Helicobacter Pylori Eradication Therapy in Portugal: Prospective, Randomized, Blind and Multicentre Trial on the Efficacy of Quadruple Therapies and Their Clinical Impact, and Immunological and Gut Microbiota Changes
Phase: NA
Conditions: Helicobacter Pylori, Gut Microbiota, Immunology
Sponsor: Unidade Local de Saúde de Coimbra, EPE

Helicobacter Pylori Eradication Therapy in Portugal: 
Prospective, randomized, blind and multicentre trial on 
the efficacy of quadruple therapies and their clinical 
impact, and immunological and gut microbiota changes 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study Protocol and Statistical 
Analysis Plan 
 
 
 
February 19th, 2021 

Helicobacter Pylori Eradication Therapy in Portugal 
Helicobacter Pylori Eradication Therapy in Portugal: Prospective, randomized, blind and 
multicentre trial on the efficacy of quadruple therapies and their clinical impact, and 
immunological and gut microbiota changes 
_________________________________ 
Keywords: Helicobacter pylori, Eradication therapy, Gut microbiota, Immunology 
 
Abstract 
Introduction: Helicobacter pylori (H. pylori) infection remains a major public health problem, 
with an estimated prevalence of over 50% worldwide and 60-86% for Portugal. H. pylori is 
associated with significant morbidity and mortality from peptic ulcerative disease to gastric 
cancer, whose eradication therapy has proven to be effective in preventing these complications. 
Factors involved in the development of these conditions include H. pylori virulence, host genetic 
factors and gut microbiota. Given the increasing pattern of antibiotic resistance evidenced by 
this bacterium and the scarcity of available antibiotic therapy, both in Portugal and worldwide, 
there is not enough evidence on the best eradication strategy. Regarding the uncertainties about 
the potential negative impact of indiscriminate use of eradication therapy on gut microbiota, 
either by proton pump inhibitors or by antibiotics per se, there is an overriding need for evidence 
about the real impact of this therapy on gut flora and possible clinical consequences in 
immunological, metabolic, nutritional and oncological terms. Objectives: Comparative 
evaluation of the efficacy of the different quadruple therapy regimens recommended for the H. 
pylori eradication. Comparative evaluation of the safety profile in terms of clinical, 
immunological and gut microbiota impact of the different therapies for the H. pylori eradication. 
Methods: Prospective longitudinal multicentre study of total of patients with gastric infection 
by H. pylori, diagnosed by 13C-urea breath test or histological analysis of gastric biopsies and 
clinical indication for its eradication, referred to the different participating Portuguese hospital 
units and a blind randomized controlled clinical trial of the efficacy and safety of the different 
quadruple therapy regimes recommended for the H. pylori eradication. This study will be carried 
out in 4 phases: Phase 1 - Recruitment and randomization of patients by the different quadruple 
eradication schemes with and without bismuth (5 parallel arms); Phase 2 - H. pylori eradication 
with evaluation of the efficacy and safety rates at 1 month and the absence of reinfection at 12 
months after treatment and collection of stool samples before and after the eradication therapy 
for evaluation of changes in gut microbiota; Phase 3 - Analysis of richness, diversity and 
uniformity of gut microbiota by DNA sequencing using the hypervariable region of the ribosomal 
16S bacteria gene as a taxonomic identification marker and their clinical impact on immunology, 
metabolism and nutrition at 12 months after the H. pylori eradication therapy; and Phase 4 - 
Analysis of immunological changes through the study of cell populations by flow cytometry 
(CD4+, CD8+, B-cell, T-cell, natural killer cells, cells ratio) and cytokines, chemokines and growth 
factors by xMAP/Luminex before and 12 months after the H. pylori eradication therapy. 
Expected results, impact and scientific outputs: Given the high rate of triple therapy inefficacy, 
high antibiotic resistance and the scarcity and controversy of existing literature on quadruple 
regimens, there may be relevant differences in the approved quadruple regimens for the H. 
pylori eradication, being necessary to define which is the most effective and safe in Portugal, 
decreasing the rate of ineffectiveness and exposure to multiple antibiotics. The homeostasis of 
gut microbiota is significantly changed after H. pylori eradication and this modification may be 

substantially different according to the therapeutic scheme used, with clinical implications on 
immunology, metabolism and nutrition. Thus, a randomized trial to compare quadruple 
regimens is need, allowing in the future, an individualized selection of the H. pylori eradication 
regimen, taking into account the higher efficacy and safety and lower gut dysbiosis and its 
systemic consequences, in short and long term. Modulating gut microbiota therapies, including 
prebiotics, probiotics, symbiotics, fecal microbiota transplantation and perhaps targeted-
immunotherapy may be beneficial as adjuvant therapy to existing H. pylori eradication 
regimens, in a systematic way or for some therapeutic regimes or risk groups. 
 
