- Email: [email protected]
Levofloxacin, bismuth, amoxicillin and esomeprazole as second-line Helicobacter pylori therapy after failure of non-bismuth quadruple therapy
Accepted Manuscript Title: Levofloxacin, bismuth, amoxicillin and esomeprazole as second-line Helicobacter pylori therapy after failure of non-bismuth quadruple therapy Author: Zhiqiang Song Liya Zhou Jianzhong Zhang Lihua He Peng Bai Yan Xue PII: DOI: Reference:
S1590-8658(15)30365-0 http://dx.doi.org/doi:10.1016/j.dld.2016.01.002 YDLD 3056
To appear in:
Digestive and Liver Disease
Received date: Revised date: Accepted date:
16-11-2015 22-12-2015 5-1-2016
Please cite this article as: Song Z, Zhou L, Zhang J, He L, Bai P, Xue Y, Levofloxacin, bismuth, amoxicillin and esomeprazole as second-line Helicobacter pylori therapy after failure of non-bismuth quadruple therapy, Digestive and Liver Disease (2016), http://dx.doi.org/10.1016/j.dld.2016.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page Title Levofloxacin, bismuth, amoxicillin and esomeprazole as second-line Helicobacter
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pylori therapy after failure of non-bismuth quadruple therapy
Short title
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LBAE as second-line H. pylori therapy
Authors and affiliations
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Zhiqiang Song1, MD, Liya Zhou1,*, BS, Jianzhong Zhang2, PhD, Lihua He2, BS, Peng Bai1, MD, Yan Xue1, MD
2
Department of Gastroenterology, Peking University Third Hospital, Beijing, China
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1
State Key Laboratory for Infectious Disease Prevention and Control, National
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Institute for Communicable Disease Control and Prevention, Chinese Center for
Ac ce pt e
Disease Control and Prevention, Beijing, China * Corresponding author
Electronic word count 3398 words
Correspondence Liya Zhou Department of Gastroenterology, Peking University Third Hospital, Beijing, China Tel: +86-18910192576 Fax: +86-10-82265021
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Email: [email protected]
Financial support This study was supported by the National Science & Technology Pillar Program of
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twelfth Five-Year Plan in China (2012BAI06B02), the clinical key projects of Peking University Third Hospital (Y76493-03) and key laboratory for Helicobacter pylori
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infection and upper gastrointestinal diseases in Beijing (No. BZ0371). The sponsor of
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the study had no role in study design, data collection, data analysis, data interpretation,
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or writing of the report.
ABSTRACT
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None declared.
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Financial disclosure
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Background: The best rescue therapy for Helicobacter pylori (H. pylori) infection following failure of non-bismuth quadruple therapy (NBQT) remains unanswered. Aims: To determine the efficacy, safety and compliance of levofloxacin, bismuth, amoxicillin and esomeprazole (LBAE) regimen following failure of NBQT. Methods: 132 patients with H. pylori infection refractory to first-line NBQT received LBAE regimen (levofloxacin 500mg once/day, bismuth potassium citrate 220mg twice/day, amoxicillin 1000mg twice/day and esomeprazole 20mg twice/day for 14 days). Gastric mucosal biopsy was obtained for H. pylori culture, antimicrobial sensitivity test and cytochrome P450 isoenzyme 2C19 polymorphism analysis. Results: LBAE therapy achieved eradication rates of 73.5% [95% confidence intervals (CI) 65.9%-81.1%] in intention-to-treat and 78.5% (71.1%-85.9%) in
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per-protocol analyses in patients with high antibiotic resistance (amoxicillin 8.3%, clarithromycin 55.6%, metronidazole 73.6% and levofloxacin 36.1%). Adverse effects were found in 19.2% and compliance in 96.1% of the treated patients. Multivariate analyses identified levofloxacin resistance [odds ratio (OR) 7.183, 95%
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CI 1.616-31.914, P=0.010] and history of quinolone intake (4.844, 1.174-19.983, P=0.029) as independent predictors of treatment failure. The eradication rate of
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patients with dual amoxicillin and levofloxacin resistance was significantly decreased
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(33.3%, P=0.006).
Conclusions: In populations with high levofloxacin resistance, 14-day second-line
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LBAE regimen resulted in an unsatisfactory efficacy in patients resistant to NBQT
Keywords
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despite good safety and compliance.
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quadruple therapy.
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Helicobacter pylori, levofloxacin, bismuth, second-line, eradication, non-bismuth
1. Introduction
With the continuous increase in resistance to antibiotics, the efficacy of standard
triple therapy for Helicobacter pylori (H. pylori) infection is unsatisfactory in many regions worldwide [1]. Non-bismuth quadruple therapy (NBQT), including sequential, concomitant and hybrid therapies, is widely used in clinical practice due to good eradication efficacy, safety profile and compliance [1, 2]. Maastricht IV consensus also recommended NBQT as the empiric first-line regimen for the eradication of H. pylori in regions with high resistance to clarithromycin [2]. However, the best rescue therapy following failure of NBQT remains unanswered, as these patients have
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limited options for further therapy because they have already received three different relevant antibiotics: amoxicillin, clarithromycin and metronidazole. Bismuth quadruple therapy, comprising proton pumper inhibitor (PPI), bismuth, tetracycline and metronidazole, has been recommended as the second-line rescue
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approach [2, 3]. However, it is greatly restricted due to the difficulty in obtaining tetracycline in many regions, complex administration and high incidence of adverse
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reactions. Furthermore, the traditional quadruple regimen fails to eradicate H. pylori
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in approximately 20%-30% of cases [3, 4].
Levofloxacin-containing triple regimen, comprising PPI, amoxicillin and
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levofloxacin for 10 days, has also been recommended in Maastricht IV consensus as a second-line therapy [2]. However, recent studies suggested that the efficacy of
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levofloxacin-containing therapy is decreasing, most likely due to increased quinolone resistance [5-8]. A recent meta-analysis showed that the eradication rate of 10-day
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levofloxacin-amoxicillin-PPI therapy after failure of concomitant and sequential
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therapies was only 78% and 81%, respectively [9]. Bismuth is one of the few antimicrobials to which resistance is not developed [10,
11]. In addition, bismuth has a synergistic effect with antibiotics and partially overcomes levofloxacin and clarithromycin resistance [10, 12]. Thus, combining bismuth and levofloxacin in the same regimen may be a good option as rescue regimen.
Up to now, a total of six papers on the efficacies of H. pylori eradication with the
quadruple regimen of levofloxacin, bismuth, amoxicillin and PPI have been published [4, 12-16]. Although there have been some related study reports, as the second-line rescue therapy after failure of initial NBQT, only one paper has been published. Gisbert JP, et al. reported the use of quadruple therapy comprising levofloxacin,
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bismuth, amoxicillin and esomeprazole (LBAE) for the patients refractory to first-line NBQT regimen with good efficacy [90.0% in intention-to-treat (ITT) analysis and 91.1% in per-protocol (PP) analysis], safety and compliance [4]. However, there are some limitations in the study, such as relatively small sample size, lack of H. pylori
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culture and antimicrobial sensitivity test, absence of comprehensive analysis of the risk factors and in the region with low levofloxacin resistance. So, more investigations
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are urgently needed to determine the efficacy of this regimen, especially in regions
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with high antibiotic resistance.
Therefore, the primary objective of this study was to determine the efficacy of
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the second-line LBAE regimen following failure of NBQT. The secondary objectives were to address the incidence of adverse effects and compliance. We also attempted to
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analyze the factors influencing eradication efficacy and investigated the effects of
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different patterns of antibiotic resistance on H. pylori eradication.
