techniques

Traditionally, culture-based techniques, such as the agar dilution method, gradient
diffusion susceptibility testing (E-test), broth microdilution method and disc diffusion
method, are used as the gold standard for the AST of H. pylori, through which antibiotic
susceptibility or resistance is inferred from the minimal inhibitory concentration (MIC) of
the antibiotic being tested [99,100]. Broth microdilution and disc diffusion are not routinely
used for H. pylori AST because of issues with standardization and accuracy in slow-growing
bacteria such as H. pylori [33,101,102]. Agar dilution is a reliable method that can be adapted
to test multiple H. pylori strains simultaneously and was recommended as a reference assay
for evaluating the accuracy of other methods [100,103]. E-test allows quantification of the
disc diffusion method and has good correlation with that of the agar dilution method for
most antibiotics, except metronidazole because of the lack of anaerobic preincubation of
plates used [101,102,104]. In the routine clinical setting, E-test also has the advantage of
being cheaper and less time consuming than agar dilution [104].
Although culture-based methods currently serve as the gold standard for AST, they
require gastric biopsy samples, which can only be obtained through invasive endoscopy
procedures. Moreover, their reliability is greatly affected by the samples provided and
the conditions under which they are performed, hence rendering the process costly, labor
intensive and time consuming, providing results after one to two weeks at best [33,105].
Examples of such factors included the site of biopsy, the number of samples obtained, the
quality of the sample (e.g., whether gut commensal microbiota were present), the time
interval between sampling and culture and the transport conditions (e.g., temperature, air
exposure, etc.) [105,106]. The culture of H. pylori itself is also challenging, as it requires
a microaerophilic environment and an appropriate medium that would not interfere with
the AST (e.g., redox variations in the medium may affect metronidazole susceptibility
testing) [35,106]. Therefore, in routine clinical practice, there is only a 60–80% chance of
successful isolation of the bacterium for testing [106], and even if results are obtained,
its interpretation is also subjective and dependent on experiment conditions [107]. A re-
cent systemic review found that bacterial growth failed in around 20% of the patients
included [108]. It also found that while culture-tailored treatment offered a higher erad-
ication rate than empirical treatment, it was only able to achieve the suggested optimal
eradication rate of >90% [4,13] in culture-positive patients when it was adopted before
first- and second-line regimens but not if it was done after two or more eradication fail-
ures [108], further highlighting the limitations of the culture-based approach to AST. It
should be noted that the first- and second-line empirical regimens compared in this sys-
temic review were mostly non-bismuth-based. A meta-analysis that included 54 studies
found that a susceptibility-guided strategy had slightly better efficacy than an empirical
clarithromycin-based triple therapy for first-line treatment but not for first-line quadruple
empirical regimens (both with and without bismuth) [29].
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3.2. Molecular-Based Techniques

Molecular-based techniques work by detecting specific mutations in H. pylori that
encode resistance mechanisms and, in general, offer advantages such as a higher degree of
standardization and reproducibility, a shorter time required for testing (often producing
results within the same day) and the possibility of using specimens that can be obtained
through non-invasive means and do not require immediate processing [105]. Molecular-
based techniques can be applied to a variety of specimens, including fresh, frozen or
paraffin-embedded formalin-fixed (FFPE) gastric biopsy samples, stool samples or gastric
juice [33]. Current molecular-based methods are largely divided into PCR-based assays
and next-generation sequencing (NGS) techniques, and their performances in different
studies are summarized in Table 2.
Table 2. Summary of studies assessing the performance of polymerase chain-reaction-based assays

and next-generation sequencing in predicting H. pylori antibiotic resistance.
Sample Size and

Study Methods Tested Antibiotics Tested Results
Specimen Samples
PCR-based assays
Sensitivity: 99.1%
223 gastric biopsy samples GenoType HelicoDR assay (Hain Clarithromycin
Fernandez-Caso Specificity: 80.0%
studied for Life Science, Nehren, Germany)
et al., 2022 [92]
antibiotic susceptibility Control: E-test Sensitivity: 100%
Levofloxacin
Specificity: 100%
Sensitivity: 94%
Clarithromycin Specificity: 99%
92 clinical strains and GenoType HelicoDR assay (Hain Concordance score: 0.96
Cambau et al.,
105 gastric biopsy samples Life Science, Germany)
2009 [93] Sensitivity: 87%
Control: E-test
Levofloxacin Specificity: 98.5%
Concordance score: 0.94
Sensitivity: 100%
Specificity: 86.2%
Clarithromycin
PPV: 89.7%
64 biopsy GenoType HelicoDR assay (Hain NPV: 100%
Deyi et al., 2011
samples compared Life Science, Germany)
[94] Sensitivity: 82.6%
Control: E-test
Specificity: 95.1%
Levofloxacin
PPV: 90.5%
NPV: 90.7%
GenoType HelicoDR assay (Hain
55 biopsy specimens Clarithromycin Concordance: 52.9%
Life Science, Germany) on stool
Brennan et al., and 66 stool specimens
2016 [97] specimens compared Control: GenoType HelicoDR Levofloxacin Concordance: 35.3%
assay on biopsy specimens
Lightmix RT-PCR (TIB Molbiol, 95% concordance rate
Redondo et al.,
60 gastric biopsies Berlin, Germany) Clarithromycin between RT-PCR
2018 [109]
Control: E-test and E-test
Sensitivity: 100% (biopsy),
100% (stool)
TaqMan RT-PCR (Meridian Specificity: 82% (biopsy),
Schaberiter- 45 isolates (biopsy and
Bioscience, Newtown, OH, 73% (stool)
Gurtner et al., stool samples) studied for Clarithromycin
United States) PPV: 100% (biopsy),
2004 [95] antibiotic susceptibility
Control: E-test 100% (stool)
NPV: 94% (biopsy),
92% (stool)
Int. J. Mol. Sci. 2023, 24, 11708 10 of 33
Table 2. Cont.
Sample Size and

