Eradication of Helicobacter Pylori with Triple Therapy Comprising Probiotic & Proton Pump Inhibitor

Objectives: The current clinical trial compared the effects of conventional triple therapy and probiotic (Lactobacillus reuteri) plus omeprazole combination in peptic ulcer patients. The secondary objectives included estimating the effects of these regimens on safety and tolerability. Study Design: Randomized clinical trial Abode and Period of Study: This was a six month research study conducted at the National Medical Centre, Karachi, Pakistan in October 2020 – March 2021. Materials & Methods: A total of 100 patients were recruited, had baseline positive stool antigen test. All the participants were separated into dual groups: conventional triple remedy (group A1) and probiotic with omeprazole combination (group B1). The study's primary endpoint was stool antigen assay and secondary included change in Hb, LFTs and renal function test. Original Research Article Amjad et al.; JPRI, 33(60A): 703-709, 2021; Article no.JPRI.78630 704 Results: The primary endpoints for combination therapy led to significantly greater reductions in positive stool antigen assay than triple therapy. This means that combination therapy is far better than triple therapy. The stool antigen test showed 56.5% positive and 43.5% negative in group A1 while in group B1 34.8% positive while 65.2% negative after treatment were seen with statistically significant difference (p=0.036). Insignificant findings were observed for level of Hb, LFTs and renal function test between both groups during the entire study. Conclusion: This is the first randomized clinical trial in peptic ulcer patients of Pakistan treated with probiotic plus omeprazole combination. Combination therapy was generally well-tolerated and effective in eradicating the Helicobacter pylori after initiation of therapy.


INTRODUCTION
Pakistan is one of the developing countries in South Asia with occurrence of peptic ulcer disorder, which is mainly caused by Helicobacter pylori bacteria is found to be 85.1%. this shows that peptic ulcer is mainly associated with the presence of helicobacter pylori [1]. Hence, this emerged as the focus of Pakistani researchers' attention in identifying effective therapeutic agents to control peptic ulcer disease which can lead to serious complications like stomach bleeding, perforation, penetration to a surrounding organ and obstruction from fibrotic structuring [2]. At the time of 90s era, the conventional triple therapy was considered the benchmark in the cure of peptic ulcer disorder. The conventional triple regimen comprises of PPI, clarithromycin and amoxil. This triple therapy was recommended at the first Maastricht conference in 1996 and has been widely used for twenty years [3]. The raise in the incidence of resistance to these drugs especially to the main antibiotics, clarithromycin and metronidazole has reduced the efficacy of therapy [4]. Because of increased level of resistance and side effects of the two main antibiotics (clarithromycin and metronidazole) substitute approaches are being employed in medical practice to cure the Helicobacter pylori resilient strains [5].
The latest Maastricht V/Florence consensus 2016 suggested the bismuth-containing quadruple therapy in those regions where resistance to clarithromycin is very high [6]. Other remedy is Levofloxacin-based therapy; this remedy has replaced clarithromycin in standard triple regimen [7]. But due to high development of resistance to fluoroquinolone this regimen is also not used [8]. Due to these reasons, the exploration of new drug to treat H. pylori infection is needed.
Probiotics have affirmative impact on the abolition rates and avoidance of side effects associated with antibiotics [9]. They have confirmed to be helpful in decreasing the side effects of antibiotics and improve the patient compliance [10]. The mainly studied probiotics used for treating stomach disorders are lactic acid bacteria [11]. Lactobacillus reuteri have been revealed to decrease the gastric symptoms and also have antimicrobial and immunomodulatory effect [12]. Probiotic like Lactobacillus reuteri is a newly discovered probiotic and gain attention in recent years because of its safety. This clinical randomized trial will focus on Lactobacillus reuteri + proton pump inhibitor combination therapy in peptic ulcer patients.

Clinical Study Design
The current randomized clinical trial was of 6 months, conducted at the National medical centre, Karachi, Pakistan. 100 patients having peptic ulcer with positive stool antigen test for H. pylori were recruited in the study, 92 patients successfully completed the study. They were divided into two groups based on their treatment regimens. In the group A, patients were given triple remedy (Omeprazole 20 mg two times daily + Clarithromycin 500 mg two times daily + Amoxicillin 1 gm two times daily) for two weeks, whereas the group B patients were given (Lactobacillus reuteri 100 mg twice daily + Omeprazole 20 mg twice daily) for two weeks. where Z α/2 is the critical value of the Normal distribution at α/2 (e.g. for a confidence level of 95%, α is 0.05 and the critical value is 1.96), Z β is the critical value of the Normal distribution at β (e.g. for a power of 80%, β is 0.2 and the critical value is 0.84), σ 2 is the population variance, and d is the difference you would like to detect.

Statistical Analysis
The calculated sample size was 70 with 35 patients in each group. However, we have enrolled 100 patients with 50 patients in each group.

Sampling technique:
Systemic random sampling (randomized sampling technique)

Patients
All the peptic ulcer patients were aged 18-50 years, had epigastric pain of 6 weeks or more duration with positive H. pylori stool antigen assay test. Patients were excluded if they had negative test for H. pylori, those who had history of cancer or other chronic diseases. Patient who are regular user of non-steroidal antiinflammatory drugs or bisphosphonates and also pregnant or lactating females were also excluded.

Endpoints and Assessments
The sample size calculation of the peptic ulcer population was estimated using Open Epi, Version 3. The primary efficacy endpoint, i.e., stool antigen assay was estimated at different intervals i.e. at baseline and after 2 weeks. The study's key secondary endpoints were complete blood count (Hb level), LFTs and renal function test. The change in liver function test (Aspartate transaminase and alanine aminotransferase), renal function test (serum urea and serum creatinine) and hemoglobin levels were quantified at baseline and after 2 weeks.

