doi:10.1111/j.1440-1746.2010.06477.

GASTROENTEROLOGY

jgh_6477

44..48

Effects of multistrain probiotic-containing yogurt on second-line triple therapy for Helicobacter pylori infection
Hyuk Yoon,* Nayoung Kim,*, Ji Yeon Kim,* So Youn Park,* Ju Hee Park,* Hyun Chae Jung* and In Sung Song*
*Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul; and Department of Internal medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, Korea

Key words drug therapy, eradication, Helicobacter pylori infection, moxioxacin, probiotics. Accepted for publication 31 July 2010. Correspondence Dr Nayoung Kim, Department of Internal Medicine, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam, Gyeonggi-do 463-707, South Korea. Email: nayoungkim49@empal.com

Abstract
Background and Aims: The adjuvant effects of probiotic-containing yogurt on secondline triple therapy for Helicobacter pylori (H. pylori) infection have not been evaluated. Methods: A total of 337 patients with persistent H. pylori infection, after rst-line triple therapy, were randomly assigned to receive either triple therapy with (yogurt group, n = 151) or without (control group, n = 186) Will yogurt. Triple therapy consisted of 400 mg moxioxacin q.d., 1000 mg amoxicillin b.i.d., and 20 mg esomeprazole b.i.d. for 14 days. Will yogurt contains Lactobacillus acidophilus, Lactobacillus casei, Bidobacterium longum, and Streptococcus thermophilus. H. pylori eradication was evaluated by the 13 C-urea breath test, histology, or the rapid urease test. Results: The eradication rates by intention-to-treat analysis were 66.7% and 68.9% in the control and yogurt groups, respectively (P = 0.667). The eradication rates by per-protocol analysis were 78.5% and 86% in the control and the yogurt groups, respectively (P = 0.110). The adverse event rates were 25.3% and 28.5% in the control group and yogurt group, respectively (P = 0.508). Conclusions: The addition of yogurt containing probiotics to moxioxacin-containing second-line treatment neither improved H. pylori eradication rates nor reduced the adverse events of treatment.

Introduction
The eradication of Helicobacter pylori (H. pylori) has attempted to prevent the development of gastrointestinal diseases, such as peptic ulcers and gastric malignancies. However, the eradication rates with conventional proton-pump inhibitor (PPI)-based triple therapy have become unacceptably low over the last decade;1 this is thought to be mainly due to increasing antibiotic resistance.2,3 To overcome this problem, alternative therapies, including quadruple therapy, sequential therapy, and triple therapy using new antimicrobials, such as levooxacin, have been introduced.4 In addition, other strategies to increase eradication rates include adjuvant therapies added to conventional treatments, such as probiotics. Although there is some controversy as to whether supplementation with probiotics improves the H. pylori eradication rates,511 several meta-analyses and review articles have suggested that probiotics can improve the H. pylori eradication rate by approximately 510%.1215 We also previously reported that the addition of probiotic-containing yogurt to triple therapy signicantly increased H. pylori eradication rates by per-protocol (PP) analy44

sis (87.5% versus 78.7%, P = 0.037) in Korea.16 However, there have been very few reports on the effects of probiotics on second-line treatment for H. pylori infection, and current studies report conicting results. That is, a report from Italy showed that 10-day quadruple therapy with probiotic supplementation did not improve H. pylori eradication rates (PP/intention to treat [ITT]: 97.1%/94.3% with probiotics vs 93.8%/85.7% without probiotics, P = not signicant).17 By contrast, a study from Taiwan concluded that pretreatment with probiotics improved the efcacy of 7-day quadruple therapy (PP/ITT: 90.8%/85% vs 76.6%/71.1%, P < 0.05).18 Furthermore, instead of quadruple therapy, we frequently use moxioxacin-containing triple therapy as a secondline treatment regimen; this is because this regimen has been shown to have a similar efcacy and is better tolerated than quadruple therapy.19,20 Therefore, a randomized, prospective, open-labeled study, with a relatively large population, was undertaken to evaluate whether the addition of a commercially-available yogurt containing multistrain probiotics to a 14-day moxioxacin-containing triple therapy, after initial treatment failure, could improve H. pylori eradication rates and reduce the adverse effects of treatment.

