Efficacy of Lactobacillus-supplemented triple therapy for H. pylori eradication: A meta-analysis of randomized controlled trials

Aim To assess the effect of Lactobacillus supplementation on Helicobacter pylori eradication rates and side effects of the triple therapy. Methods PubMed, Embase, Web of Science and Cochrane Library were searched for articles published up to July, 2019. Review Manager 5.3 and Stata 12.0 were used for statistical analyses. Results The initial database search resulted in 852 articles. Through exclusion and screening, 11 randomized controlled trials involving a total of 724 patients were finally included in this meta-analysis. The H. pylori elimination rate in the Lactobacillus supplement group was significantly higher than that in the control group (RR 1.16, 95% CI 1.08–1.25, P<0.0001). Subgroup analysis showed that the eradication rates were significantly enhanced in both adults and children group, and no significant difference was detected between Asia and Europe group. In addition, sub-analysis based on duration of Lactobacillus supplementation showed the pooled RRs in the long-term and short-term groups were 1.17 (95%CI 1.06–1.30) and 1.16 (95% CI 1.04–1.30), respectively. Regarding the Lactobacillus strains, the pooled RR was 1.33 (95% CI 1.10–1.62) in the L. casei group, 1.18 (95% CI 1.03–1.34) in the L. reuteri group while 1.02 (95% CI 0.87–1.21) in the Lactobacillus GG group. As for the total side effects, Lactobacillus supplementation significantly reduced the incidence of taste disturbance (RR = 0.36, 95% CI 0.17–0.74, P = 0.005). Conclusions Lactobacillus supplementation during the treatment of Helicobacter pylori infection can effectively improve the eradication rates, and reduce the incidence of therapy-related taste disturbance.


Introduction
Helicobacter pylori, a microaerophilic gram-negative bacterium, has quickly gained widespread attention since its discovery. H. pylori infection is closely associated with the incidence of gastrointestinal diseases [1,2]. In some developing countries, the prevalence of H. pylori is about 80-90% [3]. H. pylori infection has become a notable worldwide health problem. In 2016, The Toronto Consensus Reports (2016) reported the role of H. pylori in pathogenesis of gastrointestinal diseases [4]. Traditional triple therapy includes the proton pump inhibitor (PPI), clarithromycin and amoxicillin. The eradication rate of the traditional triple therapy could be 90% in early 1990s [5]. However, the eradication rate markedly decreased in recent decades, due to the increasing antibiotic resistance. In some countries, eradication failure rates currently exceed 20%, and they might be even higher in the areas with high prevalence of resistant H. pylori strains. [6][7]. Additionally, the therapies based on long-term or repeated use of antibiotics have also encountered problems on other side effects like alteration of gut microbiota [8]. Thus, it is necessary to improve the therapies or adjunctive treatments.
As microorganisms in mutually beneficial relation to human, probiotics can provide benefit in regulating gastrointestinal flora [9]. Some reviews showed that probiotics might have antagonistic effect on Helicobacter pylori [10]. Recently, some researches have used multistrain probiotics supplementation therapy for H. pylori eradication [11,12]. However, it seems that not all kinds of probiotics are effective for H. pylori eradication [13]. An alternative solution is to choose a clearly defined strain of probiotics that has been confirmed effective in inhibiting H. pylori in clinical trials. Studies have shown that some Lactobacillus strains can exhibit anti-H. pylori effects in vitro or in animal models [14][15][16][17][18]. A study by García et al. showed that the co-existence of Lactobacillus and H. pylori was low in the gastric biopsies of symptomatic patients [19]. If the co-existence of both species is reestablished, the growth of H. pylori may be inhibited. A network meta-analysis by Shi et al. showed that Lactobacillus might be a better choice of probiotic strains for eradicating H. pylori [20]. However, trials based on the efficacy of Lactobacillus in H. pylori eradication therapy were dramatically different in quality. Meanwhile, studies on the efficacy of Lactobacillus supplementation to the standard triple therapy are scant and lead to controversial conclusions.
Therefore, this meta-analysis was conducted to analyze whether the Lactobacillus -supplemented standard triple therapy can significantly increase the H. pylori eradication rates and reduce incidences of the side effects, aiming to provide more reliable evidences for clinical decision.

Materials and methods
The Cochrane Handbook for Systematic Reviews of Interventions [21] and the PRISMA statement for reporting systematic reviews and meta-analysis [22] were followed for our meta-analysis. randomization methods; (2) patients with H. pylori infection diagnosed by histology, culture, rapid urease test, urea breath test, or H. pylori stool antigen test; (3) studies comparing at least two treatment groups including (a) triple therapy (a proton pump inhibitor and two antibiotics) with placebo or not and (b) the same regimen plus Lactobacillus; (4) confirmation of eradication outcomes at least four weeks after eradication therapy; (5) the full text was available; (6) the data of the eradication rates and/or side effects were available.
The following studies were excluded from the analysis: (1) case reports, comments, letters or reviews; (2) use of agents other than Lactobacillus as the adjuvant therapy for H. pylori infection in the experimental group; (3) use of oral antibiotics and/or PPIs and/or H2-antagonists during the 2 weeks prior to ingestion of the study product; (4) not RCTs.
In case of duplicate reports, or studies clearly reported results from the same study population, the latest or most complete one was selected.