2. Patients and methods 2.1. Patients
This prospective study was conducted in the gastroenterology clinic at a tertiary
hospital located in Beijing, China, between January 2013 and September 2015. Patients with dyspepsia were eligible for enrolment if their H. pylori infections were resistant with first-line NBQT. Previous failure was defined as a positive result of 13
C-urea breath test (UBT) 8-12 weeks after completion of treatment. Patients with any one of the following criteria were excluded from the study: age
younger than 18 years or older than 70 years; taking any drug that could influence the study results such as PPIs, H2-receptor blockers, bismuth salts and antibiotics in the previous four weeks; gastrointestinal malignancy; previous gastric or esophageal
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surgery; severe concomitant diseases; history of allergy to any of the study drugs; currently pregnant or lactating; currently abusing alcohol or the presence of any other clinically significant medical condition that could increase the risk of adverse effects. 2.2. Methods
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Patients were enrolled by the medical staff in Gastroenterology Unit after the assessment of inclusion and exclusion criteria. Before the second-line eradication
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therapy, all the patients were mobilized to underwent gastroscopy to assess the
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condition of upper digestive tract and obtain gastric mucosal biopsy for H. pylori culture, antimicrobial sensitivity test and cytochrome P450 isoenzyme 2C19
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(CYP2C19) polymorphism analysis. All the examination and analyses were provided free of charge, and the patient was completely voluntary to decide whether to accept
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them. Patients deciding not to undergo gastroscopy were directly referred to LBAE therapy. The information of antibiotic resistance and CYP2C19 genotypes was used to
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analyze the factors influencing eradication efficacy, not to guide the selection of the
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second-line therapy drugs. If the second-line eradication fails, the information will be used for guiding the choice of drugs for the third-line eradication regimen. The medical staff explained the therapeutic regimen to patients in detail. Patients were informed of potential adverse effects during the treatment period and asked to return within three days after treatment to assess therapeutic compliance and determine the incidence of adverse effects. H. pylori eradication efficacy was determined 8-12 weeks after treatment with
13
C-UBT. Drugs that affected the study results were
prohibited during the study. Treatment allocation was not blinded. Adverse effects were evaluated using open-ended questions by patient self-reports. Adverse events were classified as mild (not interfering with daily routine), moderate (affecting daily routine), severe (markedly affecting the daily routine and
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discontinued medications) and serious (death, hospitalization, disability or require intervention to prevent permanent damage). Compliance determined by pill counts was defined as good when ≥ 80% of the prescribed drugs was taken. Patients who took < 80% of the treatment drugs were
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considered poorly compliant. 2.3. Ethical consideration
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Written informed consent was obtained from all patients. The study was
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approved by the ethics committee of Peking University Third Hospital and conducted according to the principles of the Declaration of Helsinki and the standards of Good
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Clinical Practice. 2.4. Interventions
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LBAE regimen comprised levofloxacin 500mg once/day, bismuth potassium citrate 220mg twice daily, amoxicillin 1000mg twice daily and esomeprazole 20mg
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twice daily for 14 days. Bismuth potassium citrate, amoxicillin and esomeprazole
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were administered together about 30 minutes after breakfast and dinner, and levofloxacin about 30 minutes only after breakfast. In the first-line NBQT, sequential therapy consisted of esomeprazole 20mg and
amoxicillin 1000mg taken twice daily for 5 days, followed by esomeprazole 20mg, clarithromycin 500mg and tinidazole 500mg taken twice daily for 5 days. Concomitant therapy consisted of esomeprazole 20mg, amoxicillin 1000mg, clarithromycin 500mg and tinidazole 500mg all twice daily for 10 days. Hybrid therapy consisted of esomeprazole 20mg and amoxicillin 1000mg taken twice daily for 14 days, supplemented with clarithromycin 500mg and tinidazole 500mg twice daily for the final 7 days. 2.5. H. pylori detection
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A 13C-UBT (UCBT Kit, Atom High Tech, Beijing, China) was used to confirm the presence of H. pylori infection 8-12 weeks after treatment. H. pylori infection was considered eradicated if the result of
13
C-UBT was negative. PPIs, H2-receptor
blockers, bismuth salts or antibiotics were discontinued for at least four weeks before C-UBT was performed.
13
C-UBT is performed after an overnight fast. A baseline
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13
breath sample is obtained by blowing through a disposable plastic straw into a 20ml
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container, and a capsule containing 75mg of 13C-urea is given to patients with 100ml
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water. Another breath sample is collected 30 min later. The test is considered positive if the difference between the baseline sample and the 30-min sample exceeds 4.0 13
CO2 analyzed using the gas isotope ratio mass spectrometer
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parts/1000 of
(GIRMSZC-202, Wan Yi Sci& Tech, Anhui, China).
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2.6. Gastroscopy
Gastroscopy was performed after obtaining the written informed consent. After
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the extensive observation of upper digestive tract, two mucosal biopsy specimens (one
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each from the antrum and corpus) were obtained and put into the same vial to culture H. pylori strains. One additional specimen (from the antrum) was obtained for the analysis of CYP2C19 polymorphism.
2.7. H. pylori culture and antimicrobial resistance H. pylori strains were isolated from gastric mucosal samples and in vitro
antibiotic resistance was tested by Epsilometer test (E-test, AB Biodisk, Solna, Sweden) as previously described [17]. According to the clinical guidelines for H. pylori proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [18], the resistance breakpoints for amoxicillin, clarithromycin, metronidazole and levofloxacin were > 0.12mg/L, > 0.5mg/L, > 8mg/L and > 1mg/L, respectively. All the cultures and susceptibility tests were conducted at the National
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Institute for Communicable Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. 2.8. CYP2C19 polymorphism The CYP2C19 polymorphism was analyzed to characterize PPI metabolism.
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Genotyping of the two mutated CYP2C19 genes was performed using real-time polymerase chain reaction. DNA was extracted from gastric mucosal samples using a
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QIAamp Mini Kit (Qiagen, Düsseldorf, Nordrhein-Westfalen, Germany). Two pairs
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of probes were designed to distinguish the wild and mutated CYP2C19 gene alleles. Probe specificity was validated using the sequence method. Patients were genotyped
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into three groups by identifying the CYP2C19 wild-type (CYP2C19 *1) gene and the two mutated alleles (CYP2C19 *2 and CYP2C19 *3). Patients without a mutation
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(*1/*1) were designated as homozygous extensive metabolizers, those with one mutation (*1/*2 or *1/*3) as heterozygous extensive metabolizers, and those with two
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2.9. Statistical analysis
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mutations (*2/*2, *3/*3 or *2/*3) as poor metabolizers.
The sample size was determined using a reported efficacy of 91.1% [4] and a
specified precision of ± 5%. A sample size of 124 patients was necessary, producing a 95% confidence interval. As the probability of loss to follow-up was estimated at around 5%, the final size of the sample was at least 131 patients. Statistical analysis was performed using SPSS for Windows (version 18, SPSS
Inc., Chicago, IL, USA). The primary outcomes included the eradication rates of LBAE regimen evaluated using both ITT (including all eligible patients enrolled in the study and taking at least one dose of medicine regardless of compliance with the study protocol) and PP (including only patients fully adherent to the protocol and excluding patients with poor compliance or unavailable data) analyses. Secondary
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outcome variables were adverse effect rates and compliance. Values of P<0.05 were considered statistically significant. Categorical variables are described with frequency and percentage, and continuous variables are described with mean and standard deviation. The eradication
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rates and their 95% confidence intervals (CI) were calculated. Between-group differences were evaluated using Pearson Chi-square or Fisher’s exact test for
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categorical variables.
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Univariate analysis was performed to evaluate significant predictive variables for eradication of H. pylori. A multiple logistic regression analysis was performed using
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significant variables in the univariate analysis. We used a backward modeling strategy and the likelihood ratio was the statistic for model comparison. The magnitude of the
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effect was described with an odds ratio (OR) and 95% CI. The dependent variable was eradication of H. pylori and independent variables were gender (female and male),
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age (≤ 35, 35-55 and ≥ 55years), smoking (yes and no), diagnosis (peptic ulcer
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disease and non-ulcer/uninvestigated dyspepsia), first-line eradication regimen (sequential therapy, concomitant therapy and hybrid therapy), history of quinolone intake (yes and no), compliance (good and poor), antimicrobial (amoxicillin and levofloxacin) resistance (susceptible and resistant) and CYP2C19 polymorphism (heterozygous extensive metabolizer, homozygous extensive metabolizer and poor metabolizer).