Liver Function Test (LFT)
Storage and stability: Reagents were stored at +2 to +8°C. Mix after 1 minute add start reagent 125 µl. Mix after approx. 1minute measure the decrease in absorption every minute for 3 minutes.

Calculations
The COBAS 6000/8000 system was automatically calculated the GGT activity of each patient sample. Mix after 1 minute add start reagent 125 µl. Mix after approx. 1minute measure the decrease in absorption every minute for 3 minutes.
Assess the absorbance level of sample (A S ) and standard (A St ) after 60 sec.

RESULTS
The current randomized clinical trial enrolled 100 peptic ulcer patients from Karachi, Pakistan. 92 patients completed the study, 46 patients per group. Male and female between the age group of 18 years to 50 years accomplishing the inclusion criteria were involved in the research after taking the consent.
The prime objective of this research was to find out the effectiveness of conventional triple remedy vs. Lactobacillus reuteri + proton pump inhibitors combination in peptic ulcer patients of Karachi. Followed by given respective treatment regimens, stool antigen test was done. The results revealed that group A showed 56.5% positive test results and 43.5% negative test results while group B showed 34.8% positive test results and 65.2% negative test results ( Table 1).
The safety and tolerability were identified by measuring Hb level, LFTS and Renal Function Test. Statistically insignificant change in hemoglobin level was found between groups at the end of the study (2-week) by showing p-value >0.05, as represented in Table 2. Furthermore, no clinically substantial levels of LFT and RFT were observed at the baseline and after 2 weeks between both groups, as shown in Tables 3 and  4.

DISCUSSION
The peptic ulcer disease can cause significant damage to the stomach mucosa and can lead to perforation and penetration of stomach mucosa and can even cause gastric carcinoma [13]. Therefore, to prevent these complications, eradication of Helicobacter pylori is very important. Although many treatment regimens have been proposed but conventional triple treatment is still main therapy used worldwide. But unluckily the efficacy of this management becoming reduce with time due to resistance of antibiotics mainly clarithromycin and also due to the side effects caused by antibiotics [14]. So, because of these reasons there was a strong need to find out alternative treatment regimens. Many treatment regimens have been proposed but probiotics have found to be the most suitable ones according to previous researches held on different regions of the world. That is why this research was designed to identify the effectiveness of conventional triple therapy (Amoxicillin + Clarithromycin + Proton Pump Inhibitor) versus Probiotics Lactobacillus reuteri + Proton Pump Inhibitor combination therapy in Pakistani population having peptic ulcer disease. As far as our knowledge, this is the first study which address the probiotic combination in the population of Pakistan.
Our study revealed that both of these interventions effectively eradicate the H. pylori in the Pakistani peptic ulcer population. But comparatively, the proportion of achieving a greater eradication rate was significantly more in combination therapy than conventional therapy. Our findings are in line with the prospective, randomized placebo trial conducted in Iran in 176 patients which disclosed that the eradication percentage of H. pylori was found to be considerably greater in combination therapy [15]. Another retrospective study was conducted in South Korea from March 2013 to February 2014 which showed similar results [16]. Similar outcomes were detected by Hafeez et al. in his study. He observed that the combination of probiotics with the triple therapy enhanced the effectiveness up to 85% and the gastric symptoms were reduced from 76% to 15% (p=0.002) [17]. A further study of 40 RCTs were conducted in which 5792 participants were recruited and they evaluated the efficacy of probiotic when used as an adjuvant to standard therapy and found that by adding the probiotic to the therapy the elimination rate of H. pylori has increased up to 10% [18]. In another multicentre, prospective, double-blind, placebo-controlled trial, similar results were observed. It was noted that when probiotic was combined with triple regimen the elimination rate was significantly increased (RR = 1.13; 95%CI: 1.10-1.16; P < 0.001) [19].
Besides the efficacy of combination, another most important challenge is to prevent the onset of comorbidity or drug-induced toxicity. Therefore, in this study, the effect of both interventions on hemoglobin level was identified. The test showed insignificant mean difference in both the groups at baseline and after 14 days and the p-value was also found to be insignificant between both the groups (0.741). A former study conducted by Hassan et al., 2019 also showed no significant change between the two groups before and after treatment [20]. similar results was also showed by [21].
Next, we had identified the safety profile of these treatment regimens by liver function test (LFT).
The results revealed similar findings between groups. Identifying its effect on kidney function is vital before its use. Regarding this we had checked the serum urea & creatinine levels and found that there was no change in the serum levels of urea and serum creatinine in both group A and group B at baseline and after 14 days of treatment and it showed insignificant p value for urea (p=0.520) and also for creatinine (p=0.858).
A key strength of this study is the comparison of first-line therapy, conventional triple therapy with probiotic (Lactobacillus reuteri) plus omeprazole combination for peptic ulcer treatment. Combination therapy have shown better efficacy and also helped to restore the normal microflora in the stomach. The safety profile in both groups with respect to haemoglobin, liver functions and renal functions were monitored closely. The possible limitations of the present study are the designed time period to study the effect was short and the population size of the study was also limited.

CONCLUSION
Probiotic (Lactobacillus reuteri) is clinically effective for the treatment of peptic ulcer and it has also proven to be effective in preventing the side effects associated with antibiotics in conventional triple therapy. None of the interventions elevates the incidence of cardiovascular disorder, liver toxicity and renal impairment. Probiotic (Lactobacillus reuteri) therapy can be used as an alternative for the treatment of peptic ulcer and for thorough eradication of Helicobacter pylori without producing major side effects in the population of Pakistan.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

ETHICAL APPROVAL AND CONSENT
The clinical trial was conducted after authorization by the Ethical Research Committee (ERC) of BUMDC, Karachi, Pakistan. All the patients provided written informed consent.