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Methods
Patients
Between January 2007 and January 2010, patients with persistent H. pylori infection, after a course of rst-line triple therapy, were enrolled at Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, Korea. Triple therapy included 40 mg esomeprazole b.i.d., 500 mg clarithromycin b.i.d., and 1000 mg amoxicillin b.i.d. for 7 days. Persistent H. pylori infection was dened based on the results of at least one of the following: (i) a positive13C-urea breath test (UBT); (ii) histological evidence of H. pylori by modied Giemsa staining in the stomach; and (iii) a positive rapid urease test (CLO test; Delta West, Bentley, WA, Australia) by gastric mucosal biopsy. Patients that had a history of having used PPI, histamine-2 receptor antagonists, or antibiotics within the previous 2 months were excluded. All patients provided informed consent, and the study was approved by the institutional review board of Seoul National University Bundang Hospital.

trometer (UBiT-IR300; Otsuka Pharmaceutical, Japan). The cutoff value used for H. pylori eradication was 2.5.

Statistical analysis
The eradication rates of H. pylori were determined by ITT and PP bases. All enrolled patients were included in the ITT analysis. However, for the PP analysis, patients that were lost to follow up, had taken less than 85% of the prescribed drugs or yogurt, or those that had dropped out due to severe adverse events were excluded. SPSS for Windows (version 16.0; SPSS, Chicago, IL, USA) was used for the statistical analysis. Continuous variables were analyzed using the Students t-test, and categorical variables using the c2-test or Fishers exact test. All results were considered statistically signicant when the P-values were less than 0.05.

Results
Patient population

Study design
Patients were enrolled in this randomized, prospective, openlabeled study. The patients were randomly allocated to one of two groups using a computer-generated table and received either moxioxacin-containing triple therapy (moxioxacin + esomeprazole + amoxicillin) with (MEA + yogurt group) or without (MEA-only group) Will yogurt (Korea Yakult; Chungnam, South Korea). Moxioxacin-containing triple therapy consisted of 400 mg moxioxacin q.d., 1000 mg amoxicillin b.i.d., and 20 mg esomeprazole b.i.d. for 14 days. One bottle (150 mL) of Will yogurt before breakfast, started on the same day as triple therapy, was ingested for 4 weeks by the MEA + yogurt group members. Will yogurt contains Lactobacillus acidophilus (L. acidophilus) HY 2177 (> 105 c.f.u/mL), Lactobacillus casei (L. casei) HY 2743 (> 105 c.f.u/mL), Bidobacterium longum HY 8001 (> 106 c.f.u/ mL), and Streptococcus thermophilus B-1 (> 108 c.f.u/mL). Patients were instructed to refrain from antibiotics for at least 4 weeks and from PPI for at least 2 weeks before testing for H. pylori eradication to minimize the chance of false negative results. Four weeks after completing triple therapy, H. pylori eradication was evaluated by a UBT. However, modied Giemsa staining and the rapid urease test (CLO test) were performed in the patients with peptic ulcer disease, gastric dysplasia, or cancer, for whom a follow-up endoscopic examination was necessary. At this time, compliance was assessed by a physician by direct questioning, and patients were interviewed for adverse events. Compliance was considered to be satisfactory when drug intake and ingestion of yogurt exceeded 85%. Each adverse event was scored according to severity from 1 to 5.

Figure 1 shows a schematic diagram of this study. A total of 337 patients entered into the study. Among these patients, 186 were assigned to the MEA-only group, and 151 patients to the MEA + yogurt group. The difference in the number of patients in each group was due to 17 patients, who were initially assigned to the MEA + yogurt group, refusing the yogurt due to underlying conditions, such as diabetes mellitus or lactose intolerance; these patients were then assigned to the MEA-only group. The demographic and clinical characteristics of the two study groups are summarized in Table 1. Sex, the mean age of the patients, and disease proles of the two groups were similar. There was no difference in the drop-out rate between the MEA + yogurt group and the MEA-only group.

H. pylori eradication rates

The H. pylori eradication rates are shown in Table 2. The eradication rates by ITT analysis were similar in the two groups (MEAonly group: 66.7% [95% condence interval [CI]; 59.973.5%] vs MEA + yogurt group: 68.9% [95% CI; 61.576.3%], P = 0.667). In the MEA + yogurt group, the eradication rate by PP analysis was higher than in the MEA-only group (MEA-only group: 78.5% [95% CI; 72.184.9%] vs MEA + yogurt group: 86% [95% CI; 79.792.2%]); the difference between the two groups was not signicant (P = 0.110).

