Efficacy of Antibiotic Susceptibility-based Tailored Versus Empiric Therapy for Helicobacter Pylori First-line Treatment：a Randomized Clinical Trial
Phase: PHASE4
Conditions: Helicobacter Pylori Infection
Sponsor: Shanghai Jiao Tong University School of Medicine

 
 
 
Clinical Trial Protocol and Statistical 
Analysis Plan 
 
 
 
Contents  
1. Original clinical trial protocol ························································· 2 
2. Final clinical trial protocol ····························································· 26 
3. Summary of clinical trial protocol changes ········································· 50 
4. Original statistical analysis plan ······················································ 56 
5. Final statistical analysis plan ·························································· 67 
6. Summary of statistical analysis plan changes ······································ 77 
 
 
1 
 

Original Protocol 
 
 
Original Clinical Trial Protocol 
 
Efficacy of Clarithromycin Susceptibility-based Tailored 
Versus Empiric Therapy for Helicobacter pylori First-line 
Treatment：a Multicenter Randomized Controlled Trial 
 
 
Protocol version: 1.0 
Approved Date: Jul 8, 2016 
Funding Source: Study On Helicobacter pylori Eradication Treatment and 
Infection Related Risk Factor project funded by Clinical Research Center, 
Shanghai Jiao Tong University School of Medicine, DLY201608. 
 
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Original Protocol 
Table of Contents 
Investigators and Statistician ····························································· 4 
Protocol Abstract ············································································ 5 
Protocol Schedule············································································ 9 
1. Background ················································································ 10 
2. Objectives ·················································································· 11 
3. Flow diagram ·············································································· 11 
4. Study design ··············································································· 12 
5. Study Population ········································································· 12 
6. Allocation and Treatment ······························································· 14 
7. H. pylori culture and antibiotic susceptibility testing ···························· 15 
8. Safety evaluation ········································································· 16 
9. Outcome measures ······································································· 18 
10. Statistical Methods ····································································· 19 
11. Ethical principles ········································································ 20 
12. References ················································································ 21 
Signature Page ··············································································· 23 
 
 
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Original Protocol 
Principal Investigator 
Hong Lu, Division of Gastroenterology and Hepatology, Shanghai Institute of 
Digestive Disease; Key Laboratory of Gastroenterology & Hepatology, Ministry of 
Health; Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 
China. 
 
Co-investigator 
1. Yunwei Sun, Department of Gastroenterology, Ruijin Hospital, Shanghai Jiao Tong 
University School of Medicine, Shanghai, China.  
2. Hong Gao, Department of Gastroenterology, Zhongshan Hospital, Fudan University, 
Shanghai, China. 
3. Yan Zhan, Department of Gastroenterology, Shanghai Tenth People's Hospital, 
Tongji University School of Medicine, Shanghai, China. 
4. Gang Xu, Department of Gastroenterology and Hepatology, Shanghai General 
Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 
 
Statistician 
Yanyan Song, Department of Biostatistics, Institute of Medical Sciences, Shanghai 
Jiao Tong University School of Medicine, Shanghai, China 
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Original Protocol 
Protocol Abstract 
Title 
Efficacy of Clarithromycin Susceptibility-based Tailored Versus 
Empiric Therapy for Helicobacter pylori First-line Treatment ：a 
Multicenter Randomized Controlled Trial 
Trial 
registration 
number 
ClinicalTrials.gov, NCT02935010 
Objective 
Comparing the efficacy, safety and compliance of PCR molecular 
method and genotypic clarithromycin susceptibility-based tailored 
therapy with empiric modified bismuth quadruple therapy in treating 
naïve patients with H. pylori infection 
Condition 
H. pylori infection naïve to treatment  
Study Design 
This study is a prospective, superiority-designed, multi-center, open-
label, randomized controlled trial. Eligible patients will be randomly 
allocated to receive either clarithromycin susceptibility-based tailored 
therapy (tailored therapy) or empirical bismuth quadruple therapy 
(empirical therapy) in a 3:1 ratio. Six weeks after completion of 
therapy, H. pylori eradication will be assessed by ¹³C-urea breath 
testing.  
Sample size 
500 
Inclusion 
Criteria 
1. Participants with non-ulcer functional dyspepsia or scarred peptic 
ulcer disease； 
2. Ability and willingness to participate in the study and to sign and 
give informed consent； 
3. confirmed H. pylori infection with positive results on both rapid 
urease testing and culture 
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Original Protocol 
Exclusion 
Criteria 
1. Previous H. pylori eradication therapy 
2. Less than 18 years old 
3. With history of H. pylori infection treatment 
4. With previous gastric surgery 
5. Major systemic diseases 
6. Pregnancy or lactation 
7. Allergy to any of the study drugs 
8. Administration of antibiotics, bismuth, antisecretory drugs in 8 
weeks prior to inclusion 
Arms and 
Interventions 
1. Empirical therapy 
After review of clarithromycin-taking medical history, if have used 
clarithromycin, give esomeprazole 20mg bid, bismuth potassium 
citrate 600mg bid, amoxicillin 1000mg bid and metronidazole 400mg 
bid for 14 days; if have not used clarithromycin, give esomeprazole 
20mg bid, bismuth potassium citrate 600mg bid, amoxicillin 1000mg 
bid and clarithromycin 500mg bid.  
2. Tailored therapy  
After molecular genetic assays for identification of resistance to 
clarithromycin of Helicobacter pylori from biopsy samples, if 
resistance to clarithromycin, give esomeprazole 20mg bid, bismuth 
potassium citrate 600mg bid, amoxicillin 1000mg bid and 
metronidazole 400mg bid for 14 days; if susceptible to clarithromycin, 
give esomeprazole 20mg bid, bismuth potassium citrate 600mg bid, 
amoxicillin 1000mg bid and clarithromycin 500mg bid. 
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Original Protocol 
Schedule 
1. Enrollment 
 Obtain written informed form 
 Check eligibility criteria 
 Collect baseline characteristics 
 Undergo upper endoscopy with gastric biopsy and rapid urease 
testing 
 Conduct bacterial culture and antibiotic susceptibility testing 
2. Allocation 
After the written informed consents would be obtained from eligible 
patients, the independent research assistant will open envelopes by 
randomization sequence and telephone study staﬀ to give them each 
patient’s treatment allocation. Eligible patients will be randomly 
allocated to receive either tailored therapy or empirical therapy in a 3:1 
ratio. 
3. treatment 
Study treatment regimens were all given 14 days. During the treatment 
period, the subjects kept a diary to monitor compliance and symptoms. 
4. Follow-up 
After the end of treatment, adverse events and compliance was 
surveyed. Six weeks after completion of therapy, H. pylori eradication 
was assessed by ¹³C-urea breath testing. 
Outcome 
Measures 
Primary Outcome Measure: 
1. Helicobacter pylori eradication rate 
Six weeks after completion of therapy, H. pylori eradication success is 
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Original Protocol 
defined as negative result from urea breath test (<4‰) 
Secondary Outcome Measure 
1. Rate of adverse events 
2. Compliance rate 
Other Pre-specified Outcome Measures: 
1. H. pylori eradication rate of patients with clarithromycin resistance 
strains 
2. H. pylori eradication rate of patients with clarithromycin sensitive 
strains 
3. Sensitivity, specificity, positive predictive value and negative 
predictive value of molecular genetic assays in clarithromycin 
resistance identification 
4. Medical cost per patient of tailored or empiric therapy 
5. Ratio of medical cost to H. pylori eradication rate of each therapy 
6. Ratio of incremental medical cost to incremental H. pylori 
eradication rate of tailored compared with empiric therapy 
 
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Original Protocol 
Protocol Schedule 
Study Period 
Enrollment Allocation Treatment 
Follow-up 
Visit 
1st 
2nd 
 
3rd 
4th 
Time point (day) 
-14th 
0 
0-14 
15th 
56th 
Eligibility screen 
√ 
 
 
 
 
Baseline characteristics 
√ 
 
 
 
 
Endoscopy with biopsy 
and RUT 
√ 
 
 
 
 
H. pylori culture and 
susceptibility testing 
√ 
 
 
 
 
PCR molecular method 
√ 
 
 
 
 
Written informed content 
√ 
 
 
 
 
Therapy Allocation 
 
√ 
 
 
 
Treatment 
 
 
√ 
 
 
Adverse events 
 
 
 
√ 
 
compliance assessment 
 
 
 
√ 
 
¹³C-urea breath test 
 
 
 
 
√ 
Medical cost 
 
 
 
 
√ 
 
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Original Protocol 
1. Background 
Helicobacter pylori (H. pylori) resistance to antibiotics was main challenge to previous 
efficacy antibiotic regimens such as standard triple therapy(1). The prevalence of 
antibiotic resistance have been markedly increasing with time in most parts of the 
world. Concordantly, the antimicrobial eradication rate of H. pylori has been declining 
globally.(2) In China, clarithromycin, metronidazole and levofloxacin resistance show 
similar trends, and resistance rates have now reached 21∼52% for clarithromycin, 
61~95% for metronidazole, 20~54% for levofloxacin, and 21~37% for both 
clarithromycin and metronidazole.(3–5) Standard triple therapy had not been suitable 
for first-line therapy in China because the pooled eradication rate had declined to 74.5% 
as a meta-analysis showed.(6)  
As a general rule, therapy for an infectious disease starts with identification of 
potentially useful antimicrobials and is largely based on the results of susceptibility 
testing. Systematic reviews or meta-analysis had proved susceptibility-based therapy 
was superior to 7–10 day triple therapies as ﬁrst-line treatment.(7–9) Therefore, there 
is a critical need to antimicrobial susceptibility testing (AST) for individualized analysis 
of antibiotic resistance prior to deﬁnitive treatment.(2)  
Now culture-based susceptibility testing for H pylori is rarely performed in standard 
practice. This may be due to several major limitations: they are time-consuming, costly, 
and invasive procedures, culture difficulty, and et al. In the age of molecular 
diagnostics, assays based on genetic material identification have become common as 
they enable acquiring rapid and accurate results at relatively low costs. The mechanism 
of resistance to clarithromycin in H. pylori is attributed to a decreased potency in 
binding of macrolides to the ribosome, caused by point mutations within the rrl gene in 
the peptidyl transferase- encoding region of the 23S rRNA gene. Three major point 
mutations in two positions, A2142C, A2142G, and A2143G, is the main cause of 
resistance to clarithromycin.(10) 
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Original Protocol 
So in this study, we will evaluate the efficacy, safety and compliance of PCR molecular 
method and genotypic clarithromycin susceptibility-based tailored therapy to treat 
naïve patients with H. pylori infection. The comparator is novel bismuth quadruple 
therapy which has been proved highly effective.(11) 
 
2. Objective 
2.1 The primary objective 
To Comparing the efficacy of tailored therapy with empiric modified bismuth quadruple 
therapy in treating naïve patients with H. pylori infection. 
2.2 The secondary objective 
(1) To compare the safety and compliance of tailored therapy with empiric modified 
bismuth quadruple therapy; 
(2) To evaluate the impact of clarithromycin resistance on eradication rates; 
(3) To evaluate the diagnosis accuracy of molecular genetic assays in clarithromycin 
resistance identification 
(4) To evaluate the cost-effectiveness of tailored therapy in treating naïve patients with 
H. pylori infection. 
3. Follow Diagram 
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Original Protocol 
 