Data extraction
Two reviewers used the standardized data abstraction sheets to extract the data independently. Disagreements were resolved by consulting another reviewer. The following information was extracted from each included study: the first author's name, published year, location, baseline features of the involved patients, diagnostic methods of testing H. pylori infection before enrolling and after completing study, H. pylori eradication regimen, Lactobacillus regimen, follow-up time, eradication rate as the primary outcome, and side effect rate as the secondary outcome.

Quality assessment
The Cochrane risk of bias assessment tool [21] was used to evaluate the quality of the included studies, which include the following seven parameters: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. Each study was divided into three levels (low risk, unclear risk, and high risk). Review Manager 5.3 statistical software was used to assess these studies, and the results of them were showed as risk of bias graph and risk of bias summary. In addition, the quality of evidence for key outcomes was assessed using GRADEpro GDT. The GRADE system classifies quality of evidence as high, moderate, low, or very low according to factors that include the study methodology, consistency and precision of the results, and directness of the evidence [23]. Two independent reviewers completed the entire quality assessment. Consensus on quality assessment was reached via discussion.

Statistical analysis
This meta-analysis was conducted using Review Manager 5.3 and Stata 12.0. The eradication and side effects rates were analyzed based on intent-to-treat (ITT) principle, and the dichotomous outcomes were expressed as relative risk (RR) with 95% confidence interval (CI). P < 0.05 was considered as statistically significant. Heterogeneity among studies was evaluated via chi-square tests and the inconsistency statistic. It indicated that there was significant heterogeneity if I 2 > 50% and/or P < 0.1. Besides, the levels of heterogeneity assessed by I 2 were as follows: 0-25% meant homogeneity; 25-50% meant low heterogeneity; 50-75% meant moderate heterogeneity, and >75% meant high heterogeneity [24]. The fixed-effects model (Mantel-Haensze) was used to analyze the data if no heterogeneity was present, while the randomeffects model was used if I 2 > 50% [25]. Subgroup analyses were conducted based on the ages, trial locations, duration, dose and species of Lactobacillus supplementation. In addition, Begg's and Egger's tests were used to detect potential publication bias. P values < 0.05 were considered as statistically significant.

Eradication rates
Among the involved cases, 483 patients were in the experimental group, 489 patients in the control group (Fig 2). The Mantel-Haensze fixed-effects model was used because of the low heterogeneity (I 2 = 0%). The eradication rates in the Lactobacillus supplementation group were 80.3%, while 69.1% in the control group. The pooled RR was 1.16 (95% CI 1.08-1.25, P<0.0001), as showed via intention-to-treat analysis.

Side effects
Six RCTs including 659 patients provided the side effect rates [26,27,31,[33][34][35]. The total side effect rates in the Lactobacillus supplementation group was 25.9%, while 34.3% in the control group. There was no significant difference between these two groups (RR = 0.77, 95% CI 0.55-1.07, P = 0.12) (Fig 5). Occurrence of certain specific symptoms resultant from the side effects, such as diarrhea, taste disturbance, loss of appetite and abdominal distension were also analyzed, respectively, in this study (Fig 5).  group. Other individual side effects, such as nausea, constipation etc. were not analyzed here due to the limitation of the data obtained.

Sensitivity analyses and publication bias
By removing one study at a time, none of the studies changed the pooled risk of the H. pylori eradication rates substantially, indicating the results of this study were reliable. The funnel plot obtained by an intentional analysis of the eradication rates revealed a slightly asymmetrical distribution (Fig 6). Nevertheless, no significant publication bias was detected by the Begg's test (P = 1.000) and the Egger's test (P = 0.931).

Risk assessment of bias in the studies
All the included studies had adequate and clear random sequence generation and blinding methods. One study [28] was judged as high risk of blinding of participants and personnel because the trial outcomes could be affected by the lack of blinding methods. Fig 7 shows the whole risk of bias summary. As for the quality of evidences, the reports on that the eradication rates, as the primary outcome, were enhanced by using Lactobacillus combined with traditional triple therapy eradication were graded as being of moderate quality, and evaluated as critical. Meanwhile, Lactobacillus supplementation reducing the chances of side effects as the second outcome was also graded as moderate, and evaluated as important. Table 2 details the GRADE quality of findings.