Specimen Samples
PCR-based assays
Sensitivity in detecting
TaqMan RT-PCR (Meridian H. pylori DNA: 93.8%
Beckman et al., Bioscience, United States) Correlation between
Total of 294 stool samples Clarithromycin
2017 [23] Reference: Treatment outcome as genotype prediction by
tested by UBT PCR and eradication of
infection: 86%
For H. pylori detection:
Sensitivity: 63%
Specificity: 100%
PPV: 100%
ClariRes RT-PCR (Ingenetix,
Lottspeich et al., Total of 100 stool samples NPV: 76.1%
Vienna, Austria) Clarithromycin
2007 [24] from children For clarithromycin
Control: E-test
susceptibility testing:
2 false negative results out
of 6 clarithromycin
resistant cases
Sensitivity: 83.8%
Specificity: 98.4%
PPV: 98.5%
ClariRes RT-PCR (Ingenetix, NPV: 82.7%
Vecsei et al., 2009 Total of 143 stool samples
Vienna, Austria) Clarithromycin For clarithromycin
[25] from children
Control: E-test susceptibility testing:
Sensitivity: 89.2%
Specificity: 100%
PPV: 100%
NPV: 88.2%
Sensitivity: 69%
Specificity: 100%
ClariRes RT-PCR (Ingenetix, Test accuracy: 93.9%
Scaletsky et al., Total of 217 stool samples
Vienna, Austria) Clarithromycin For clarithromycin
2011 [26] from children
Control: E-test susceptibility testing:
Sensitivity: 83.3%
Specificity: 100%
Test accuracy: 95.6%
Next-Generation Sequencing
Sensitivity: 93.3%
Specificity: 94.3%
PPV: 77.8%
Clarithromycin NPV: 98.5%
Accuracy: 94.1%
Agreement (k coefficient):
0.81236 (p < 0.0001)
170 H. pylori clinical
isolates and FFPE gastric Sensitivity: 70.1%
biopsies for amoxicillin, Specificity: 86.3%
PyloriAR NGS (American
clarithromycin and PPV: 87.2%
Hulten et al., Molecular Laboratories, Vernon
metronidazole Metronidazole NPV: 68.5%
2021 [96] Hills, IL, United States)
57 H. pylori clinical isolates Accuracy: 77.1%
Control: Agar dilution
and FFPE gastric biopsies Agreement (k coefficient):
for levofloxacin and 0.54645 (p < 0.0001)
tetracycline
Sensitivity: 87.9%
Specificity: 87.5%
PPV: 90.6%
Levofloxacin NPV: 84.0%
Accuracy: 87.7%
0.74953 (p < 0.0001)
Int. J. Mol. Sci. 2023, 24, 11708 11 of 33
Table 2. Cont.
Sample Size and

Specimen Samples
Next-Generation Sequencing
Sensitivity: 12.5%
Specificity: 100%
PPV: 100%
Amoxicillin NPV: 95.9%
Accuracy: 95.9%
0.21400 (p = 0.0051)
Only 1 out of 57 samples
tested resistant to
tetracycline by
Tetracycline agar dilution;
none of the 57 samples
were tested tetracycline
resistant by PyloriAR NGS
Clarithromycin
0.94 (95% CI: 0.90–1.00)
Metronidazole
PyloriAR NGS (American 0.89 (95% CI: 0.76–1.00)
64 fresh gastric biopsy
Molecular Laboratories, United
Moss et al., samples and stool samples Agreement (k coefficient):
States) on stool samples Levofloxacin
2022 [98] compared for antibiotic 0.88 (95% CI: 0.75–1.00)
Control: PyloriAR NGS on gastric
susceptibility prediction
biopsy samples Agreement
Amoxicillin
(k coefficient): 1.00
Agreement
Tetracycline
(k coefficient): 1.00
Abbreviations: RT-PCR, real-time polymerase chain reaction; NGS, next-generation sequencing; FFPE, formalin-
fixed paraffin-embedded.
(1) PCR-based assays