4. Study Design 
This study is a prospective, superiority-designed, multi-center, open-label, randomized 
controlled trial conducted in five hospitals in Shanghai, China including Renji Hospital, 
Ruijin Hospital, Zhongshan Hospital, Shanghai general Hospital, and Shanghai Tenth 
People
's Hospital. 
5. Study Population 
5.1 Inclusion criteria 
(1) Participants with non-ulcer functional dyspepsia or scarred peptic ulcer disease； 
(2) Ability and willingness to participate in the study and to sign and give informed 
consent； 
(3) confirmed H. pylori infection with positive results on both rapid urease testing and 
culture 
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Original Protocol 
5.2 Exclusion criteria 
(1) Previous H. pylori eradication therapy 
(2) Less than 18 years old 
(3) With history of H. pylori infection treatment 
(4) With previous gastric surgery 
(5) Major systemic diseases 
(6) Pregnancy or lactation 
(7) Allergy to any of the study drugs 
(8) Administration of antibiotics, bismuth, antisecretory drugs in 8 weeks prior to 
inclusion 
5.3 Drop out standard 
Subjects who sign the informed consent and do not complete the prescribed observation 
period regardless of when and where to exit, are called drop out. 
(1)Patients found in the study course who do not meet the inclusion criteria or do follow 
the exclusion standards. 
(2)Patients who get other irrelevant diseases that could affect the assessment on the 
efficiency and safety of the drug during the test. 
(3)Patients who receive any drug that influences the assessment on the drug. 
(4)Patients who do not strictly take standard medication because of any reason (e.g. 
severe side effects). 
5.4 Termination standard 
Test termination refers to that clinical trials stop before the end, for the purpose to 
protect the rights and interests of subjects and to ensure the test quality. 
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Original Protocol 
(1)Sever safety issues occur during the trials; 
(2)Intervention drug was proved poor or no efficacy; 
(3)Serious medical errors in protocol that makes estimation impossible; 
(4) Administration reasons; 
(5)Withdraw by State food and drug administration. 
6. Allocation and Treatment 
6.1 Randomization and blinding 
Eligible patients will be randomly allocated to receive either susceptibility-based 
tailored therapy (tailored therapy) or empirical bismuth quadruple therapy (empirical 
therapy) in a 3:1 ratio. An independent statistician at Shanghai Jiao Tong University 
College of Basic Medical Sciences generates the computerized random number 
sequence and used a permuted block randomization with a block size of eight. All 
investigators is masked to the randomization sequence. Allocation is concealed in an 
opaque envelope until the intervention was assigned. Envelopes will be kept at Renji 
Hospital. After the written informed consents would be obtained from eligible patients, 
the independent research assistant will open envelopes by randomization sequence and 
telephone study staﬀ to give them each patient’s treatment allocation. This study is an 
open-labeled trial and patients are not blinded. The technicians, who perform the H. 
pylori tests (rapid urease test, culture, antimicrobial susceptibility testing, and urea 
breath test) or ﬁll in the questionnaires, will be blinded to treatment allocation. 
6.2 Intervention 
(1) Empirical therapy 
After review of clarithromycin-taking medical history, if have used clarithromycin, give 
esomeprazole 20mg bid, bismuth potassium citrate 600mg bid, amoxicillin 1000mg bid 
and metronidazole 400mg bid for 14 days; if have not used clarithromycin, give 
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Original Protocol 
esomeprazole 20mg bid, bismuth potassium citrate 600mg bid, amoxicillin 1000mg bid 
and clarithromycin 500mg bid for 14 days. 
(2) Tailored therapy  
After molecular genetic assays for identification of resistance to clarithromycin of 
Helicobacter pylori from biopsy samples, if resistance to clarithromycin, give 
esomeprazole 20mg bid, bismuth potassium citrate 600mg bid, amoxicillin 1000mg 
bid and metronidazole 400mg bid for 14 days; if susceptible to clarithromycin, give 
esomeprazole 20mg bid, bismuth potassium citrate 600mg bid, amoxicillin 1000mg 
bid and clarithromycin 500mg bid for 14 days. 
6.3 Drugs 
Esomeprazole, 20mg/tablet, AstraZeneca AB, Sodertalje, Sweden. 
Bismuth potassium citrate, 300mg/capsule (110mg elemental bismuth), Dawnrays 
Pharmaceutical Limited, Suzhou, Jiangsu, China. 
Amoxicillin, 250mg/capsule, Ruiyang Pharmaceutical Co. Ltd., Shandong, China. 
Clarithromycin, 250mg/tablet, Shanghai Abbott Laboratories Co. Ltd., Shanghai, 
China. 
Metronidazole, 200mg/tablet, Shanghai Xinyi Wanxiang Pharmaceutical Industry 
Company Limited, Shanghai, China. 
 
7. H. pylori real-time PCR and Culture Based Antibiotic 
Susceptibility Testing 
Tissue samples will be obtained from gastroscopic biopsy.  
DNAs will be isolated from biopsy specimens and mutation of clarithromycin 
resistance will be identified by standard molecular (real-time PCR) instrument systems 
and The Helicobacter pylori Analyte Specific Reagents (BIOLINE USA Inc., MA, 
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Original Protocol 
USA) according to the manufacturer’s guidelines. 
The biopsy specimens will be cultured and maintained on brain heart infusion agar 
medium (OXOID, Basingstoke, UK) containing 5% defibrinated sheep blood under 
microaerophilic conditions (85% N2, 10% CO2, 5% O2) at 37℃. All isolates will be 
stored in brain heart infusion broth (Difco Laboratory, Detroit, MI, USA) supplemented 
with 30% glycerol at -80℃. Clinical isolates will be also identified as H. pylori using 
positive tests for urease, oxidase, catalase and Gram staining. 
Minimal inhibitory concentrations (MIC) of clarithromycin and metronidazole will be 
determined by the two-fold agar dilution method. H. pylori is suspended in saline and 
measured using a spectrophotometer. The bacterial suspensions (108 colony forming 
units per milliliter) are then plated with an inoculator (Sakuma Seisaku, Tokyo, Japan) 
onto agar plates containing various concentrations of above antibiotics. After three days 
of microaerophilic incubation, MIC is defined as the lowest drug concentration that 
prevented visible growth of bacteria. ATCC43504 is used as the quality control. 
Clarithromycin >2 µg/mL, and metronidazole >8 µg/mL is defined as resistance 
breakpoints. 
8. Safety evaluation 
From the beginning of that patients signed the informed consent and are selected for 
trial to one month after the end of treatment, any adverse medical events, regardless of 
whether a causal relationship with the study medication, will be judged to be Adverse 
Event (AE). 
8.1 AE’s degree 
Mild: patients are easier to accept without induced questions, or patients have only mild 
discomfort which does not affect their daily lives and there is no need for clinical 
treatment.  
Moderate: patients actively describe the symptoms that affect the life, but they can 
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Original Protocol 
tolerate, which doesn’t need clinical treatment.  
Severe: patients have objective manifestations, which significantly affect the life, but 
patients can or can’t bear, which needs clinical treatment. 
8.2 Records and follow-up of AE 
During the study, AE will be accurately recorded, including the time of occurrence, 
severity, duration, the measures taken and the outcome. Researchers will follow all the 
AE until the symptoms of patients disappeared or condition become stable. SAE should 
be tracked until a proper solution is found even though the study is over. 
8.3 Serious adverse event (SAE) 
(1)The judgments of SAE:  
 Death, Life-threatening,  
 Leading to hospitalization or prolong hospitalization time,  
 Permanent or severe disability,  
 Congenital Anomaly/Birth Defect.  
(2)SAE report system:  
Principal investigator and the hospital ethics committee should be reported and SAE 
report form should be filled in within 24 hours by phone no matter whether any kinds 
of SAE are related with the drug in 30 days after the treatment. And the form should be 
reported to national drug administration in time by principal investigator. SAE should 
be promptly handled, closely tracked until it is properly solved.  
Contact method:  
1) Contact persons: Hong Lu 86-13611958022 
2) SFDA Safety Supervision 
9. Outcome Measures 
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Original Protocol 
9.1 Primary Outcome Measure: 
1. Helicobacter pylori eradication rate 
Six weeks after completion of therapy, H. pylori eradication success is defined as 
negative result from urea breath test (<4‰). If patients would be lost to follow-up urea 
breath testing, they will be scored as treatment failures in the primary ITT analysis. The 
eradication rate is defined as the number of patients who successful eradicate H. pylori 
divided by the total number of population for analysis. 
9.2 Secondary Outcome Measure 
(1) Rate of adverse events 
During the 14-day treatment period, the subjects will keep a diary to score any possible 
side effects or discomforts. The subjects will be asked to grade the severity of adverse 
events according to their influence on daily activities, experienced as “mild” (transient 
and well tolerated), “moderate” (causing discomfort and partially interfering with daily 
activities), or “severe” (causing considerable interference with daily activities). The 
side effect score recorded is based on the most severe event. Rate of adverse events is 
defined as the number of patients with any adverse event divided by the total number 
of population for analysis. 
(2) Compliance rate 
Compliance was deﬁned as either poor when they had taken less than 80% of the total 
medication or good when they had taken more than 80% medication. Compliance rate 
is defined as the number of patients with good compliance divided by the total number 
of population for analysis. 
9.3 Other Pre-specified Outcome Measures: 
(1) H. pylori eradication rate of patients with clarithromycin resistance strains 
(2) H. pylori eradication rate of patients with clarithromycin sensitive strains 
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Original Protocol 
(3) Sensitivity, specificity, positive predictive value and negative predictive value of 
molecular genetic assays in clarithromycin resistance identification 
(4) Medical cost per patient of tailored or empiric therapy 
(5) Ratio of medical cost to H. pylori eradication rate of each therapy 
(6) Ratio of incremental medical cost to incremental H. pylori eradication rate of 
tailored compared with empiric therapy 
10. Statistical Methods 
10.1 Sample size 
Assuming 95.7% eradication rate of tailored therapy in our trial, 88.8% eradication rate 
of empirical therapy, a superiority margin of >0, a power of 80%, an alpha of 0.05 (one-
sided), a ratio of 3:1, at least 340 subjects in susceptibility-based therapy and 114 
subjects in empirical therapy will be required to show superiority of susceptibility-
based therapy over empirical therapy on the basis of H. pylori eradication rate. Taking 
into consideration of 10% lost to follow-up, at least 500 participants (374 for tailored 
therapy and 126 for empiric therapy) is expected to be recruited for the study. 
10.2 Statistical Analysis Data 
Intention--to-treat (ITT) Analysis population: According to the Intention-to-treat 
principle: All randomized patients will be included in the ITT analysis. Patients who 
did not return for a follow-up 13C-UBT will be recorded as treatment failures. 
Per-protocol (PP) analysis population: All individuals who violated the study 
protocol, such as patients not taking at least 80% of treatment drugs, or with unknown 
post-treatment H. pylori status will be excluded from the PP analysis.  
Safety analysis population: The patients with missing safety data in ITT population 
will excluded from the safety analysis. 
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Original Protocol 
10.3 Statistical methods 
Comparative superiority of the two groups was assessed through hypothesis testing 
(one-sided Z test) and derivation of a two-sided 95% confidence interval (CI) of 
difference based on H. pylori eradication rate. If p-value of the testing less than 0.025 
and the lower bound of the 95% CI greater than zero, superiority of susceptibility-based 
therapy over empirical therapy could be concluded.  
Between-group differences were evaluated using Student’s t-test for continuous 
variables and Pearson’s χ2/corrected χ2 or Fisher’s exact test for categorical variables, 
as appropriate. All p-values were two-sided except the testing of superiority, and were 
considered statistically significant if p-value less than 0.05. 
Subgroup analyses of eradication efficacy were performed based on the results of 
antibiotic susceptibility test and compliance assessment.  
11 Ethical principles 
The study is registered in ClinicalTrials.gov. The study protocol is approved by the 
Ethics Committee at all institutions, and the study will be performed according to good 
clinical practice and the Declaration of Helsinki.  
Before each patient is selected into the study, the physician of the study have the 
responsibility to tell patients or their designated agents the purpose, process, completion 
and comprehensively introduce possible adverse reactions, risks to bear, possible 
benefits and other information all-round and detailed, making the patients know their 
rights. We are ordered to inform the patients of having the right to decide whether to 
participate in the study, as well as the right to withdraw the study at any time without 
any discrimination. The patients or their legal representatives should sign the informed 
consent after carefully reading and fully understanding, and retain the signature page of 
the copy. 
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12 References 
1. Malfertheiner P, Megraud F, O’Morain CA, et al. Management of Helicobacter pylori 
infection—the Maastricht IV/Florence Consensus Report. Gut 2012;61:646–64. 
2. Thung I, Aramin H, Vavinskaya V, Gupta S, Park JY, Crowe SE, et al. Review article: 
the global emergence of Helicobacter pylori antibiotic resistance. Aliment Pharmacol 
Ther. 2016 Feb 1;43(4):514–33.  
3. Su P, Li Y, Li H, Zhang J, Lin L, Wang Q, et al. Antibiotic Resistance of Helicobacter 
Pylori Isolated in the Southeast Coastal Region of China. Helicobacter. 2013 Aug 
1;18(4):274–9.  
4. Bai P, Zhou LY, Xiao XM, Luo Y, Ding Y. Susceptibility of Helicobacter pylori to 
antibiotics in Chinese patients. J Dig Dis. 2015 Aug;16(8):464–70.  
5. Zhang Y-X, Zhou L-Y, Song Z-Q, Zhang J-Z, He L-H, Ding Y. Primary antibiotic 
resistance of Helicobacter pylori strains isolated from patients with dyspeptic 
symptoms in Beijing: A prospective serial study. World J Gastroenterol WJG. 2015 Mar 
7;21(9):2786–92.  
6. Wang B, Lv Z-F, Wang Y-H, Wang H, Liu X-Q, Xie Y, et al. Standard triple therapy 
for Helicobacter pylori infection in China: A meta-analysis. World J Gastroenterol. 
2014 Oct 28;20(40):14973–85.  
7. Wenzhen Y, Yumin L, Quanlin G, Kehu Y, Lei J, Donghai W, et al. Is Antimicrobial 
Susceptibility Testing Necessary Before First-line Treatment for Helicobacter pylori 
Infection? -Meta-analysis of Randomized Controlled Trials-. Intern Med. 
2010;49(12):1103–9.  
8. López-Góngora S, Puig I, Calvet X, Villoria A, Baylina M, Muñoz N, et al. 
Systematic review and meta-analysis: susceptibility-guided versus empirical antibiotic 
treatment for Helicobacter pylori infection. J Antimicrob Chemother. 2015 
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Sep;70(9):2447–55.  
9. Chen H, Dang Y, Zhou X, Liu B, Liu S, Zhang G. Tailored Therapy Versus Empiric 
Chosen Treatment for Helicobacter pylori Eradication: A Meta-Analysis. Medicine 
(Baltimore). 2016 Feb;95(7):e2750.  
10. De Francesco V, Zullo A, Ierardi E, et al. Phenotypic and genotypic Helicobacter 
pylori clarithromycin resistance and therapeutic outcome: benefits and limits. J 
Antimicrob Chemother. 2010;65:327-332.  
11. Zhang W, Chen Q, Liang X, Liu W, Xiao S, Graham DY, et al. Bismuth, 
lansoprazole, amoxicillin and metronidazole or clarithromycin as first-line 
Helicobacter pylori therapy. Gut. 2015 Nov;64(11):1715–20.  
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Signature Page 
Protocol Title:  
Efficacy of Antibiotic Susceptibility-based Tailored Versus Empiric Therapy for 
Helicobacter pylori First-line Treatment：a Multicenter Randomized Controlled 
Trial 
 
