Yini Dang1, Jan D. Reinhardt2,3,4, Xiaoying Zhou1, Guoxin Zhang1*

1 Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China, and First Clinical Medical College of Nanjing Medical University, Nanjing, China, 2 Institute for Disaster Management and Reconstruction, Sichuan University, Chengdu, China, and Hong Kong Polytechnical University, Hung Hom, Hong Kong, China, 3 Department of Health Sciences, University of Lucerne, Lucerne, Switzerland, 4 Swiss Paraplegic Research, Nottwil, Switzerland

Introduction

‘Helicobacter pylori (H. pylori) is a common bacterium infecting about half of the world’s population. It is causally associated with a diverse spectrum of gastrointestinal disorders’ [1]. Eradication of H. pylori is necessary for the management of H. pylori-related complications. The recommended first approach for H. pylori eradication is standard triple antibiotic therapy. Other choices include sequential therapy and quadruple therapy [2]. However, due to antibiotic resistance and patient non-compliance, several studies showed widespread failure of antibiotic therapy [3,4]. Driven by the growing necessity for alternative solutions to

eradication regimens, some studies have started to focus on probiotics [5], i.e. ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ [6]. The most commonly used probiotic bacteria belong to the genera Lactobacillus and Bifidobacterium, and these also include several yeasts such as Saccharomyces boulardii [7]. It has been hypothesized that probiotics could improve H. pylori eradication and reduce side effects during therapy.

Although seven meta-analyses [8–14] on this topic have been conducted over the past seven years, we perform an additional meta-analysis based on the following considerations: 1) The number of articles included in six of the seven previously

1. Figure 1. Flow of article selection.

1. doi:10.1371/journal.pone.0111030.g001

conducted meta-analysis was ten or less [9–14]. In 2010–2014, many new studies were performed to evaluate the effectiveness of probiotics supplementation increasing the evidence base. 2) There are various genera of probiotics used in clinical practice and most meta-analyses [9,11,12,13] solely concentrated on one specific strain. 3) Opposite conclusions were drawn regarding side effects: Sachdeva [10], Zou [9], and Zheng [13] report no significant reduction of overall side effects in probiotics groups. However, Tong [8], Szajewska [11], Wang [12], and Li [14] arrive at opposite conclusions. Moreover, no sensitivity analysis regarding side effects has been conducted so far. 4) While all meta-analysis conducted so far reported increased eradication rates due to probiotics supplementation, no sensitivity analysis with regard to the effectiveness of the antibiotic therapy has been conducted, i.e. it is conceivable that probiotics supplementation will solely increase effectiveness in relatively ineffective antibiotic regimens.

This meta-analysis is thus designed to evaluate the current evidence regarding effects of probiotics supplementation compared with eradication triple therapy only on H. pylori eradication rates and side effects. Moreover, we aim to compare different strains of probiotics as well as differentially effective antibiotic therapies and to evaluate the quality of the trials conducted so far.

Methods Search Strategy

We performed a literature search in Pubmed and Web of Science, covering papers published up to May 2014. A combination of the following keywords was used: (probiotics OR prebiotics OR Bifidobacterium OR Lactobacillus OR Saccharomyces) AND (Helicobacter pylori OR H. pylori). The reference lists of the selected papers and the seven previous meta-analyses were also screened for other eligible articles that may have been missed in the initial search. Next we scanned the titles and abstracts of the trials identified in the computerized search to exclude studies that were obviously irrelevant and scrutinized the full-texts of the remaining studies.