Most currently available assays are PCR-based assays that test for the most common
point mutations in 23S rRNA for clarithromycin resistance and in gyrA for levofloxacin
resistance [33,92,105]. In contrast, the development of assays for detecting metronidazole
and amoxicillin resistance is challenging because of the complex underlying mechanisms of
resistance [33]. In particular, assays for detecting clarithromycin resistance have been more
well developed, and these assays could be applied to both biopsy and stool specimens
and be used to detect H. pylori presence and clarithromycin resistance at the same time,
with some even able to distinguish high or low levels of resistance based on the point
mutation [95,109]. These real-time PCR assays also have a much faster turnaround time
of around 2 to 6 h compared to culture-based techniques [105]. However, although stool
sampling enabled the possibility of non-invasive testing, the sensitivity of molecular assays
in detecting H. pylori using stool samples was not consistent in different studies, with one
study finding a good sensitivity of 93.8% [23], while others found lower sensitivity (ranging
from 63% to 84%) compared to traditional biopsy specimens [24–26]. The GenoType Heli-
coDR assay (Hain Life Science, Germany) has high sensitivity in detecting clarithromycin
(ranging from 94–100%) and levofloxacin (ranging from 82.6–100%) when conducted on
gastric biopsy samples [92–94], but one study found low concordance (clarithromycin:
52.9%; levofloxacin: 35.3%) of results obtained from stool samples with that from biopsy
samples [97]. Such variance in sensitivity was likely dependent on the DNA extraction
method and molecular assay used. Even if biopsy samples were used, the accuracy of
such assays were also affected by the quality of the sample, as well as purity and condition
of the DNA. False-negative results could arise if paraffin-embedded gastric biopsy sam-
ples were used as the DNA was broken into small pieces by the fixative [110–112]. The
traditional Sanger sequencing method for identifying mutations after PCR also only has
a limited coverage of nucleotides and cannot identify complex structural variants related
Int. J. Mol. Sci. 2023, 24, 11708 12 of 33
to antibiotic resistance and may also not be cost effective if it is intended for use in routine
settings because of its relatively higher price compared to conventional culture-based
methods [33,92,110].
(2) Next-generation sequencing
Next-generation sequencing (NGS)-based methods have emerged as a potential al-
ternative to both culture-based methods and current PCR-based assays, as they allow
the identification of much more complex genetic variants that are involved in antibiotic
resistance. NGS refers to methods that are developed after Sanger and Maxam–Gilbert
sequencing and that allow massive parallel sequencing of DNA and RNA at a relatively low
cost [113]. Whole-genome sequencing (WGS) is one of the applications of NGS technology
in sequencing the entire genome of an organism and has enabled both the prediction of
antibiotic resistance based on point mutations detected on target genes [35], as well as the
detection of novel genetic mutations in clinical isolates. This is in stark contrast to conven-
tional PCR-based assays that only detect known mutations that are most prevalent. The
use of WGS to detect novel resistance-related mutations has not only been applied to clar-
ithromycin and levofloxacin [36,43] but also to metronidazole [114] and amoxicillin [36,115].
This is particularly important for discovering new potential mechanisms that can explain
resistance, as well as to provide new candidate genes that confer a better genotypic–
phenotypic correlation than existing ones. A recent study conducted in Shenzhen, the
southern part of China, detected a novel Gln31Arg mutation in fliJ in clarithromycin-
resistant strains and levofloxacin-resistant strains, as well as a Ser176Thr/Ala mutation
in cheA in levofloxacin-resistant strains, thus identifying fliJ and cheA as new candidate
genes that might predict clarithromycin and levofloxacin resistance in H. pylori [43]. The
same study also found no significant difference in the genotype of gyrA between H. pylori
with phenotypic levofloxacin-resistance and those without, which was in concordance
with a previous study finding inconsistent genotype-to-phenotype correlation of gyrA in
predicting levofloxacin resistance [116], further highlighting the importance of continuous
detection of novel mutations. Another recent study conducted in China detected the novel
mutations N118K in Fur and Q242K in Ribf, as well as K219Q/N and H705fs in Omp11
in metronidazole-resistant H. pylori strains [114]. Two studies found a total of seven new
putative genotypes in pbp1A that might cause amoxicillin resistance [36,115]. A recent
study conducted on 112 H. pylori strains from China even identified as many as 75, 5 and
13 new unique genes in metronidazole, clarithromycin and levofloxacin-resistant categories,
respectively [117].
The accuracy of using NGS to predict antibiotic resistance was previously shown to be
satisfactory and largely similar to that of culture-based methods, which are considered the
gold standard for AST. Hulten et al. conducted paired comparisons between NGS and MICs
obtained from culture using agar dilution for their determination of antibiotic resistance to
clarithromycin, metronidazole, levofloxacin and amoxicillin [96]. When performed on clini-
cal isolates, NGS showed good agreement with the agar dilution method for clarithromycin
(k = 0.90012, p < 0.0001) and levofloxacin (k = 0.78161, p < 0.0001), while agreement with
agar dilution for metronidazole (k = 0.5588, p < 0.0001) and amoxicillin (k = 0.21400,
p = 0.0051) was less satisfactory. The accuracy of NGS in predicting resistance in clinical
isolates was 97.1% for clarithromycin, 89.5% for levofloxacin, 77.6% for metronidazole
and 95.9% for amoxicillin. Similarly, when performed on FFPE gastric biopsies, NGS
showed good agreement with agar dilution for clarithromycin (k = 0.81236, p < 0.0001) and
levofloxacin (k = 0.74953, p < 0.0001), with less satisfactory agreement for metronidazole
(k = 0.54645, p < 0.0001) and amoxicillin (k = 0.21400, p = 0.0051). The accuracy of NGS in
predicting resistance in FFPE biopsies was 94.1% for clarithromycin, 87.7% for levofloxacin,
77.1% for metronidazole and 95.9% for amoxicillin. Based on this, Moss et al. recently
expanded further to investigate the accuracy of NGS in predicting antibiotic resistance
in stool samples as well and found that the results obtained from stool samples was con-
cordant with that from FFPE gastric biopsies in 91.4% of cases and that from fresh gastric
specimens in 92.2% of cases [98]. The agreement between stool and fresh gastric samples
Int. J. Mol. Sci. 2023, 24, 11708 13 of 33
was good for clarithromycin (k = 0.94), levofloxacin (k = 0.88) and metronidazole (k = 0.89).
This was in stark contrast with conventional PCR assays, which might not be sensitive
enough if performed on stool samples, hence highlighting NGS as a potentially reliable
tool that can enable non-invasive means of AST. More studies on this subject, however, will
have to be conducted to verify the performance of NGS on different clinical samples and in
different geographical regions.
The use of NGS, in particular WGS, for AST in H. pylori strains faces several limitations.
First, the accuracy of WGS when applied to gastric biopsies might be hampered by high
human DNA background and low bacterial DNA content [36,118,119]; hence, suitable
DNA extraction methods need to be carefully chosen and used. Second, as genotypic and
phenotypic resistance may not always be correlated, further studies are required to verify
the predictive accuracy of novel genotypes detected by NGS using phenotypic outcomes or
clinical observations, or through retrospective analysis of the sequencing data [33,120–122].
Studies are also required to assess the relative importance of each novel mutation in
causing resistance, which is important for routine clinical explanations. The explanation
of novel genotypes in causing resistance will in part rely on an existing understanding of
the molecular basis of causing resistance, and new mechanisms will need to be verified in
follow-up studies, such as through knock-out studies.
In addition, current studies that have assessed the accuracy of NGS or WGS in pre-
dicting antibiotic resistance are limited by small sample sizes for certain antibiotics (e.g.,
tetracycline and rifabutin) because of low prevalence of antibiotic resistance [35,96]. Hence,
studies with wider gene coverages, larger sample sizes or multicenter design from different
geographical regions are warranted. Standardized and user-friendly computational soft-
ware and tools need to be developed so that NGS data can be easily analyzed and applied
in routine clinical settings [33,36]. NGS databases also require regular and continuous
updating of novel mutations detected; otherwise, underestimation of resistance mecha-
nisms may occur because of them not being represented in the databases [122]. Despite
these limitations, advancements in sequencing technology and increasing knowledge of
molecular mechanisms of resistance will undoubtedly boost the role of NGS as a fast and
reliable tool for AST in the context of H. pylori infection in the future.
4. Antibiotic Stewardship in the Treatment of Helicobacter pylori Infection

4.1. Issues of the “Better-than Approach” in H. pylori Treatment and Research
Subtherapeutic levels of an antibiotic in H. pylori treatment is the main culprit for the
development of resistance against it, as it favors the survival and natural selection of these
resistant strains [33]. Prior antibiotic usage within 180 days was found to be associated
with higher risk of needing retreatment for H. pylori [123]. Antibiotic stewardship has
been increasingly promoted as an effective solution to this growing problem. In essence,
antibiotic stewardship refers to sets of actions that (1) promote and monitor the responsible
use of antibiotics, (2) promote the selection of optimal treatment regimens and (3) ensure the
sustainable access to antibiotics by those in need [13,124,125]. This is especially important
in H. pylori treatment as antibiotic combinations in treating H. pylori have long been based
on trial and error, and treatment regimens were chosen because they were relatively “better
than” traditional empirical regimens but not because they had an acceptable absolute cure
rate [13,125]. The adaptation of antibiotic stewardship principles in H. pylori treatment
will mean that the effectiveness of treatment should instead be determined as whether the
treatment can achieve a prespecified success rate in both routine clinical settings and in
clinical trials.
The use of the “better-than” approach in clinical trials had several issues. On one
hand, its value might be undermined if at least one or even all of the treatment arms
tested failed to achieve the optimal success rate stipulated by antibiotic stewardship [125].
On the other, ethical concerns might arise as participants were often not informed of
any possible suboptimal eradication rate, which was actually predictable based on prior
experience [125–127]. Similarly, overfocus on relative comparisons in meta-analysis means
Int. J. Mol. Sci. 2023, 24, 11708 14 of 33
that the results may be misguided by inherent variations in the clinical trials included, such
as geographical variations of resistance patterns [125]. With antibiotic stewardship, only
therapies with reliable high cure rates will remain of interest to physicians, and comparisons
will only be made among them [13].
4.2. Susceptibility-Guided Therapy vs. Empirical Therapy in Achieving Antibiotic Stewardship