After reviewing the protocol, each investigator and statistician signed this page as an 
attachment. 
 
Investigator: 
I will earnestly fulfill my duties as an investigator in accordance with the Chinese Good 
Clinical Practice (GCP). 
This study will be conducted in accordance with the principles of morality, ethics and 
science laid down in the Helsinki Declaration and the Chinese GCP. I agree to carry out 
this clinical trial in accordance with this protocol. 
I will be responsible for making timely and appropriate medical decisions for adverse 
events that occur in subjects during the study period. I know the requirements for 
correctly reporting serious adverse events and I will record and report these events. 
I guarantee that the data will be accurately, completely, timely, and legally filled in the 
Case Report Form. I will accept the inspections to ensure the quality of the study. 
I agree that the findings of the study are published. 
 
Principal Investigator: Hong Lu 
Institution: Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 
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Original Protocol 
Shanghai, China. 
Signature: ____________________________ 
 
Co-investigator: Yunwei Sun 
Institution: Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 
Shanghai, China.  
Signature: ____________________________ 
 
Co-investigator: Hong Gao 
Institution: Zhongshan Hospital, Fudan University, Shanghai, China 
Signature: ____________________________ 
 
Co-investigator: Yan Zhan 
Institution: Shanghai Tenth People's Hospital, Tongji University School of Medicine, 
Shanghai, China. 
Signature: ____________________________ 
 
Co-investigator: Gang Xu 
Institution: Department of Gastroenterology and Hepatology, Shanghai General 
Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 
Signature: ____________________________ 
 
 
24 

Original Protocol 
Statistical unit 
We will conduct statistical analysis based on the Chinese "guideline of Biostatistics for 
Clinical Trials of Chemical Drugs and Biological Products" and related regulations. 
Statistician: Yanyan Song 
Institution: Institute of Medical Sciences, Shanghai Jiao Tong University School of 
Medicine, Shanghai, China 
Signature: ____________________________ 
 
 
25 

Final Protocol 
 
 
Final Clinical Trial Protocol 
 
Efficacy of Antibiotic Susceptibility-based Tailored Versus 
Empiric Therapy for Helicobacter pylori First-line 
Treatment：a Multicenter Randomized Controlled Trial 
 
 
Protocol version: 1.1 
Revised Date: Jan 10, 2017 
Funding Source: Study On Helicobacter Pylori Eradication Treatment and 
Related Risk Factors of Infection project funded by Clinical Research 
Center, Shanghai Jiao Tong University School of Medicine, DLY201608. 
 
26 
 

Final Protocol 
Table of Contents 
Investigators and Statistician ····························································· 28 
Protocol Abstract ············································································ 29 
Protocol Schedule············································································ 33 
1. Background ················································································ 34 
2. Objectives ·················································································· 35 
3. Flow diagram ·············································································· 35 
4. Study design ··············································································· 36 
5. Study Population ········································································· 36 
6. Allocation and Treatment ······························································· 38 
7. H. pylori culture and antibiotic susceptibility testing ···························· 40 
8. Safety evaluation ········································································· 40 
9. Outcome measures ······································································· 42 
10. Statistical Methods ····································································· 43 
11. Ethical principles ········································································ 44 
12. References ················································································ 45 
Signature Page ··············································································· 47 
 
 
27 

Final Protocol 
Principal Investigator 
Hong Lu, Division of Gastroenterology and Hepatology, Shanghai Institute of 
Digestive Disease; Key Laboratory of Gastroenterology & Hepatology, Ministry of 
Health; Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 
China. 
 
Co-investigator 
1. Yunwei Sun, Department of Gastroenterology, Ruijin Hospital, Shanghai Jiao Tong 
University School of Medicine, Shanghai, China.  
2. Hong Gao, Department of Gastroenterology, Zhongshan Hospital, Fudan University, 
Shanghai, China. 
3. Yan Zhan, Department of Gastroenterology, Shanghai Tenth People's Hospital, 
Tongji University School of Medicine, Shanghai, China. 
4. Gang Xu, Department of Gastroenterology and Hepatology, Shanghai General 
Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 
 
Statistician 
Yanyan Song, Department of Biostatistics, Institute of Medical Sciences, Shanghai 
Jiao Tong University School of Medicine, Shanghai, China 
28 

Final Protocol 
Protocol Abstract 
Title 
Efficacy of Antibiotic Susceptibility-based Tailored Versus Empiric 
Therapy for Helicobacter pylori First-line Treatment：a Multicenter 
Randomized Controlled Trial 
Trial 
registration 
number 
ClinicalTrials.gov, NCT02935010 
Objective 
Comparing the efficacy, safety and compliance of pretreatment culture 
and susceptibility-based tailored therapy with empiric modified 
bismuth quadruple therapy in treating naïve patients with H. pylori 
infection 
Condition 
H. pylori infection naïve to treatment  
Study Design 
This study is a prospective, superiority-designed, multi-center, open-
label, randomized controlled trial. Eligible patients will be randomly 
allocated to receive either susceptibility-based tailored therapy 
(tailored therapy) or empirical bismuth quadruple therapy (empirical 
therapy) in a 3:1 ratio. Six weeks after completion of therapy, H. pylori 
eradication will be assessed by ¹³C-urea breath testing.  
Sample size 
382 
Inclusion 
Criteria 
1. Participants with non-ulcer functional dyspepsia or scarred peptic 
ulcer disease； 
2. Ability and willingness to participate in the study and to sign and 
give informed consent； 
3. confirmed H. pylori infection with positive results on both rapid 
urease testing and culture 
29 

Final Protocol 
Exclusion 
Criteria 
1. Previous H. pylori eradication therapy 
2. Less than 18 years old 
3. With history of H. pylori infection treatment 
4. With previous gastric surgery 
5. Major systemic diseases 
6. Pregnancy or lactation 
7. Allergy to any of the study drugs 
8. Administration of antibiotics, bismuth, antisecretory drugs in 8 
weeks prior to inclusion 
Arms and 
Interventions 
1. Empirical therapy 
Esomeprazole 20 mg, bismuth potassium citrate 600 mg taken twice a 
day, amoxicillin 1 g, and metronidazole 500 mg taken three times a day 
for 14 days.  
2. Tailored therapy  
Tailored therapy is based on antimicrobial susceptibility of 
clarithromycin, metronidazole and levofloxacin. according to 
antibiotic resistance pattern included esomeprazole 20mg bid, 
amoxicillin 1g bid, with a third drug (clarithromycin 500mg bid, 
metronidazole 400mg bid, or levofloxacin 500mg qd) for susceptible 
strains, or with bismuth 220mg bid plus metronidazole 400mg qid for 
triple-resistant strains. Study treatment regimens were all given 14 
days. 
Schedule 
1. Enrollment 
 Obtain written informed form 
30 

Final Protocol 
 Check eligibility criteria 
 Collect baseline characteristics 
 Undergo upper endoscopy with gastric biopsy and rapid urease 
testing 
 Conduct bacterial culture and antibiotic susceptibility testing 
2. Allocation 
After the written informed consents would be obtained from eligible 
patients, the independent research assistant will open envelopes by 
randomization sequence and telephone study staﬀ to give them each 
patient’s treatment allocation. Eligible patients will be randomly 
allocated to receive either tailored therapy or empirical therapy in a 3:1 
ratio. 
3. treatment 
Study treatment regimens were all given 14 days. During the treatment 
period, the subjects kept a diary to monitor compliance and symptoms. 
4. Follow-up 
After the end of treatment, adverse events and compliance was 
surveyed. Six weeks after completion of therapy, H. pylori eradication 
was assessed by ¹³C-urea breath testing. 
Outcome 
Measures 
Primary Outcome Measure: 
1. Helicobacter pylori eradication rate 
Six weeks after completion of therapy, H. pylori eradication success is 
defined as negative result from urea breath test (<4‰) 
Secondary Outcome Measure 
31 

Final Protocol 
1. Rate of adverse events 
2. Compliance rate 
Other Pre-specified Outcome Measures: 
1. Medical cost per patient of tailored or empiric therapy 
2. Ratio of medical cost to H. pylori eradication rate of each therapy 
3. Ratio of incremental medical cost to incremental H. pylori 
eradication rate of tailored compared with empiric therapy 
 
32 

Final Protocol 
Protocol Schedule 
Study Period 
Enrollment Allocation Treatment 
Follow-up 
Visit 
1st 
2nd 
 
3rd 
4th 
Time point (day) 
-14th 
0 
0-14 
15th 
56th 
Eligibility screen 
√ 
 
 
 
 
Baseline characteristics 
√ 
 
 
 
 
Endoscopy with biopsy 
and RUT 
√ 
 
 
 
 
H. pylori culture and 
susceptibility testing 
√ 
 
 
 
 
Written informed content 
√ 
 
 
 
 
Therapy Allocation 
 
√ 
 
 
 
Treatment 
 
 
√ 
 
 
Adverse events 
 
 
 
√ 
 
compliance assessment 
 
 
 
√ 
 
¹³C-urea breath test 
 
 
 