Smoking was defined as consumption of more than one pack of cigarettes a week
in the previous three months. Patients with duodenal and/or gastric ulcer found in upper endoscopy were diagnosed with peptic ulcer disease, while those without ulcer were considered as non-ulcer dyspeptic patients. Uninvestigated dyspeptic patients included those with dyspeptic symptoms but did not receive gastroscopy. The history
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of quinolone intake was obtained via patients’ memories (including oral intake, muscular injection and intravenous injection).
3. Results
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Figure 1 outlines the patient flowchart. A total of 147 patients who failed the initial NBQT therapy were enrolled, including 12 who refused to participate and 3
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who met the exclusion criteria. Finally, 132 patients who had been treated with
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sequential therapy (n=40), concomitant therapy (n=49) and hybrid therapy (n=43) were enrolled into second-line LBAE therapy. A total of 11 patients were excluded
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from the PP analysis because of poor compliance (n=5), loss to follow-up (n=3), intolerance to medications (n=2) and protocol violation (n=1).
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Table 1 shows the baseline characteristics of enrolled patients. Among the 132 patients included, 89 (67.4%) consented to undergo gastroscopy and analysis for
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CYP2C19 polymorphism and H. pylori culture. The percentage of patients carrying
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homozygous extensive metabolizer, heterozygous extensive metabolizer and poor metabolizer CYP2C19 genotypes were 37.1%, 48.3% and 14.6%, respectively. H. pylori culture and antimicrobial sensitivity test were successful in 72 patients (success rate: 80.9%). The rate of resistance to amoxicillin, clarithromycin, metronidazole and levofloxacin was 8.3%, 55.6%, 73.6% and 36.1%, respectively. 3.1. Eradication rates
LBAE therapy achieved the eradication rates of 73.5% (95% CI 65.9%-81.1%;
97 of 132 patients) in ITT analysis and 78.5% (95% CI 71.1%-85.9%; 95 of 121 patients) in PP analysis. 3.2. Adverse effects and compliance 25 patients (19.2%) had adverse effects including 16, 7 and 2 with mild,
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moderate and severe adverse responses, respectively. No serious adverse effects were reported. The list and proportion of adverse effects are shown in Table 2. Good compliance was attained in 123 (96.1%) patients. 3.3. Predictors of successful H. pylori eradication
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Univariate analysis indicated that the eradication rate was significantly higher in patients without a history of quinolone intake. The eradication rate was also affected
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by levofloxacin resistance. There was no significant effect of gender, age, smoking,
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diagnosis, first-line eradication regimen, compliance, CYP2C19 polymorphism or amoxicillin resistance on the eradication rates (Table 3).
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Multivariate analysis identified levofloxacin resistance and history of quinolone intake as independent predictors of treatment failure (levofloxacin resistance: OR
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7.183, 95%CI 1.616-31.914, P=0.010; history of quinolone intake: 4.844, 1.174-19.983, P=0.029).
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3.4. Effect of amoxicillin and levofloxacin resistance on eradication rates
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Figure 2 illustrates the effects of amoxicillin and levofloxacin resistance on the
eradication rates. Significant differences were found in the eradication rates among patients with dual amoxicillin and levofloxacin resistance, isolated resistance and dual susceptibility.
4. Discussion
Compared with the second-line 10-day levofloxacin-amoxicillin-PPI regimen
recommended in Maastricht IV consensus, the 14-day LBAE regimen used in this study potentially increases the eradication efficacy as follows: (1) Antibiotic effects of bismuth Bismuth inhibits H. pylori via multiple mechanisms, such as reducing the
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viscosity of mucus, binding to the toxins produced by H. pylori, preventing the adherence of H. pylori with gastric epithelial cells, and decreasing the number of H. pylori [10, 19]. Up to now, studies reporting H. pylori resistant to bismuth are lacking [10, 11]. Combined use of bismuth and antibiotics, such as levofloxacin and
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clarithromycin, results in synergistic or additive effects [10, 12]. (2) Bismuth reducing the resistance to levofloxacin
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A recent study reported the efficacy and effect of fluoroquinolone resistance on
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levofloxacin-containing therapy [12]. Patients were randomized to receive PPI, amoxicillin and levofloxacin with or without bismuth for 14 days. For
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levofloxacin-resistant strains, the bismuth combination was still relatively effective (71%) while the non-bismuth regimen resulted in H. pylori eradication in only 37% of
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the patients. These findings suggest that bismuth improved the sensitivity to levofloxacin and thus reduced the resistance to levofloxacin.
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(3) Prolonging eradication therapy to 14 days
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Maastricht IV consensus reported that prolonging PPI-clarithromycin-containing
triple therapy from 7 days to 10-14 days improve the eradication rate by about 5% [2]. Regarding the duration of levofloxacin-containing regimens, meta-analyses also reported higher cure rates with 10-day than 7-day regimens [20-22]. A study performed by Tai et al. [7] showed that the eradication rate of 14-day levofloxacin-containing triple therapy was higher than that of 10-day therapy. In a review published by Lu et al., the authors suggest that the treatment duration was a critical determinant of outcome with bismuth quadruple therapy [11]. However in the present study, LBAE therapy did not achieve good eradication efficacy unlike in patients from Spain and Italy, which is mainly due to the high levofloxacin resistance. Multivariate analyses revealed that levofloxacin resistance
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and past quinolone intake were independent risk factors for LBAE eradication failure. Although bismuth supplementation to levofloxacin-amoxicillin-PPI regimen and extension of treatment duration enabled better eradication outcomes in regions with relatively high levofloxacin resistance, the improvement was still limited. For
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example, the cut-off level of resistance at which PP success dropped below 90% has been calculated: treatment success decreased below 90% with a 14-day
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fluoroquinolone triple therapy when fluoroquinolone resistance rates exceeded
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approximately 12% [4]; whereas 14-day bismuth-containing fluoroquinolone quadruple therapy could be used in areas with a fluoroquinolone resistance of up to
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approximately 26% [12]. However, the rate of levofloxacin resistance in the present study was as high as 36.1%, while the eradication effects were poor.
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In the study performed by Gisbert JP et al., a high dose of PPI, namely esomeprazole 40mg twice daily, may also have contributed to the effectiveness of the
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LBAE regimen [4]. A previous meta-analysis showed that high-dose PPI increased
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cure rates by approximately 6%-10% in comparison with standard doses [23]. However in quinolone-containing eradication regimens, the effects of high-dose PPI on the eradication rate are still unclear. In the present study, a standard dose of PPI, namely esomeprazole 20mg twice daily, was used due to restrictions of Chinese Insurance Policy, reimbursing patients only for using standard dose, twice daily. In addition, related studies also showed that double-dose PPI mainly improved the eradication rate in patients with CYP2C19 fast metabolizer genotype, while for patients with moderate and poor metabolizer genotypes, the effects were not significant [24-26]. The percentage of CYP2C19 fast metabolizer genotype in Asians is only about 30-40%, which is far lower than in Europeans and Americans (70%-85%) [24, 25, 27]. Therefore, we speculated that the effects of double-dose PPI
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in improving the eradication rate in Asians were not as pronounced as in Europeans and Americans. As a second-line regimen, only one study used LBAE regimen in NBQT-resistant patients with a good eradication efficacy [4]. However, the study had
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several limitations. First, the number of NBQT-failed patients was relatively low (n=69), and patients refractory to standard triple therapy were also included. Second,
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the information of antibiotic resistance to H. pylori was not available, and therefore
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the impact of antibiotic resistance to levofloxacin in the rescue therapy was not evaluated. It has been established that primary resistance of H. pylori to levofloxacin
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significantly reduces the eradication rate [7]. According to the recent drug resistance data, Spain and Italy showed relatively low levofloxacin resistance rate, at 13% [28]
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and 4%-8% [29, 30], respectively. Finally, the factors influencing LBAE therapy were not extensively investigated, except for the effects of region, diagnosis and previous
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treatment regimens. The present study focused specifically on patients with a previous
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H. pylori eradication with NBQT with a larger sample size. In addition, H. pylori culture, antimicrobial sensitivity tests, CYP2C19 genotyping and other extensive factor analyses were performed, which are the highlights of this study. So, we believe that this study has some important information and innovations, which are unavailable in other related papers and very helpful to the clinical treatment of Helicobacter pylori infection.