Antibiotic stewardship also entails the principle that only antibiotics to which the
bacteria are susceptible should be used in treatment, and ideally, only directly (based on
AST) or indirectly (based on test-of-cure results) susceptibility-guided therapy (SGT) opti-
mized for the population being treated should be used [13]. However, empirical therapies
used by physicians for treating H. pylori are not often based on the latest information of
local antibiotic resistance patterns because such data are either not available or not shared
with physicians systematically. When optimized, empirical therapy can be effective in
H. pylori treatment. The results of five meta-analyses found that SGT was superior to
empirical regimens in first-line treatment [22,27–29,128]. However, the studies included
were highly heterogeneous, and there might be no significant difference when each em-
pirical regimen was analyzed separately (Table 3). The most recent meta-analysis, which
included the most studies, found that SGT (using either culture or PCR) was superior to
empirical clarithromycin-containing triple therapy for first-line treatment in high (>20%)
clarithromycin resistance areas (RR: 1.13, 95% CI: 1.03–1.25) and also in low clarithromycin
resistance areas (RR: 1.24, 95% CI: 1.15, 1.32) [29]. However, there was no significant differ-
ence in eradication rates between SGT and first-line quadruple therapy [29]. Paradoxically,
there was also no significant difference between SGT and empirical regimens in rescue
treatments, similar to two other meta-analysis [27–29]. This interesting phenomenon might
be due to the paucity and high heterogeneity of the studies included [128] or because the
efficacy of rescue SGT could have been affected by a previous first-line regimen choice [28].
The lack of evidence in the use of SGT in rescue therapies was acknowledged by the Maas-
tricht VI report, though it recommended the routine monitoring of antibiotic resistance
patterns by AST or eradication rate data to optimize empirical rescue therapies [4].
In recent years, there have been more studies that compared PCR-based SGT to
empirical regimens. A study in France found that PCR-based SGT had a significantly higher
eradication rate than empirical triple therapy [129]. Similar findings were seen in a Taiwan
study with PCR conducted on gastric juice samples [130] and several Korean studies
that used dual priming oligonucleotide (DPO)-based PCR on gastric biopsies [131,132].
However, other Korean studies found no significant differences when DPO-PCR-based
SGT was compared to quadruple therapies, both bismuth- [30,31,133] or non-bismuth-
containing [134,135]. A Taiwan study also found no significant difference in eradication
rates of PCR-based SGT and empirical therapy based on medication history in patients
with ≥2 failed eradication attempts [136]. Another Taiwan study showed that SGT based
on PCR on gastric biopsies could achieve a similar eradication rate as culture-based SGT
in first-line treatment (86% vs. 87%, p = 0.81) and in third-line treatment (88% vs. 87%,
p = 0.74) [137], hence supporting the use of PCR methods to substitute culture as the method
of AST if SGT strategy is to be used.
Int. J. Mol. Sci. 2023, 24, 11708 15 of 33
Table 3. Summary of meta-analysis comparing eradication rates of susceptibility-guided therapy with empirical therapy.
Meta-Analysis No. of Studies Included in Meta-Analysis Results

First-line treatment:
Wenzhen et al., 2010 [22] 5 RCTs (I2 = 0%) Superiority of SGT (Method of AST: culture-based; 2 antibiotics based on AST + PPI or
Bismuth) over empirical triple therapy: RR: 1.19, 95% CI: 1.11–1.30, p < 0.001
Superiority of SGT (methods including culture-based methods and fecal PCR) to
empirical treatment
Total: 8 RCTs, 4 quasi-RCTs
• Including all studies: RR 1.16, 95% CI 1.10–1.23, p < 0.001
• First-line treatment: 5 RCTs, 4 quasi-RCTs; mild to moderate heterogeneity
Lopez-Gongora et al., 2015 [27] • Including RCTs only: RR 1.15, 95% CI 1.07–1.24, p < 0.001
(I2 = 23–54%) • Including studies using empirical triple therapy only: RR 1.18, 95% CI 1.11–1.26,
• Second-line treatment: 4 RCTs; high heterogeneity (I2 = 87%) p < 0.001
Second-line treatment:
No significant difference between SGT (culture-based methods) and empirical treatment
(none are bismuth quadruple therapy): RR 1.11, 95% CI: 0.86–1.50, p = 0.38
Pooled analysis:
Superiority of SGT to empirical treatment: RR 1.16, 95% CI 1.11–1.22
Total: 10 RCTs, 3 non-randomized controlled clinical trials; high pooled heterogeneity Superiority of SGT to empirical treatment: pooled RR 1.18, 95% CI 1.14–1.22
(I2 = 57.1%) Rescue treatment:
• First-line treatment: 10 studies No significant difference between SGT and empirical treatment: pooled RR 1.16,
• Rescue treatment: 4 studies 95% CI 0.96–1.39
Subgroup analysis:
• Method of AST:
# Culture-based: 10 studies
• Method of AST: Superiority of SGT to empirical treatment for both culture-based
and PCR
# PCR: 3 studies
Chen et al., 2016 [28] # Culture-based: pooled RR 1.14, 95% CI 1.08–1.21
• Empirical regimen: # PCR: pooled RR 1.23, 95% CI 1.11–1.35
# Standard triple therapy: 7 studies • Empirical regimens:
n 7-day duration: 5 studies Superiority of SGT to:
n 10-day duration: 2 studies
• 7-day standard triple therapy (pooled RR 1.22, 95% CI 1.16–1.29)
# Bismuth quadruple therapy: 3 studies • Bismuth quadruple therapy (pooled RR 1.15, 95% CI 1.08–1.22)
# Sequential therapy: 2 studies • 14-day moxifloxacin-containing triple therapy (pooled RR 1.27, 95% CI 1.08–1.51)
# 14-day moxifloxacin-containing triple therapy: 1 study • No significant difference between SGT and:
• 10-day standard triple therapy (pooled RR 1.03, 95% CI 0.76–1.41)
• Sequential therapy (pooled RR 1.01, 95% CI 0.79–1.30)
Int. J. Mol. Sci. 2023, 24, 11708 16 of 33
Table 3. Cont.