 
√ 
Medical cost 
 
 
 
 
√ 
 
33 

Final Protocol 
1. Background 
Helicobacter pylori (H. pylori) resistance to antibiotics was main challenge to previous 
efficacy antibiotic regimens such as standard triple therapy(1). The prevalence of 
antibiotic resistance have been markedly increasing with time in most parts of the 
world. Concordantly, the antimicrobial eradication rate of H. pylori has been declining 
globally.(2) In China, clarithromycin, metronidazole and levofloxacin resistance show 
similar trends, and resistance rates have now reached 21∼52% for clarithromycin, 
61~95% for metronidazole, 20~54% for levofloxacin, and 21~37% for both 
clarithromycin and metronidazole.(3–5) Standard triple therapy had not been suitable 
for first-line therapy in China because the pooled eradication rate had declined to 74.5% 
as a meta-analysis showed.(6)  
As a general rule, therapy for an infectious disease starts with identification of 
potentially useful antimicrobials and is largely based on the results of susceptibility 
testing. Systematic reviews or meta-analysis had proved susceptibility-based therapy 
was superior to 7–10 day triple therapies as ﬁrst-line treatment.(7–9) Therefore, there 
is a critical need to antimicrobial susceptibility testing (AST) for individualized analysis 
of antibiotic resistance prior to deﬁnitive treatment.(2)  
Now culture-based susceptibility testing for H pylori is rarely performed in standard 
practice. This may be due to several major limitations: they are time-consuming, costly, 
and invasive procedures, culture difficulty, and et al. So empirical therapy would be 
needed for most patients over the world. In the settings with high antibiotic resistance, 
only bismuth quadruple therapy (BQT) is the recommended first-line treatment, and 
any non-BQT therapy (triple, concomitant, hybrid, or sequential) would not be more 
effective option.(1) Our previous study showed that substitution of amoxicillin for 
tetracycline in a 14-day BQT containing 1,600mg/d of metronidazole eradicated 
96.4%(54/56) of metronidazole susceptible strains and 93.3%(42/45) of metronidazole 
resistant strains when used as first-line H. pylori therapy.(10). Fewer studies had 
34 

Final Protocol 
compared the effect of susceptibility-based therapy versus BQT for first-line H. pylori 
eradication, except one.(11) 
In this study, we will evaluate the efficacy, safety and compliance of pretreatment 
culture and susceptibility-based tailored therapy to treat naïve patients with H. pylori 
infection. Susceptibility-based therapy will use Pref. Graham DY et al’s 
recommendation, in which proton pump inhibitors, amoxicillin, and third susceptible 
drug triple therapies will be given after testing the susceptibility to clarithromycin, 
metronidazole and levofloxacin, or bismuth quadruple therapy was given if no 
susceptible antibiotics.(12) The comparator is novel bismuth quadruple therapy in 
which tetracycline was replaced by amoxicillin, without reference to antimicrobial 
susceptibility. 
 
2. Objective 
2.1 The primary objective 
To Comparing the efficacy of tailored therapy with empiric modified bismuth quadruple 
therapy in treating naïve patients with H. pylori infection. 
2.2 The secondary objective 
(1) To compare the safety and compliance of tailored therapy with empiric modified 
bismuth quadruple therapy; 
(2) To evaluate the impact of antibiotic resistance on eradication rates; 
(3) To evaluate the cost-effectiveness of susceptibility-based tailored therapy in treating 
naïve patients with H. pylori infection. 
3. Follow Diagram 
35 

Final Protocol 
 
4. Study Design 
This study is a prospective, superiority-designed, multi-center, open-label, randomized 
controlled trial conducted in five hospitals in Shanghai, China including Renji Hospital, 
Ruijin Hospital, Zhongshan Hospital, Shanghai general Hospital, and Shanghai Tenth 
People
's Hospital. 
5. Study population 
5.1 Inclusion criteria 
(1) Participants with non-ulcer functional dyspepsia or scarred peptic ulcer disease； 
(2) Ability and willingness to participate in the study and to sign and give informed 
consent； 
(3) confirmed H. pylori infection with positive results on both rapid urease testing and 
culture 
36 

Final Protocol 
5.2 Exclusion criteria 
(1) Previous H. pylori eradication therapy 
(2) Less than 18 years old 
(3) With history of H. pylori infection treatment 
(4) With previous gastric surgery 
(5) Major systemic diseases 
(6) Pregnancy or lactation 
(7) Allergy to any of the study drugs 
(8) Administration of antibiotics, bismuth, antisecretory drugs in 8 weeks prior to 
inclusion 
5.3 Drop out standard 
Subjects who sign the informed consent and do not complete the prescribed observation 
period regardless of when and where to exit, are called drop out. 
(1)Patients found in the study course who do not meet the inclusion criteria or do follow 
the exclusion standards. 
(2)Patients who get other irrelevant diseases that could affect the assessment on the 
efficiency and safety of the drug during the test. 
(3)Patients who receive any drug that influences the assessment on the drug. 
(4)Patients who do not strictly take standard medication because of any reason (e.g. 
severe side effects). 
5.4 Termination standard 
Test termination refers to that clinical trials stop before the end, for the purpose to 
protect the rights and interests of subjects and to ensure the test quality. 
37 

Final Protocol 
(1)Sever safety issues occur during the trials; 
(2)Intervention drug was proved poor or no efficacy; 
(3)Serious medical errors in protocol that makes estimation impossible; 
(4) Administration reasons; 
(5)Withdraw by State food and drug administration. 
6. Allocation and Treatment 
6.1 Randomization and blinding 
Eligible patients will be randomly allocated to receive either susceptibility-based 
tailored therapy (tailored therapy) or empirical bismuth quadruple therapy (empirical 
therapy) in a 3:1 ratio. An independent statistician at Shanghai Jiao Tong University 
College of Basic Medical Sciences generates the computerized random number 
sequence and used a permuted block randomization with a block size of eight. All 
investigators is masked to the randomization sequence. Allocation is concealed in an 
opaque envelope until the intervention was assigned. Envelopes will be kept at Renji 
Hospital. After the written informed consents would be obtained from eligible patients, 
the independent research assistant will open envelopes by randomization sequence and 
telephone study staﬀ to give them each patient’s treatment allocation. This study is an 
open-labeled trial and patients are not blinded. The technicians, who perform the H. 
pylori tests (rapid urease test, culture, antimicrobial susceptibility testing, and urea 
breath test) or ﬁll in the questionnaires, will be blinded to treatment allocation. 
6.2 Intervention 
(1) Empirical therapy 
Esomeprazole 20 mg, bismuth potassium citrate 600 mg taken twice a day, amoxicillin 
1 g, and metronidazole 500 mg taken three times a day for 14 days.  
38 

Final Protocol 
(2) Tailored therapy  
Tailored therapy is based on antimicrobial susceptibility of clarithromycin, 
metronidazole and levofloxacin. a) If the H. pylori strain of one patient was sensitive to 
clarithromycin, clarithromycin triple therapy (EAC) will be given: esomeprazole 20 
mg, amoxicillin 1 g, and clarithromycin 500 mg taken twice a day; b) If the strain was 
resistant to clarithromycin but sensitive to metronidazole, metronidazole triple therapy 
(EAM) will be given: esomeprazole 20 mg, amoxicillin 1 g, and metronidazole 500 mg 
taken twice a day. c) If the strain was resistant to both clarithromycin and metronidazole 
but sensitive to levofloxacin, levofloxacin triple therapy (EAL) will be given: 
esomeprazole 20 mg, amoxicillin 1 g taken twice a day, and levofloxacin 500 mg taken 
once a day. d) If the strain was resistant to all three antibiotics, bismuth quadruple 
therapy (EBA2M4) will be given: esomeprazole 20 mg, bismuth potassium citrate 600 
mg, amoxicillin 1 g taken twice a day, and metronidazole 500 mg taken four times a 
day. Study treatment regimens will be all given 14 days. 
6.3 Drugs 
Esomeprazole, 20mg/tablet, AstraZeneca AB, Sodertalje, Sweden. 
Bismuth potassium citrate, 300mg/capsule (110mg elemental bismuth), Dawnrays 
Pharmaceutical Limited, Suzhou, Jiangsu, China. 
Amoxicillin, 250mg/capsule, Ruiyang Pharmaceutical Co. Ltd., Shandong, China. 
Clarithromycin, 250mg/tablet, Shanghai Abbott Laboratories Co. Ltd., Shanghai, 
China. 
Metronidazole, 200mg/tablet, Shanghai Xinyi Wanxiang Pharmaceutical Industry 
Company Limited, Shanghai, China. 
Levofloxacin, 500mg/tablet, Daiichi Sankyo Pharmaceutical Beijing Co., Ltd., Beijing, 
China. 
 
39 

Final Protocol 
7. H. pylori Culture and Antibiotic Susceptibility Testing 
Tissue samples will be obtained from gastroscopic biopsy. The biopsy specimens will 
be cultured and maintained on brain heart infusion agar medium (OXOID, Basingstoke, 
UK) containing 5% defibrinated sheep blood under microaerophilic conditions (85% 
N2, 10% CO2, 5% O2) at 37℃. All isolates will be stored in brain heart infusion broth 
(Difco Laboratory, Detroit, MI, USA) supplemented with 30% glycerol at -80℃. 
Clinical isolates will be also identified as H. pylori using positive tests for urease, 
oxidase, catalase and Gram staining. 
Minimal inhibitory concentrations (MIC) of clarithromycin, metronidazole, and 
levofloxacin will be determined by the two-fold agar dilution method. H. pylori is 
suspended in saline and measured using a spectrophotometer. The bacterial suspensions 
(108 colony forming units per milliliter) are then plated with an inoculator (Sakuma 
Seisaku, Tokyo, Japan) onto agar plates containing various concentrations of above 
antibiotics. After three days of microaerophilic incubation, MIC is defined as the lowest 
drug concentration that prevented visible growth of bacteria. ATCC43504 is used as the 
quality control. Clarithromycin >2 µg/mL, metronidazole >8 µg/mL, levofloxacin >2 
µg/mL is defined as resistance breakpoints. 
8. Safety evaluation 
From the beginning of that patients signed the informed consent and are selected for 
trial to one month after the end of treatment, any adverse medical events, regardless of 
whether a causal relationship with the study medication, will be judged to be Adverse 
Event (AE). 
8.1 AE’s degree 
Mild: patients are easier to accept without induced questions, or patients have only mild 
discomfort which does not affect their daily lives and there is no need for clinical 
treatment.  
40 

Final Protocol 
Moderate: patients actively describe the symptoms that affect the life, but they can 
tolerate, which doesn’t need clinical treatment.  
Severe: patients have objective manifestations, which significantly affect the life, but 
patients can or can’t bear, which needs clinical treatment. 
8.2 Records and follow-up of AE 
During the study, AE will be accurately recorded, including the time of occurrence, 
severity, duration, the measures taken and the outcome. Researchers will follow all the 
AE until the symptoms of patients disappeared or condition become stable. SAE should 
be tracked until a proper solution is found even though the study is over. 
8.3 Serious adverse event (SAE) 
(1)The judgments of SAE:  
 Death, Life-threatening,  
 Leading to hospitalization or prolong hospitalization time,  
 Permanent or severe disability,  
 Congenital Anomaly/Birth Defect.  
(2)SAE report system:  
Principal investigator and the hospital ethics committee should be reported and SAE 
report form should be filled in within 24 hours by phone no matter whether any kinds 
of SAE are related with the drug in 30 days after the treatment. And the form should be 
reported to national drug administration in time by principal investigator. SAE should 
be promptly handled, closely tracked until it is properly solved.  
Contact method:  
1) Contact persons: Hong Lu 86-13611958022 
2) SFDA Safety Supervision 
41 