Literature Review  
Helicobacter pylori (H. pylori) infection remains a major public health problem, affecting over 
50% of the worldwide population and 60-86.4% of the Portuguese population.1,2 This bacterium 
is responsible for chronic active gastritis, likely to develop into peptic ulcerative disease, atrophic 
gastritis, gastric adenocarcinoma and mucosal-associated lymphoid tissue lymphoma.3,4 
Factors involved in the development of these conditions include bacterial virulence, host genetic 
factors and gut microbiota.3,4 H. pylori eradication therapy has been shown to be effective in 
preventing the development or recurrence of these complications.3,5 However, the increase in 
primary and secondary resistance of this bacterium to most antibiotics has reduced the efficacy 
of this therapeutic strategy. As in the rest of Europe, overall resistance to multiple antibiotics is 
high in Portugal, with a prevalence of 42-50% for clarithromycin, 25-34.4% for metronidazole, 
18-33.9% for levofloxacin, 0.2-0.6% for tetracycline, 0.1-0.6% for amoxicillin and 20% for 
clarithromycin and metronidazole.6-8 Taking into account the pattern of H. pylori antibiotic 
resistance in Portugal and the selection flowchart proposed by [GrLeWu14],9 the currently 
recommended therapeutic regimens include quadruple therapies: sequential, hybrid, 
concomitant with bismuth and concomitant bismuth-free.3,5,7,10-12 However, some studies 
including quadruple schemes have shown unsatisfactory results, not exceeding 90% considered 
as the minimum efficacy rate.12,13 Given the high rate of resistance of H. pylori to 
nitroimidazoles registered in Portugal, it would be worth considering therapeutic optimization 
with metronidazole 500mg tid and not only bid.3 Thus, at present, there are no solid data that 
allow us to state the best therapeutic option in this country, being essential to conduct a 
randomized study to definitively resolve this uncertain. Animal and human studies have shown 
that H. pylori results in changes in gut microbiota.4,14,15 Human gut microbiota represents a 
dynamic and complex ecosystem in close linking with the immune system, regulating 
gastrointestinal homeostasis. It is composed mostly of Gram-negative anaerobic bacilli, mainly 
Bacteroidetes and Fusobacteria. Gut dysbiosis has been associated with immune, metabolic, 
nutritional and oncological changes, such as the development of several gastrointestinal and 
systemic conditions such as inflammatory bowel disease, obesity, diabetes mellitus, metabolic 
syndrome, dyslipidemia, asthma and allergies.16-18 However, the interactions between H. 
pylori, gut microbiota and host pathophysiology are still far from being understood. Similarly, 
there are also uncertainties regarding the potential negative impact of indiscriminate use of H. 
pylori eradication therapy on the gut microbiota, mediated by either proton pump inhibitors or 
antibiotherapy.17,19 In some recent studies, eradication of H. pylori has been associated with a 
decrease in butyrate-producing bacteria (such as Faecalibacterium prausnitzii), Proteobacteria, 
Actinobacteria and Firmicutes, and an increase in several potentially harmful bacteria such as 
Bacteroidetes and the Bacteroidetes:Firmicutes ratio.4,19 These changes persist until 12-18 
months after H. pylori eradication therapy,16 although in one study changes in gut microbiota 

were documented up to 4 years after eradication.20 Given that only approximately 20% of H. 
pylori infected individuals will develop complications21 and the potentially harmful effects of 
antibiotherapy use on the gut microbiota, further studies are needed to understand the complex 
interactions between the H. pylori-gut microbiota-host axis in order to identify individuals at 
increased risk of long-term sequelae.19 H. pylori gastric infection can induces humoral and 
cellular immune responses. Some studies showed an increase of Th1 and Th17 cells into the 
gastric mucosa and activation of humoral immunity, Th2 polarization and some cytokines such 
as IL1ᵦ, Il4 and IL5 in the peripheral blood, during H. pylori infection.22-26 Data on 
immunological changes after eradication of H. pylori are very scarce, with one study showing a 
downregulation of the Th1 cytokines, a prolonged Th2 response with persistent raised IL4 levels 
and markedly reduction of the T-regulatory response.27 To date, there is a lack of evidence on 
the impact of H. pylori eradication therapy, as well as the comparative impact of different 
therapeutic regimens used in gut microbiota, immunological changes and their implication in 
clinical practice. 
 
