Efficacy and Tolerability of Two Quadruple Regimens: Bismuth, Omeprazole, Metronidazole with Amoxicillin or Tetracycline as First-Line Treatment for Eradication of Helicobacter Pylori in Patients with Duodenal Ulcer: A Randomized Clinical Trial

Aim To evaluate the efficacy and tolerability of tetracycline vs. high-dose amoxicillin in bismuth-based quadruple therapy for Helicobacter pylori(H. pylori) eradication. Methods This randomized, open-label clinical trial included 228 patients with H.pylori infection and duodenal ulcer without a history of H.pylori treatment. Patients were randomly divided into two groups. The amoxicillin group received metronidazole 500mg, bismuth subcitrate 240mg, and amoxicillin 1000mg, all three times a day, plus omeprazole 20 mg twice a day, for 14 days. The tetracycline group received metronidazole 500mg three times a day; bismuth subcitrate240mg and tetracycline HCl 500mg, both four times a day; and omeprazole 20 mg twice a day, for 14 days. Evaluation for compliance and drug-relatedadverse effects were evaluated at the end of two weeks. Eight weeks after the end of treatment, the rate of H.pylori eradication was assessed by the C13urease breath test. Results There were no significant demographic differences between the two groups. Eradication rate was higher with the amoxicillin-containing regimen than the tetracycline-containing regimen: 105/110 (95.51%; 95% confidence interval, 91.5%–99.3%) vs. 88/105 (83.8%; 95%CI, 76.7%–90.8%) by per-protocol analysis (p = 0.005) and 92.9% (95%CI, 88.1%–97.6%) vs. 76.5% (95%CI, 68.7%–84.2%) by intention-to-treat analysis (ITT, p = 0.001). Adverse effects were significant higher in the tetracycline groupthan in the amoxicillin group (65.2% vs. 43.4%; p = 0.001). Conclusion Bismuth-based quadruple therapy including high-dose amoxicillin and metronidazole achieved an acceptable rate of H.pylori infection eradication with good tolerance in patients with duodenal ulcer. This regimen can overcome treatment resistance in areas with high prevalence of metronidazole and clarithromycin resistance. Trial registration The Thai Clinical Trial Registry (TCTR) 20170623004


Introduction
Helicobacter pylori(H.pylori) is a gram-negative bacterium that infects approximately 50% of people in industrialized nations and up to 80% in less-developed countries [1].H. pyloricauses peptic ulcer disease, chronic gastritis, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma [2].Although the bacterium is susceptible to most antimicrobial agentsinvitro, successful treatment of H.pyloriremains a challenge [3].Factors contributing to success inH.pylori treatment are drug efficacy, host compliance, and bacterial resistance [1,2].Resistance to commonly used drugs such as metronidazole and clarithromycin, is the most important reason for treatment failure of current regimens [1].Emerging evidence shows high resistance to clarithromycin in countries with high consumption of macrolidederivatives [4], whereas regimen containing clarithromycin was reported to achieve 80% eradication rate [5].Previous studies from our region (Iran)revealed that more than 20% of H. pylori isolates were resistant to clarithromycin and that over 50% of the H. pylori isolates were resistant to metronidazole [6][7][8].
Due to high resistance to metronidazole and clarithromycin, regimens containing these antibiotics are not efficient [9].In some studies on patients with metronidazole-and clarithromycin-resistantH. pylori, bismuth-based quadruple therapy was reported as a preferable regimen for eradication of H. pylori [10,11].This bismuth-based quadruple therapy includesbismuth, a proton pump inhibitor (PPI), and tetracycline together with metronidazole or tinidazole, with proper doses and duration.Quadruple regimen with suboptimal metronidazole doses (<1500 mg/day) was reported to achieve an overall eradication rate of 70% [3,4].Increasing the dose of metronidazole in bismuth-based quadruple therapy was the first step in increasing eradication rates to acceptable levels, and bismuth compound was reported to be necessary for such response [12,13].However, low compliance,as well as increased side effects, are major issues that arise if full-dose metronidazole and tetracycline are used.There is also some evidence suggesting an increase in H.pylori resistance to tetracycline [14][15][16].
Outcomes with substitution of tetracycline with amoxicillin in bismuth-based quadruple therapy have not been widely studied, especially in countries with high H.pylori resistance to metronidazole and clarithromycin such as Iran.Our pilot study revealed that a very good H. pylori eradication rate could be achieved with a modified bismuth-based quadruple therapy containing high-dose amoxicillin (3 g/day), adequate-dose metronidazole (1.5 g/day), and a PPI.Therefore, this open-label, randomized clinical trial was designed to compare the classic bismuth-based quadruple regimen containing metronidazole (1500mg/day) with a modified bismuth-based quadruple therapy containing high-dose amoxicillin (3 g/day), metronidazole