Final Protocol 
9. Outcome Measures 
9.1 Primary Outcome Measure: 
1. Helicobacter pylori eradication rate 
Six weeks after completion of therapy, H. pylori eradication success is defined as 
negative result from urea breath test (<4‰). If patients would be lost to follow-up urea 
breath testing, they will be scored as treatment failures in the ITT analysis. The 
eradication rate is defined as the number of patients who successful eradicate H. pylori 
divided by the total number of population for analysis. 
9.2 Secondary Outcome Measure 
(1) Rate of adverse events 
During the 14-day treatment period, the subjects will keep a diary to score any possible 
side effects or discomforts. The subjects will be asked to grade the severity of adverse 
events according to their influence on daily activities, experienced as “mild” (transient 
and well tolerated), “moderate” (causing discomfort and partially interfering with daily 
activities), or “severe” (causing considerable interference with daily activities). The 
side effect score recorded is based on the most severe event. Rate of adverse events is 
defined as the number of patients with any adverse event divided by the total number 
of population for analysis. 
(2) Compliance rate 
Compliance was deﬁned as either poor when they had taken less than 80% of the total 
medication or good when they had taken more than 80% medication. Compliance rate 
is defined as the number of patients with good compliance divided by the total number 
of population for analysis. 
9.3 Other Pre-specified Outcome Measures: 
(1) Medical cost per patient of tailored or empiric therapy 
42 

Final Protocol 
Medical cost of one patient is overall cost including visit, endoscopy, H. pylori culture, 
susceptibility testing, eradication treatment, and adverse event treatment. 
(2) Ratio of medical cost to H. pylori eradication rate of each therapy 
(3)Ratio of incremental medical cost to incremental H. pylori eradication rate of tailored 
compared with empiric therapy 
10. Statistical Methods 
10.1 Sample size 
Assuming 96.5% eradication rate of tailored therapy in our trial, 88.9% eradication rate 
of empirical therapy, a superiority margin of >0, a power of 80%, an alpha of 0.05 (one-
sided), a ratio of 3:1, at least 260 subjects in susceptibility-based therapy and 87 subjects 
in empirical therapy will be required to show superiority of susceptibility-based therapy 
over empirical therapy on the basis of H. pylori eradication rate. Taking into 
consideration of 10% lost to follow-up, at least 382 participants (286 for tailored 
therapy and 96 for empiric therapy) is expected to be recruited for the study. 
10.2 Statistical Analysis Data 
Intention--to-treat (ITT) Analysis population: According to the Intention-to-treat 
principle: All randomized patients will be included in the ITT analysis. Patients who 
did not return for a follow-up 13C-UBT will be recorded as treatment failures. 
Per-protocol (PP) analysis population: All individuals who violated the study 
protocol, such as patients not taking at least 80% of treatment drugs, or with unknown 
post-treatment H. pylori status will be excluded from the PP analysis.  
Safety analysis population: The patients with missing safety data in ITT population 
will excluded from the safety analysis. 
10.3 Statistical methods 
43 

Final Protocol 
Comparative superiority of the two groups was assessed through hypothesis testing 
(one-sided Z test) and derivation of a two-sided 95% confidence interval (CI) of 
difference based on H. pylori eradication rate. If p-value of the testing less than 0.025 
and the lower bound of the 95% CI greater than zero, superiority of susceptibility-based 
therapy over empirical therapy could be concluded.  
Between-group differences were evaluated using Student’s t-test for continuous 
variables and Pearson’s χ2/corrected χ2 or Fisher’s exact test for categorical variables, 
as appropriate. All p-values were two-sided except the testing of superiority, and were 
considered statistically significant if p-value less than 0.05. 
Subgroup analyses of eradication efficacy were performed based on the results of 
antibiotic susceptibility test and compliance assessment.  
11 Ethical principles 
The study is registered in ClinicalTrials.gov. The study protocol is approved by the 
Ethics Committee at all institutions, and the study will be performed according to good 
clinical practice and the Declaration of Helsinki.  
Before each patient is selected into the study, the physician of the study have the 
responsibility to tell patients or their designated agents the purpose, process, completion 
and comprehensively introduce possible adverse reactions, risks to bear, possible 
benefits and other information all-round and detailed, making the patients know their 
rights. We are ordered to inform the patients of having the right to decide whether to 
participate in the study, as well as the right to withdraw the study at any time without 
any discrimination. The patients or their legal representatives should sign the informed 
consent after carefully reading and fully understanding, and retain the signature page of 
the copy. 
  
44 

Final Protocol 
12 References 
1.  Malfertheiner P, Megraud F, O’Morain CA, et al. Management of Helicobacter 
pylori infection—the Maastricht IV/Florence Consensus Report. Gut 2012;61:646–64.  
2.  Thung I, Aramin H, Vavinskaya V, Gupta S, Park JY, Crowe SE, et al. Review 
article: the global emergence of Helicobacter pylori antibiotic resistance. Aliment 
Pharmacol Ther. 2016 Feb 1;43(4):514–33.  
3.  Su P, Li Y, Li H, Zhang J, Lin L, Wang Q, et al. Antibiotic Resistance of 
Helicobacter Pylori Isolated in the Southeast Coastal Region of China. Helicobacter. 
2013 Aug 1;18(4):274–9.  
4.  Bai P, Zhou LY, Xiao XM, Luo Y, Ding Y. Susceptibility of Helicobacter pylori to 
antibiotics in Chinese patients. J Dig Dis. 2015 Aug;16(8):464–70.  
5.  Zhang Y-X, Zhou L-Y, Song Z-Q, Zhang J-Z, He L-H, Ding Y. Primary antibiotic 
resistance of Helicobacter pylori strains isolated from patients with dyspeptic 
symptoms in Beijing: A prospective serial study. World J Gastroenterol WJG. 2015 Mar 
7;21(9):2786–92.  
6.  Wang B, Lv Z-F, Wang Y-H, Wang H, Liu X-Q, Xie Y, et al. Standard triple therapy 
for Helicobacter pylori infection in China: A meta-analysis. World J Gastroenterol. 
2014 Oct 28;20(40):14973–85.  
7.  Wenzhen Y, Yumin L, Quanlin G, Kehu Y, Lei J, Donghai W, et al. Is Antimicrobial 
Susceptibility Testing Necessary Before First-line Treatment for Helicobacter pylori 
Infection? -Meta-analysis of Randomized Controlled Trials-. Intern Med. 
2010;49(12):1103–9.  
8.  López-Góngora S, Puig I, Calvet X, Villoria A, Baylina M, Muñoz N, et al. 
Systematic review and meta-analysis: susceptibility-guided versus empirical antibiotic 
treatment for Helicobacter pylori infection. J Antimicrob Chemother. 2015 
Sep;70(9):2447–55.  
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Final Protocol 
9.  Chen H, Dang Y, Zhou X, Liu B, Liu S, Zhang G. Tailored Therapy Versus Empiric 
Chosen Treatment for Helicobacter pylori Eradication: A Meta-Analysis. Medicine 
(Baltimore). 2016 Feb;95(7):e2750.  
10.  
Zhang W, Chen Q, Liang X, Liu W, Xiao S, Graham DY, et al. Bismuth, 
lansoprazole, amoxicillin and metronidazole or clarithromycin as first-line 
Helicobacter pylori therapy. Gut. 2015 Nov;64(11):1715–20.  
11.  Zhou L, Zhang J, Song Z, He L, Li Y, Qian J, et al. Tailored versus Triple plus 
Bismuth or Concomitant Therapy as Initial Helicobacter pylori Treatment: A 
Randomized Trial. Helicobacter. 2016 Apr 1;21(2):91–9.  
12.  
Graham DY, Dore MP. Helicobacter pylori therapy: a paradigm shift. Expert 
Rev Anti Infect Ther. 2016 Jun;14(6):577–85.  
46 

Final Protocol 
Signature Page 
Protocol Title:  
Efficacy of Antibiotic Susceptibility-based Tailored Versus Empiric Therapy for 
Helicobacter pylori First-line Treatment：a Multicenter Randomized Controlled 
Trial 
 
After reviewing the protocol, each investigator and statistician signed this page as an 
attachment. 
 
Investigator: 
I will earnestly fulfill my duties as an investigator in accordance with the Chinese Good 
Clinical Practice (GCP). 
This study will be conducted in accordance with the principles of morality, ethics and 
science laid down in the Helsinki Declaration and the Chinese GCP. I agree to carry out 
this clinical trial in accordance with this protocol. 
I will be responsible for making timely and appropriate medical decisions for adverse 
events that occur in subjects during the study period. I know the requirements for 
correctly reporting serious adverse events and I will record and report these events. 
I guarantee that the data will be accurately, completely, timely, and legally filled in the 
Case Report Form. I will accept the inspections to ensure the quality of the study. 
I agree that the findings of the study are published. 
 
Principal Investigator: Hong Lu 
Institution: Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 
47 

Final Protocol 
Shanghai, China. 
Signature: ____________________________ 
 
Co-investigator: Yunwei Sun 
Institution: Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 
Shanghai, China.  
Signature: ____________________________ 
 
Co-investigator: Hong Gao 
Institution: Zhongshan Hospital, Fudan University, Shanghai, China 
Signature: ____________________________ 
 
Co-investigator: Yan Zhan 
Institution: Shanghai Tenth People's Hospital, Tongji University School of Medicine, 
Shanghai, China. 
Signature: ____________________________ 
 
Co-investigator: Gang Xu 
Institution: Department of Gastroenterology and Hepatology, Shanghai General 
Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 
Signature: ____________________________ 
 
 
48 

Final Protocol 
Statistical unit 
We will conduct statistical analysis based on the Chinese "guideline of Biostatistics for 
Clinical Trials of Chemical Drugs and Biological Products" and related regulations. 
Statistician: Yanyan Song 
Institution: Institute of Medical Sciences, Shanghai Jiao Tong University School of 
Medicine, Shanghai, China 
Signature: ____________________________ 
49 

Summary of protocol changes 
Summary of protocol changes 
 
Change 
Original 
Final 
Title 
Efficacy of Clarithromycin Susceptibility-based Tailored Versus 
Empiric Therapy for Helicobacter pylori First-line Treatment ：a 
Multicenter Randomized Controlled Trial 
Efficacy of Antibiotic Susceptibility-based Tailored Versus Empiric 
Therapy for Helicobacter pylori First-line Treatment ：a Multicenter 
Randomized Controlled Trial 
2.2 The 
secondary 
objective 
(1) To compare the safety and compliance of tailored therapy with 
empiric modified bismuth quadruple therapy; 
(2) To evaluate the impact of clarithromycin resistance on eradication 
rates; 
(3) To evaluate the diagnosis accuracy of molecular genetic assays in 
clarithromycin resistance identification 
(4) To evaluate the cost-effectiveness of tailored therapy in treating 
(1) To compare the safety and compliance of tailored therapy with empiric 
modified bismuth quadruple therapy; 
(2) To evaluate the impact of antibiotic resistance on eradication rates; 
(3) To evaluate the cost-effectiveness of susceptibility-based tailored therapy 
in treating naïve patients with H. pylori infection. 
50 
 

Summary of protocol changes 
naïve patients with H. pylori infection. 
6.2 
Intervention 
(1) Empirical therapy 
After review of clarithromycin-taking medical history, if have used 
clarithromycin, give esomeprazole 20mg bid, bismuth potassium citrate 
600mg bid, amoxicillin 1000mg bid and metronidazole 400mg bid for 
14 days; if have not used clarithromycin, give esomeprazole 20mg bid, 
bismuth potassium citrate 600mg bid, amoxicillin 1000mg bid and 
clarithromycin 500mg bid for 14 days. 
(2) Tailored therapy  
After molecular genetic assays for identification of resistance to 
clarithromycin of Helicobacter pylori from biopsy samples, if 
resistance to clarithromycin, give esomeprazole 20mg bid, bismuth 
potassium citrate 600mg bid, amoxicillin 1000mg bid and 
metronidazole 400mg bid for 14 days; if susceptible to clarithromycin, 
give esomeprazole 20mg bid, bismuth potassium citrate 600mg bid, 
amoxicillin 1000mg bid and clarithromycin 500mg bid for 14 days. 
(1) Empirical therapy 
Esomeprazole 20 mg, bismuth potassium citrate 600 mg taken twice a day, 
amoxicillin 1 g, and metronidazole 500 mg taken three times a day for 14 
days.  
(2) Tailored therapy  
Tailored therapy is based on antimicrobial susceptibility of clarithromycin, 
metronidazole and levofloxacin. a) If the H. pylori strain of one patient was 
sensitive to clarithromycin, clarithromycin triple therapy (EAC) will be 
given: esomeprazole 20 mg, amoxicillin 1 g, and clarithromycin 500 mg 
taken twice a day; b) If the strain was resistant to clarithromycin but sensitive 
to metronidazole, metronidazole triple therapy (EAM) will be given: 
esomeprazole 20 mg, amoxicillin 1 g, and metronidazole 500 mg taken twice 
a day. c) If the strain was resistant to both clarithromycin and metronidazole 
but sensitive to levofloxacin, levofloxacin triple therapy (EAL) will be given: 
51 

