Antibiotic Resistance of Helicobacter Pylori in Nanjing: A Cross-Sectional Study
Conditions: HELICOBACTER PYLORI INFECTIONS
Sponsor: Nanjing First Hospital, Nanjing Medical University

Antibiotic Resistance of Helicobacter pylori in
Nanjing: A Cross-Sectional Study
NCT Number:
Document Date: January 8, 2026

1. Introduction
Since its discovery, Helicobacter pylori has become one of the most prevalent
microorganisms globally, with an estimated worldwide infection rate of 44.3%. The
burden is notably higher in developing countries (50.8%) compared to developed
nations (34.7%) [1]. Beyond its established role in chronic active gastritis and peptic
ulcer disease, H. pylori is now recognized as a primary risk factor for gastric cancer
and mucosa-associated lymphoid tissue lymphoma. This is particularly significant in
China, where gastric cancer ranks as the third most common malignancy in men and
the second in women [2]. Consequently, H. pylori infection constitutes a major public
health
challenge[3].
The
escalating
threat
of
antimicrobial
resistance
further
underscores the urgent need to improve eradication strategies[4, 5].
The Maastricht VI/Florence Consensus Report recommends routine antibiotic
susceptibility testing to guide the selection of precise treatment regimens prior to H.
pylori eradication[6]. Currently, only a limited number of antibiotics—including
amoxicillin,
clarithromycin,
metronidazole,
levofloxacin,
tetracycline,
and
furazolidone—remain effective. However, their widespread use has accelerated the
emergence of resistance[7, 8]. China, with its high burden of infection, faces a
particularly complex landscape of antibiotic resistance [9].
Regimens containing clarithromycin and levofloxacin are effective strategies for both
first-line and salvage therapy[10]. A meta-analysis highlighted that a 14-day
levofloxacin-based sequential therapy is one of the most effective regimens
worldwide[11]. However, levofloxacin susceptibility shows significant geographic
variation. For instance, the average primary resistance rate in the Asia-Pacific region
is approximately 18% [12], while in China it has risen to 34.21% [13].
Faced with continuously rising resistance rates to clarithromycin and levofloxacin, it
is crucial to delineate two aspects: first, the dynamic trends in phenotypic resistance to
these antibiotics in our region in recent years; and second, the characteristics of gene
mutations associated with this resistance.
The increasingly severe resistance situation necessitates the establishment of
continuous surveillance and the accumulation of local data. Our center has previously

published cross-sectional studies analyzing H. pylori resistance in Nanjing for the
periods 2018–2021 and 2018–2023, focusing on the genotypic resistance of
clarithromycin and levofloxacin. However, data on resistance trends for other
antimicrobials, CYP2C19 gene polymorphisms, and their potential mechanisms
remain limited. Therefore, this study systematically reviews the evolution of
phenotypic and genotypic resistance rates of H. pylori to six commonly used
antibiotics over the past seven years.
Beyond phenotypic analysis, this study investigates the genotypic resistance
mechanisms for amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline,
and furazolidone. Resistance in H. pylori primarily results from mutations in genes
encoding
drug
targets,
leading
to
reduced
efficacy. Evidence
indicates
that
susceptibility can be effectively assessed by detecting mutations in the 23S rRNA
gene (primarily point mutations A2143G, A2142G, and A2142C in domain V) for
clarithromycin resistance; the gyrA gene for quinolone resistance; penicillin-binding
protein genes for amoxicillin resistance; the 16S rRNA gene for tetracycline
resistance; and the rdxA, frxA, and frxB genes for metronidazole resistance.
This study aims to provide an updated analysis of H. pylori resistance patterns in the
Nanjing region, focusing on the current status of phenotypic and genotypic resistance
to these six antimicrobial agents. The findings are expected to provide a scientific
basis for the rapid diagnosis and personalized treatment of H. pylori infection.
2. Objectives
This project aims to analyze H. pylori resistance patterns in Nanjing through a
retrospective
review
of
medical
records
of
outpatients
who
underwent
H.
pylori culture and genetic testing between 2018 and 2025. The focus is on
characterizing the current status of phenotypic and genotypic resistance to amoxicillin,
clarithromycin, metronidazole, levofloxacin, tetracycline, and furazolidone. The
findings are intended to assist clinicians in the rapid diagnosis and individualized
treatment of H. pylori infection.

