The Efficacy, Safety, and Tolerability of Levofloxacin Quadruple Therapy for Helicobacter pylori Eradication: A Randomized, Double-Blind Clinical Trial

The incidence of microbial resistance is growing, and new rescue regimens are needed to treat Helicobacter pylori (H. pylori) infection. This study aimed to evaluate levofloxacin-based quadruple therapies' efficacy, safety, and tolerability in eradicating H. pylori. In a randomized, double-blind clinical trial, 220 patients with dyspepsia and H. pylori infection were randomly assigned to receive either bismuth subcitrate 240 mg, pantoprazole 20 mg, amoxicillin 1000 mg twice a day, and levofloxacin 500 mg daily for seven days (BPAL-7) or ten days (BPAL-10). The eradication of H. pylori was evaluated two months after the end of treatment, and adverse drug reactions (ADRs) were assessed during the intervention. According to intention-to-treat and per-protocol, the eradication rate was significantly lower in the BPAL-7 regimen at 49.1% (95% CI: 39.3–57.8) and 47.6% (95% CI: 39.7–58.4), respectively, compared to the BPAL-10 regimen at 62.7% (95% CI: 53.6–72.8) and 62.4% (95% CI: 55.1–72.8), respectively. The ADR incidence was not statistically significant between the groups of BPAL-7 (33.6%) and BPAL-10 (36.7%). Although the ADRs were negligible in both groups, these regimens could not be an ideal alternative therapy for H. pylori because of their low eradication rates compared to standard regimens. Trial Registration. The study was reviewed and approved by the Iranian Registry of Clinical Trials (IRCT201406141155N19).


Introduction
Helicobacter pylori (H. pylori) afects about 80% of the total population in developing countries and 20-50% in developed countries [1]. Tis infection, as a worldwide health problem, is associated with acute and chronic gastritis, peptic ulcer disease, and gastric cancer [2][3][4]. Te eradication of H. pylori has a main role in the prevention and treatment of these complications [5,6]. However, the major concerns with H. pylori treatment are low compliance of patients, adverse drug reactions (ADRs), and microbial resistance to medicines [7,8].
Te efcacy of present pharmacological treatments for H. pylori infection has been evaluated in diferent studies [9][10][11][12][13][14][15]. Te co-administration of a proton-pump inhibitor (PPI) and two antibiotics (triple therapy) or the addition of bismuth salts to the triple therapy (bismuth-based quadruple therapy) for a period of 14 days is the most common regimens [16]. Among antibacterial agents, amoxicillin or metronidazole, along with clarithromycin, is the frst-line treatment for the eradication of H. pylori [10,17]. Nevertheless, microbial resistance is one of the challenging factors of treatment, which causes the low efcacy of these regimens. Hence, efective antibiotics should be selected according to the regional pattern of H. pylori resistance [18,19].
Te prevalence of H. pylori infection is relatively high among the Iranian population [20,21]. Moreover, novel strains that resist clarithromycin and metronidazole are prevalent in some geographical parts of Iran [22,23]. Terefore, evidence suggested new antibiotic regimens with higher efcacy (eradication rates of >80%), lower ADRs and cost, and acceptable patient compliance. Te efcacy of levofoxacin as a new-generation quinolone in the treatment of H. pylori infection has been assessed in several studies, and levofoxacin-containing regimens have shown various eradication rates [24][25][26][27][28][29][30].
Te current study aimed to evaluate the efcacy, safety, and tolerability of a seven-and ten-day period of bismuth subcitrate, pantoprazole, amoxicillin, and levofoxacin regimens in the eradication of H. pylori among patients referred to the Gastrointestinal Outpatient Clinic of Razi Hospital, Rasht, Iran.

Trial Design.
Tis study was a phase III open-label, randomized, double-blind, controlled trial carried out between July 2015 and November 2015 at the Gastrointestinal and Liver Diseases Research Center (GLDRC), Rasht, Iran. All included participants were randomly divided into one of the therapeutic regimens and signed informed consent. Te eradication rate of H. pylori was assessed two months after the end of the intervention, and the incidence of ADRs and patient compliance were assessed at the end of treatment.