Inclusion and Exclusion Criteria

The following criteria were used for the selection of relevant articles: 1) studies should be randomized controlled trials (RCTs); 2) study populations should never have been treated for H. pylori infection before; 3) studies should include at least two branches of treatment consisting of (a) studies should have patients in a control group who received antibiotic therapy, (b) there should be patients

(57.1% (12/20)

79.2% (19/24) 86.9% (20/23)

91.7% (22/24)

57.1% (12/20)

(24/30) 40.0% (12/30)

33.3% (10/30)

80.7% (46/57) 43.3% (26/60)

61.7% (37/60)

57.1% (12/20)

74.0% (37/50) 74.0% (40/54)

82.7% (43/50)

21.3% (10/47)

patients) (treatment/

80.0% (16/20) 23.8% (5/21)

71.2% (42/59) 10.0% (6/60)

10.0% (6/60)

80.0% (16/20) 14.3% (3/21)

80.0% (16/20) 13.6% (3/22)

79.2% (27/44) 23.1% (9/39)

control)

87.5% (63/72) /

% Rradication

of Control

(19/24) PP

(63/80) PP

(16/21) PP

(42/60) PP

(42/60) PP

(16/21) PP

(16/21) PP

(37/52) PP

(27/47) PP

91.3% (21/23) ITT 79.2%

94.8% (73/77) ITT 78.8%

85.7% (18/21) ITT 76.2%

89.7% (52/58) ITT 70.0%

(25/30) ITT 80.0%

80.0% (48/60) ITT 70.0%

76.2% (16/21) ITT 76.2%

85.0% (17/20) ITT 76.2%

78.0% (39/50) ITT 71.2%

91.7% (33/36) ITT 57.4%

(no. of

% Eradication

of Treatment

(21/23) PP

(73/80) PP

(18/21) PP

(52/60) PP

(48/60) PP

(16/21) PP

(17/22) PP

(39/54) PP

(33/39) PP

patients)

ITT 91.3%

Corp., Tainan, Taiwan), bid, 5 weeks ITT 91.3%

ITT 85.7%

per capsule, tid, 10 days ITT 86.7%

Euroterapici S.p.A, Milan, Italy), bid, 14 days ITT 83.3%

Euroterapici S.p.A, Milan, Italy), bid, 14 days ITT 80.0%

Euroterapici, Milan, Italy), bid, 14 days ITT 76.2%

Beecham, Milan, Italy), bid, 14 days ITT 77.3%

OTC, Milan, Italy), tid, 14 days ITT 72.2%

14 days ITT 84.6%

(no. of

Bb99andPropionibacterium freudenreichii ssp.

