Effect of Rebamipide on Fecal Helicobacter pylori Antigen Test

INTRODUCTION

In developing countries, more than 50|% of the population is infected with Helicobacter pylori (H. pylori), largely due to widespread exposure to risk factors such as inadequate access to clean water, poor sanitation, and overcrowded living conditions—circumstances that facilitate fecal-oral transmission [1]. Helicobacter pylori is classified as a gastric cancer Group 1 carcinogen [2], a malignancy that ranks as the fourth most common cancer and the second leading cause of cancer-related deaths globally [3].

Although the precise risk of gastric cancer in developing countries remains uncertain, evidence from Japanese men—a population with relatively low H. pylori prevalence—indicates that infection may result in a lifetime gastric cancer risk of up to 17%, compared to just 1% among uninfected individuals [4].

Fecal antigen testing for H. pylori is a simple, non-invasive, and cost-effective diagnostic method that plays a critical role in screening high-risk populations and may facilitate earlier detection, ultimately contributing to a reduction in gastric cancer incidence, prevalence, and mortality [5].

Rebamipide is widely used as a protective agent against mucosa in East Asia for managing different upper gastrointestinal (UGI) symptoms [6].

Several studies have reported that Rebamipide may improve upper gastrointestinal (UGI) symptoms in Asian patients with chronic gastritis [11] and type 2 diabetes mellitus [12]. Also, continuous co-prescription of Rebamipide significantly reduces the risk of upper gastrointestinal bleeding in new users of non-steroidal anti-inflammatory drugs (NSAIDs) [13].

Rebamipide has been linked to symptom relief in cases of organic dyspepsia, though evidence supporting its effectiveness in functional dyspepsia (FD) remains less conclusive. The therapeutic benefit is likely due to the resolution of chronic gastritis, a condition that is often difficult to detect using standard endoscopic techniques in clinical practice. Given increasing concerns about the long-term safety of proton pump inhibitors [14], Rebamipide may represent a viable alternative for managing FD or for patients susceptible to recurrent NSAID-related peptic ulcers [15].

In addition, Nishizawa et al., 2014 found in a meta-analysis that supplementation with Rebamipide might be effective in increasing H. pylori eradication rates of PPI–amoxicillin dual therapy [16]. In the current study, we aim to estimate the accuracy of fecal H pylori Ag testing while on Rebamipide therapy. To the best of our knowledge, no prior research has assessed the impact of Rebamipide on fecal H. pylori Ag levels, making this study a novel contribution to the field.

PATIENTS AND METHODS

Study Design and Participants:

A quasi-experimental study "before-and-after study" was conducted between 11th March 2025 and 11th April 2025. A total of 73 patients with dyspepsia and positive fecal H. pylori Ag test results were enrolled. Participants were recruited from Elkharga Specialized Hospital, New Valley University, and Aswan University Hospital. All of them received Rebamipide 100 mg three times daily for 14 days without any other additional therapy. At the end of this period, they were retested for fecal H pylori Ag.

Inclusion Criteria: 

1. Adults (≥18 years).
2. Presence of dyspeptic symptoms. 
3. Positive Fecal H. pylori Ag test at baseline. 
4. Willingness to provide informed consent. 

Exclusion Criteria: 

1. 1.                  Use of PPIs, H2 blockers, or antibiotics within 4 weeks.
2. 2.                  Patients who are indicated for immediate therapy with antisecretory drugs [PPI or potassium-competitive acid blocker (PCAB)] or antibiotics for any reason.
3. 3.                  Red-flag symptoms (e.g., weight loss, hematemesis) require urgent endoscopy. 
4. 4.                  Pregnancy, lactation, or severe comorbidities (e.g., liver/kidney failure).
5. 5.                  Patients who had previous experience of any allergic reactions (itching or rash, etc.) to any drugs or food.
6. 6.                  Patients refusing to participate in the study

Fecal H. Pylori Ag Detection by ELISA 

The EDIT™ Fecal H. Pylori Antigen ELISA Kit (Epitope Diagnostics, Cat. No. KT 826) was used for qualitative and quantitative detection of H. pylori antigen in stool samples. 

1. Sample Collection and Preparation

a)         Collection: Fresh stool samples (1–2 g solid or 1–2 mL liquid) were collected in sterile containers and stored at –20°C if not processed within 24 hours. 

b)         Homogenization: 

-           Forty milligrams of stool were suspended in 1 mL of 1X Assay Buffer provided in the kit.

-           Vortexed for 1–2 minutes, then centrifuged at 3,000 × g for 5 minutes. A supernatant was used for testing. 

1. ELISA Procedure (Quantitative) 

a)         Coating: 

   - Added 100 µL of calibrators (Cat. No. 31141–31146), controls (Cat. No. 31147–31148), or patient samples to antibody-coated microplate wells (Cat. No. 30665). 

   - Incubated at room temperature for 1 hour.

b)         Washing: 

   - Washed 5× with 350 µL/well of diluted Wash Buffer (Cat. No. 10010). 

c)         Detection:

   - Added 100 µL of HRP-conjugated anti-H. Pylori tracer antibody (Cat. No. 30666). 

   - Incubated at RT for 30 minutes. 

d)         Substrate Reaction:

   - Added 100 µL of TMB substrate (Cat. No. 10020), and incubated for 20 minutes (protected from light). 

e)         Stop & Measurement:

   - Stopped with 100 µL of sulfuric acid (Cat. No. 10030). 

   - Absorbance was measured at 450/620 nm using a microplate reader.

Interpretation 

- Quantitative: 

H. pylori concentration (ng/mL) calculated from a standard curve (Calibrators 1–6). 

- Positive cutoff: ≥3 ng/mL. 

- Qualitative: 

- Positive: OD ≥ 1.1 × (mean negative control + 0.10). 

1. Quality Control 

- Intra-assay CV < 10%. 

- Controls: Positive control OD > 0.8; negative control OD < 0.18.

This protocol aligns with the manufacturer’s guidelines (Epitope Diagnostics, KT 826) andclinical standards for H. pylori detection. 

Sample size:

The sample size was calculated using OpenEpi, version 3, an open-source calculator considering the outcome frequency as 95%, confidence limits of 5%, and design effect 1. Sample size equation: n = [DEFF*NP (1-p)]/ [(d2/Z21-α/2*(N-1) +p*(1-p)]. The calculated sample was 73 participants with 95% confidence interval [17].

Statistical analysis:

The data was collected and entered the Statistical Package for Social Science (SPSS) version 25; the qualitative data was presented as numbers and percentages, while quantitative data was presented as mean, standard deviations, range, and median with inter-quartile range and according to their distribution the suitable test was used. The confidence interval was 95%, and the accepted error margin was 5%. When the p-value was less than 0.05, it was considered significant.

RESULTS

Seventy-three patients were enrolled in this quasi-experimental study "before-and-after study". Among them, 38 (52.1%) were female and 35 (47.9%) were male. The mean age of the study group was 38.93±14.268 (15-71). After 2 weeks of treatment with three times daily Rebamipide 100mg alone without additional therapy, 43 patients (58.9%) showed no dyspepsia, and 30 patients (41.1%) still had dyspepsia (fig.1). Among the enrolled patients, 12 patients (16.4%) showed negative results of the fecal Helicobacter pylori test after 2 weeks therapy with three times daily Rebamipide 100mg and 61 patients (83.6%) showed positive results (fig.2). There was no significant difference between those with positive and negative results regarding age, gender or improvement of dyspepsia (p = 0.343, 0.274, 0.221 respectively).

DISCUSSION