The findings of the present study also showed that the safety and compliance of
the 14-day LBAE regimen were good. Adverse responses were seen only in 19.2% of the patients, most of which were transient mild or moderate. In light of the previous findings and our results, levofloxacin-bismuth-containing regimens are safe and well-tolerated [12, 20, 31-33].
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At present, the estimated primary clarithromycin resistance rate is about 35-40% in my center [17]. In this study, the clarithromycin resistance rate of the patients after failure of initial NBQT treatment was 55.6%, about 15-20% increase in comparison to primary clarithromycin resistance rate. Although the difference is not as obvious as
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reported in some other studies [34, 35], the disparity is relatively reasonable. Of course, random sampling error should also be considered.
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One limitation of the current study is that it was performed in a single center and
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the results will need to be confirmed in different regions and population all over China. However, in a recent nation-wide multi-center survey, we found a substantially
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and consistently increased antibiotic resistance in many regions of China [17]. In summary, despite the good safety and compliance, the 14-day second-line
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LBAE therapy has poor eradication efficacy in the first-line NBQT-refractory patients in regions with high rates of levofloxacin resistance. Resistance to levofloxacin and
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past use of quinolones are risk factors for failure of LBAE therapy.
Appendices No.
Acknowledgements None.
Author Contributions Zhiqiang Song contributed to the study concept, study design, clinical studies and manuscript editing. Liya Zhou contributed to the study concept, study design, clinical studies and manuscript editing. Jianzhong Zhang contributed to the experimental
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studies. Lihua He contributed to the experimental studies. Peng Bai contributed to the clinical studies. Yan Xue contributed to the clinical studies. Liya Zhou had full access to all the data in the study and takes responsibility for the integrity of the data and
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accuracy of the data analysis.
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quadruple therapy was inferior to traditional quadruple therapy in the treatment of resistant Helicobacter pylori infection. Aliment Pharmacol Ther 2007;26:1063-7.
[15]
Hsu PI, Wu DC, Chen A, et al. Quadruple rescue therapy for Helicobacter pylori infection after two treatment failures. Eur J Clin Invest 2008;38:404-9.
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[16]
Gao XZ, Qiao XL, Song WC, et al. Standard triple, bismuth pectin quadruple and sequential therapies for Helicobacter pylori eradication. World J Gastroenterol 2010;16:4357-62.
[17]
Song Z, Zhang J, He L, et al. Prospective multi-region study on primary
patients. Dig Liver Dis 2014;46:1077-81. Clinical
breakpoints-bacteria
(v
cr
[18]
3.1).
files/Breakpoint
us
http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST tables/Breakpoint table v 3.1.pdf .
Wagstaff AJ, Benfield P, Monk JP. Colloidal bismuth subcitrate. A review of
an
[19]
ip t
antibiotic resistance of Helicobacter pylori strains isolated from Chinese
its pharmacodynamic and pharmacokinetic properties, and its therapeutic use
[20]
Gisbert
JP,
Morena
F.
M
in peptic ulcer disease. Drugs 1988;36:132-57. Systematic
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meta-analysis:
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levofloxacin-based rescue regimens after Helicobacter pylori treatment failure.
Ac ce pt e
Aliment Pharmacol Ther 2006;23:35-44.
[21]
Saad RJ, Schoenfeld P, Kim HM, et al. Levofloxacin-based triple therapy
versus bismuth-based quadruple therapy for persistent Helicobacter pylori infection: a meta-analysis. Am J Gastroenterol 2006;101:488-96.
[22]
Li Y, Huang X, Yao L, et al. Advantages of Moxifloxacin and
Levofloxacin-based triple therapy for second-line treatments of persistent Helicobacter pylori infection: a meta analysis. Wien Klin Wochenschr 2010;122:413-22.
[23]
Villoria A. [Acid-related diseases: are higher doses of proton pump inhibitors more effective in the treatment of Helicobacter pylori infection?]. Gastroenterol Hepatol 2008;31:546-7.
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[24]
Yang
JC,
Lin
CJ.
CYP2C19
genotypes
in
the
pharmacokinetics/pharmacodynamics of proton pump inhibitor-based therapy of Helicobacter pylori infection. Expert Opin Drug Metab Toxicol 2010;6:29-41. Hagymasi K, Mullner K, Herszenyi L, et al. Update on the pharmacogenomics
ip t
[25]
of proton pump inhibitors. Pharmacogenomics 2011;12:873-88.
Furuta T, Graham DY. Pharmacologic aspects of eradication therapy for
cr
[26]
[27]
us
Helicobacter pylori Infection. Gastroenterol Clin North Am 2010;39:465-80. Zhou L, Zhang J, Song Z, et al. Tailored versus Triple plus Bismuth or
an
Concomitant Therapy as Initial Helicobacter pylori Treatment: A Randomized Trial. Helicobacter 2015.
Megraud F, Coenen S, Versporten A, et al. Helicobacter pylori resistance to
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[28]
2013;62:34-42.
Romano M, Cuomo A, Gravina AG, et al. Empirical levofloxacin-containing
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[29]
d
antibiotics in Europe and its relationship to antibiotic consumption. Gut
versus clarithromycin-containing sequential therapy for Helicobacter pylori eradication: a randomised trial. Gut 2010;59:1465-70.
[30]
Federico A, Nardone G, Gravina AG, et al. Efficacy of 5-day
levofloxacin-containing concomitant therapy in eradication of Helicobacter pylori infection. Gastroenterology 2012;143:55-61.e1; quize e13-4.
[31]
Gao XZ, Qiao XL, Song WC, et al. Standard triple, bismuth pectin quadruple
and sequential therapies for Helicobacter pylori eradication. World J Gastroenterol 2010;16:4357-62. [32]
Yee YK, Cheung TK, Chu KM, et al. Clinical trial: levofloxacin-based quadruple therapy was inferior to traditional quadruple therapy in the
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treatment of resistant Helicobacter pylori infection. Aliment Pharmacol Ther 2007;26:1063-7. [33]
Ford AC, Malfertheiner P, Giguere M, et al. Adverse events with bismuth salts for Helicobacter pylori eradication: systematic review and meta-analysis.
[34]
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World J Gastroenterol 2008;14:7361-70. Liou JM, Chang CY, Chen MJ, et al. The Primary Resistance of Helicobacter
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pylori in Taiwan after the National Policy to Restrict Antibiotic Consumption
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and Its Relation to Virulence Factors-A Nationwide Study. PLoS One 2015;10:e0124199.
Lee JW, Kim N, Kim JM, et al. Prevalence of primary and secondary
an
[35]
antimicrobial resistance of Helicobacter pylori in Korea from 2003 through
Figure legends
Ac ce pt e
Figure 1. Patient flowchart
d
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2012. Helicobacter 2013;18:206-14.
Figure 2. Effect of amoxicillin and levofloxacin resistance on Helicobacter pylori eradication rates
Table 1 Baseline characteristics
Variables
Gender (female/male) Age (years) Smoking (yes/no) Diagnosis (peptic ulcer disease/non-ulcer or uninvestigated dyspepsia) First-line eradication regimen (sequential therapy/concomitant therapy/hybrid therapy) History of quinolone intake (yes/no) Cytochrome P450 isoenzyme 2C19 polymorphism (homozygous extensive metabolizer/heterozygous
Patients enrolled (n=132) 61/71 44.4±14.2 (18-70) 21/111 20/112 40/49/43 40/92 33/43/13
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extensive metabolizer/poor metabolizer) Amoxicillin resistance Clarithromycin resistance Metronidazole resistance Levofloxacin resistance
8.3% 55.6% 73.6% 36.1%
In the 132 enrolled patients, H. pylori culture and antimicrobial sensitivity tests were
ip t
successful in 72 patients, and cytochrome P450 isoenzyme 2C19 polymorphism was
Ac ce pt e
d
M
an
us
cr
analyzed in 89 patients.