Ethical Consideration.
Te protocol of the study was approved by the local ethical committee of Guilan University of Medical Sciences (Ethics Committee Code: 1930175713) and also performed according to the Declaration of Helsinki for human subjects. Te study was reviewed and approved by the Iranian Registry of Clinical Trials (IRCT201406141155N19).

Patients.
Patients complaining of dyspeptic symptoms referred to the Gastrointestinal Outpatient Clinic of Razi Hospital, Rasht, Iran, were enrolled. Te main inclusion criteria were a positive rapid urease test and being 15 to 65 years old. Patients with coexisting serious illnesses, including liver cirrhosis, renal failure, heart failure, gastrointestinal malignancies, a history of seizure or hematologic diseases, an allergy to medications, and an previous incomplete treatment course, as well as pregnant or nursing mothers, were excluded.

Randomization and Intervention.
A block randomization method was used in order to assign patients randomly into permuted treatment blocks and ensure equal numbers in each group of treatments. One of these groups received a seven-day period of bismuth subcitrate 240 mg, pantoprazole 20 mg, amoxicillin 1000 mg twice a day, and levofoxacin 500 mg daily (BPAL-7), and the other received a ten-day period of the same regimen (BPAL-10).
Patients were recommended to take bismuth subcitrate, pantoprazole, levofoxacin before meals and amoxicillin after meals at the scheduled times. It was also advised to avoid smoking, drinking alcoholic or cafeinated beverages, eating spicy foods, and taking nonsteroidal antiinfammatory drugs or medications containing a monoamine oxidize inhibitor.
Demographic criteria, including age, gender, cigarette smoking, and alcohol drinking, were recorded.

Evaluation of H. pylori Eradication.
Two months after the end of the intervention, a Heliprobe 14C-Urea Breath Test (14C-UBT, Kibion AB, Uppsala, Sweden) with a 95% sensitivity and 100% specifcity was performed to evaluate the H. pylori eradication. Treatment success was defned as the result of <50 disintegrations per minute (DPM). Taking the medicines was stopped during these two months to avoid interaction with the results of 14C-UBT.

Assessment of ADRs and Patient Compliance.
All patients were informed about potential ADRs, including pyrosis, anorexia, nausea, vomiting, bitter taste, abdominal colic, epigastric pain, headache, and back pain. Te ADRs were evaluated during the intervention using a 0-10 scoring system (mild: 0-3, the ADRs exist but are intolerable; moderate: 4-6, ADRs sometimes interfere with daily life activity; severe: 7-10, ADRs interfere continuously with daily life activities). Acceptable patient compliance was defned as the consumption of >80% of the prescribed medicines [31].

Statistical Analyses.
Te eradication rates of the two regimens were determined using intention-to-treat (ITT) and per-protocol (PP) analysis. Patients who declined to proceed with the treatment or those with poor compliance were excluded from PP analysis; however, all patients, including patients who used the treatments out of the protocols or dropouts, were included in ITT analysis. Te eradication rate of H. pylori, odd ratios, and 95% confdence interval were evaluated for each regimen. Chi-square and ttests were employed to compare qualitative and quantitative variables between the two treatment groups, respectively. All statistical analyses were performed using SPSS, version 18.0 software (SPSS Inc., Chicago, IL, United States). A P < 0.05 was considered statistically signifcant.

Characteristics of the Patients.
A total of 220 patients with H. pylori infection were randomly assigned to either the BPAL-7 (n � 110) or BPAL-10 (n � 110) groups. Tree patients in the BPAL-7 group (one female with severe epigastric pain and diarrhoea and two males with severe anorexia and back pain) and one patient in the BPAL-10 group (one female with severe epigastric pain and nausea) were excluded from the trial because of drug intolerance and low compliance. A fow diagram describing patient selection and study outcomes is shown in Figure 1.
No signifcant diferences were observed between the two groups regarding age, gender, cigarette smoking, and alcohol drinking (P > 0.05). Te basic demographic characteristics of the patients are demonstrated in Table 1.
Te compliance rates were 97.3% and 99.1% for the BPAL-7 and BPAL-10 regimens, respectively. Tree patients in the BPAL-7 regimen and one in the BPAL-10 regimen were excluded from the PP analysis because they failed to ingest >80% of the medications.

