Fourteen-Day Sequential Therapy Containing Rabeprazole Versus 10 And 14-Day Concomitant Therapy in The First Line Treatment of Helicobacter Pylori Infection: A Randomized, Open-Label Clinical Trial

To compare an optimized sequential therapy to 10 and 14-day non-Bismuth quadruple therapies currently recommended, in terms of ecacy, incidence of adverse effects and cost. This open-label prospective study randomized patients with conrmed Helicobacter pylori (H.Pylori) infection to 3 groups (1:1:1): The rst group received quadruple therapy of twice-daily (bid) Omeprazole 20mg, Amoxicillin 1g, Clarithromycin 500mg and Metronidazole 500mg for 10days (QT-10), the second group received a 14 day quadruple therapy following the same regimen (QT-14), and the third received an optimized sequential therapy consisting on a bid Rabeprazole 20 mg plus amoxicillin 1g for 7 days, followed by bid Rabeprazole 20 mg, clarithromycin 500 mg and metronidazole 500mg for the next 7 days (OST-14). Adverse events (AEs) were recorded throughout the study, and the H.Pylori eradication rate was determined 4 to 6 weeks after treatment using the 13C urea breath test.

group received a 14 day quadruple therapy following the same regimen (QT- 14), and the third received an optimized sequential therapy consisting on a bid Rabeprazole 20 mg plus amoxicillin 1g for 7 days, followed by bid Rabeprazole 20 mg, clarithromycin 500 mg and metronidazole 500mg for the next 7 days (OST- 14). Adverse events (AEs) were recorded throughout the study, and the H.Pylori eradication rate was determined 4 to 6 weeks after treatment using the 13C urea breath test.
The overall incidence of AEs was signi cantly lower in the OST-14 group (p=0,01). Furthermore, the OST-14 was the most cost-effective among the three groups. Conclusion: 14-day optimized sequential therapy is a safe and effective alternative that allows a higher eradication rate compared to 10 and 14-days quadruple therapies while causing less adverse effects and allowing a gain in term of cost.
Despite the H.Pylori infection management is evolving, no regimen can currently achieve a cure rate of 100%, the last Maastricht V consensus recommend non-Bismuth quadruple therapies (Amoxicillin 1g bid, Clarithromycin 500mg bid, Metronidazole 500 mg bid and pomp inhibitor (PPI) bid) for 10 to 14 days or Bismuth therapy for 10 days to achieve cure rate of at least 90% despite a signi cant rate of adverse effects 7 . However, it's observed that the prevalence of antimicrobial resistance is widely rising which become a problem of great interest worldwide 8 ; In Morocco, a previous study showed a Clarithromycin resistance of more than 15% 9 . Therefore, the best strategy to increase the eradication rate of H.Pylori would be a personalized treatment according to antibiotics susceptibility 10 .
However, this strategy is not possible in many developing countries. Therefore, many studies have focused on optimizing the recommended regimens 11 . It can be either an optimization by extending the length of the protocol 12 , using higher dose and/or second-generation PPIs substances, or switch to Vonoprazan 13,14 , modifying antibiotic therapy used and their posology, or supplementation with other molecules such as probiotics 15 .
Optimizing sequential therapy seems to be the most attractive strategy in terms of e cacy, tolerability and cost. The most common sequential therapy consists of a dual therapy (PPI and Amoxicillin) for the rst period (5 to 7 days), followed by a triple therapy for the second period (PPI, Clarithromycin and Metronidazole). PPIs play a key role in maintaining a gastric PH allowing an optimal e cacy of antibiotics 16 , hence the idea of the use of new generation molecules.
The primary aim of this study was to compare the e cacy of 14-day sequential therapy optimized by using a second-generation PPI to the standard non-bismuth quadruple therapies of 10 and 14 days. The secondary aims were to compare the tolerability and adverse effects occurrence among the groups, as well as their cost-effectiveness.  [17], and then studied for the presence of H.Pylori using Hematoxylin and eosin staining at the pathology laboratory of our hospital. Patients who received eradication therapy previously, PPI, H 2blockers, NSAIDS or Bismuth containing compounds 4 weeks prior to the study, and/or who were allergic to prescribed antibiotics were excluded from the study. Additionally, pregnant and breastfeeding females as well as patients with history of gastric surgery, kidney or liver failure, or severe psychiatric conditions were also excluded. All included patients provided a written informed consent to participate in the study.
The protocol followed Helsinki Declaration guidelines and was approved by our local scienti c committee at Mohamed V Military Teaching Hospital of Rabat. Our clinical trial was registered in the Pan African Clinical Trial Registry (www.pactr.org) on 12/07/2021 / registration number: PACTR202112632957229.