Summary of protocol changes 
esomeprazole 20 mg, amoxicillin 1 g taken twice a day, and levofloxacin 500 
mg taken once a day. d) If the strain was resistant to all three antibiotics, 
bismuth quadruple therapy (EBA2M4) will be given: esomeprazole 20 mg, 
bismuth potassium citrate 600 mg, amoxicillin 1 g taken twice a day, and 
metronidazole 500 mg taken four times a day. Study treatment regimens will 
be all given 14 days. 
6.3 Drugs 
Esomeprazole, 20mg/tablet, AstraZeneca AB, Sodertalje, Sweden. 
Bismuth potassium citrate, 300mg/capsule (110mg elemental 
bismuth), Dawnrays Pharmaceutical Limited, Suzhou, Jiangsu, China. 
Amoxicillin, 250mg/capsule, Ruiyang Pharmaceutical Co. Ltd., 
Shandong, China. 
Clarithromycin, 250mg/tablet, Shanghai Abbott Laboratories Co. Ltd., 
Shanghai, China. 
Metronidazole, 
200mg/tablet, 
Shanghai 
Xinyi 
Wanxiang 
Esomeprazole, 20mg/tablet, AstraZeneca AB, Sodertalje, Sweden. 
Bismuth potassium citrate, 300mg/capsule (110mg elemental bismuth), 
Dawnrays Pharmaceutical Limited, Suzhou, Jiangsu, China. 
Amoxicillin, 250mg/capsule, Ruiyang Pharmaceutical Co. Ltd., Shandong, 
China. 
Clarithromycin, 250mg/tablet, Shanghai Abbott Laboratories Co. Ltd., 
Shanghai, China. 
Metronidazole, 200mg/tablet, Shanghai Xinyi Wanxiang Pharmaceutical 
52 

Summary of protocol changes 
Pharmaceutical Industry Company Limited, Shanghai, China. 
Industry Company Limited, Shanghai, China. 
Levofloxacin, 500mg/tablet, Daiichi Sankyo Pharmaceutical Beijing Co., 
Ltd., Beijing, China. 
7. 
Antibiotic 
Susceptibility 
Testing 
Tissue samples will be obtained from gastroscopic biopsy. DNAs will 
be isolated from biopsy specimens and mutation of clarithromycin 
resistance will be identified by standard molecular (real-time PCR) 
instrument systems and The Helicobacter pylori Analyte Specific 
Reagents (BIOLINE USA Inc., MA, USA) according to the 
manufacturer’s guidelines. The biopsy specimens will be cultured and 
maintained on brain heart infusion agar medium (OXOID, Basingstoke, 
UK) containing 5% defibrinated sheep blood under microaerophilic 
conditions (85% N2, 10% CO2, 5% O2) at 37℃. 
Tissue samples will be obtained from gastroscopic biopsy. The biopsy 
specimens will be cultured and maintained on brain heart infusion agar 
medium (OXOID, Basingstoke, UK) containing 5% defibrinated sheep blood 
under microaerophilic conditions (85% N2, 10% CO2, 5% O2) at 37℃. 
9.3 Other Pre-
specified 
Outcome 
(1) H. pylori eradication rate of patients with clarithromycin resistance 
strains 
(2) H. pylori eradication rate of patients with clarithromycin sensitive 
(1) Medical cost per patient of tailored or empiric therapy 
Medical cost of one patient is overall cost including visit, endoscopy, H. 
pylori culture, susceptibility testing, eradication treatment, and adverse event 
53 

Summary of protocol changes 
Measures: 
strains 
(3) Sensitivity, specificity, positive predictive value and negative 
predictive value of molecular genetic assays in clarithromycin 
resistance identification 
(4) Medical cost per patient of tailored or empiric therapy 
(5) Ratio of medical cost to H. pylori eradication rate of each therapy 
(6) Ratio of incremental medical cost to incremental H. pylori 
eradication rate of tailored compared with empiric therapy 
treatment. 
(2) Ratio of medical cost to H. pylori eradication rate of each therapy 
(3)Ratio of incremental medical cost to incremental H. pylori eradication rate 
of tailored compared with empiric therapy 
10.1 Sample 
size 
Assuming 95.7% eradication rate of tailored therapy in our trial, 88.8% 
eradication rate of empirical therapy, a superiority margin of >0, a power 
of 80%, an alpha of 0.05 (one-sided), a ratio of 3:1, at least 340 subjects 
in susceptibility-based therapy and 114 subjects in empirical therapy 
will be required to show superiority of susceptibility-based therapy over 
empirical therapy on the basis of H. pylori eradication rate. Taking into 
Assuming 96.5% eradication rate of tailored therapy in our trial, 88.9% 
eradication rate of empirical therapy, a superiority margin of >0, a power of 
80%, an alpha of 0.05 (one-sided), a ratio of 3:1, at least 260 subjects in 
susceptibility-based therapy and 87 subjects in empirical therapy will be 
required to show superiority of susceptibility-based therapy over empirical 
therapy on the basis of H. pylori eradication rate. Taking into consideration 
54 

Summary of protocol changes 
consideration of 10% lost to follow-up, at least 500 participants (374 for 
tailored therapy and 126 for empiric therapy) is expected to be recruited 
for the study. 
of 10% lost to follow-up, at least 382 participants (286 for tailored therapy 
and 96 for empiric therapy) is expected to be recruited for the study 
 
55 

Original SAP 
Original Statistical Analysis Plan 
 
This statistical analysis plan includes the details of study sample size calculation and 
statistical methods for analysis of outcomes and safety. 
Table of Contents 
1. Sample size estimation ·································································· 57 
2. Definition of populations for analysis ················································ 57 
3. Evaluation of baseline characteristics ················································ 57 
4. Evaluation of primary outcomes ······················································ 59 
5. Evaluation of secondary outcomes ···················································· 60 
6. Additional analyses ······································································· 61 
7. Table Design ··············································································· 63 
56 
 

Original SAP 
1. Sample size estimation 
Assuming 95.7% eradication rate of tailored therapy in our trial, 88.8% eradication rate 
of empirical therapy, a superiority margin of >0, a power of 80%, an alpha of 0.05 (one-
sided), a ratio of 3:1, at least 340 subjects in susceptibility-based therapy and 114 
subjects in empirical therapy will be required to show superiority of susceptibility-
based therapy over empirical therapy on the basis of H. pylori eradication rate. Taking 
into consideration of 10% lost to follow-up, at least 500 participants (374 for tailored 
therapy and 126 for empiric therapy) is expected to be recruited for the study. 
2. Definition of populations for analysis 
2.1 Intention--to-treat (ITT) Analysis population 
According to the Intention-to-treat principle: All randomized patients will be included 
in the ITT analysis. Patients who did not return for a follow-up 13C-UBT will be 
recorded as treatment failures. 
2.2 Per-protocol (PP) analysis population 
All individuals who violated the study protocol, such as patients not taking at least 80% 
of treatment drugs, or with unknown post-treatment H. pylori status will be excluded 
from the PP analysis.  
2.3 Safety analysis population 
The patients with missing safety data in ITT population will excluded from the safety 
analysis. 
 
3. Evaluation of baseline characteristics 
3.1 presentation of baseline characteristics 
57 

Original SAP 
Variables of baseline characteristics collected are age (years), gender (male/female), 
diagnosis (Dyspepsia/PUD), clarithromycin-taking history, 23S rRNA mutation, 
antibiotic resistance pattern, poor adherence, and loss of follow-up. The following table 
shows the types and presentation of variables.    
Variable 
Type of variable 
Presentation 
Age (years) 
Continuous   
Mean ± standard deviation (range) 
Gender (male/female) 
Categorical 
Number (percentage) 
Diagnosis (Dyspepsia/PUD) 
Categorical 
Number (percentage) 
Clarithromycin-taking 
history 
Categorical 
Number (percentage) 
23S rRNA mutation 
Categorical 
Number (percentage) 
Antibiotic resistance pattern (Phenotypic) 
Clarithromycin, n (%) 
Categorical 
Number (percentage) 
Metronidazole, n (%) 
Categorical 
Number (percentage) 
Levofloxacin, n (%) 
Categorical 
Number (percentage) 
All-susceptible, n (%) 
Categorical 
Number (percentage) 
Triple-resistant, n (%) 
Categorical 
Number (percentage) 
Poor adherence 
Categorical 
Number 
Loss of follow-up 
Categorical 
Number 
 
3.2 Statistical methods for baseline characteristics analyses 
Comparisons of baseline characteristics between the tailored therapy and empiric 
therapy will be performed in the ITT analysis populations. Comparisons between the 
both groups will be performed using the Pearson’s χ2, corrected χ2 or Fisher’s exact 
test for categorical variables and using the Student t test for continuous variables.   
58 

Original SAP 
 
4. Evaluation of primary outcomes 
4.1 Helicobacter pylori eradication rate 
Six weeks after completion of therapy, H. pylori eradication success is defined as 
negative result from urea breath test (<4‰). If patients would be lost to follow-up urea 
breath testing, they will be scored as treatment failures in the ITT analysis.  
The eradication rate is defined as the number of patients who successful eradicate H. 
pylori divided by the total number of population for analysis. These data are presented 
with number, percentage, and 95% confidence interval (CI). 
4.2 Testing the differences of eradication rates 
Testing the differences of eradication rates between the tailored therapy and empiric 
therapy will be performed using the Pearson’s χ2, corrected χ2 or Fisher’s exact test in 
the ITT and PP analysis populations.  
4.3 Testing the superiority of the tailored therapy 
Testing the superiority of tailored therapy group to empiric therapy group will be 
performed in the ITT and PP analysis populations using hypothesis testing (one-sided 
Z test) and derivation of a two-sided 95% confidence interval (CI) of difference based 
on H. pylori eradication rate. If p-value of the testing less than 0.025 and the lower 
bound of the 95% CI greater than zero, superiority of susceptibility-based therapy over 
empirical therapy could be concluded. 
 
5. Evaluation of secondary outcomes 
5.1 Rate of adverse events 
59 

Original SAP 
During the 14-day treatment period, the subjects will keep a diary to score any possible 
side effects or discomforts. The subjects will be asked to grade the severity of adverse 
events according to their influence on daily activities, experienced as “mild” (transient 
and well tolerated), “moderate” (causing discomfort and partially interfering with daily 
activities), or “severe” (causing considerable interference with daily activities). The 
side effect score recorded is based on the most severe event. Rate of adverse events is 
defined as the number of patients with any adverse event divided by the total number 
of population for analysis. These data are presented with number, percentage.  
Comparisons of presence of each adverse event and any adverse event between the 
tailored therapy and empiric therapy will be performed in the safety analysis 
populations using the Pearson’s χ2, corrected χ2 or Fisher’s exact test. 
 
5.2 Compliance rate 
Compliance was deﬁned as either poor when they had taken less than 80% of the total 
medication or good when they had taken more than 80% medication. Compliance rate 
is defined as the number of patients with good compliance divided by the total number 
of population for analysis. These data are presented with number, percentage.  
Comparisons of presence of each adverse event and any adverse event between the 
tailored therapy and empiric therapy will be performed in the ITT analysis populations 
using the Pearson’s χ2, corrected χ2 or Fisher’s exact test. 
 