Pooled analysis:
Superiority of SGT over empirical therapy: RR 1.14, 95% CI: 1.07–1.21, p < 0.0001
Superiority of SGT over empirical therapy: RR 1.14, 95% CI: 1.07–1.21, p < 0.001
Subgroup analysis:
Total: 16 RCTs (I2 = 75%) • Empirical regimen:
• First-line treatment: 13 studies; high heterogeneity (I2 = 75%) # Superiority of SGT to empirical triple therapy: RR 1.19, 95% CI: 1.13–1.26,
• Second-line treatment: 3 studies; high heterogeneity (I2 = 84%), hence meta-analysis p < 0.001
not done # No significant difference between SGT and empirical
Gingold-Belfer et al., 2021 [128]
• Method of AST: quadruple therapy
# Culture-based: 14 studies • Clarithromycin resistance:
# PCR: 5 studies # No significant difference between SGT and clarithromycin triple therapy
when the prevalence of clarithromycin resistance is <20% (RR: 1.10,
95% CI 0.95–1.28, p = 0.199)
# Superiority of SGT over clarithromycin triple therapy when the
prevalence of clarithromycin resistance is >20% (RR: 1.18,
95% CI 1.07–1.30, p = 0.001)
All studies:
• Total: 31 RCT comparisons, 23 non-RCTs; high heterogeneity (I2 = 83%) A. All studies
• First-line treatment: 30 studies; high heterogeneity (I2 = 83%)
• Rescue treatment (more than one treatment failure): 16 studies (I2 = 78%) Pooled analysis:
• Method of AST: Superiority of SGT to empirical treatment: RR 1.12, 95% CI: 1.08–1.17
# Culture-based: 36 studies (I2 = 83%) Superiority of SGT to empirical treatment: RR 1.13, 95% CI: 1.08–1.17
# PCR: 16 studies (I2 = 84%) Rescue treatment:
No significant difference between SGT and empirical treatment: RR 1.07,
• Empirical regimen: 95% CI: 0.97–1.18
# First-line quadruple therapy (both with and without bismuth): 12 studies Subgroup analysis:
(I2 = 72%) • Method of AST: Superiority of SGT to empirical treatment for both culture-based
Nyssen et al., 2022 [29] RCTs only: and PCR
# Culture-based: pooled RR 1.11, 95% CI 1.05–1.16
• Total: 31 RCT comparisons (from 27 RCT studies) *; high heterogeneity (I2 = 74%)
# PCR: pooled RR 1.08, 95% CI 1.01–1.16
• First-line treatment: 21 comparisons; high heterogeneity (I2 = 75%)
• Rescue treatment (more than one treatment failure): 8 comparisons (I2 = 84%) • Empirical regimen:
• Method of AST: # Superiority of SGT to first-line clarithromycin triple therapy in areas with:
# Culture-based: 24 RCTs (I2 = 65%) n High (>20%) clarithromycin resistance: RR 1.13,
# PCR: 8 RCTs (I2 = 85%) 95% CI: 1.08–1.17
n Low clarithromycin resistance: RR 1.24, 95% CI: 1.15–1.32
• Empirical regimen:
# No significant difference between SGT and first-line quadruple therapy:
# First-line quadruple therapy (both with and without bismuth): 8 studies RR 1.04, 95% CI: 0.99–1.09
(I2 = 77%)
Int. J. Mol. Sci. 2023, 24, 11708 17 of 33
Table 3. Cont.
B. RCTs only
Pooled analysis:
Superiority of SGT to empirical treatment: RR 1.13, 95% CI: 1.07–1.18
Superiority of SGT to empirical treatment: RR 1.14, 95% CI: 1.08–1.20
Rescue treatment:
No significant difference between SGT and empirical treatment: RR 1.10,
95% CI: 0.85–1.42
Subgroup analysis:
• Method of AST:
# Superiority of SGT to empirical treatment for culture-based methods in:
n Pooled analysis: RR 1.13, 95% CI 1.08–1.19
n First-line treatment: RR 1.13, 95% CI: 1.07–1.20
# No significant difference of SGT to empirical treatment for:
n Culture-based methods in second-line treatment: RR 1.05, 95%
CI: 0.95–1.17
n PCR: pooled RR 1.10, 95% CI 0.99–1.24
• Empirical regimen:
# No significant difference between SGT and first-line quadruple therapy:
RR 1.05, 95% CI: 0.99–1.12
* Different treatment groups in same RCT are meta-analyzed separately. Abbreviation: RCT, randomized clinical trials; RR, risk ratio; CI, confidence interval; SGT, susceptibility
testing-guided therapy; AST, antibiotic susceptibility testing; PCR, polymerase chain reaction.
Int. J. Mol. Sci. 2023, 24, 11708 18 of 33
Alternatively, it was recently shown that a treatment regimen derived from routine
evaluation of mutations associated with antibiotic resistance using NGS on gastric biopsy
samples offered 4.4-greater odds of eradication than using empirical regimens [138]. That
being said, comparative studies of NGS on other samples, such as stool, are limited.
More studies are also needed to compare molecular-testing-guided treatment to empirical
bismuth quadruple treatment (BQT), which can achieve approximately 90% eradication
rates in the Western population [139].
Apart from efficacy, the cost effectiveness of the susceptibility-guided strategy is
another factor that must be taken into consideration. However, as in the case of efficacy,
studies that have attempted to compare the cost effectiveness of a susceptibility-guided
strategy with empirical treatment did not find consistent conclusions [106]. The continuous
improvement of molecular-based methods may be able to offer a lower cost than culture-
based testing and raise the cost effectiveness of susceptibility-guided strategy in the future,
though further studies are required to provide clinical evidence [4,140]. Current evidence
suggests that PCR-based SGT might not be more cost effective than an up-to-date empirical
treatment, such as BQT. Two Korean studies found that DPO-PCR-based SGT would
offer lower costs than empirical triple therapy only when the latter’s eradication rate
dropped to below 75.3% to [132] 80% [131]. In contrast, one Korean study found that the
total medical cost was lower for SGT compared to empirical treatment (including triple
therapy, sequential therapy and BQT) because of significantly higher treatment costs in the
empirical group [141]. When only BQT was under comparison, two Korean studies found
the empirical BQT was more cost effective than DPO-PCR-based SGT [30,31]. In these
studies, the cost of PCR-based SGT ranged from USD 90.3 [31] to USD 503.5 [30]. A Taiwan
study also found that compared to empirical treatment based on medication history, PCR-
based SGT required USD 6920 to additionally cure one patient with refractory H. pylori
infection, which undermined SGT’s cost effectiveness. The use of NGS for designing SGT
in routine clinical practice is also hindered by the need of specialized machines, hardware
and software for handling large genomic data, as well as trained staff for operation, which
together made NGS very expensive (reaching USD 2000–3000 per sample merely for
pathogen detection) and not suitable for routine use in the short term [142].
Therefore, empirical regimens, if based on latest local antibiotic susceptibility pat-
terns, are likely more cost effective than SGT. The performance of empirical therapies
should be regularly monitored so that they can quickly adapt to changes in antibiotic
susceptibility patterns. In recent years, using vonoprazan, a potassium-competitive acid
blocker with a higher acid suppressive potency than PPIs, in eradication treatment has
been shown to achieve a >90% eradication rate in several clinical trials in Asia but not in
the West (Table 4) [143–150]. Whether empirical use of a vonoprazan-based regimen is
non-inferior to a susceptibility-guided strategy in terms of H. pylori eradication warrants
further investigation.
Int. J. Mol. Sci. 2023, 24, 11708 19 of 33
Table 4. Summary of randomized clinical trials comparing vonoprazan-based therapies to empirical eradication therapies (arranged in decreasing order of
eradication rate by vonoprazan-based therapies).
Sample Special Patient Results (Eradication Rates of H. pylori)