Adverse events
The percentage of patients with adverse events were 25.3% and 28.5% for the MEA-only group and MEA + yogurt group, respectively; there was no signicant difference between the groups (P = 0.508). Details of the adverse events are shown in Table 3. The most common adverse event was diarrhea in both groups. It was more frequently observed in the MEA + yogurt group than in the MEA-only group; however, the difference was not statistically signicant (P = 0.231). The other adverse events were similar in both groups. Severe adverse events (with a score of 4 or 5) occurred at a rate of 8.1% (15/186) and 7.3% (11/151) in the
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UBT
Patients fasted for 4 h before testing. Then, 100 mg of13C-urea powder (UBiTkit; Otsuka Pharmaceutical, Tokyo, Japan) was dissolved in 100 mL water and administered orally; a second breath sample was collected 20 min later. The collected samples were analyzed using an isotope-selective, non-dispersive infrared spec-

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Figure 1 Flow schematic of the study included in intention-to-treat (ITT) and perprotocol (PP) analyses. MEA + yogurt, moxioxacin-containing triple therapy (moxioxacin + esomeprazole + amoxicillin) with Will yogurt; MEA only, moxioxacincontaining triple therapy without Will yogurt.

Table 1

Patient baseline demographics MEA only MEA + yogurt 151 66/85 53.7 11.1 20 36 15 80 30 8 19 3 (13.2%) (23.8%) (9.9%) (53.0%) (19.9%) (5.3%) (12.6%) (2.0%) P-value

Table 2

Helicobacter pylori eradication rates MEA only MEA + yogurt P-value

Included in ITT analysis (n) Male/female (n) Mean age SD (years) Disease, n (%) Benign gastric ulcer Duodenal ulcer Gastric dysplasia or cancer Non-ulcer dyspepsia Drop out, n (%) Non-compliance Follow-up loss Discontinued therapy because of adverse events

186 85/101 55.0 12.5 15 38 32 101 28 5 20 3 (8.1%) (20.4%) (17.2%) (54.3%) (15.1%) (2.7%) (10.8%) (1.6%)

0.715 0.313 0.123

ITT analysis Eradication rate 95% CI PP analysis Eradication rate 95% CI

66.7% (124/186) 59.973.5 78.5% (124/158) 72.184.9

68.9% (104/151) 61.576.3 86.0% (104/121) 79.792.2

0.667

0.110

0.244 0.216 0.601 1.0

CI, condence interval; ITT, intention-to-treat; MEA + yogurt, moxioxacin-containing triple therapy (moxioxacin + esomeprazole + amoxicillin) with Will yogurt; MEA only, moxioxacin-containing triple therapy without Will yogurt; PP, per-protocol.

ITT, intention-to-treat; MEA + yogurt, moxioxacin-containing triple therapy (moxioxacin + esomeprazole + amoxicillin) with Will yogurt; MEA only, moxioxacin-containing triple therapy without Will yogurt.

MEA-only group and MEA + yogurt group, respectively (P = 0.790). The percentage of patients who discontinued therapy because of adverse events was similar in both groups (Table 1).

Discussion
In this study, the PP eradication rate of the MEA + yogurt group (86%) was higher than that of the MEA-only group (78.5%); however, this difference was not statistically signicant. This result is different from that of our previous study, which added the same commercial yogurt to PPIclarithromycinamoxicillin therapy.16 This could be explained by several factors: (i) it might be related to an insufcient sample size. The enrolled number of patients in the MEA + yogurt group was 151, which was smaller
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than the 186 in the MEA-only group; (ii) the different results might be explained by the different antibiotics used for the triple therapy. That is, moxioxacin was used for the second-line therapy in this study instead of clarithromycin; this suggests that the interaction of the probiotics with moxioxacin might be less synergistic than with clarithromycin. In Taiwan, Sheu et al. demonstrated that supplementation with the same probiotic-containing yogurt (AB yogurt), which increased the eradication rate of rst-line treatment by ITT analysis,6 also improved the efcacy of secondline quadruple therapy.18 The different response to the addition of probiotic-containing yogurt with quadruple therapy and moxioxacin containing triple therapy might be related to different trends in antibiotic resistance. That is, the resistance to metronidazole in Korea has decreased from 34.8% in 20032005 to 27.6% in 20072009.21 By contrast, in the case of moxioxacin, the resistance rate has rapidly increased from 5.6% in 2004 to 12% in 20052006, and 28.2% in 20072008 in Korea.22 Thus, the adjuvant effect of probiotics might be too modest to overcome the increased moxioxacin resistance rate of H. pylori. To resolve this issue of resistance to moxioxacin, it is suggested that more potent quinolones, such as satioxacin, be tried, as they have the lowest