Randomization and treatment:
Patients were randomly assigned in 1:1:1 ratio using a computer-generated table into three groups: QT-14, QT-10 and OST-14. Allocations were concealed in a sealed opaque envelope which was to be opened on the consultation day. The QT-14 and QT-10 groups received Omeprazole 20mg, Amoxicillin 1g, Clarithromycin 500mg and Metronidazole 500mg, all twice daily for 14 and 10 days, respectively. The OST-14 group received an optimized sequential therapy consisting on twice daily Rabeprazole 20mg and Amoxicillin 1g during 7days, followed by Rabeprazole 20mg, Clarithromycin 500mg and Metronidazole 500mg, all twice daily for the remaining 7days.
PPI was administered 30 minutes before breakfast and supper, whereas antibiotics were administered every 12 hours after meals.
Follow-up and outcomes: The H.Pylori eradication was determined, at least 4 weeks after the last day of the treatment, using the 13C-urea Breath test (UBT), which was performed blindly at the same laboratory for all patients. The cutoff value for the UBT was 2,5 per thousand. The patients didn't undergo additional therapy with PPI, after completion of eradication therapy.
All patients were evaluated two weeks after the start of the treatment and at its end to assess adverse effects and compliance. Drugs compliance was de ned by taking at least 90% of the prescribed protocol drugs and was assessed at the end of the protocol. Adverse effects (AEs) were assessed by using a preestablished structured questionnaire. The questionnaire consisted of dichotomous questions about the occurrence and intensity of AEs including diarrhea, nausea and/or vomiting, gastralgia, metallic taste, dysgeusia, symptoms related to allergic reaction, headache, dizziness, asthenia, or any other AE.
The cost-effectiveness analysis was assessed by comparing the overall cost of each protocol. The cost of every drug was calculated using a national website: www.medicament.ma. The cost-effectiveness ratio for each regimen was calculated by dividing the total cost of 100 patients treated by the percent of patients treated.
Statistical analysis: The eradication rate of QT-10 was assumed to be 83,3% from a previous meta-analysis 18 . We assumed that other groups would show non-inferiority with a margin of 15%, an expected level for the primary outcome of 85% with a strength of 0,8 and bilateral signi cance level of 0,05. With a presumed drop-out rate of 20% in each group, at least 120 patients were required in each group.
The primary endpoint of the study was the eradication rate of H.Pylori, which was assessed by intention to treat (ITT) and per-protocol (PP) analyses. The safety population included all randomized patients who received at least one treatment dose during the study, ITT population included all patients who received at least one treatment dose during the study and who were examined during the rst visit, while the PP population included only patients who completed the study. Therapeutic failure was recorded as outcome for patients with missing data due to incomplete treatment. The secondary outcomes were the incidence of AEs, therapeutic compliance and cost-effectiveness of the protocols.
Descriptive and inferential statistical analyses were performed using Software Package Social Science Results: Population characteristics A total of 360 patients met the inclusion criteria and were enrolled in safety population.
Thirty-two patients refused to participate to the study. We therefore included 328 patients in the ITT analysis who were randomly assigned to the 3 groups. After eliminating the dropped-out patients from the study, the PP analysis included 317 patients. The ow chart of patients of different groups is resumed on gure 1. Demographic and clinical characteristics of different groups are shown in Table 1 and were not signi cantly different between the groups.