Study Country Study Design Treatment Regimen
Size Characteristics ITT PP
A. Vonoprazan dual or triple therapy vs. PPI triple therapy
In treatment-naive patients
1. 20 mg VPZ twice daily + 1 g AMX

twice daily + 500 mg CLA twice
• Open-label daily for 7 days VPZ triple: 96.7% VPZ triple: 98.3%
Bunchorntavakul
Thailand 122 • Single-center / 2. 20 mg OPZ twice daily + 1 g AMX PPI triple: 88.5% PPI triple: 93.1%
et al., 2021 [143]
twice daily + 500 mg CLA twice p = 0.083 p = 0.159
daily for 14 days
1. 20 mg VPZ twice daily + 750 mg

AMX twice daily + 200 mg or
400 mg CLA twice daily for
7 days; or VPZ-triple: 95.8% VPZ-triple: 95.7%
Maruyama • Single-blind
Japan 141 / 2. 20 mg RPZ or 30 mg LPZ twice PPI-triple: 69.6% PPI-triple: 71.4%
et al., 2017 [145] • Single-center
daily + 750 mg AMX twice daily + p < 0.001 p < 0.001
200 mg (if RPZ) or 400 mg (if RPZ
or LPZ) CLA twice daily for
7 days

AMX twice daily + 200 mg or VPZ triple (regardless of
400 mg CLA twice daily for CLA dose): 92.6%
• Double-blind All patients had
Murakami et al., 7 days LPZ triple (regardless of
Japan 650 • Multi-center history of gastric or NA
2016 [146] 2. 30 mg LPZ twice daily + 750 mg CLA dose): 75.9%
• Noninferiority duodenal ulcer AMX twice daily + 200 mg or noninferiority
400 mg CLA twice daily for p < 0.0001
7 days
Int. J. Mol. Sci. 2023, 24, 11708 20 of 33
Table 4. Cont.


• Open-label daily for 7 days VPZ triple: 87.4% VPZ triple: 96.3%
Ang et al., 2022 • Single-center 2. 20 mg OPZ or 20 mg EPZ or
Singapore 252 / PPI triple: 88.0% PPI triple: 94.0%
[151] • Noninferiority 20 mg RPZ twice daily + 1 g AMX noninferiority p < 0.05 noninferiority p < 0.05
daily for 14 days
1. Open-label VPZ dual therapy

• Open-label or (20 mg VPZ twice daily + 1 g AMX VPZ triple: 84.7% VPZ triple: 90.4%
double-blind 3 times daily) for 14 days VPZ dual: 78.5% VPZ dual: 81.2%
dependent on All patients were not 2. Double-blind triple therapy twice LPZ triple: 78.8% LPZ triple: 82.1%
Chey et al., USA,
1046 treatment group resistant to CLA a day (20 mg VPZ twice daily or VPZ triple vs. LPZ triple: VPZ triple vs. LPZ triple:
2022 [152] Europe
• Multi-center or AMX 30 mg LPZ twice daily + 1 g AMX noninferiority p < 0.001 noninferiority p < 0.001
• Noninferiority twice daily + 500 g CLA twice VPZ dual vs. LPZ triple: VPZ dual vs. LPZ triple:
daily) for 14 days noninferiority p < 0.05 noninferiority p < 0.05

AMX twice daily + 200 mg or
400 mg CLA twice daily for 7 days In CLA-susceptible patients: In CLA-susceptible patients:
(VPZ triple therapy for VPZ triple: 87.3% VPZ triple: 88.9%
CLA-susceptible and PPI triple: 76.5% PPI triple: 86.7%
Sue et al., 2018 • Open-label CLA-resistant patients) p = 0.21 p = 0.77
Japan 63 • Multi-center / 2. 30 mg LPZ or 10 mg RPZ or 20 mg
[153] In CLA-resistant patients: In CLA-resistant patients:
EPZ twice daily + 750 mg AMX VPZ triple: 82.9% VPZ triple: 82.9%
twice daily + 200 mg or 400 mg (no comparison with (no comparison with
CLA twice daily for 7 days (PPI PPI triple) PPI triple)
triple therapy for CLA-susceptible
patients only)
Int. J. Mol. Sci. 2023, 24, 11708 21 of 33
Table 4. Cont.