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Table 3

Adverse events MEA only (n = 186) 17 15 11 6 5 2 1 1 2 2 1 1 47 (9.1%) (8.1%) (5.9%) (3.2%) (2.7%) (1.1%) (0.5%) (0.5%) (1.1%) (1.1%) (0.5%) (0.5%) (25.3%) MEA + yogurt (n = 151) 20 9 9 3 2 2 2 2 43 (13.2%) (6.0%) (6.0%) (2.0%) (1.3%) (1.3%) (1.3%) (1.3%) P-value

Adverse events

Diarrhea Dyspepsia Nausea or vomiting Metallic taste Abdominal pain Anorexia Abdominal distension Itching or rash Constipation Headache Regurgitation Dizziness Total

(28.5%)

0.231 0.455 0.986 0.736 0.466 1.0 0.589 0.589 0.504 0.504 1.0 1.0 0.508

difference in the total adverse events between the two groups; the frequency of diarrhea did not differ. In conclusion, the addition of a commercial yogurt containing multistrain probiotics to moxioxacin-containing triple therapy as a second-line treatment for H. pylori infection neither improved H. pylori eradication rates nor reduced the adverse events of treatment.

Acknowledgments
This work was supported by a grant from the Seoul National University Bundang Hospital Research Fund (no. 06-2009-026).

References
1 Graham DY, Lu H, Yamaoka Y. A report card to grade Helicobacter pylori therapy. Helicobacter 2007; 12: 2758. 2 Kim JM, Kim JS, Jung HC, Kim N, Kim YJ, Song IS. Distribution of antibiotic MICs for Helicobacter pylori strains over a 16-year period in patients from Seoul, South Korea. Antimicrob. Agents Chemother. 2004; 48: 48437. 3 Kim N, Kim JM, Kim CH et al. Institutional difference of antibiotic resistance of Helicobacter pylori strains in Korea. J. Clin. Gastroenterol. 2006; 40: 6837. 4 Vakil N, Megraud F. Eradication therapy for Helicobacter pylori. Gastroenterology 2007; 133: 9851001. 5 Canducci F, Armuzzi A, Cremonini F et al. A lyophilized and inactivated culture of Lactobacillus acidophilus increases Helicobacter pylori eradication rates. Aliment. Pharmacol. Ther. 2000; 14: 16259. 6 Sheu BS, Wu JJ, Lo CY et al. Impact of supplement with Lactobacillus- and Bidobacterium-containing yogurt on triple therapy for Helicobacter pylori eradication. Aliment. Pharmacol. Ther. 2002; 16: 166975. 7 Armuzzi A, Cremonini F, Ojetti V et al. Effect of Lactobacillus GG supplementation on antibiotic-associated gastrointestinal side effects during Helicobacter pylori eradication therapy: a pilot study. Digestion 2001; 63: 17. 8 Armuzzi A, Cremonini F, Bartolozzi F et al. The effect of oral administration of Lactobacillus GG on antibiotic-associated gastrointestinal side-effects during Helicobacter pylori eradication therapy. Aliment. Pharmacol. Ther. 2001; 15: 1639. 9 Cremonini F, Di Caro S, Covino M et al. Effect of different probiotic preparations on anti-Helicobacter pylori therapy-related side effects: a parallel group, triple blind, placebo-controlled study. Am. J. Gastroenterol. 2002; 97: 27449. 10 Nista EC, Candelli M, Cremonini F et al. Bacillus clausii therapy to reduce side-effects of anti-Helicobacter pylori treatment: randomized, double-blind, placebo controlled trial. Aliment. Pharmacol. Ther. 2004; 20: 11818. 11 Myllyluoma E, Veijola L, Ahlroos T et al. Probiotic supplementation improves tolerance to Helicobacter pylori eradication therapya placebo-controlled, double-blind randomized pilot study. Aliment. Pharmacol. Ther. 2005; 21: 126372. 12 Tong JL, Ran ZH, Shen J, Zhang CX, Xiao SD. Meta-analysis: the effect of supplementation with probiotics on eradication rates and adverse events during Helicobacter pylori eradication therapy. Aliment. Pharmacol. Ther. 2007; 25: 15568. 13 Lesbros-Pantoickova D, Corthesy-Theulaz I, Blum AL. Helicobacter pylori and probiotics. J. Nutr. 2007; 137: 812S8S. 14 Sachdeva A, Nagpal J. Effect of fermented milk-based probiotic preparations on Helicobacter pylori eradication: a systematic review

