Meta-analysis: High-dose vs. low-dose metronidazole-containing therapies for Helicobacter pylori eradication treatment

Objective The purpose of this study was to evaluate the efficacy of high dose of metronidazole in the treatment of Helicobacter pylori (H. pylori) infection. Methods Studies were identified from databases (Pubmed, Embase, Cochrane Library, ClinicalTrials.gov) searched from January 1990 to September 2017 using a battery of keywords. We included randomized controlled trials (RCTs) of H. pylori treatment comparing the high-dose and low-dose metronidazole-containing therapies (high-dose and low-dose therapies). Two reviewers independently selected studies, extracted relevant data and assessed study quality. A meta-analysis was performed by using Review Manager 5.3. Dichotomous data were pooled to obtain the relative risk (RR) of the eradication rate, with 95% confidence intervals (CIs). Results Four randomized controlled trials, a total of 612 patients with a diagnosis of H. pylori infection were included. Overall the meta-analysis showed that both high-dose and low-dose therapies achieved similar efficacy of intention-to-treat (ITT) eradication rate 82% vs. 76%, RR 1.12 (95%CI: 0.96 to 1.30), P = 0.15, and adherence 94% vs. 94%, RR 1.00 (95%CI: 0.97 to 1.04), P = 0.81, but side effects were more likely in high-dose therapies [32% vs. 17%, RR 1.84 (95%CI: 1.17 to 2.88), P = 0.008]. In subgroup analysis, increasing the dose of metronidazole enhanced eradication rates in areas with high metronidazole resistance [74% vs 52%, RR 1.40 (95%CI: 1.08 to 1.82), P = 0.01] and in individuals with metronidazole-resistant strains [71% vs. 46%, RR 1.50 (95%CI: 1.02 to 2.19), P = 0.04]. Conclusions Both high-dose and low-dose therapies can achieve similar eradication rates and adherence and generally low-dose therapies cause fewer side effects. In populations with high metronidazole resistance, high dose of metronidazole can increase the eradication rates of H. pylori infection.

Introduction Therapy with either proton pump inhibitors (PPIs) or colloidal bismuth subcitrate (CBS) plus two antibiotics (metronidazole, tetracycline, amoxicillin, clarithromycin, levofloxacin or rifabutin) have been used as effective and economical therapeutic regimes for curing Helicobacter pylori (H. pylori) infections [1,2]. However, according to the recent Maastricht V/Florence Consensus Report guidelines [3], increasing H. pylori resistance to antibiotics [4][5][6][7][8][9], especially to clarithromycin, levofloxacin and metronidazole, has undermined the efficacy of triple therapies containing these drugs. Resistance varies from country to country; for most developed countries [5,10], clarithromycin resistance has reached high level (15-40%), while the metronidazole resistance remains lower (<40%). For most developing countries, metronidazole resistance can reach 60% or more [6,11]. For individuals who have taken clarithromycin and/ or metronidazole before are also at high risk of antibiotic resistance regardless of their population expectations [3].
To improve the eradication rate for H. pylori, several studies have been conducted to examine alternative strategies to increase efficacy, for example, by extending the treatment duration to 14 days [12], using sequential therapy instead of continuous therapy [13], using high dose or new generation of PPIs [14,15], using tailored therapy instead of empiric chosen treatment [16], and using bismuth-containing quadruple therapies [17,18]. However, there is no metaanalysis reporting whether increasing the dose of metronidazole can enhance the eradication rate of H. pylori. Currently, there are few publications of studies having assessed the role of high dose of metronidazole in H. pylori treatment, as well as the side effects and their conclusion are inconsistent [19][20][21][22]. Therefore, we conducted a meta-analysis to compare high-dose metronidazole-containing therapies (high-dose therapies) with low-dose metronidazole-containing therapies (low-dose therapies) not only regarding therapeutic effectiveness, but also on the adherence and side effects during the treatments.

Literature search
A systematic review of the evidence was performed in September 2017, including the period from January 1990 to September 2017. The electronic databases of PubMed, Embase and Cochrane Library were searched through the combination of a series of logic keywords and text words related to Helicobacter pylori, metronidazole, therapy, and randomized controlled trails (RCTs) (S2 File). The references of the most recent guidelines were identified manually [3]. Besides, we've also searched the following website manually to retrieve unpublished and ongoing studies: ClinicalTrails.gov (http://www.clinicaltrails.gov/).