Plan and Methods  
A. Research hypothesis  
The authors hypothesize that there are relevant differences in the quadruple schemes that can 
be used in the H. pylori eradication therapy, being necessary to define which of them is the most 
effective in Portugal, in order to decrease the failure rate and subsequent exposure to 
potentially harmful effects of antibiotics. Additionally, the authors assume that homeostasis of 
gut microbiota is significantly changed after H. pylori eradication therapy and these alterations 
may not be similar among the different recommended therapeutic regimes. Likewise, these 
alterations may have clinical implications in the immunological, metabolic and nutritional status 
of individuals submitted to this therapy. Hypothesis: There is a quadruple H. pylori eradication 
therapy associated with a higher efficacy and safety than other currently approved quadruple 
regimens in terms of clinical, immunological and microbiota changes in H. pylori gastric 
infection.  
B. Objectives  
• To determine and compare the efficacy of the different therapeutic regimens recommended 
as H. pylori eradication therapy;  
• Comparative evaluation of the safety profile in terms of clinical, immunological and gut 
microbiota impact of the different recommended therapies for the H. pylori eradication:  
• To evaluate the changes in gut microbiota after the H. pylori eradication therapy;  
• Compare the changes in gut microbiota induced by the different therapeutic regimens for H. 
pylori eradication and evaluate their clinical implications on immunology, nutrition and 
metabolism;  
• To evaluate the immunological changes after the H. pylori eradication therapy;  
• Compare the immunological changes induced by the different therapeutic regimens for H. 
pylori eradication and evaluate their clinical implications.  
C. Expected results, study clinical impact and scientific outputs  

Given the high rate of triple therapy inefficacy, high antibiotic resistance and the scarcity and 
controversy of existing literature on quadruple regimens, there may be relevant differences in 
the approved quadruple regimens for the H. pylori eradication, being necessary to define which 
is the most effective and safe in Portugal, decreasing the rate of ineffectiveness and exposure 
to multiple antibiotics. The homeostasis of gut microbiota is significantly changed after H. pylori 
eradication and this modification may be substantially different according to the therapeutic 
scheme used, with clinical implications on immunology, metabolism and nutrition. Thus, a 
randomized trial to compare quadruple regimens is need, allowing in the future, an 
individualized selection of the H. pylori eradication regimen, taking into account the higher 
efficacy and safety and lower gut dysbiosis and its systemic consequences, in short and long 
term. Modulating gut microbiota therapies, including prebiotics, probiotics, symbiotics, fecal 
microbiota transplantation and perhaps targeted-immunotherapy may be beneficial as adjuvant 
therapy to existing H. pylori eradication regimens, in a systematic way or for some therapeutic 
regimes or risk groups.  
D. Methodology and Phases of project development  
D.1 Type of study  
Prospective longitudinal multicentre study and a blind randomized controlled trial involving 
several hospital units in Portugal. Regarding the evaluation of gut microbiota and immunological 
changes, the study will be blinded, since the researchers responsible for sequencing and 
immunological analysis will not know the therapeutic scheme applied. The trial will be registered 
on the https://clinicaltrials.gov platform.  
D.2 Study population  
Patients with gastric infection by H. pylori, diagnosed by 13C-urea breath test or histological 
analysis of gastric biopsies and clinical indication for its eradication.  
D.3 Study design  
This project will be carried out in 4 distinct phases:  
Phase 1. Recruitment and randomization of patients by the different H. pylori eradication 
schemes (5 parallel arms)  
A total of patients with gastric infection by H. pylori and clinical indication for its eradication, 
who have never had previous H. pylori eradication therapy, referred to the Gastroenterology 
Departments of the different participating Portuguese hospital centres (Centro Hospitalar e 
Universitário de Coimbra, Francisco Gentil Portuguese Oncology Institute of Coimbra and Leiria 
Hospital Centre), will be included in the study. Patients’ selection will taking into account the 
inclusion and exclusion criteria and the minimum number of patients required in order to obtain 
statistical power. Inclusion criteria: Gastric infection by H. pylori. H. pylori status will be 
determined by histological examination of gastric biopsies or carbon 13-labeled urea breath 
test. The breath test will be considered positive if [13CO2] exceeds the baseline value by > 4 
parts per thousand (> 0.4%) after 30 minutes. Histological examination for H. pylori will be 
considered positive when this bacterium is detected in ≥1 biopsies (staining with hematoxylin-
eosin, modified Giemsa and/or Warthin-Starry). Exclusion criteria: Age < 18 years; Pregnant, 
breast-feeding or women of childbearing age who do not comply with effective anticonception 
measures; History of allergy, hypersensitivity or contraindication to the use of H. pylori 
eradication drugs (antibiotics or proton pump inhibitors); History of previous gastrointestinal 