Registration
Ethics approval for this study was obtained from Yazd University Medical Sciences ethics committee on September 20, 2014, prior to patient enrollment.The clinical trial registration was completed retrospectively several months after patient enrollment was started, due to a miscommunication with the institution.We emphasize that our study begins and progress with complete consideration of ethicalrules.The authors confirm that all ongoing and related trials related to this study are registered.

Intervention
Patients were enrolled between October 20, 2014 and July 15, 2016.After the patients provided consent to participate in this trial, they were randomly assigned at a 1:1 ratio to receive one of the following two treatment regimens.The amoxicillin group (group I) received metronidazole 500mg, amoxicillin 1000mg, bismuth subcitrate 240mg, all three times a day, plus omeprazole 20mg twice a day, for 14 days.The tetracycline group (group II) received omeprazole 20mg twice a day; bismuth 240 mg, and tetracycline HCl500 mg, both four times a day;and metronidazole 500 mg three times a day, for 14 days.Omeprazole and bismuth were taken before meals, and antibiotics were used after meals.All patients were instructed on potential adverse effects and kept under observation during treatment for evaluation of adverse effects and compliance.All patients were requested to record any adverse effects that occurred during therapy, including bad taste, diarrhea, dizziness, weakness, nausea, loss of appetite, vomiting, fatigue, fever, and skin rash.Severe adverse effects were defined as those that would be considered to disrupt daily activities that required treatment discontinuation by the patient.

Outcomes
For evaluation for the primary outcome of H. pylori eradication rate, patients were asked to stop the PPI or the H2 blocker for at least four weeks before follow-up evaluation.Eight weeks after conclusion of the two-week study treatment, patients were assessed by the C 13 urease breath test by personnel who were blinded to the treatment, and a value of less than 4% was defined as successfulH.pylori eradication.For evaluation of secondary outcomes, data on adverse effects were collected through a standard sideeffect questionnaire, and good compliance was defined as ingesting more than 80% of the total number of doses included in the regimen.

Sampling and blinding
Sample size for the trial was calculated based on the following assumptions.Average rate of successfully achieved eradication of H.pyloriwith the standard quadruple therapy was 80% [17].Based onourpilot study results showing an H.pylori eradication rate of 94% with the modified bismuth-based quadruple therapy containing high-dose amoxicillin (3 g/day),adequatedose metronidazole dose (1.5 g/day) and omeprazole, we chose a two-sided alpha value of 0.05, and a power of 80%.Based on these assumptions at least 208 participates (104 subjects in each groups) would be required.In order to accommodate a 10% rate of lost to follow-up, we enrolled 228 patients.

Statistical methods
All registered data were analyzed using SPSS software version 22 for Windows (SPSS, Chicago, IL).Data were presented as means with standard deviation (SD), frequencies, and percentages.The chi-square and Fisher's exact tests were used for comparison of categorical data between the two groups.P values of less than 0.05 were considered significant for all analyses.ITT and per-protocol (PP)analyses were performed to calculate eradication rates.The ITT analysis included all randomized patients.Individuals who did not take at least 80% of the drugs andthosewith unknown post-treatment H. pylori status were excluded from the PP analysis.Odds ratios with 95% confidence intervals (CIs) were calculated where appropriate.