Summary of evidence
In this meta-analysis, the eradication rates of H. pylori infection increased by 8.4% after the addition of Lactobacillus (pooled RR = 1.16, P<0.0001), which indicated Lactobacillus supplementation could significantly improve the eradication efficacy. Furthermore, in the subgroup analysis, the eradication rates in the Lactobacillus supplementation group increased by more than ten percent in both adult/children group and Asia/Europe group. In addition, the eradication rates of the Lactobacillus supplementation increased in both long-term/short-term groups and the high-dose/low-dose groups. With respect to Lactobacillus species, Lactobacillus GG couldn't improve the eradication rates, L. casei and L. reuteri seemed to be more effective in H. pylori eradication therapy. As for the adverse events, Lactobacillus supplementation could significantly reduce the risk of taste disturbance. However, the incidences of the total side effects might not decrease significantly. All the included RCTs featured clear and adequate randomization methods, and their results remained stable. All evidence for eradication rates and side effects was graded as moderate quality in our study according to the GRADE assessment. The results provide certain clinical implications for future researches. Because of the misuse or overuse of antibiotics in H. pylori eradication therapy, the antibiotics kill not only H. pylori, but also the normal flora, which could lead to the bacterial superinfection and H. pylori infection recurrence. At this point, the Lactobacillus supplementation might be helpful to reconstruct the micro-ecological environment.

Experiments supporting the findings
Early studies showed that Lactobacillus strains could inhibit the attachment of H. pylori to gastric cell lines in vitro [37]. It has also been demonstrated in animal experiments that Lactobacillus could alleviate gastritis in mice infected with H. pylori [18]. At the same time, Sung et al. (2014) found the anti-H. pylori activity of the Lactobacillus strains isolated from baikkimchi [38]. Francavilla et al. found that L. reuteri could secrete reuterin, which has the anti-H. pylori effect [39]. Another study showed that L. reuteri could interfere with the binding of H. pylori to epithelial cell receptors through producing cell-surface proteins [40]. In addition, studies evidenced that Lactobacillus GG can inhibit the cell membrane leakage induced by Helicobacter pylori through improving the epithelial barrier [41]. Moreover, Lactobacillus might play a role in regulating the immune response. L. acidophilus could mitigate gastritis associated with H. pylori infection by inhibiting NFκB pathways [15], and Lactobacillus GG could also in vitro reduce the release of IL-8 induced by H. pylori [42]. Meanwhile, Lactobacillus acidophilus can Effect of Lactobacillus-supplemented triple therapy on H. pylori eradication also activate opioid and cannabinoid receptors, thus alleviating the discomfort caused by antibiotics and possibly reducing side effects [43].

Strengths and limitations
The present study used the well-defined probiotics (Lactobacillus), which has been confirmed effective for inhibiting H. pylori in clinical trials, to investigate the association of the bacteria of the same genus with the therapeutic efficacy. Besides, the RCTs included in our meta-analysis covered adults and children from Asia, Europe and Africa. Moreover, the Cochrane risk of bias assessment tool was used for quality assessment of the included studies, and the GRADE system was applied to evaluate the evidences of the key outcomes. The design of this study could improve the reliability of the conclusions. However, this meta-analysis also has some limitations. First, the study included only 11 RCTs, which might influence the credibility of the outcomes. Next, the variety of population characteristics and the different duration of Lactobacillus supplementation may lead to clinical heterogeneity. Although subgroup analysis was conducted for breaking through this limitation, its influence may still be incompletely controlled. In addition, not all the studies included the side effect rates, and the severity of the adverse events was not evaluated here since relevant data was not mentioned in the included studies.

Comparison with other studies
Our results proved Lactobacillus supplementation could significantly improve H. pylori eradication rate of the triple therapy, which showed accordance with some previous studies [44][45][46]. Feng et al.(2017) [47] identified that L. casei was more appropriate to supplement triple therapy for improving H. pylori eradication compared with other probiotic regimens, which was consistent with our findings. A study by Cindoruk et al. also showed the addition of Lactobacillus GG did not improve the eradication rate [48]. Besides, several probiotics, including Saccharomyces boulardii and Bifidobacterium, have also been used in combination with traditional therapies for eradication of H. pylori infection. Compared to standard triple therapies, although addition of S. boulardii reduced the incidence of diarrhea, it did not significantly improve the H. pylori eradication rate [49]. In another study, addition of the combination of Lactobacillus acidophilus and Bifidobacterium also failed to show an improvement in H. pylori infection eradication [50]. Subgroup analysis based on dose of Lactobacillus supplementation suggested that both low and high dose could enhance the efficacy of H. pylori eradication, which was consistent with Zheng et al. [44]. However, Fang et al. [46] drew inconsistent conclusions. As for the therapy-related adverse events, our results revealed Lactobacillus supplementation did not significantly reduce the incidence of total side effects, which was in accordance with Fang et al. [46] and Zheng et al. [44]. Furthermore, our results identified that Lactobacillus supplementation could reduce the incidence of taste disturbance, which was inconsistent with Lv et al. [51] and Li et al. [52]. A possible explanation for inconsistencies with other studies might be that the age distribution of the patients involved in the study was significantly different, and the duration and species of supplementation with Lactobacillus or probiotics might also be an important factor. Considering these factors, well-designed trials are needed to get more credible evidences.

Conclusion
In conclusion, current evidences suggest that Lactobacillus supplementation during treatment with standard triple therapy for H. pylori infection can significantly improve the eradication rates, and reduce the incidence of the taste disturbance. Future studies should focus on clarifying the optimal duration of Lactobacillus administration and an effective way to reduce the adverse events for enhancing the curative efficacy of the antibiotic-based therapies for H. pylori infection.
Supporting information S1