In patients with possible/confirmed prior eradication treatment

twice daily for 14 days VPZ dual: 93.5%
Zuberi et al., • Single-center Prior treatment 2. 20 mg OPZ twice daily + 1 g AMX
Pakistan 192 NA PPI triple: 83.9%
2022 [149] status unknown twice daily + 500 mg CLA twice p = 0.042
daily for 14 days

Patients failed AMX twice daily + 100 mg
first-line (PPI or VPZ sitafloxacin twice daily for 7 days
• Open-label VPZ group: 75.8% VPZ group: 83.3%
Sue et al., 2019 + AMX + CLA) and 2. 30 mg LPZ or 10 mg RPZ or 20 mg
Japan 147 • Single-center PPI group: 53.3% PPI group: 57.1%
[154] second-line (PPI or EPZ twice daily + 750 mg AMX p = 0.071 p = 0.043
VPZ + AMX + twice daily + 100 mg sitafloxacin
MNZ) regimens twice daily for 7 days
1. 20 mg VPZ twice daily +750 mg

Patients failed AMX twice daily + 250 mg MNZ
• Open-label first-line eradication twice daily for 7 days VPZ group: 73.9% VPZ group: 89.5%
Hojo et al., 2020
Japan 46 • Multi-center therapy consisting of 2. 10 mg RPZ twice daily + 750 mg PPI group: 82.6% PPI group: 86.4%
[155]
VPZ or AMX twice daily + 250 mg MNZ p = 0.72 p = 1.00
PPI + AMX + CLA twice daily for 7 days
Int. J. Mol. Sci. 2023, 24, 11708 22 of 33
Table 4. Cont.

B. Vonoprazan dual or triple therapy vs. PPI-bismuth quadruple therapy

AMX 4 times daily for 10 days
(VHA-dual) VHA-dual: 92.7% VHA-dual: 93.4%
2. 20 mg VPZ twice daily + 750 mg VA-dual: 84.4% VA-dual: 85.4%
• Open-label BQT: 89.4% BQT: 90.9%
Qian et al., 2023 AMX 4 times daily or 1 g AMX
China 375 • Single-center / VHA-dual vs. BQT: VHA-dual vs. BQT:
[148] twice daily for 10 days (VA-dual)
• Noninferiority noninferiority p < 0.001 noninferiority p < 0.001
3. 20 mg EPZ twice daily + 200 mg
colloidal bismuth twice daily + 1 g VA-dual vs. BQT: VA-dual vs. BQT:
AMX twice daily + 500 mg CLA noninferiority p > 0.05 noninferiority p > 0.05
twice daily for 10 days (BQT)

doxycycline twice daily + 100 mg
furazolidone twice daily for VDF-triple: 88.1% VDF-triple: 97.9%
14 days (VDF triple) VDA-triple: 87.5% VDA-triple: 96.6%
2. 20 mg VPZ twice daily + 100 mg EBDF-quadruple: 80.0% EBDF-quadruple: 91.4%
doxycycline twice daily + 1 g EBDA-quadruple: 75.0% EBDA-quadruple: 90.2%
AMX twice daily for 14 days VDF-triple vs. VDF-triple vs.
(VDA triple) VDA-triple: p = 0.864 VDA-triple: p = 0.723
• Open-labelr 3. 20 mg EPZ twice daily + 220 mg VDF-triple vs. VDF-triple vs.
Zhang et al.,
China 640 • Single-center / bismuth twice daily + 100 mg EBDF-quadruple: EBDF-quadruple:
[156]
doxycycline twice daily + 100 mg p = 0.047 p = 0.017
furazolidone twice daily for VDA-triple vs. VDA-triple vs.
14 days (EBDF quadruple) EBDA-quadruple: EBDA-quadruple:
4. 20 mg EPZ twice daily + 220 mg p = 0.004 p = 0.049
bismuth twice daily + 100 mg EBDF-quadruple vs. EBDF-quadruple vs.
doxycycline twice daily + 1 g EBDA-quadruple: EBDA-quadruple:
AMX twice daily for 14 days p = 0.284 p = 0.730
(EBDA quadruple)
Int. J. Mol. Sci. 2023, 24, 11708 23 of 33
Table 4. Cont.

C. Vonoprazan-bismuth quadruple therapy vs. PPI-bismuth quadruple therapy

daily + 220 mg bismuth twice
• Double-blind Patients were daily for 14 days;
Huh et al., 2021 VPZ quadruple: 100%
Korea 30 • Single-center not necessarily 2. 30 mg LPZ twice daily + 1 g AMX NA
[150] PPI quadruple: 100%
treatment-naive twice daily + 500 mg CLA twice
daily + 220 mg bismuth twice
daily for 14 days

twice daily + 100 mg furazolidone
twice daily + 200 mg colloidal V10 group: 96.2% V10 group: 98.6%
bismuth twice daily for 10 days V14 group: 94.9% V14 group: 97.4%
• Open-label (V10 group) or 14 days E14 group: 93.6% E14 group: 94.8%
Lu et al., 2023 • Single-center (V14 group)
China 234 / V10 vs. E14 group: V10 vs. E14 group:
[144] • Noninferiority 2. 20 mg EPZ twice daily + 1 g AMX noninferiority p < 0.05 noninferiority p < 0.05
twice daily + 100 mg furazolidone V14 vs. E14 group: V14 vs. E14 group:
twice daily + 200 mg colloidal noninferiority p < 0.05 noninferiority p < 0.05
bismuth twice daily for 14 days
(E14 group)
Int. J. Mol. Sci. 2023, 24, 11708 24 of 33
Table 4. Cont.

In H. pylori-positive patients:
• Double-blind 1. 20 mg VPZ twice daily + bismuth
China • Multi-center 600 mg twice daily + 1 g AMX
• Noninferiority All patients had twice daily + 500 mg CLA twice
including VPZ quadruple: 91.5%
Hou et al., 2022 • (* primary duodenal ulcer daily for 14 days
Taiwan, 533 LPZ quadruple: 86.8% NA
[147] outcome was (85.4% were 2. 30 mg LPZ twice daily + bismuth
South noninferiority p < 0.05
duodenal ulcer H. pylori-positive) 600 mg twice daily + 1 g AMX
Korea
healing) twice daily + 500 mg CLA twice
daily for 14 days
* It is to highlight the that unlike other studies, the primary outcome is dudenal ulcer healing instead of HP eradication in this study. Abbreviations: ITT, intention-to-treat analysis;
PP: per protocol analysis; CI: confidence interval; PPI, proton pump inhibitor; VPZ, vonoprazan; AMX, amoxicillin; CLA, clarithromycin; LPZ, lansoprazole; OPZ, omeprazole; EPZ,
esomeprazole; RPZ, rabeprazole; MNZ, metronidazole; NA, not available.
Int. J. Mol. Sci. 2023, 24, 11708 25 of 33
4.3. Measures to Achieve Antibiotic Stewardship in H. pylori Treatment