Study selection and eligibility criteria
After combining search results from different databases and removing duplicates by using EndNote reference manager, two investigators (Yingjie Ji and Hong Lu) independently reviewed all the retrieved abstracts and full texts to remove ineligible studies. If any disagreement was raised, it was worked out by consultation and discussion with another researcher until the difference was resolved. Inclusion criteria for papers in the meta-analysis were: (1) papers or abstracts had to report the results of comparative, randomized trials; (2) studies had to include at least two branches of treatment comparing a low dose of metronidazole with a higher dose in similar therapies; (3) therapies should include the combination of a PPI or CBS, metronidazole and either tetracycline or amoxicillin lasting for at least 7 days; (4) H. pylori infection had to be determined by biopsy and/or urea breath test (UBT) prior to treatment; (5) eradication had to be evaluated by biopsy and/or UBT at least 4 weeks after the end of treatment [23]; (6) studies had to report the eradication outcome and side effects of patients. For each selected publication, the data extracted were: publication year, area, study design, patient characteristics (treatment experience), number of patients in each treatment arm, drug regimen, duration of treatment, tests used to confirm H. pylori infection and eradication of infection, number of patients in whom H. pylori infection was successfully eradicated [intent-totreat (ITT) and per-protocol (PP)analyses], number of patients who discontinued therapy due to side effects, and number of patients with side effects as defined within each included trial.

Quality assessment
Two researchers (Yingjie Ji and Hong Lu) evaluated the methodological quality of the included studies independently, using the modified Jadad score [24], a scale ranging from 0 to 7 according to the descriptions of randomization (0-2 points), concealment allocation (0-2 points), blinding method (0-2 points) and reporting of patient withdrawals (0-1 points), in which points are awarded if: the study is described as randomized (+1) and double blinded (+1) with allocation concealment (+1), of means of which is described appropriately in detail (+1 for each point mentioned above) and there is a description of withdrawals giving number and reason in both groups (+1). The scores range from 1 to 3 was considered to be low quality, while scores range from 4 to 7 was presumed to be high quality. Discrepancies in the interpretation were resolved by consensus.

Outcome measures
The primary goal of this study was to compare the effectiveness of H. pylori eradication (ITT and PP eradication rates), adherence and the incidence of side effects of high-dose vs. lowdose therapies. Subgroup analyses were performed to evaluate: (1) the efficacy of the therapies in areas with different metronidazole resistance; (2) the efficacy of the therapies for metronidazole-resistant and sensitive strains; (3) the side effects and adherence of the therapies on the eradication rates.

Statistical analyses
The results of the studies were analyzed by using RevMan software version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration, Copenhagen, 2014). To evaluate heterogeneity between pooled studies, we used both the Chi 2 (χ2) test [p-value (P) less than 0.10 indicates significant heterogeneity] and inconsistency index (I 2 ) statistic (a value of less than 40% represents low heterogeneity and a value of 75% or more indicates high heterogeneity) [25]. Statistical heterogeneity was measured by sensitivity analyses to illustrate if any clinical heterogeneity was responsible for such statistical difference. We conducted not only an ITT analysis, but also a PP analysis to assess clinical outcomes. We summarized dichotomous outcome measures as the relative risk (RR) along with 95% confidence intervals (95%CI) by RevMan 5.3. The outcomes were pooled using random effects model. All P<0.05 were considered statistically significant.

Study characteristics and methodologies
The search of electronic databases and other sources in September 2017 resulted in 2896 studies. After combining the results, removing duplicates and selections based on the title and abstract, 19 full-text studies remained. Fifteen studies were excluded after reviewing the full text. Seven were excluded because they were not RCTs [26][27][28][29][30][31][32], eight were excluded because they were not comparison of high-dose vs. low-dose therapies [33][34][35][36][37][38][39][40]. After reviewing the potentially useful abstracts for the analysis, four studies, including 612 patients, with no publication bias (Egger's test, P = 0.563, Begg's test, P = 0.734), finally fulfilled the inclusion criteria and were included in this meta-analysis (Fig 1) [19][20][21][22]. The funnel plot of these trails is shown in S1 Fig Among them, 297 patients received high-dose therapies, whereas 315 received lower-dose therapies. In terms of areas, two studies were reported from Iran [19,20], other two from Europe [21] and Japan [22]. In terms of durations, two studies were given 7 days [21,22], while other two 14 days [19,20]. In brief, one of them compared metronidazole 250 mg three times a day (tid) to metronidazole 125 mg tid [19], one metronidazole 800 mg twice a day (bid) to metronidazole 300 mg bid [21], one metronidazole 500 mg tid to metronidazole 250 mg tid [20], and one metronidazole 250 mg tid to metronidazole 250 mg bid [22]. One study used a combination of CBS, tetracycline and metronidazole [19], other three combined a PPI (omeprazole 20 mg bid, lansoprazole 30 mg bid, or rabeprazole 10 mg bid), clarithromycin and metronidazole [20][21][22]. Eradication was tested by UBT or endoscopy [rapid urease test (RUT), histology and culture] at least 4 weeks after the treatment. Two studies showed a significant improvement in eradication rates when using high-dose therapies [19,20], meanwhile another two showed no differences in eradication rates [21,22]. The methodological quality assessment (Table 3) showed that two studies belonged to low quality (score 3) [19,21], and two belonged to high quality (score 4 or 5) [20,22].