Table 2 Adverse effects and compliance Variables, n/N (%) Nausea Diarrhea Abdominal pain Taste distortion Dizziness Anorexia Fatigue Headache Insomnia Skin rash Vomiting Patients with adverse effects Discontinued medication because of adverse effects Compliance
Second-line treatment 12/130 (9.2%) 11/130 (8.5%) 6/130 (4.6%) 4/130 (3.1%) 3/130 (2.3%) 3/130 (2.3%) 3/130 (2.3%) 3/130 (2.3%) 1/130 (0.8%) 1/130 (0.8%) 1/130 (0.8%) 25/130 (19.2%) 2/130 (1.5%) 123/128 (96.1%)
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Ac ce pt e
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Table 3 Univariate analysis of factors influencing eradication efficacy Eradication Variables, n/N (%) efficacy Gender: female 44/57 (77.2%) male 53/69 (76.8%) Age: ≤ 35 years 36/47 (76.6%) 35-55 years 30/38 (78.9%) ≥ 55 years 31/41 (75.6%) Smoking: yes 15/19 (78.9%) no 82/107 (76.6%) Diagnosis: peptic ulcer disease 15/18 (83.3%) non-ulcer or uninvestigated dyspepsia 82/108 (75.9%) First-line eradication regimen: sequential therapy 30/38 (78.9%) concomitant therapy 36/47 (76.6%) hybrid therapy 31/41 (75.6%) History of quinolone intake: yes 23/37 (62.2%) no 74/89 (83.1%) Compliance: good 95/121 (78.5%) poor 2/5 (40.0%) Cytochrome P450 isoenzyme 2C19 polymorphism: homozygous extensive metabolizer 23/31 (74.2%) heterozygous extensive metabolizer 32/42 (76.2%)
P value 0.960 0.937 1.000 0.697 0.937 0.011 0.144
0.974 23
Page 23 of 26
poor metabolizer Amoxicillin sensitivity: susceptible resistant Levofloxacin sensitivity: susceptible resistant
10/13 (76.9%) 54/63 (85.7%) 3/6 (50.0%) 40/43 (93.0%) 17/26 (65.4%)
0.101 0.009
In the 126 patients who underwent urea breath test after second-line treatment, H.
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pylori culture and antimicrobial sensitivity tests were successful in 69 patients, and
Ac ce pt e
d
M
an
us
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cytochrome P450 isoenzyme 2C19 polymorphism was analyzed in 86 patients.
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d te ep Ac c
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S1590-8658(15)30365-0 http://dx.doi.org/doi:10.1016/j.dld.2016.01.002 YDLD 3056
To appear in:
Digestive and Liver Disease
Received date: Revised date: Accepted date:
16-11-2015 22-12-2015 5-1-2016
Please cite this article as: Song Z, Zhou L, Zhang J, He L, Bai P, Xue Y, Levofloxacin, bismuth, amoxicillin and esomeprazole as second-line Helicobacter pylori therapy after failure of non-bismuth quadruple therapy, Digestive and Liver Disease (2016), http://dx.doi.org/10.1016/j.dld.2016.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page Title Levofloxacin, bismuth, amoxicillin and esomeprazole as second-line Helicobacter
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pylori therapy after failure of non-bismuth quadruple therapy
Short title
us
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LBAE as second-line H. pylori therapy
Authors and affiliations
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Zhiqiang Song1, MD, Liya Zhou1,*, BS, Jianzhong Zhang2, PhD, Lihua He2, BS, Peng Bai1, MD, Yan Xue1, MD
2
Department of Gastroenterology, Peking University Third Hospital, Beijing, China
M
1
State Key Laboratory for Infectious Disease Prevention and Control, National
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Institute for Communicable Disease Control and Prevention, Chinese Center for
Ac ce pt e
Disease Control and Prevention, Beijing, China * Corresponding author
Electronic word count 3398 words
Correspondence Liya Zhou Department of Gastroenterology, Peking University Third Hospital, Beijing, China Tel: +86-18910192576 Fax: +86-10-82265021
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Page 1 of 26
Email: [email protected]
Financial support This study was supported by the National Science & Technology Pillar Program of
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twelfth Five-Year Plan in China (2012BAI06B02), the clinical key projects of Peking University Third Hospital (Y76493-03) and key laboratory for Helicobacter pylori
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infection and upper gastrointestinal diseases in Beijing (No. BZ0371). The sponsor of
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the study had no role in study design, data collection, data analysis, data interpretation,
an
or writing of the report.
ABSTRACT
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None declared.
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Financial disclosure
Ac ce pt e
Background: The best rescue therapy for Helicobacter pylori (H. pylori) infection following failure of non-bismuth quadruple therapy (NBQT) remains unanswered. Aims: To determine the efficacy, safety and compliance of levofloxacin, bismuth, amoxicillin and esomeprazole (LBAE) regimen following failure of NBQT. Methods: 132 patients with H. pylori infection refractory to first-line NBQT received LBAE regimen (levofloxacin 500mg once/day, bismuth potassium citrate 220mg twice/day, amoxicillin 1000mg twice/day and esomeprazole 20mg twice/day for 14 days). Gastric mucosal biopsy was obtained for H. pylori culture, antimicrobial sensitivity test and cytochrome P450 isoenzyme 2C19 polymorphism analysis. Results: LBAE therapy achieved eradication rates of 73.5% [95% confidence intervals (CI) 65.9%-81.1%] in intention-to-treat and 78.5% (71.1%-85.9%) in
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per-protocol analyses in patients with high antibiotic resistance (amoxicillin 8.3%, clarithromycin 55.6%, metronidazole 73.6% and levofloxacin 36.1%). Adverse effects were found in 19.2% and compliance in 96.1% of the treated patients. Multivariate analyses identified levofloxacin resistance [odds ratio (OR) 7.183, 95%
ip t
CI 1.616-31.914, P=0.010] and history of quinolone intake (4.844, 1.174-19.983, P=0.029) as independent predictors of treatment failure. The eradication rate of
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patients with dual amoxicillin and levofloxacin resistance was significantly decreased
us
(33.3%, P=0.006).
Conclusions: In populations with high levofloxacin resistance, 14-day second-line
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LBAE regimen resulted in an unsatisfactory efficacy in patients resistant to NBQT
Keywords
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despite good safety and compliance.
Ac ce pt e
quadruple therapy.
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Helicobacter pylori, levofloxacin, bismuth, second-line, eradication, non-bismuth
1. Introduction
With the continuous increase in resistance to antibiotics, the efficacy of standard
triple therapy for Helicobacter pylori (H. pylori) infection is unsatisfactory in many regions worldwide [1]. Non-bismuth quadruple therapy (NBQT), including sequential, concomitant and hybrid therapies, is widely used in clinical practice due to good eradication efficacy, safety profile and compliance [1, 2]. Maastricht IV consensus also recommended NBQT as the empiric first-line regimen for the eradication of H. pylori in regions with high resistance to clarithromycin [2]. However, the best rescue therapy following failure of NBQT remains unanswered, as these patients have
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limited options for further therapy because they have already received three different relevant antibiotics: amoxicillin, clarithromycin and metronidazole. Bismuth quadruple therapy, comprising proton pumper inhibitor (PPI), bismuth, tetracycline and metronidazole, has been recommended as the second-line rescue
ip t
approach [2, 3]. However, it is greatly restricted due to the difficulty in obtaining tetracycline in many regions, complex administration and high incidence of adverse
cr
reactions. Furthermore, the traditional quadruple regimen fails to eradicate H. pylori
us
in approximately 20%-30% of cases [3, 4].
Levofloxacin-containing triple regimen, comprising PPI, amoxicillin and
an
levofloxacin for 10 days, has also been recommended in Maastricht IV consensus as a second-line therapy [2]. However, recent studies suggested that the efficacy of
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levofloxacin-containing therapy is decreasing, most likely due to increased quinolone resistance [5-8]. A recent meta-analysis showed that the eradication rate of 10-day
d
levofloxacin-amoxicillin-PPI therapy after failure of concomitant and sequential
Ac ce pt e
therapies was only 78% and 81%, respectively [9]. Bismuth is one of the few antimicrobials to which resistance is not developed [10,
11]. In addition, bismuth has a synergistic effect with antibiotics and partially overcomes levofloxacin and clarithromycin resistance [10, 12]. Thus, combining bismuth and levofloxacin in the same regimen may be a good option as rescue regimen.