Ethical consideration
This study was approved by the Ethics Committee of ShahidSadoughiUniversityof Medical Sciences in Yazd, Iran and registered with the protocol number "Ir.ssu.rec.1394.13712"onSeptember 20, 2014.Participants provided written informed consent and were included in the study after they were provided information on treatment methods.This trial was also registered with Thai Clinical Trial Registry (Number: TCTR20170623004).The Consort 2010 checklist, the study protocol and the Ethic approval of study are given S1 Checklist, S1 File and S2 File. http://dx.doi.org/10.17504/protocols.io.mdjc24n

Patient characteristics and compliance
A total of 228 patients who were eligible were recruited from the SSU Gastroenterology Department.There were no significant differences in baseline characteristics including age and sex between the two treatment groups (Table 1).All patients except those who were lost to follow-up and those who could not tolerate the regimen were assessed by the C 13

Adverse effects and compliance
Overall, 4 patients in the tetracycline group and 2 patients in the amoxicillin group were lost to follow-up, and no outcome data were available for these patients.Additionally, 6 patients in the tetracycline group and 1 patient in the amoxicillin group could not tolerate the medication, i.e.,failed to take at least 80% of the prescribed medication, due to severe adverse effects that started a few days after initiation of the treatment.Adverse effects included tolerable diarrhea, bad taste, dizziness, weakness, nausea, skin rash, fever, fatigue, and vomiting, which were reported by 43.4% (49 of 113) of the patients in the amoxicillin group and 65.2% (75 of 115) of the patients in the tetracycline group, and adverse effects were significantly more frequent in the tetracycline group (p = 0.001).Bad taste was the most common side effect in both groups, whereas the frequency of vomiting was significantly higher in the tetracycline treatment group (4 vs. 0 in tetracycline vs.amoxicillin groups; p = 0.04).A summary of adverse effects in this study is presented in Table 3.