MEA + yogurt, moxioxacin-containing triple therapy (moxioxacin + esomeprazole + amoxicillin) with Will yogurt; MEA only, moxioxacin-containing triple therapy without Will yogurt.

minimal inhibitory concentration of quinolones;23 (iii) the treatment duration was different. That is, the duration of moxioxacin containing triple therapy was 14 days, which is longer than the 7 days of rst-line triple therapy. Several mechanisms have been proposed to explain the inhibition of H. pylori by probiotics.13 Among these, in the competition for nutrients or adhesion sites in gastric epithelial cells, the viability of probiotics during antibiotic treatment is important for the successful inhibition of H. pylori. One study reported that the administration of moxioxacin to healthy patients for 7 days did not markedly affect the number of lactobacilli and bidobacteria in the feces.24 However, as moxioxacin treatment was for 14 days in this study, we cannot completely exclude the possibility that the probiotics were suppressed by moxioxacin; and (iv) it is possible that the dose of probiotics administered was not sufcient. Although there is no scientic evidence, the minimal effective dose of probiotic bacteria has been frequently reported to be at least 108109 c.f.u/day.25 In fact, in the randomized, controlled trials in previous meta-analyses,12,14,15 the amount of probiotics administered was generally more than 109 c.f.u/day. However, in this study, the dose of L. acidophilus and L. casei given to the MEA + yogurt group of patients were both only 1.5 107 c.f.u/day. Although supplementation with the same dose of lactobacilli improved the H. pylori eradication rate of rst-line treatment by PP analysis in the previous study,16 there is the possibility that this amount of lactobacilli might be relatively insufcient in the present study, in which the moxioxacin resistance rate of H. pylori was as high as 28.2%. There is controversy as to whether the addition of probiotics to eradication treatment can reduce the adverse events of treatment. Although probiotic supplementation had a positive impact on adverse events during H. pylori eradication treatment in a metaanalysis reported in 2007,12 subsequent meta-analyses reported that the effect of probiotics on adverse events varies or is not signicant.14,15 Since the alteration of the intestinal microora is associated with diarrhea during H. pylori eradication treatment, the administration of probiotics might decrease the frequency of diarrhea. However, in the present study, there was no signicant

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20 Kang JM, Kim N, Lee DH et al. Second-line treatment for Helicobacter pylori infection: 10-day moxioxacin-based triple therapy versus 2-week quadruple therapy. Helicobacter 2007; 12: 6238. 21 Hwang TJ, Kim N, Kim HB et al. Change in antibiotic resistance of Helicobacter pylori strains and the effect of A2143G point mutation of 23S rRNA on the eradication of H. pylori in a single center of Korea. J. Clin. Gastroenterol. 2010. 22 Yoon H, Kim N, Lee BH et al. Moxioxacin-containing triple therapy as second-line treatment for Helicobacter pylori infection: effect of treatment duration and antibiotic resistance on the eradication rate. Helicobacter 2009; 14: 7785. 23 Suzuki H, Nishizawa T, Muraoka H, Hibi T. Sitaoxacin and garenoxacin may overcome the antibiotic resistance of Helicobacter pylori with gyrA mutation. Antimicrob. Agents Chemother. 2009; 53: 17201. 24 Edlund C, Beyer G, Hiemer-Bau M, Ziege S, Lode H, Nord CE. Comparative effects of moxioxacin and clarithromycin on the normal intestinal microora. Scand. J. Infect. Dis. 2000; 32: 815. 25 de Vrese M, Schrezenmeir J. Probiotics, prebiotics, and synbiotics. Adv. Biochem. Eng. Biotechnol. 2008; 111: 166.

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