Up to now, a total of six papers on the efficacies of H. pylori eradication with the
quadruple regimen of levofloxacin, bismuth, amoxicillin and PPI have been published [4, 12-16]. Although there have been some related study reports, as the second-line rescue therapy after failure of initial NBQT, only one paper has been published. Gisbert JP, et al. reported the use of quadruple therapy comprising levofloxacin,
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bismuth, amoxicillin and esomeprazole (LBAE) for the patients refractory to first-line NBQT regimen with good efficacy [90.0% in intention-to-treat (ITT) analysis and 91.1% in per-protocol (PP) analysis], safety and compliance [4]. However, there are some limitations in the study, such as relatively small sample size, lack of H. pylori
ip t
culture and antimicrobial sensitivity test, absence of comprehensive analysis of the risk factors and in the region with low levofloxacin resistance. So, more investigations
cr
are urgently needed to determine the efficacy of this regimen, especially in regions
us
with high antibiotic resistance.
Therefore, the primary objective of this study was to determine the efficacy of
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the second-line LBAE regimen following failure of NBQT. The secondary objectives were to address the incidence of adverse effects and compliance. We also attempted to
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analyze the factors influencing eradication efficacy and investigated the effects of
Ac ce pt e
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different patterns of antibiotic resistance on H. pylori eradication.
2. Patients and methods 2.1. Patients
This prospective study was conducted in the gastroenterology clinic at a tertiary
hospital located in Beijing, China, between January 2013 and September 2015. Patients with dyspepsia were eligible for enrolment if their H. pylori infections were resistant with first-line NBQT. Previous failure was defined as a positive result of 13
C-urea breath test (UBT) 8-12 weeks after completion of treatment. Patients with any one of the following criteria were excluded from the study: age
younger than 18 years or older than 70 years; taking any drug that could influence the study results such as PPIs, H2-receptor blockers, bismuth salts and antibiotics in the previous four weeks; gastrointestinal malignancy; previous gastric or esophageal
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surgery; severe concomitant diseases; history of allergy to any of the study drugs; currently pregnant or lactating; currently abusing alcohol or the presence of any other clinically significant medical condition that could increase the risk of adverse effects. 2.2. Methods
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Patients were enrolled by the medical staff in Gastroenterology Unit after the assessment of inclusion and exclusion criteria. Before the second-line eradication
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therapy, all the patients were mobilized to underwent gastroscopy to assess the
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condition of upper digestive tract and obtain gastric mucosal biopsy for H. pylori culture, antimicrobial sensitivity test and cytochrome P450 isoenzyme 2C19
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(CYP2C19) polymorphism analysis. All the examination and analyses were provided free of charge, and the patient was completely voluntary to decide whether to accept
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them. Patients deciding not to undergo gastroscopy were directly referred to LBAE therapy. The information of antibiotic resistance and CYP2C19 genotypes was used to
d
analyze the factors influencing eradication efficacy, not to guide the selection of the
Ac ce pt e
second-line therapy drugs. If the second-line eradication fails, the information will be used for guiding the choice of drugs for the third-line eradication regimen. The medical staff explained the therapeutic regimen to patients in detail. Patients were informed of potential adverse effects during the treatment period and asked to return within three days after treatment to assess therapeutic compliance and determine the incidence of adverse effects. H. pylori eradication efficacy was determined 8-12 weeks after treatment with
13
C-UBT. Drugs that affected the study results were
prohibited during the study. Treatment allocation was not blinded. Adverse effects were evaluated using open-ended questions by patient self-reports. Adverse events were classified as mild (not interfering with daily routine), moderate (affecting daily routine), severe (markedly affecting the daily routine and
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discontinued medications) and serious (death, hospitalization, disability or require intervention to prevent permanent damage). Compliance determined by pill counts was defined as good when ≥ 80% of the prescribed drugs was taken. Patients who took < 80% of the treatment drugs were
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considered poorly compliant. 2.3. Ethical consideration
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Written informed consent was obtained from all patients. The study was
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approved by the ethics committee of Peking University Third Hospital and conducted according to the principles of the Declaration of Helsinki and the standards of Good
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Clinical Practice. 2.4. Interventions
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LBAE regimen comprised levofloxacin 500mg once/day, bismuth potassium citrate 220mg twice daily, amoxicillin 1000mg twice daily and esomeprazole 20mg
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twice daily for 14 days. Bismuth potassium citrate, amoxicillin and esomeprazole
Ac ce pt e
were administered together about 30 minutes after breakfast and dinner, and levofloxacin about 30 minutes only after breakfast. In the first-line NBQT, sequential therapy consisted of esomeprazole 20mg and
amoxicillin 1000mg taken twice daily for 5 days, followed by esomeprazole 20mg, clarithromycin 500mg and tinidazole 500mg taken twice daily for 5 days. Concomitant therapy consisted of esomeprazole 20mg, amoxicillin 1000mg, clarithromycin 500mg and tinidazole 500mg all twice daily for 10 days. Hybrid therapy consisted of esomeprazole 20mg and amoxicillin 1000mg taken twice daily for 14 days, supplemented with clarithromycin 500mg and tinidazole 500mg twice daily for the final 7 days. 2.5. H. pylori detection
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A 13C-UBT (UCBT Kit, Atom High Tech, Beijing, China) was used to confirm the presence of H. pylori infection 8-12 weeks after treatment. H. pylori infection was considered eradicated if the result of
13
C-UBT was negative. PPIs, H2-receptor
blockers, bismuth salts or antibiotics were discontinued for at least four weeks before C-UBT was performed.
13
C-UBT is performed after an overnight fast. A baseline
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13
breath sample is obtained by blowing through a disposable plastic straw into a 20ml
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container, and a capsule containing 75mg of 13C-urea is given to patients with 100ml
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water. Another breath sample is collected 30 min later. The test is considered positive if the difference between the baseline sample and the 30-min sample exceeds 4.0 13
CO2 analyzed using the gas isotope ratio mass spectrometer
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parts/1000 of
(GIRMSZC-202, Wan Yi Sci& Tech, Anhui, China).
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2.6. Gastroscopy
Gastroscopy was performed after obtaining the written informed consent. After
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the extensive observation of upper digestive tract, two mucosal biopsy specimens (one
Ac ce pt e
each from the antrum and corpus) were obtained and put into the same vial to culture H. pylori strains. One additional specimen (from the antrum) was obtained for the analysis of CYP2C19 polymorphism.
2.7. H. pylori culture and antimicrobial resistance H. pylori strains were isolated from gastric mucosal samples and in vitro
antibiotic resistance was tested by Epsilometer test (E-test, AB Biodisk, Solna, Sweden) as previously described [17]. According to the clinical guidelines for H. pylori proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [18], the resistance breakpoints for amoxicillin, clarithromycin, metronidazole and levofloxacin were > 0.12mg/L, > 0.5mg/L, > 8mg/L and > 1mg/L, respectively. All the cultures and susceptibility tests were conducted at the National
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Institute for Communicable Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. 2.8. CYP2C19 polymorphism The CYP2C19 polymorphism was analyzed to characterize PPI metabolism.
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Genotyping of the two mutated CYP2C19 genes was performed using real-time polymerase chain reaction. DNA was extracted from gastric mucosal samples using a
cr
QIAamp Mini Kit (Qiagen, Düsseldorf, Nordrhein-Westfalen, Germany). Two pairs
us
of probes were designed to distinguish the wild and mutated CYP2C19 gene alleles. Probe specificity was validated using the sequence method. Patients were genotyped
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into three groups by identifying the CYP2C19 wild-type (CYP2C19 *1) gene and the two mutated alleles (CYP2C19 *2 and CYP2C19 *3). Patients without a mutation
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(*1/*1) were designated as homozygous extensive metabolizers, those with one mutation (*1/*2 or *1/*3) as heterozygous extensive metabolizers, and those with two
Ac ce pt e
2.9. Statistical analysis
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mutations (*2/*2, *3/*3 or *2/*3) as poor metabolizers.