surgery or neoplasia; Previous H. pylori eradication therapies; Antibiotic or probiotic therapies 
in the month prior to recruitment; Use of proton pump inhibitors, other antacids or gastric 
mucosal protection agents in the 2 weeks prior to recruitment; Corticosteroids or 
immunomodulatory therapy in the month prior to recruitment; Immunodeficiency; Insulin-
treated diabetes mellitus; Obesity (Body mass index ≥30Kg/m2); Use of laxative therapy in the 
15 days prior to recruitment; Decompensated heart, liver, kidney or respiratory diseases and; 
Refusal or inability to give informed consent. H. pylori eradication schemes: Each patient will 
randomly follow one of the five H. pylori eradication schemes (5 parallel arms): sequential, 
hybrid, concomitant with bismuth and two concomitants bismuth-free. Sample size calculation: 
Assuming the estimated prevalence of H. pylori gastric infection in Portugal in adults is 86.4%1, 
it was determined that for a 95% confidence level and a 10% sampling error it will be necessary 
to include 46 patients in each of the 5 arms of the study, making a total of 230 patients. 
Phase 2. Eradication of H. pylori and collection of stool an blood samples  
Stool samples shall be collected from patients and analyzed on 3 occasions: before (baseline), 
after stopping treatment (Day 10 or 14) and 1 month after treatment (Day 40 or 44). Blood 
samples will be collected and analyzed on 2 occasions: before (baseline) and 12 months after 
treatment. Efficacy and safety rates of H. pylori eradication will be determined for each 
therapeutic regimen. Carbon 13-labeled urea breath test or upper gastrointestinal endoscopy 
with biopsies will be performed at 1 month (D40 or D44) and 12 months after treatment. The 
clinical data, adherence and therapy-related adverse events will be recorded at baseline and 
during follow-up at 1 and 12 months post-eradication.  
Phase 3. Analysis of gut microbiota changes by DNA sequencing after H. pylori eradication 
therapy and their clinical impact on immunology, metabolism and nutrition  
The impact of H. pylori eradication therapy on gut microbiota will be studied by next generation 
genome sequencing techniques. A comparative analysis will be performed for each therapeutic 
regimen used. Microbiota changes found will be compared with clinical and analytical data in 
order to establish their impact on immunology, metabolism and nutrition status at 1 and 12 
months post-eradication. Phase 4. Analysis of immunological changes of cell populations, 
cytokines, chemokines and growth factors after H. pylori eradication therapy and their clinical 
impact The impact of H. pylori eradication therapy on humoral and cellular immunity will be 
studied by flow cytometry and xAMP/Luminex. A comparative analysis will be performed for 
each therapeutic regimen used. Immunological changes found will be compared with clinical 
and analytical data in order to establish their clinical impact at 12 months post-eradication.  
D.4 Statistical Analysis  
Using the Statistical Package for the Social Sciences (SPSS) version 22.0 (IL, USA). The level of 
statistical significance will be for a p-value < 0.05. The normality of data distribution will be 
evaluated by Kolmogorov-Smirnov or Shapiro-Wilk tests. Continuous variables will be expressed 
as mean and standard deviation or median and interquartile range and categorical variables as 
frequency and percentage. The comparison of continuous variables will be made using the 
Kruskal-Wallis test or Anova test and categorical variables using the Qui-squared test. For the 
comparison of paired data the Wilcoxon test or Friedman test and the t-Student or Anova test 
will be used. Further statistical analysis of H. pylori eradication efficacy, gut microbiota and 
immunological changes is described in Tasks section. The genome sequences will be grouped in 
OTU according to their similarity. The alpha taxonomic richness and diversity of fecal samples 
will be expressed in observed species, Sobs index and Shannon index. The composition of fecal 

microbiota will be expressed in rarefaction curves and the comparison of the differences in OTU 
richness with the t-Welch test. Immunological findings will be expressed by radar charts and 
representative FACS dot-plots. Additional statistical analyses will be defined throughout the 
study. 
D.5 Ethical aspects  
The project will be subject to the standards of good clinical practice and will at all times comply 
with the ethical precepts in the World Medical Association Helsinki’s Declaration and updates, 
including the Oviedo agreement. This study was approved by Ethical Committee of University of 
Coimbra (ref.n.º CE-001/2019). Written informed consent will be obtained from all patients. The 
confidentiality of the data will be respected at all times, by means of the anonymity of the data 
in the database, in accordance with General Data Protection (UE 2016/679) and updates. 
 
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