Sensitivity analyses
We performed a sensitivity analysis in which we excluded one study at a time. The sensitivity analyses did not change either the direction or the statistical significance of any of the RRs or the level of heterogeneity in any of the analyses, which was attributed to the insufficient sample size of all the included publications.

Summary of evidence
In the last 30 years the standard H. pylori regimen which includes a PPI and/or bismuth plus clarithromycin and amoxicillin or metronidazole was often regarded as the first choice of treatment for eradication of H. pylori [44,45]. However, due to increasing primary resistance to these antibiotics, the eradication rates have generally declined to unacceptable levels [46,47], especially for the dual clarithromycin and metronidazole-resistant strains. It has led some researchers to find new ways to enhance the eradication rates, for example, by increasing the dosage of metronidazole. This meta-analysis included four prospective RCTs with 612 patients (297 in the high-dose therapies, and 315 in the low-dose therapies). The eradication rates (ITT, 82% vs. 76%, P = 0.15; PP, 85% vs. 79%, P = 0.21) and adherence (94% vs. 95%, P = 0.81) were not significantly different between these two groups. This finding suggest that regimens with both high dose and low dose of metronidazole might be considered equally effective. However, high dose of metronidazole resulted in more adverse events than that of low-dose groups (32% vs. 17%, P = 0.008). Subgroup analysis found that the eradication rates of these groups tend to be similar (ITT, 85% vs. 82%, P = 0.51) in areas like Europe or Japan [21,22], where resistance to metronidazole was lower. In contrary, The ITT eradication rates of high-dose therapies was superior to that of low-dose therapies (74% vs. 52%, P = 0.01) in the areas like Iran, in which the prevalence of metronidazole-resistant strains is higher. Besides, the results of the present studies showed that the eradication rates of the high-dose therapies in Iran is 74%, lower than that in Europe and Japan. Thus, we propose that in the areas with high metronidazole resistance, higher dose of metronidazole would achieve higher eradication rates than that of lowdose ones, while in areas with low metronidazole resistance, low dose of metronidazole can also reach high eradication rates.
Antibiotic resistance is considered to be one of the main reasons for eradication failure [48,49]. Thus, it is important to perform antimicrobial susceptibility test before treatment. Two of our included studies involving 343 patients conducted an antimicrobial susceptibility test [19,21]. In the metronidazole-resistant subgroup (79 strains), we found that high-dose therapies reached a higher eradication rate than low-dose ones (71% vs. 46%, P = 0.04), with obvious statistical significance. But for the metronidazole-susceptible subgroup (264 strains), the eradication rates proved to be more similar between the two groups (82% vs. 75%, P = 0.38). The results shows that high dose of metronidazole can partially overcome the metronidazole resistance and reach a higher eradication rate, especially in areas with high metronidazole resistance or for individuals who had taken metronidazole before without antimicrobial susceptibility test.      Adherence is also one of the factors that determine the efficacy of eradication therapy [50]. Overall, no difference emerged among both groups (94% vs. 95%, P = 0.81) as good adherence (>94%) was observed in patients in all four involved studies. However, our meta-analysis found that high-dose therapies would cause higher incidence of side effects than low-dose therapies (total events, 32% vs. 17%, P = 0.008). Although the side effects appear more frequently in high-dose therapies, there was no difference in the adherence of both groups, which means that the patients using high dose of metronidazole can tolerate all the side effects mentioned above and can also complete the entire course of the treatment.

Strengths and limitations
To diminish bias, the study selection, data extraction and evaluation of the study quality were performed by two reviewers separately. We comprehensively analyzed the efficacy of highdose therapies. Sensitive analyses helped to make the outcomes of our meta-analysis reliable, and the subgroup analysis helped us to investigate the impact of high dose of metronidazole in population with different antibiotic resistance.
There were also several potential limitations of our meta-analysis. First, some well-designed studies were excluded because they were not published in English. Second, the sample size was small as only 4 RCTs were included in our meta-analysis. Although the studies were from different geographic locations that spanned Europe, Middle East and Asia, the results might have been more convincing if more RCTs had been analyzed. Third, the bias of the publications might have affected the validity of our conclusions, such as lack of double blinding. Fourth, individual studies included in our analysis differed in a few respects, such as inclusion and exclusion criteria, different PPIs, duration and combined antibiotics. Furthermore, largescale randomized controlled trials are warranted to compare therapeutic efficacy between high-dose and low-dose therapies.

Conclusions
In summary, in some regions low dose metronidazole-containing therapies can achieve good eradication rates, with good adherence and fewer side effects. But in areas with high metronidazole resistance or for individuals who had taken metronidazole before without antimicrobial susceptibility test, increasing the dose of metronidazole can partially overcome the resistance and result in a higher eradication rates than use of low-dose therapies with the same adherence.
Supporting information S1 File.