Safety
The treatment tolerance was better in the OST-14 group, with an incidence of AEs of 24,7% compared to 42,7% and 39% in the QT-14 and QT-10 respectively (p=0,03). (Table 2) However, the treatment was globally well tolerated among the three groups, and adverse effects were mild to moderate in all patients.
The drugs compliance was excellent among the three groups: 97%, 95% and 98,9% in the QT-10, QT-14 and OST-14 respectively (p=0,48). Cost-effectiveness The overall cost was lower in the OST-14 group (427,10dhs), comparing to QT-14 and QT-10 groups (691,90dhs and 587,10dhs respectively). Considering the effectiveness of different regimens, the costeffectiveness ratio was lower in the OST-14 group as shown in Table 3.  According to the Maastricht V consensus, the most recommended regimens for Helicobacter Pylori infection are non-Bismuth quadruple concomitant therapy and Bismuth quadruple therapy 7 . However, eradication rates vary widely by geographic area, due to varying antimicrobial resistance, especially to Clarithromycin and Metronidazole 19 . Therefore, several regimens have been tested to improve the management of Helicobacter Pylori infection 20,21 . One of them is the modi ed sequential therapy 12,22 . In the present study, we aimed to compare the results of the standard 10-and 14-day non-bismuth quadruple therapies to a modi ed sequential therapy by using a second-generation PPI.
Overall, we found that the 14-day optimized sequential regimen using Rabeprazole achieves a higher cure rate compared to the standard non-bismuth quadruple therapy for 10 or 14 days (95,4%, 85,5% and 91,8% in the OST-14, QT-10 and QT-14 respectively, p=0,03). The OST-14 allowed a great tolerance with a lower adverse effects rate compared to quadruple therapies (p=0,01), and there was no difference in term of drugs compliance between the three groups. Furthermore, the cost-effectiveness ratio was lower in the OST-14 group.
The sequential therapy was introduced for the rst time in 2000 in Italy by Zullo 23 . We personally demonstrated its superiority compared to the standard triple therapy in a previous study 24 .
A recent metanalysis by Youhua wang et al showed that there is no difference in term of eradication rate between 14-day sequential and 14-day concomitant therapy 25 . Another study showed that 14-day sequential therapy is equivalent to 10day bismuth quadruple therapy in term of eradication rate (91,3% and 91,6%) but with more adverse effects with the second regimen 26 . The same team demonstrated in a metanalysis that 14-day sequential therapy is more effective than 14-day triple therapy 27 .
In the present study, the gain in terms of eradication rate can be explained by the use of a second generation PPI (Rabeprazole 20mg bid) in the OST-14 group. In fact, the last Maastricht consensus states that switching Omeprazole 20mg twice daily to Rabeprazole 20mg bid or Esomeprazole 40mg bid may increase eradication rate by 8-12% 7 . the advantage of PPIs lies in the fact that the majority of proposed regimens are PH dependent and become less effective when the intragastric PH is low 28 , hence the use of higher dose of PPIs and second-generation substances. A possible explanation for the superiority of second-generation PPIs (Rabeprazole and Esomeprazole) can be their metabolism, which is less dependent on CYP2C19 genetic variables and their higher acid inhibition power 29 . A further metanalysis by McNichol et al con rmed that both Esomeprazole and Rabeprazole lead to higher eradication rates compared to rst generation PPIs (Omeprazole, Lansoprazole and Pantoprazole) 30 .
All therapeutic regimens currently recommended are associated with gastrointestinal adverse effects 31 .
Herein, the OST-14 allowed a gain in term of adverse effects incidence compared to quadruple concomitant therapies. These ndings con rm those of previous studies [32][33][34] .
The 14-day sequential therapy consists of the same antibiotics as the 14-day concomitant regimen but for a shorter duration. It should therefore be responsible of less adverse effects. This was the case in our study with a bene t of 18% in term of AEs occurrence (31,3% vs 49,5%; p=0,03). Moreover, the absence of drugs adverse effects was a predictive factor of successful treatment (OR=2,48; 95%IC=1,09-7,38; p=0,03).
Because the treatment cost is a determining factor especially in developing countries, we carried-out a cost-effectiveness analysis and showed that the OST-14 is the most cost-effective among the study three groups. The same result was reported by Nagwan S et al previously who found that 14-day sequential therapy is cheaper than 14 triple therapy 33 , Kate V et al. con rmed in a metanalysis that sequential therapies are cheaper than standard therapies 35 . Other cost-analysis studies have showed the same results and found that sequential therapy is the most economically attractive option 36, 37 .
This bene ce can be explained by the fact that clarithromycin is the most expensive drug used in different protocols, and will be used for a shorter duration in the sequential therapy.
One of the limitations of this study is that we didn't performed Helicobacter Pylori cultures and didn't have data about antibiotic susceptibility. However, a previous recent study showed that the local primary resistance to Clarithromycin was 29% in Morocco, 40% to Metronidazole and 0% to Amoxicillin 9 .
Another potential limitation is that second line treatments were not included, which makes hard to interpret the cost effectiveness analysis. Additionally, it's di cult to generalize our results to other areas as the study was conducted in a single center. Nonetheless, the sample size was reasonable allowing correct statistical analysis. Though our results have to be completed by further studies in different geographic areas to have them validated. In conclusion, the results of the present study showed that 14day sequential therapy using Rabeprazole is an optimal therapy leading to an excellent cure rate, and demonstrate its superiority to the standard non-bismuth quadruple therapies particularly in terms of tolerance and adverse effects occurrence.

Declarations
Data Availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Additional information

Competing interests
The author(s) declare no competing interests.
Funding sources.
Mohamed V Military teaching Hospital of Rabat Figure 1 Flow-chart of patients in the study Figure 2 Helicobacter pylori eradication rates with Intention to Treat and Per-Protocol analyses among the three groups