Discussion
Te present study's fndings showed a lower rate of H. pylori eradication in the BPAL-7 regimen than in the BPAL-10 regimen among patients with dyspepsia and H. pylori infection. Both BPAL-7 and BPAL-10 regimens were well tolerated and had acceptable patient compliance.
Recent guidelines suggest the eradication rate of triple therapy with a PPI, amoxicillin, or metronidazole and clarithromycin as frst-line regimens has decreased, particularly due to increased microbial resistance to clarithromycin [2]. Moreover, some recommended bismuthbased quadruple therapy regimens also have an eradication rate of less than 80% [32]. Terefore, more efective regimens are needed to eradicate the H. pylori infection.
A study compared a triple therapy consisting of esomeprazole, levofoxacin (500 mg daily), and amoxicillin with a standard regimen consisting of esomeprazole, metronidazole, bismuth, and tetracycline for 14 days. Te eradication rate of the levofoxacin-containing regimen (96.3%) was higher than of the standard regimen (86.0%) in ITT analysis [25].
Gisbert et al. from Spain demonstrated the eradication rate of ranitidine bismuth citrate, levofoxacin, and amoxicillin as frst-line triple therapy for H. pylori was 84.4, and ADRs, mainly including diarrhea, were reported in 9.5% of the patients [36]. Tis group performed a trial with 1000 patients on the efcacy of omeprazole, levofoxacin, and amoxicillin for ten days. Tey reported an eradication rate of 73.8% and ADRs of 20%, which most commonly included nausea, a metallic taste, and abdominal pain [26]. Te levofoxacin dosage was higher in these studies (500 mg twice daily). In a recent multicenter study conducted by Gisbert et al., 14 days of treatment with esomeprazole, amoxicillin, levofoxacin (500 mg daily), and bismuth as a rescue therapy achieved more than 90% of the eradiation rate [27]. In another Spanish study, the eradication rate of omeprazole, levofoxacin (500 mg twice a day), and amoxicillin for ten days also had about a 20% failure rate [37].
A study from Kosovo randomized 105 patients to undergo either seven or ten days of levofoxacin-based regimens, including omeprazole, levofoxacin (500 mg daily), and amoxicillin. Te eradication rates were 86.2% and 93.6%, respectively. About 5% of patients experienced ADRs, mainly nausea and diarrhea [28].
Several Taiwanese studies evaluated the efcacy of levofoxacin-containing regimens as rescue therapy for eradicating H. pylori. Kuo et al. reported the eradication rate of esomeprazole, amoxicillin, and levofoxacin (500 mg daily) regimen for seven days is similar to a second-  Evidence-Based Complementary and Alternative Medicine linequadruple standard regimen (about 60-70%) [38]. Four years later, the same group published a study comparing levofoxacin-containing quadruple therapy (esomeprazole, bismuth, tetracycline, and 500 mg of levofoxacin once daily) for ten days with a high-dosemetronidazole-containing standard quadruple regimen. Based on their fndings, eradication rates of about 80% were reached for both regimens [39]. On the other hand, Hsu et al. showed a ten-day quadruple therapy consisting of esomeprazole, bismuth, tetracycline, and levofoxacin (500 mg daily) achieved a very high eradication rate (95.8%) in this geographical area. ADRs were detected in 25.0% of patients [40]. In another study, the efcacy of adding bismuth to a levofoxacin-based triple regimen (rabeprazole, bismuth, amoxicillin, and 500 mg of levofoxacin daily) was assessed. Te results revealed no further signifcant eradication rate (67.6%), but no more ADRs were noted [41].
According to the results of the present study, the BPAL-7 and PBAL-10 regimens were not clinically successful in the treatment of H. pylori, with an eradication rate of 62.7% and 49.1% by ITT analyses, respectively. Both regimens achieved neither the cut point of an ideal regimen (eradication rates of >90%) nor the acceptable eradication rates of Maastricht and other guidelines (>80%) [42]. Te low efcacy of these regimens is likely related to the regional pattern of bacterial resistance. Te results of these regimens are consistent with a previous Iranian study evaluating the efectiveness of a 14day triple therapy comprising omeprazole, levofoxacin (500 mg daily), and amoxicillin with a success rate of 75% [43]. However, studies conducted in other parts of Iran revealed fuoroquinolone-containing regimens, including a 14-day of omeprazole, levofoxacin (250 mg twice a day), and amoxicillin, as well as a 14-day of omeprazole, levofoxacin (500 mg twice a day), and amoxicillin as rescue therapies for H. pylori, had eradication rates of 90% and 86.7%, respectively [44,45]. Moreover, some other Iranian investigations evaluated the efcacy of levofoxacin-based, sequential therapy in treating H. pylori, and the eradication rate ranged from 70.8% to 85.1% [46][47][48]. Values were demonstrated as numbers (percentages) and mean ± SD for categorical and continuous variables, respectively. Chi-square and t-tests were performed to compare categorical and continuous variables between groups, respectively. BPAL-7 and BPAL-10: bismuth-based quadruple therapies containing pantoprazole, amoxicillin, and levofoxacin for seven and ten days, respectively. Values were demonstrated as numbers (percentages). 14 C-urea breath test was conducted to confrm the eradication of H. pylori two months after the end of treatment. A Chi-square test was performed to compare variables between groups. BPAL-7 and BPAL-10: bismuth-based quadruple therapies containing pantoprazole, amoxicillin, and levofoxacin for seven and ten days, respectively.  Values were demonstrated as numbers (percentages). Adverse drug reactions were evaluated during the study. Chi-square test was performed to compare variables between groups. None of variables were shown statistically signifcant diferences between two regimens in each category. ADRs: adverse drug reactions; BPAL-7 and BPAL-10: bismuth-based quadruple therapies containing pantoprazole, amoxicillin, and levofoxacin for seven and ten days, respectively.