The sample size was determined using a reported efficacy of 91.1% [4] and a
specified precision of ± 5%. A sample size of 124 patients was necessary, producing a 95% confidence interval. As the probability of loss to follow-up was estimated at around 5%, the final size of the sample was at least 131 patients. Statistical analysis was performed using SPSS for Windows (version 18, SPSS
Inc., Chicago, IL, USA). The primary outcomes included the eradication rates of LBAE regimen evaluated using both ITT (including all eligible patients enrolled in the study and taking at least one dose of medicine regardless of compliance with the study protocol) and PP (including only patients fully adherent to the protocol and excluding patients with poor compliance or unavailable data) analyses. Secondary
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outcome variables were adverse effect rates and compliance. Values of P<0.05 were considered statistically significant. Categorical variables are described with frequency and percentage, and continuous variables are described with mean and standard deviation. The eradication
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rates and their 95% confidence intervals (CI) were calculated. Between-group differences were evaluated using Pearson Chi-square or Fisher’s exact test for
cr
categorical variables.
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Univariate analysis was performed to evaluate significant predictive variables for eradication of H. pylori. A multiple logistic regression analysis was performed using
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significant variables in the univariate analysis. We used a backward modeling strategy and the likelihood ratio was the statistic for model comparison. The magnitude of the
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effect was described with an odds ratio (OR) and 95% CI. The dependent variable was eradication of H. pylori and independent variables were gender (female and male),
d
age (≤ 35, 35-55 and ≥ 55years), smoking (yes and no), diagnosis (peptic ulcer
Ac ce pt e
disease and non-ulcer/uninvestigated dyspepsia), first-line eradication regimen (sequential therapy, concomitant therapy and hybrid therapy), history of quinolone intake (yes and no), compliance (good and poor), antimicrobial (amoxicillin and levofloxacin) resistance (susceptible and resistant) and CYP2C19 polymorphism (heterozygous extensive metabolizer, homozygous extensive metabolizer and poor metabolizer).
Smoking was defined as consumption of more than one pack of cigarettes a week
in the previous three months. Patients with duodenal and/or gastric ulcer found in upper endoscopy were diagnosed with peptic ulcer disease, while those without ulcer were considered as non-ulcer dyspeptic patients. Uninvestigated dyspeptic patients included those with dyspeptic symptoms but did not receive gastroscopy. The history
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of quinolone intake was obtained via patients’ memories (including oral intake, muscular injection and intravenous injection).
3. Results
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Figure 1 outlines the patient flowchart. A total of 147 patients who failed the initial NBQT therapy were enrolled, including 12 who refused to participate and 3
cr
who met the exclusion criteria. Finally, 132 patients who had been treated with
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sequential therapy (n=40), concomitant therapy (n=49) and hybrid therapy (n=43) were enrolled into second-line LBAE therapy. A total of 11 patients were excluded
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from the PP analysis because of poor compliance (n=5), loss to follow-up (n=3), intolerance to medications (n=2) and protocol violation (n=1).
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Table 1 shows the baseline characteristics of enrolled patients. Among the 132 patients included, 89 (67.4%) consented to undergo gastroscopy and analysis for
d
CYP2C19 polymorphism and H. pylori culture. The percentage of patients carrying
Ac ce pt e
homozygous extensive metabolizer, heterozygous extensive metabolizer and poor metabolizer CYP2C19 genotypes were 37.1%, 48.3% and 14.6%, respectively. H. pylori culture and antimicrobial sensitivity test were successful in 72 patients (success rate: 80.9%). The rate of resistance to amoxicillin, clarithromycin, metronidazole and levofloxacin was 8.3%, 55.6%, 73.6% and 36.1%, respectively. 3.1. Eradication rates
LBAE therapy achieved the eradication rates of 73.5% (95% CI 65.9%-81.1%;
97 of 132 patients) in ITT analysis and 78.5% (95% CI 71.1%-85.9%; 95 of 121 patients) in PP analysis. 3.2. Adverse effects and compliance 25 patients (19.2%) had adverse effects including 16, 7 and 2 with mild,
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moderate and severe adverse responses, respectively. No serious adverse effects were reported. The list and proportion of adverse effects are shown in Table 2. Good compliance was attained in 123 (96.1%) patients. 3.3. Predictors of successful H. pylori eradication
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Univariate analysis indicated that the eradication rate was significantly higher in patients without a history of quinolone intake. The eradication rate was also affected
cr
by levofloxacin resistance. There was no significant effect of gender, age, smoking,
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diagnosis, first-line eradication regimen, compliance, CYP2C19 polymorphism or amoxicillin resistance on the eradication rates (Table 3).
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Multivariate analysis identified levofloxacin resistance and history of quinolone intake as independent predictors of treatment failure (levofloxacin resistance: OR
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7.183, 95%CI 1.616-31.914, P=0.010; history of quinolone intake: 4.844, 1.174-19.983, P=0.029).
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3.4. Effect of amoxicillin and levofloxacin resistance on eradication rates
Ac ce pt e
Figure 2 illustrates the effects of amoxicillin and levofloxacin resistance on the
eradication rates. Significant differences were found in the eradication rates among patients with dual amoxicillin and levofloxacin resistance, isolated resistance and dual susceptibility.
4. Discussion
Compared with the second-line 10-day levofloxacin-amoxicillin-PPI regimen
recommended in Maastricht IV consensus, the 14-day LBAE regimen used in this study potentially increases the eradication efficacy as follows: (1) Antibiotic effects of bismuth Bismuth inhibits H. pylori via multiple mechanisms, such as reducing the
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viscosity of mucus, binding to the toxins produced by H. pylori, preventing the adherence of H. pylori with gastric epithelial cells, and decreasing the number of H. pylori [10, 19]. Up to now, studies reporting H. pylori resistant to bismuth are lacking [10, 11]. Combined use of bismuth and antibiotics, such as levofloxacin and
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clarithromycin, results in synergistic or additive effects [10, 12]. (2) Bismuth reducing the resistance to levofloxacin
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A recent study reported the efficacy and effect of fluoroquinolone resistance on
us
levofloxacin-containing therapy [12]. Patients were randomized to receive PPI, amoxicillin and levofloxacin with or without bismuth for 14 days. For
an
levofloxacin-resistant strains, the bismuth combination was still relatively effective (71%) while the non-bismuth regimen resulted in H. pylori eradication in only 37% of
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the patients. These findings suggest that bismuth improved the sensitivity to levofloxacin and thus reduced the resistance to levofloxacin.
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(3) Prolonging eradication therapy to 14 days
Ac ce pt e
Maastricht IV consensus reported that prolonging PPI-clarithromycin-containing
triple therapy from 7 days to 10-14 days improve the eradication rate by about 5% [2]. Regarding the duration of levofloxacin-containing regimens, meta-analyses also reported higher cure rates with 10-day than 7-day regimens [20-22]. A study performed by Tai et al. [7] showed that the eradication rate of 14-day levofloxacin-containing triple therapy was higher than that of 10-day therapy. In a review published by Lu et al., the authors suggest that the treatment duration was a critical determinant of outcome with bismuth quadruple therapy [11]. However in the present study, LBAE therapy did not achieve good eradication efficacy unlike in patients from Spain and Italy, which is mainly due to the high levofloxacin resistance. Multivariate analyses revealed that levofloxacin resistance
13
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and past quinolone intake were independent risk factors for LBAE eradication failure. Although bismuth supplementation to levofloxacin-amoxicillin-PPI regimen and extension of treatment duration enabled better eradication outcomes in regions with relatively high levofloxacin resistance, the improvement was still limited. For
ip t
example, the cut-off level of resistance at which PP success dropped below 90% has been calculated: treatment success decreased below 90% with a 14-day
cr
fluoroquinolone triple therapy when fluoroquinolone resistance rates exceeded
us
approximately 12% [4]; whereas 14-day bismuth-containing fluoroquinolone quadruple therapy could be used in areas with a fluoroquinolone resistance of up to
an
approximately 26% [12]. However, the rate of levofloxacin resistance in the present study was as high as 36.1%, while the eradication effects were poor.