5.3 Other Pre-specified Outcome Measures: 
(1) Medical cost per patient of tailored or empiric therapy 
Medical cost of one patient is overall cost including visit, endoscopy, H. pylori culture, 
susceptibility testing, eradication treatment, and adverse event treatment. 
60 

Original SAP 
Comparisons of medical cost between the tailored therapy and empiric therapy will be 
performed in the PP analysis populations using Student t test. 
(2) Ratio of medical cost to H. pylori eradication rate of each therapy 
Ratio of medical cost to H. pylori eradication rate of each therapy is defined as average 
medical cost of the therapy divided by H. pylori eradication rate of one therapy. 
(3)Ratio of incremental medical cost to incremental H. pylori eradication rate of 
tailored compared with empiric therapy 
If one therapy cost less and achieved higher eradication rate than the other one, it will 
be referred to as dominant strategy. If one therapy yielded higher eradication rate but 
also cost more, an incremental cost-effectiveness ratio (ICER) will be calculated. 
Obviously, the tailored therapy will cost more because of additional bacterial culture 
and antibiotic susceptibility testing. So, if eradication rate of the tailored therapy was 
higher, the ICER of the tailored therapy will be calculated as the ratio of incremental 
medical cost to incremental H. pylori eradication rate of tailored compared with empiric 
therapy (in terms of RMB per incremental eradication percentage). if eradication rate 
of the tailored therapy was equal to or lower than the empiric therapy, the tailored 
therapy will not be more cost-effective option and the economic evaluation will not be 
conducted. 
 
6. Additional analyses 
6.1 Antibiotic resistance rate 
Minimal 
inhibitory 
concentrations 
(MIC) 
of 
clarithromycin 
>2 
µg/mL, 
metronidazole >8 µg/mL, levofloxacin >2 µg/mL is defined as resistance breakpoints. 
Antibiotic resistance rate is defined as the number of patients with antibiotic resistant 
strains divided by the total number of population for analysis. These data are presented 
61 

Original SAP 
with number, percentage. 
6.2 Effect of antibiotic resistance on eradication rate  
Comparisons of eradication rate of one therapy between antibiotic susceptible and 
resistant strains will be performed in the PP analysis populations using the Pearson’s 
χ2, corrected χ2 or Fisher’s exact test. 
Additional comparisons of eradication rate of antibiotic resistant strains between the 
tailored therapy and the empiric therapy will be performed in the PP analysis 
populations using the Pearson’s χ2, corrected χ2 or Fisher’s exact test. 
6.3 Effect of 23S rRNA mutation on eradication rate 
Comparisons of eradication rate of clarithromycin-containing therapy between strains 
with 23S rRNA mutation and those without 23S rRNA mutation will be performed in 
the PP analysis populations using the Pearson’s χ2, corrected χ2 or Fisher’s exact test. 
6.4 Sensitivity, specificity, positive predictive value and negative predictive value 
of molecular genetic assays in clarithromycin resistance identification 
Using culture based antibiotic susceptibility testing as the gold standard, sensitivity, 
specificity, positive predictive value (PPV) and negative predictive value (NPV) of 23S 
rRNA mutation in identification of H. pylori resistance to clarithromycin will be 
estimated.  
𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆=
number of true positives
number of true positives + number of false negatives  
𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆=
number of true negatives
number of true negatives + number of false positives  
𝑃𝑃𝑃𝑃𝑃𝑃=
number of true positives
number of true positives + number of false positives  
𝑁𝑁𝑁𝑁𝑁𝑁=
number of true negatives
number of true negatives + number of false negatives  
True positives: the strains with 23S rRNA mutation and MIC of clarithromycin >2 
62 

Original SAP 
µg/mL 
False positives: the strains with 23S rRNA mutation but MIC of clarithromycin ≤2 
µg/mL 
True negatives: the strains without 23S rRNA mutation and MIC of clarithromycin ≤2 
µg/mL 
False negatives: the strains without 23S rRNA mutation but MIC of clarithromycin >2 
µg/mL 
6.5 Effect of presence of adverse events on compliance. 
Comparisons of compliance rate of one therapy between presence and absence of 
adverse event will be performed in the safety analysis populations using the Pearson’s 
χ2, corrected χ2 or Fisher’s exact test. 
6.6 Effect of compliance on eradication rate 
Comparisons of eradication rate of one therapy between good and poor compliance will 
be performed in the ITT analysis populations who had UBT results using the Pearson’s 
χ2, corrected χ2 or Fisher’s exact test. 
 
7. Table Design 
Table 1. Baseline characteristics 
Variables 
Tailored therapy 
Empiric therapy 
p value 
Age, years (range) 
 
 
 
Gender (male/female) 
 
 
 
Diagnosis (Dyspepsia/PUD) 
 
 
 
Clarithromycin-taking history 
 
 
 
23S rRNA mutation 
 
 
 
Antibiotic resistance pattern 
Clarithromycin, n (%) 
 
 
 
Metronidazole, n (%) 
 
 
 
63 

Original SAP 
Levofloxacin, n (%) 
 
 
 
All-susceptible, n (%) 
 
 
 
Triple-resistant, n (%) 
 
 
 
Poor adherence* 
 
 
 
Loss of follow-up 
 
 
 
NUD, non-ulcer dyspepsia; PUD, healed peptic ulcer disease. 
* Poor adherence, took less than 80% of drugs 
 
Table 2. Eradication rate of each group in ITT and PP analysis 
Analysis 
Tailored 
therapy 
Empiric 
therapy 
Difference 
p value for 
inequality* 
p 
value 
for 
superiority ** 
ITT (%, n/N) 
 
 
 
 
 
95% CI 
 
 
 
 
 
PP (%, n/N) 
 
 
 
 
 
95% CI 
 
 
 
 
 
CI= confidence interval; ITT = intention-to-treat; PP = per-protocol. 
* The p values were for testing the difference between tailored therapy group and empiric 
therapy group. 
** The p values were for testing the superiority of tailored therapy group to empiric therapy 
group. 
 
Table 3. antibiotic resistance pattern of ITT population 
antibiotic resistance pattern 
(CLA-MET-LEV) 
ITT population 
Tailored therapy 
Empiric therapy 
All susceptible 
S-S-S 
 
 
 
Single resistant 
 
 
 
R-S-S 
 
 
 
S-R-S 
 
 
 
S-S-R 
 
 
 
Dual resistant 
 
 
 
R-R-S 
 
 
 
64 

Original SAP 
R-S-R 
 
 
 
S-R-R 
 
 
 
Triple resistant 
 
 
 
R-R-R 
 
 
 
CLA, clarithromycin; LEV, levofloxacin; MET, metronidazole; R, resistant; S, susceptible. 
 
 
Table 4. Eradication rates of tailored and empiric therapy in the presence of antibiotic 
resistance in the per-protocol population 
PP population 
Tailored therapy 
Empiric therapy 
p value 
23S rRNA mutation (genotypic) 
Susceptible  
 
 
 
Resistant 
 
 
 
Clarithromycin resistance (phenotypic) 
Susceptible  
 
 
 
Resistant 
 
 
 
Metronidazole resistance (phenotypic) 
Susceptible  
 
 
 
Resistant 
 
 
 
 
 
Table 5. Adverse events and adherence in the treatment groups 
Variables 
Tailored therapy 
Empiric therapy 
p value* 
Total, n/N (%) 
 
 
 
AE grade 
None/Mild/Moderate/Severe 
 
 
 
AE variety 
Taste alteration 
 
 
 
Dyspepsia 
 
 
 
…… 
 
 
 
…… 
 
 
 
…… 
 
 
 
65 

Original SAP 
Skin rash 
 
 
 
Discontinued drugs  
because of AEs 
 
 
 
Good Adherence (Took at least 80% of drugs) 
Subjects without AEs 
 
 
 
Subjects with AEs 
 
 
 
Total 
 
 
 
AE, adverse event.  
 
 
66 

Final SAP 
Final Statistical Analysis Plan 
 
This statistical analysis plan includes the details of study sample size calculation and 
statistical methods for analysis of outcomes and safety. 
Table of Contents 
1. Sample size estimation ·································································· 68 
2. Definition of populations for analysis ················································ 68 
3. Evaluation of baseline characteristics ················································ 68 
4. Evaluation of primary outcomes ······················································ 70 
5. Evaluation of secondary outcomes ···················································· 70 
6. Additional analyses ······································································· 72 
7. Table Design ··············································································· 73 
67 
 

Final SAP 
1. Sample size estimation 
Assuming 96.5% eradication rate of susceptibility-based therapy in our trial, 88.9% 
eradication rate of empirical therapy, a superiority margin of >0, a power of 80%, an 
alpha of 0.05 (one-sided), a ratio of 3:1, at least 260 subjects in susceptibility-based 
therapy and 87 subjects in empirical therapy will be required to show superiority of 
susceptibility-based therapy over empirical therapy on the basis of H. pylori 
eradication rate. Taking into consideration of 10% lost to follow-up, at least 382 
participants (286 for tailored therapy and 96 for empiric therapy) is expected to be 
recruited for the study.  
2. Definition of populations for analysis 
2.1 Intention--to-treat (ITT) Analysis population 
According to the Intention-to-treat principle: All randomized patients will be included 
in the ITT analysis. Patients who did not return for a follow-up 13C-UBT will be 
recorded as treatment failures. 
2.2 Per-protocol (PP) analysis population 
All individuals who violated the study protocol, such as patients not taking at least 80% 
of treatment drugs, or with unknown post-treatment H. pylori status will be excluded 
from the PP analysis.  
2.3 Safety analysis population 
The patients with missing safety data in ITT population will excluded from the safety 
analysis. 
 
3. Evaluation of baseline characteristics 
3.1 presentation of baseline characteristics 
68 

Final SAP 
Variables of baseline characteristics collected are age (years), gender (male/female), 
diagnosis (Dyspepsia/PUD), antibiotic resistance pattern, poor adherence, and loss of 
follow-up. The following table shows the types and presentation of variables.    
Variable 
Type of variable 
Presentation 
Age (years) 
Continuous   
Mean ± standard deviation (range) 
Gender (male/female) 
Categorical 
Number (percentage) 
Diagnosis (Dyspepsia/PUD) 
Categorical 
Number (percentage) 
Antibiotic resistance pattern 
Clarithromycin, n (%) 
Categorical 
Number (percentage) 
Metronidazole, n (%) 
Categorical 
Number (percentage) 
Levofloxacin, n (%) 
Categorical 
Number (percentage) 
All-susceptible, n (%) 
Categorical 
Number (percentage) 
Triple-resistant, n (%) 
Categorical 
Number (percentage) 
Poor adherence* 
Categorical 
Number 
Loss of follow-up 
Categorical 
Number 
 
3.2 Statistical methods for baseline characteristics analyses 
Comparisons of baseline characteristics between the tailored therapy and empiric 
therapy will be performed in the ITT analysis populations. Comparisons between the 
both groups will be performed using the Pearson’s χ2, corrected χ2 or Fisher’s exact 
test for categorical variables and using the Student t test for continuous variables.   
 
4. Evaluation of primary outcomes 
69 

Final SAP 
4.1 Helicobacter pylori eradication rate 
Six weeks after completion of therapy, H. pylori eradication success is defined as 
negative result from urea breath test (<4‰). If patients would be lost to follow-up urea 
breath testing, they will be scored as treatment failures in the ITT analysis.  
The eradication rate is defined as the number of patients who successful eradicate H. 
pylori divided by the total number of population for analysis. These data are presented 
with number, percentage, and 95% confidence interval (CI). 
4.2 Testing the differences of eradication rates 
Testing the differences of eradication rates between the tailored therapy and empiric 
therapy will be performed using the Pearson’s χ2, corrected χ2 or Fisher’s exact test in 
the ITT and PP analysis populations.  
4.3 Testing the superiority of the tailored therapy 
Testing the superiority of tailored therapy group to empiric therapy group will be 
performed in the ITT and PP analysis populations using hypothesis testing (one-sided 
Z test) and derivation of a two-sided 95% confidence interval (CI) of difference based 
on H. pylori eradication rate. If p-value of the testing less than 0.025 and the lower 
bound of the 95% CI greater than zero, superiority of susceptibility-based therapy over 
empirical therapy could be concluded. 
 