In the meantime, analyzing routinely collected clinical records of diagnosis, treatment
and the test of a cure (such as urea breath test) conducted at least four weeks after the treat-
ment is completed [13,125,157] is an existing method that can indirectly assess antibiotic
susceptibility. Yet, despite the expected lower cost and relative ease in implementation,
these routinely collected data are often not shared with physicians to guide their clinical
decisions or not included in antibiotic surveillance programs so that they can be analyzed
and incorporated into treatment guidelines systematically [13,125]. The latest Maastricht
VI Florence consensus report stated that “it is reasonable to recommend that susceptibility
tests (molecular or after culture) are routinely performed, even before prescribing first-line
treatment, in respect to antibiotic stewardship” [4]. As molecular methods for AST become
more convenient and affordable, the long-term goal will be to adapt an individualized
approach in the design of treatment regimens, so that the highest eradication rate can be
achieved while avoiding the use of unnecessary antibiotics (i.e., antibiotics that in fact
do not contribute to treatment effectiveness) in treatment combinations to combat global
resistance [13,125,158,159].
The implementation of antibiotic stewardship in H. pylori treatment requires collective
effort from the individual, local and up to international level. At the individual level,
patient compliance is an important factor deciding treatment effectiveness. A study found
a global odds ratio (OR) of around 7 in its association with treatment effectiveness, and
that excellent compliance was the factor that was mostly associated with eradication
rates in all treatment regimens evaluated [12]. Another meta-analysis also identified that
patient compliance, with an OR of 16, was the most significant factor affecting treatment
success [160]. Therefore, the best way the individual patient can help in achieving antibiotic
stewardship is to comply with the treatment regimen prescribed by the physician. This
must be emphasized to the patient by the physician during consultation or through public
education campaigns [161,162]. Physicians must also be educated on up-to-date antibiotic
resistance patterns and relevant guidelines concerning antibiotic use [161,162]. Local
healthcare authorities should take the initiative to establish antibiotic stewardship programs
that provide AST in routine clinical setting, monitor antibiotic prescriptions and resistance
patterns and provide treatment guidelines tailored to local conditions [125,161,162]. On the
international level, consensus reports and guidelines play an important role in promoting
antibiotic stewardship measures and providing treatment suggestions based on updated
antibiotic resistance pattern, but this potential has not been fully reached up until now [125].
Existing antibiotic surveillance programs, both at local and international level, should
include H. pylori if it has not already been done [125]. Collaborative efforts from all sorts of
stakeholders are thus crucial in ensuring that antibiotic stewardship is applied to H. pylori
treatment around the globe.
5. Conclusions
Antibiotic stewardship should be practiced in view of increasing antibiotic resistance
of H. pylori worldwide to ensure the sustainability of current treatment regimens. Antibiotic
susceptibility testing becomes increasingly important to provide data on antibiotic resis-
tance patterns to guide antibiotic stewardship measures. With advancements in technology,
molecular-based sequencing techniques allow the detection of antibiotic resistance in
a rapid and convenient manner. In particular, preliminary evidence showed that stool-
based molecular tests have potential to serve as a non-invasive and cost-effective AST tool
in routine clinical practice, though more studies are needed to establish its accuracy and
costs for it to be of use in the future. Novel genetic mutations may also be discovered with
WGS, although validation studies are required. Collaboration at the individual, local and
international levels is needed to implement various measures aimed at improving treatment
effectiveness and patient compliance that are of utmost importance for the application of
antibiotic stewardship in H. pylori treatment.
Int. J. Mol. Sci. 2023, 24, 11708 26 of 33
Author Contributions: Conceptualization, H.-Y.N. and K.-S.C.; creation of figures: H.-Y.N.; drafting
of manuscript, H.-Y.N.; supervision and revision of manuscript, K.-S.C. and W.K.L. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Acknowledgments: Figures 1 and 2 were created with Biorender.com (accessed on 18 July 2023).
Conflicts of Interest: The authors declare no conflict of interest.
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International Journal of

Int. J. Mol. Sci. 2023, 24, 11708. https://doi.org/10.3390/ijms241411708 https://www.mdpi.com/journal/ijms

quadruple therapy, typically consisting of bismuth, PPI, metronidazole and tetracycline, is

2. Antibiotic Resistance in Helicobacter pylori

Figure 1. Summary of molecular mechanisms of antibiotic resistance in H. pylori. In general, antibi-

Genes Involved Resistance Mechanisms

2.1. Resistance to Clarithromycin

2.2. Resistance to Metronidazole

2.3. Resistance to Levofloxacin

2.4. Resistance to Amoxicillin

2.5. Resistance to Tetracycline

3. Antibiotic Susceptibility Testing for Helicobacter pylori

Figure 2. Comparison of culture-based and molecular-based antibiotic susceptibility testing (AST)

3.1. Culture-Based Techniques

3.2. Molecular-Based Techniques

Table 2. Summary of studies assessing the performance of polymerase chain-reaction-based assays

Sample Size and

Sample Size and

Sample Size and

(1) PCR-based assays

4. Antibiotic Stewardship in the Treatment of Helicobacter pylori Infection

4.2. Susceptibility-Guided Therapy vs. Empirical Therapy in Achieving Antibiotic Stewardship

Meta-Analysis No. of Studies Included in Meta-Analysis Results

Meta-Analysis No. of Studies Included in Meta-Analysis Results

Meta-Analysis No. of Studies Included in Meta-Analysis Results

Sample Special Patient Results (Eradication Rates of H. pylori)

A. Vonoprazan dual or triple therapy vs. PPI triple therapy

1. 20 mg VPZ twice daily + 1 g AMX

1. 20 mg VPZ twice daily + 750 mg

1. 20 mg VPZ twice daily + 750 mg

Sample Special Patient Results (Eradication Rates of H. pylori)

1. 20 mg VPZ twice daily + 1 g AMX

1. Open-label VPZ dual therapy

1. 20 mg VPZ twice daily + 750 mg

Sample Special Patient Results (Eradication Rates of H. pylori)

1. 20 mg VPZ twice daily + 1 g AMX

1. 20 mg VPZ twice daily + 750 mg

1. 20 mg VPZ twice daily +750 mg

Sample Special Patient Results (Eradication Rates of H. pylori)

B. Vonoprazan dual or triple therapy vs. PPI-bismuth quadruple therapy

1. 20 mg VPZ twice daily + 750 mg

1. 20 mg VPZ twice daily + 100 mg

Sample Special Patient Results (Eradication Rates of H. pylori)

C. Vonoprazan-bismuth quadruple therapy vs. PPI-bismuth quadruple therapy

1. 20 mg VPZ twice daily + 1 g AMX

1. 20 mg VPZ twice daily + 1 g AMX

Sample Special Patient Results (Eradication Rates of H. pylori)

4.3. Measures to Achieve Antibiotic Stewardship in H. pylori Treatment

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