3. Study Design, Methods, and Sample Collection
3.1. Study Design
This is a retrospective, single-center, cross-sectional epidemiological study. It
involves a retrospective review of medical records to analyze H. pylori resistance
patterns in Nanjing, with the goal of supporting rapid diagnosis and individualized
treatment.
3.2. Methods
Patient records from the electronic medical record system and laboratory information
system of Nanjing First Hospital were searched to identify all outpatients who
underwent H. pylori culture and genetic testing at the gastroenterology clinic between
January 1, 2018, and December 31, 2025, according to pre-defined inclusion and
exclusion criteria.
3.3. Sample Collection and Requirements
Sample Type: This study retrospectively analyzes existing laboratory results from
gastric mucosal biopsy samples stored in the hospital database. No new biological
samples were collected for this research.
Sample Collection: All original samples were gastric mucosal tissues collected by
clinicians during routine esophagogastroduodenoscopy for standard clinical care,
following standard operating procedures.
3.4. Study Procedures and Technical Pathway
(1) Study Procedures:
1. Study Initiation Meeting: The principal investigator convened all research staff to
review the study protocol, inclusion/exclusion criteria, data definitions, and extraction
rules to ensure uniform understanding.
2. Cohort Identification and Lock: The data administrator retrieved and exported all
7,228 initial records of H. pylori testing from the hospital information system for the
period from January 1, 2018, to December 31, 2025.
3. Case Screening and Enrollment: Two researchers independently screened the
cases from the initial cohort according to the uniform inclusion/exclusion criteria.
Discrepancies were resolved by a third senior researcher to finalize the list of patients

included in the analysis.
4. Data Extraction: Using a pre-designed electronic Case Report Form, the following
information was extracted from the hospital's medical record and laboratory systems:
* Basic Information: Study ID, age, sex.
* Clinical Information: Endoscopic diagnosis, relevant medical history.
*
Laboratory
Results:
H.
pylori
culture
results,
phenotypic
antimicrobial
susceptibility testing results for the six antibiotics, and mutation detection results for
resistance-related genes.
5. Data Cleaning and Database Lock: Extracted data were cleaned to address
missing values and logical errors. Key variables underwent independent double-data
entry and verification. An anonymized research database suitable for analysis was
formed as the final study cohort and locked without further changes.
6. Statistical Analysis
7. Report Writing: A comprehensive, standardized retrospective study report was
written based on the locked data and analysis results.
(2) Technical Pathway
4. Participant Selection
4.1. Inclusion Criteria
Individuals meeting all of the following criteria were included:
(1) Patients who visited the gastroenterology outpatient clinic of Nanjing First
Hospital and underwent gastroscopy with H. pylori culture and genetic testing
between 2018 and 2025.
(2) Positive for H. pylori by at least one diagnostic method.
(3) Provision of general written informed consent for the use of gastroscopy and
specimen data in research.
(4) Availability of complete medical records and laboratory test results.
4.2. Exclusion Criteria
Individuals meeting any of the following criteria were excluded:
(1) Withdrawal of informed consent; use of antibiotics, proton pump inhibitors,