5. Evaluation of secondary outcomes 
5.1 Rate of adverse events 
During the 14-day treatment period, the subjects will keep a diary to score any possible 
side effects or discomforts. The subjects will be asked to grade the severity of adverse 
events according to their influence on daily activities, experienced as “mild” (transient 
70 

Final SAP 
and well tolerated), “moderate” (causing discomfort and partially interfering with daily 
activities), or “severe” (causing considerable interference with daily activities). The 
side effect score recorded is based on the most severe event. Rate of adverse events is 
defined as the number of patients with any adverse event divided by the total number 
of population for analysis. These data are presented with number, percentage.  
Comparisons of presence of each adverse event and any adverse event between the 
tailored therapy and empiric therapy will be performed in the safety analysis 
populations using the Pearson’s χ2, corrected χ2 or Fisher’s exact test. 
 
5.2 Compliance rate 
Compliance was deﬁned as either poor when they had taken less than 80% of the total 
medication or good when they had taken more than 80% medication. Compliance rate 
is defined as the number of patients with good compliance divided by the total number 
of population for analysis. These data are presented with number, percentage.  
Comparisons of presence of each adverse event and any adverse event between the 
tailored therapy and empiric therapy will be performed in the ITT analysis populations 
using the Pearson’s χ2, corrected χ2 or Fisher’s exact test. 
 
5.3 Other Pre-specified Outcome Measures: 
(1) Medical cost per patient of tailored or empiric therapy 
Medical cost of one patient is overall cost including visit, endoscopy, H. pylori culture, 
susceptibility testing, eradication treatment, and adverse event treatment. 
Comparisons of medical cost between the tailored therapy and empiric therapy will be 
performed in the PP analysis populations using Student t test. 
(2) Ratio of medical cost to H. pylori eradication rate of each therapy 
71 

Final SAP 
Ratio of medical cost to H. pylori eradication rate of each therapy is defined as average 
medical cost of the therapy divided by H. pylori eradication rate of one therapy. 
(3)Ratio of incremental medical cost to incremental H. pylori eradication rate of 
tailored compared with empiric therapy 
If one therapy cost less and achieved higher eradication rate than the other one, it will 
be referred to as dominant strategy. If one therapy yielded higher eradication rate but 
also cost more, an incremental cost-effectiveness ratio (ICER) will be calculated. 
Obviously, the tailored therapy will cost more because of additional bacterial culture 
and antibiotic susceptibility testing. So, if eradication rate of the tailored therapy was 
higher, the ICER of the tailored therapy will be calculated as the ratio of incremental 
medical cost to incremental H. pylori eradication rate of tailored compared with empiric 
therapy (in terms of RMB per incremental eradication percentage). if eradication rate 
of the tailored therapy was equal to or lower than the empiric therapy, the tailored 
therapy will not be more cost-effective option and the economic evaluation will not be 
conducted. 
 
6. Additional analyses 
6.1 Antibiotic resistance rate 
Minimal 
inhibitory 
concentrations 
(MIC) 
of 
clarithromycin 
>2 
µg/mL, 
metronidazole >8 µg/mL, levofloxacin >2 µg/mL is defined as resistance breakpoints. 
Antibiotic resistance rate is defined as the number of patients with antibiotic resistant 
strains divided by the total number of population for analysis. These data are presented 
with number, percentage. 
6.2 Effect of antibiotic resistance on eradication rate  
Comparisons of eradication rate of one therapy between antibiotic susceptible and 
72 

Final SAP 
resistant strains will be performed in the PP analysis populations using the Pearson’s 
χ2, corrected χ2 or Fisher’s exact test. 
Additional comparisons of eradication rate of antibiotic resistant strains between the 
tailored therapy and the empiric therapy will be performed in the PP analysis 
populations using the Pearson’s χ2, corrected χ2 or Fisher’s exact test. 
6.3 Effect of presence of adverse events on compliance. 
Comparisons of compliance rate of one therapy between presence and absence of 
adverse event will be performed in the safety analysis populations using the Pearson’s 
χ2, corrected χ2 or Fisher’s exact test. 
6.4 Effect of compliance on eradication rate 
Comparisons of eradication rate of one therapy between good and poor compliance will 
be performed in the ITT analysis populations who had UBT results using the Pearson’s 
χ2, corrected χ2 or Fisher’s exact test. 
 
7. Table Design 
Table 1. Baseline characteristics 
Variables 
Tailored therapy 
Empiric therapy 
p value 
Age, years (range) 
 
 
 
Gender (male/female) 
 
 
 
Diagnosis (Dyspepsia/PUD) 
 
 
 
Antibiotic resistance pattern 
Clarithromycin, n (%) 
 
 
 
Metronidazole, n (%) 
 
 
 
Levofloxacin, n (%) 
 
 
 
All-susceptible, n (%) 
 
 
 
Triple-resistant, n (%) 
 
 
 
Poor adherence* 
 
 
 
Loss of follow-up 
 
 
 
73 

Final SAP 
NUD, non-ulcer dyspepsia; PUD, healed peptic ulcer disease. 
* Poor adherence, took less than 80% of drugs 
 
Table 2. Eradication rate of each group in ITT and PP analysis 
Analysis 
Tailored 
therapy 
Empiric 
therapy 
Difference 
p value for 
inequality* 
p 
value 
for 
superiority ** 
ITT (%, n/N) 
 
 
 
 
 
95% CI 
 
 
 
 
 
PP (%, n/N) 
 
 
 
 
 
95% CI 
 
 
 
 
 
CI= confidence interval; ITT = intention-to-treat; PP = per-protocol. 
* The p values were for testing the difference between tailored therapy group and empiric 
therapy group. 
** The p values were for testing the superiority of tailored therapy group to empiric therapy 
group. 
 
Table 3. antibiotic resistance pattern of ITT population 
antibiotic resistance pattern 
(CLA-MET-LEV) 
ITT population 
Tailored therapy 
Empiric therapy 
All susceptible 
S-S-S 
 
 
 
Single resistant 
 
 
 
R-S-S 
 
 
 
S-R-S 
 
 
 
S-S-R 
 
 
 
Dual resistant 
 
 
 
R-R-S 
 
 
 
R-S-R 
 
 
 
S-R-R 
 
 
 
Triple resistant 
 
 
 
R-R-R 
 
 
 
CLA, clarithromycin; LEV, levofloxacin; MET, metronidazole; R, resistant; S, susceptible. 
74 

Final SAP 
 
 
Table 4. Eradication rates of tailored and empiric therapy in the presence of antibiotic 
resistance in the per-protocol population 
PP population 
Tailored therapy 
Empiric therapy 
p value 
Clarithromycin resistance 
Susceptible  
 
 
 
Resistant 
 
 
 
Metronidazole resistance 
Susceptible  
 
 
 
Resistant 
 
 
 
Levofloxacin resistance 
Susceptible  
 
 
 
Resistant 
 
 
 
Resistance pattern (Clarithromycin-Metronidazole-Levofloxacin) 
S-X-X 
 
 
 
R-S-X 
 
 
 
R-R-S 
 
 
 
R-R-R 
 
 
 
R=resistant; S=susceptible; X=resistant or susceptible. 
 
Table 5. Adverse events and adherence in the treatment groups 
Variables 
Tailored therapy 
Empiric therapy 
p value* 
Total, n/N (%) 
 
 
 
AE grade 
None/Mild/Moderate/Severe 
 
 
 
AE variety 
Taste alteration 
 
 
 
Dyspepsia 
 
 
 
…… 
 
 
 
…… 
 
 
 
…… 
 
 
 
75 

Final SAP 
Skin rash 
 
 
 
Discontinued drugs  
because of AEs 
 
 
 
Good Adherence (Took at least 80% of drugs) 
Subjects without AEs 
 
 
 
Subjects with AEs 
 
 
 
Total 
 
 
 
AE, adverse event.  
76 

Summary of SAP changes 
Summary of Statistical Analysis Plan Changes 
 
Change 
Original 
Final 
1.Sample 
size 
estimation 
Assuming 95.7% eradication rate of tailored therapy in our trial, 
88.8% eradication rate of empirical therapy, a superiority margin 
of >0, a power of 80%, an alpha of 0.05 (one-sided), a ratio of 3:1, at 
least 340 subjects in susceptibility-based therapy and 114 subjects in 
empirical therapy will be required to show superiority of 
susceptibility-based therapy over empirical therapy on the basis of H. 
pylori eradication rate. Taking into consideration of 10% lost to 
follow-up, at least 500 participants (374 for tailored therapy and 126 
for empiric therapy) is expected to be recruited for the study. 
Assuming 96.5% eradication rate of tailored therapy in our trial, 88.9% 
eradication rate of empirical therapy, a superiority margin of >0, a power of 
80%, an alpha of 0.05 (one-sided), a ratio of 3:1, at least 260 subjects in 
susceptibility-based therapy and 87 subjects in empirical therapy will be 
required to show superiority of susceptibility-based therapy over empirical 
therapy on the basis of H. pylori eradication rate. Taking into consideration 
of 10% lost to follow-up, at least 382 participants (286 for tailored therapy 
and 96 for empiric therapy) is expected to be recruited for the study 
3.1 presentation of 
Variables of baseline characteristics collected are age (years), gender 
(male/female), diagnosis (Dyspepsia/PUD), clarithromycin-taking 
Variables of baseline characteristics collected are age (years), gender 
(male/female), 
diagnosis 
(Dyspepsia/PUD), 
clarithromycin-taking 
77 
 

Summary of SAP changes 
baseline 
characteristics 
history, 23S rRNA mutation, antibiotic resistance pattern, poor 
adherence, and loss of follow-up. The following table shows the 
types and presentation of variables. 
history, 23S rRNA mutation, antibiotic resistance pattern, poor adherence, 
and loss of follow-up. The following table shows the types and presentation 
of variables. 
6. Additional 
analyses 
6.3 Effect of 23S rRNA mutation on eradication rate 
Comparisons of eradication rate of clarithromycin-containing 
therapy between strains with 23S rRNA mutation and those without 
23S rRNA mutation will be performed in the PP analysis populations 
using the Pearson’s χ2, corrected χ2 or Fisher’s exact test. 
6.4 Sensitivity, specificity, positive predictive value and negative 
predictive value of molecular genetic assays in clarithromycin 
resistance identification 
Using culture based antibiotic susceptibility testing as the gold 
standard, sensitivity, specificity, positive predictive value (PPV) and 
negative predictive value (NPV) of 23S rRNA mutation in 
identification of H. pylori resistance to clarithromycin will be 
6.3 Effect of 23S rRNA mutation on eradication rate (Deleted) 
Comparisons of eradication rate of clarithromycin-containing therapy 
between strains with 23S rRNA mutation and those without 23S rRNA 
mutation will be performed in the PP analysis populations using the 
Pearson’s χ2, corrected χ2 or Fisher’s exact test. 
6.4 Sensitivity, specificity, positive predictive value and negative 
predictive value of molecular genetic assays in clarithromycin 
resistance identification (Deleted) 
Using culture based antibiotic susceptibility testing as the gold standard, 
sensitivity, specificity, positive predictive value (PPV) and negative 
predictive value (NPV) of 23S rRNA mutation in identification of H. pylori 
78 

Summary of SAP changes 
estimated.  
resistance to clarithromycin will be estimated. 
7. Table Design 
Table 1. Baseline characteristics 
Table 4. Eradication rates of tailored and empiric therapy in the 
presence of antibiotic resistance in the per-protocol population 
In Table 1, clarithromycin-taking history and 23S rRNA mutation were 
deleted. 
In Table 4, subgrouping according to 23S rRNA mutation was deleted, and 
subgrouping according to levofloxacin resistance and antibiotic resistance 
pattern (Clarithromycin-Metronidazole-Levofloxacin) was added. 
 
79