Evidence-Based Complementary and Alternative Medicine
No relationship was observed between patient demographic characteristics, including age, gender, cigarette smoking, and alcohol drinking, and the success rate of both the BPAL-7 and BPAL-10 regimens. Previous studies have also demonstrated the eradication rate of levofoxacincontaining regimens was not associated with age [45,46,48], gender [45][46][47], smoking status [25,46], and education level [46].
Although high efcacy and compatibility with the pattern of regional bacterial resistance are the main properties of an ideal pharmacological regimen for eradicating H. pylori, tolerability, patient compliance, simplicity, and cost efcacy should not be neglected [31]. Te compliance rates for the BPAL-7 and BPAL-10 regimens were 97.3% and 99.1%, respectively. Despite high compliance rates, ADRs were reported in one-third of patients. Te most-reported ADRs were epigastric pain, nausea, vomiting, and bitter taste. Generally, BPAL-7 and BPAL-10 regimens were patient-compatible and well-tolerated.

Limitations.
Te main limitations of the present study are the lack of a regional pattern of antibiotic resistance and a local estimate of the H. pylori eradication rate, as well as the small number of patients in the groups. Te absence of pretreatment susceptibility assessment for levofoxacin is another drawback. Moreover, the fnding may not apply to patients with previous treatment failure and recurrence of the disease.

Conclusion
Te BPAL-7 and BPAL-10 regimens were patientcompatible and well-tolerated. However, both regimens showed no satisfactory eradication rate. Terefore, these regimens could not be defnitive alternative therapies for H. pylori eradication in this region. It is better to evaluate the efcacy and tolerability of this regimen in other geographical areas through more extended studies with a larger sample size.

Data Availability
Te datasets analyzed during the current study are available from the corresponding author on reasonable request.

Disclosure
A preprint has previously been published [49]. Guilan University of Medical Sciences had no role in the execution of the study, patient recruitment, or data collection.

Conflicts of Interest
Te authors declare that they have no conficts of interest in this work.

Authors' Contributions
FJ and FMG designed and supervised the procedure of the research. FMG, HS, and BM enrolled participants, and collected data. FJ, SH, and HS analyzed data, and MF, FJ, MA, AMG, and HS drafted the manuscript. MF, SH, and AMG edited and revised the manuscript. All authors have read and approved the fnal draft of the paper.