Discussion
Results of this open-label randomized study revealed that substitution of tetracycline with high-dose amoxicillin in classic bismuth-based quadruple therapy can increase H. pylori eradication rate and compliance in patients with duodenal ulcer.In patients who received highdose amoxicillin in bismuth-based quadruple therapy, the eradication rates were 95.6% and 92.9% by the PP and ITT analyses, respectively.Conversely, the eradication rates in patients who received classic bismuth-based quadruple therapy were 83.3% and 75% by PP and ITT analyses, respectively.These findings indicatedthat substitution of tetracycline with high-dose amoxicillin significantly increased the eradication rate not only by the ITT analysis but also the PP analysis.As more than 50% of H. pylori strains in Iran are resistant to metronidazole [6][7][8], amoxicillin might achieve better synergistic effects with metronidazole than tetracycline for eradication of metronidazole-resistantH.pylori strains.Additionally, patients had better tolerance to bismuth-based quadruple therapy containing amoxicillin instead of tetracycline.Generally, management of disorders associated with H. pylori infection requires treatment regimens that have eradication rates of more than 90% to 95%, if possible [18].Use of similar effective first-line treatments is important not only for disease cure but also for prevention of secondary antibiotic resistance [19].
A recent review of various regimens used as primary treatment in West Asia suggested that bismuth-furazolidone-metronidazole treatment for 10 days, clarithromycin-based hybrid treatment for 14 days, and classic bismuth-based quadruple therapy including PPIs, bismuth, tetracycline, and metronidazole for 14 days,wereequally effective [20].Since the furazolidonebased regimen has many side effects, the regimen containing adequate-dose furazolidone (400mg/day) is not recommended [21,22].Due to high resistance to clarithromycin, clarithromycin-containing regimen isnot acceptable in our course [23].Potentially high resistance to levofloxacin also limits its use as front-line treatment [9,23].H. pylori culture and antibiogram are expensive, and lack of information on pretreatment susceptibility to antibiotics is the main reason for bismuth-based quadruple therapy as a primary regimen for eradication of H. pylori in our region, as recommended by the Toronto Consensus [24].Malfertheiner and colleagues showed that in regions with high resistance to clarithromycin, quadruple treatment containing bismuth, metronidazole, tetracycline, and a PPI was preferable as the primary therapy for H. pylori eradication.Quadruple therapy achieves superior eradication with safety and tolerability comparable to those obtained with standard triple therapy [25].In contrast to metronidazole, increase in clarithromycin dose was reported not to increase the eradication rate of clarithromycin-resistant H. pylori [26].
Liang et al. concluded that four different bismuth-based quadruple treatments exhibited eradication ratesof>90% against H. pylori in subjects who were unresponsive to prior therapies, including those showing resistance to clarithromycin and metronidazole [27].Therefore, the two-week bismuth-based quadruple therapy containing high-dose metronidazole, a PPI, and tetracycline is a proper first-line treatment regimen in our patients with duodenal ulcer [28][29][30].This regimen requires modifications with respect to drugs, doses, and duration to reach excellent H. pylori eradication rates.Bismuth and metronidazole are necessaryadditionsin this regimen.Increase in H. pylorieradication rate with an increased metronidazole dose is dependent on the presence of bismuth.Bismuth also improves the elimination ofH.pylori by triple therapy regimen [31].In a systematic review and meta-analysis of 35 randomized clinical trials and 4763 subjects, the safety profile of bismuth was assessed, which revealed insignificant side effects in patients undergoing bismuth-based treatment [32].Emerging evidence of increasing resistance to tetracycline reports demonstrating that adequate-dose metronidazole in combination with tetracycline results in more side effects raise concerns regarding substitution of tetracycline with another antibiotic.
To achieve increased compliance to classic bismuth-based quadruple therapy, tetracycline was replaced with high-dose amoxicillin, based on several lines of evidence:In a recent study, high-dose amoxicillin (750mg four times a day) in combination with a potent PPI achieved a very good eradication rate, suggesting that amoxicillin is a unique anti-H.pylori antibiotic that has an acceptable eradication rate when used as part of a dual therapy containing a PPI [33,34].The resistance rates to amoxicillin, metronidazole, and clarithromycin were about 2%, 44%, and 29% in America; 0.7%, 35%, and 18% in Europe; and 2%, 38%, and 21% in Asia, respectively [35].H. pyloriwas found to rapidly acquire antibiotic resistance to clarithromycin, metronidazole, and levofloxacin but not to amoxicillin after a single course of anti-H.pylori therapy [36].One potential explanation for the difference in acquired antibiotic resistance is that a single-point mutation can lead to resistance to clarithromycin, metronidazole, and levofloxacin, whereas multiple-site mutations are necessary to confer amoxicillin resistance [37][38][39].A recent study among US male patients revealed increased resistance to clarithromycin and levofloxacin but no resistance to amoxicillin [40].In Iran, 1.6%, 16.7%, and 57.5% ofH.pylori strainsshowed resistance to amoxicillin, clarithromycin, and metronidazole, respectively; however, no tetracycline resistance was reported [41].However, this sensitivity pattern was found to have changed five years later, and there is an increasing resistance to tetracycline [42].A recent study in Yazd, Iran revealed that H.pylori is more resistant to tetracycline than amoxicillin [43].In a study in China, amoxicillin, in combination with metronidazole, bismuth, and lansoprazole, eradicated metronidazole-resistantH.pylori.In their study, Zhang et al. used 2g amoxicillin per day in combination with lansoprazole, metronidazole, and bismuth [44].Lansoprazole, albeit more potent, is more expensive than omeprazole in Iran; therefore, high-dose amoxicillin with omeprazole was preferred over 2g amoxicillin with lansoprazole.
The ITT analysis revealed an eradication rate of 92.92% in the amoxicillin group compared with the rate of 76.5% in the tetracycline group, indicating that bismuth-based quadruple therapy containing tetracycline with adequate-dose metronidazole was associated with more adverse effects and intolerance.Unfortunately, severe adverse effects that lead to intolerance in the tetracycline group occurred 4-5 days after treatment initiation.In contrast, there were no severe drug-associated complications with the high-dose amoxicillin regimen, and the patient compliance was good.
Inability to evaluate the H. pyloriantimicrobial resistance patterns was one of the main limitations of this study; howevera recent regional study indicated thatH.pylori resistance to tetracycline was more common than that to amoxicillin, which might explain the 16% failure rate in the tetracycline groupcompared to the 5% failure rate in the amoxicillin group based on the PP analysis [43].

Conclusion
The results of this randomized, open-label clinical trial revealed that a two-week bismuthbased quadruple therapy including high-dose amoxicillin and metronidazole had an acceptable eradication rate with good tolerance in patients with duodenal ulcer and H. pylori infection.This therapy might overcome treatment resistance in areas with high prevalence of H. pylori resistance to metronidazole and clarithromycin.
urease breath test after the conclusion of treatments.The CONSORTflow diagram is depicted in Fig 1.