H2-receptor antagonists, or bismuth agents within four weeks prior to gastroscopy.
(2) History of gastric surgery.
(3) Incomplete medical records or test results precluding evaluation.
(4) Other factors deemed by the investigator to render the participant unsuitable for
inclusion.
4.3. Sample Size
Based on an initial search, a total of 7,228 records were identified within the specified
timeframe. All cases meeting the inclusion and exclusion criteria were included in the
analysis.
5. Alternative Diagnostic and Treatment Methods
For patients unsuitable for or unwilling to undergo gastroscopy, non-invasive
diagnostic options such as the 13C-urea breath test or stool antigen test are clinically
available.
In the absence of susceptibility testing, clinicians may empirically select eradication
regimens based on regional resistance epidemiology or choose different combinations
of salvage therapies based on the patient's prior medication history.
6. Testing Items and Time Points
Phenotypic Antimicrobial Susceptibility Testing: For isolated H. pylori strains, the
minimum inhibitory concentration for amoxicillin, clarithromycin, metronidazole,
levofloxacin, tetracycline, and furazolidone was determined using the agar dilution
method or E-test. Resistance was interpreted according to CLSI or EUCAST
standards.
Genotypic Resistance Detection: DNA from gastric tissue or isolates was analyzed
using multiplex PCR or targeted sequencing to detect mutations in the following
resistance-related genes:
* Clarithromycin: 23S rRNA gene
* Levofloxacin: gyrA gene
* Amoxicillin: PBP1A gene

* Tetracycline: 16S rRNA gene
* Metronidazole: rdxA, frxA genes
Testing
Time
Point: This
study
retrospectively
analyzes
the
results
of
the
above-mentioned tests that were completed at the time of initial diagnosis for all
included cases. No new testing was arranged.
7. Efficacy Evaluation Criteria
As this is a retrospective surveillance study of resistance patterns and does not involve
any therapeutic intervention, there are no efficacy outcomes. The primary outcome
measures are the phenotypic resistance rates and genotypic mutation rates for each
antibiotic.
8. Observation, Recording, and Management of Adverse Events
This is a retrospective data-based study that does not implement any new
interventions. Therefore, it is not expected to directly cause any adverse events. The
study only analyzes historical data and does not alter the patients' original diagnostic
or treatment processes.
9. Quality Control and Quality Assurance
Data extraction and entry were performed independently by two researchers using a
standardized Case Report Form, followed by cross-verification. Discrepancies were
adjudicated by a third party. The reviewed laboratory data originated from the
ISO-certified clinical laboratory of our hospital, where all operations followed
standard operating procedures and included regular participation in external quality
assessment schemes. All uses of retrospective data comply with hospital data security
regulations, and ethical review exemption or approval has been obtained.
10. Data Safety Monitoring
During the study, all patient personal information was replaced with a unique study ID
to ensure complete data anonymization. After study completion, the data will be

stored for at least five years to facilitate potential audits.
11. Statistical Analysis
Data analysis was performed using IBM SPSS Statistics (version 26.0). Percentages
were used to describe the rates of H. pylori resistance to antibiotics. The Kappa
consistency test was used to evaluate the agreement between antibiotic resistance
phenotypes and genotypes. In statistical analysis, the Kappa coefficient ranges from
-1 to 1. Typically, a Kappa value greater than 0.75 indicates substantial agreement.
12. Ethical Principles and Requirements
This clinical study was conducted in accordance with the Declaration of Helsinki and
the Chinese National Health and Family Planning Commission's "Ethical Review of
Biomedical Research Involving Humans." The principles of informed consent,
privacy protection, research-related free services and compensation, risk control,
protection of vulnerable subjects, and compensation for research-related injuries were
strictly followed. The study protocol was approved by the ethics committee before
initiation. Before enrollment, investigators were responsible for providing each
participant or their legal guardian with a comprehensive explanation of the study's
purpose, procedures, and potential risks, and for obtaining written informed consent.
Participants were informed that their participation was entirely voluntary and that they
could refuse to participate or withdraw from the study at any time without
discrimination or retaliation, and that their medical care and rights would not be
affected. The informed consent forms were retained as part of the clinical study
documentation to ensure the protection of personal privacy and data confidentiality.
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