M
In the study performed by Gisbert JP et al., a high dose of PPI, namely esomeprazole 40mg twice daily, may also have contributed to the effectiveness of the
d
LBAE regimen [4]. A previous meta-analysis showed that high-dose PPI increased
Ac ce pt e
cure rates by approximately 6%-10% in comparison with standard doses [23]. However in quinolone-containing eradication regimens, the effects of high-dose PPI on the eradication rate are still unclear. In the present study, a standard dose of PPI, namely esomeprazole 20mg twice daily, was used due to restrictions of Chinese Insurance Policy, reimbursing patients only for using standard dose, twice daily. In addition, related studies also showed that double-dose PPI mainly improved the eradication rate in patients with CYP2C19 fast metabolizer genotype, while for patients with moderate and poor metabolizer genotypes, the effects were not significant [24-26]. The percentage of CYP2C19 fast metabolizer genotype in Asians is only about 30-40%, which is far lower than in Europeans and Americans (70%-85%) [24, 25, 27]. Therefore, we speculated that the effects of double-dose PPI
14
Page 14 of 26
in improving the eradication rate in Asians were not as pronounced as in Europeans and Americans. As a second-line regimen, only one study used LBAE regimen in NBQT-resistant patients with a good eradication efficacy [4]. However, the study had
ip t
several limitations. First, the number of NBQT-failed patients was relatively low (n=69), and patients refractory to standard triple therapy were also included. Second,
cr
the information of antibiotic resistance to H. pylori was not available, and therefore
us
the impact of antibiotic resistance to levofloxacin in the rescue therapy was not evaluated. It has been established that primary resistance of H. pylori to levofloxacin
an
significantly reduces the eradication rate [7]. According to the recent drug resistance data, Spain and Italy showed relatively low levofloxacin resistance rate, at 13% [28]
M
and 4%-8% [29, 30], respectively. Finally, the factors influencing LBAE therapy were not extensively investigated, except for the effects of region, diagnosis and previous
d
treatment regimens. The present study focused specifically on patients with a previous
Ac ce pt e
H. pylori eradication with NBQT with a larger sample size. In addition, H. pylori culture, antimicrobial sensitivity tests, CYP2C19 genotyping and other extensive factor analyses were performed, which are the highlights of this study. So, we believe that this study has some important information and innovations, which are unavailable in other related papers and very helpful to the clinical treatment of Helicobacter pylori infection.
The findings of the present study also showed that the safety and compliance of
the 14-day LBAE regimen were good. Adverse responses were seen only in 19.2% of the patients, most of which were transient mild or moderate. In light of the previous findings and our results, levofloxacin-bismuth-containing regimens are safe and well-tolerated [12, 20, 31-33].
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At present, the estimated primary clarithromycin resistance rate is about 35-40% in my center [17]. In this study, the clarithromycin resistance rate of the patients after failure of initial NBQT treatment was 55.6%, about 15-20% increase in comparison to primary clarithromycin resistance rate. Although the difference is not as obvious as
ip t
reported in some other studies [34, 35], the disparity is relatively reasonable. Of course, random sampling error should also be considered.
cr
One limitation of the current study is that it was performed in a single center and
us
the results will need to be confirmed in different regions and population all over China. However, in a recent nation-wide multi-center survey, we found a substantially
an
and consistently increased antibiotic resistance in many regions of China [17]. In summary, despite the good safety and compliance, the 14-day second-line
M
LBAE therapy has poor eradication efficacy in the first-line NBQT-refractory patients in regions with high rates of levofloxacin resistance. Resistance to levofloxacin and
Ac ce pt e
d
past use of quinolones are risk factors for failure of LBAE therapy.
Appendices No.
Acknowledgements None.
Author Contributions Zhiqiang Song contributed to the study concept, study design, clinical studies and manuscript editing. Liya Zhou contributed to the study concept, study design, clinical studies and manuscript editing. Jianzhong Zhang contributed to the experimental
16
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studies. Lihua He contributed to the experimental studies. Peng Bai contributed to the clinical studies. Yan Xue contributed to the clinical studies. Liya Zhou had full access to all the data in the study and takes responsibility for the integrity of the data and
ip t
accuracy of the data analysis.
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Figure legends
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Figure 1. Patient flowchart
d
M
2012. Helicobacter 2013;18:206-14.
Figure 2. Effect of amoxicillin and levofloxacin resistance on Helicobacter pylori eradication rates
Table 1 Baseline characteristics
Variables
Gender (female/male) Age (years) Smoking (yes/no) Diagnosis (peptic ulcer disease/non-ulcer or uninvestigated dyspepsia) First-line eradication regimen (sequential therapy/concomitant therapy/hybrid therapy) History of quinolone intake (yes/no) Cytochrome P450 isoenzyme 2C19 polymorphism (homozygous extensive metabolizer/heterozygous
Patients enrolled (n=132) 61/71 44.4±14.2 (18-70) 21/111 20/112 40/49/43 40/92 33/43/13
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extensive metabolizer/poor metabolizer) Amoxicillin resistance Clarithromycin resistance Metronidazole resistance Levofloxacin resistance
8.3% 55.6% 73.6% 36.1%
In the 132 enrolled patients, H. pylori culture and antimicrobial sensitivity tests were
ip t
successful in 72 patients, and cytochrome P450 isoenzyme 2C19 polymorphism was
Ac ce pt e
d
M
an
us
cr
analyzed in 89 patients.
Table 2 Adverse effects and compliance Variables, n/N (%) Nausea Diarrhea Abdominal pain Taste distortion Dizziness Anorexia Fatigue Headache Insomnia Skin rash Vomiting Patients with adverse effects Discontinued medication because of adverse effects Compliance
Second-line treatment 12/130 (9.2%) 11/130 (8.5%) 6/130 (4.6%) 4/130 (3.1%) 3/130 (2.3%) 3/130 (2.3%) 3/130 (2.3%) 3/130 (2.3%) 1/130 (0.8%) 1/130 (0.8%) 1/130 (0.8%) 25/130 (19.2%) 2/130 (1.5%) 123/128 (96.1%)
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ip t cr us an M
Ac ce pt e
d
Table 3 Univariate analysis of factors influencing eradication efficacy Eradication Variables, n/N (%) efficacy Gender: female 44/57 (77.2%) male 53/69 (76.8%) Age: ≤ 35 years 36/47 (76.6%) 35-55 years 30/38 (78.9%) ≥ 55 years 31/41 (75.6%) Smoking: yes 15/19 (78.9%) no 82/107 (76.6%) Diagnosis: peptic ulcer disease 15/18 (83.3%) non-ulcer or uninvestigated dyspepsia 82/108 (75.9%) First-line eradication regimen: sequential therapy 30/38 (78.9%) concomitant therapy 36/47 (76.6%) hybrid therapy 31/41 (75.6%) History of quinolone intake: yes 23/37 (62.2%) no 74/89 (83.1%) Compliance: good 95/121 (78.5%) poor 2/5 (40.0%) Cytochrome P450 isoenzyme 2C19 polymorphism: homozygous extensive metabolizer 23/31 (74.2%) heterozygous extensive metabolizer 32/42 (76.2%)
P value 0.960 0.937 1.000 0.697 0.937 0.011 0.144
0.974 23
Page 23 of 26
poor metabolizer Amoxicillin sensitivity: susceptible resistant Levofloxacin sensitivity: susceptible resistant
10/13 (76.9%) 54/63 (85.7%) 3/6 (50.0%) 40/43 (93.0%) 17/26 (65.4%)
0.101 0.009
In the 126 patients who underwent urea breath test after second-line treatment, H.
ip t
pylori culture and antimicrobial sensitivity tests were successful in 69 patients, and
Ac ce pt e
d
M
an
us
cr
cytochrome P450 isoenzyme 2C19 polymorphism was analyzed in 86 patients.
24
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u M an ed pt ce Ac
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d te ep Ac c
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