shermanii JS(Valio Ltd, Helsinki, Finland), bid

during the eradication treatment and qd for

B. clausii(Enterogermina, Sanofi–Synthelabo

69 heat-killedL. acidophiluscontaining 510

Lacteol Fort (Bruschettini s.r.l., Genoa, Italy),

LactobacillusandBifidobacterium(President

Saccharomyces boulardii(Codex, SmithKline

‘6a probiotic preparation containing 6109

‘6a probiotic preparation containing 6109

‘69a probiotic preparation containing 610

‘69a probiotic preparation containing 510

‘69a probiotic preparation containing 210

‘6a combination of 5109 liveLactobacillus

65 mL milk-based fruit drink containing

rhamnosus LC705,Bifidobacterium breve

acidophilusandBiphidobacterium lactis

(Ferzym, Specchiasol, Milan, Italy), bid,

‘6200 mL AB-Yogurt containing 5109

‘69 cfu/mL ofLactobacillus GG,L.110

100 mL of fermented milk containing

‘10 cfu/mLL. casei DN-114001, qd,10

Lactobacillus GG(Giflorex, Errekappa

Lactobacillus GG(Giflorex, Errekappa

Lactobacillus GG(Giflorex, Errekappa

MethodsEradication TherapyProbiotics Supplementation

the following 3 weeks

14 days

(30 kg) bid, clarithromycin

7.5 mg/kg bid, amoxicillin

(6 weeks later) pantoprazole 40 mg bid,

ELISA (6 weeks later) lansoprazole 30 mg bid,

(8 weeks later) lansoprazole 30 mg bid,

bid, amoxicillin 1 g bid,

(6 weeks later) rabeprazole 20 mg bid,

(6 weeks later) rabeprazole 20 mg bid,

(6 weeks later) rabeprazole 20 mg bid,

(5–7 weeks later) rabeprazole 20 mg bid,

(5–7 weeks later) rabeprazole 20 mg bid,

(5–7 weeks later) rabeprazole 20 mg bid,

bid, amoxicillin 1 g tid,

bid, amoxicillin 1 g tid,

tid, amoxicillin 500 mg

bid, tinidazole 500 mg

bid, tinidazole 500 mg

bid, tinidazole 500 mg

bid, tinidazole 500 mg

bid, tinidazole 500 mg

clarithromycin 250 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

25 mg/kg bid, 7 days

(15–30 kg) or 20 mg

(4 weeks later) omeprazole 10 mg

bid, 7 days

bid, 7 days

bid, 7 days

bid, 7 days

bid, 7 days

tid, 7 days

7 days

7 days

7 days

+HistologyRUT/UBT

+HpSA/UBTHpSA

++UBTELISA/UBT

++HistologyRUT

Table 1.Characteristica of studies included in the meta-analysis.

+UBTELISA/UBT

+UBTELISA/UBT

+UBTHistology/

UBT-Histology

control)Patients Diagnositic

adults UBT/UBT

adults UBT/UBT

adults UBT/UBT

adults UBT/UBT

LB[18] 2002Italy42 (21/21)asymptomatic

SM[18] 2002Italy42 (22/21)asymptomatic

MS[18] 2002Italy42 (21/21)asymptomatic

Nista [20]2004Italy106 (54/52)asymptomatic

Myllyuom [21]2005Finland47 (23/24)asymptomatic

Armuzzi a[16]2001Italy60 (30/30)asymptomatic

(Taiwan) 160 (80/80)symptomatic

Sykora[22]2005UK86 (39/47)symptomatic

Canducci [15]2000Italy120 (60/60)symptomatic

Armuzzi b 17]2001Italy120 (60/60)symptomatic

children

adults

adults

adults

adults

adults

(references)Year Area Case Number

(treatment/

Sheu [19]2002China

Cremonini

Cremonini

Cremonini

Authors

40.6% (41/101)

41.1% (69/168)

26.3% (47/179)

59.7% (37/62) 22.6% (14/62)

59.7% (37/62)

(73/96) 9.5% (10/105)

patients) (treatment/

25.0% (4/16)

66.7% (10/15) 5.9% (1/17)

control)

80.0% (16/20) /

/

84.2% (16/19) /

37.5% (12/32) /

% Rradication

(93/101) ITT 72.3% (73/

101) PP 76.0%

(165/192) PP

(129/179) PP

of Control

(16/20) PP

(10/16) PP

(37/62) PP

(16/20) PP

(12/32) PP

85.0% (17/20) ITT 80.0%

52.9% (9/17) ITT 62.5%

71.0% (44/62) ITT 59.7%

96.2% (51/53) ITT 85.9%

ITT 72.1%

89.5% (17/19) ITT 80.0%

45.5% (15/33) ITT 37.5%

(165/192)

(129/164)

(no. of

85.9%

78.6%

% Eradication

of Treatment

ITT 88.6% (93/

105) PP 92.1%

(133/168) PP

(17/20) PP

(44/62) PP

(51/53) PP

(17/20) PP

(15/33) PP

patients)

55730 (Reuterin, No´os), qd, 4 weeks ITT 85.0%

55730 (Reuterin, No´os), bid, 7 days ITT 52.9%

Turkey), bid, 14 days ITT 71.0%

andLactobacillus rhamnosus, bid, 10 days ITT 96.2%

ITT 79.2%

qd, 20 days ITT 85.0%

andLactobacillus casei ITT 45.5%

(133/152)

(9/17) PP

(no. of

87.5%

.5 cfu/mL),B. longumHY 8001 (106 cfu/10

‘..5 cfu/mL),L. caseiHY 2743 (HY 2177 (10

‘.mL), andS. thermophilusB-1 (108 cfu/mL)

150 mL Will yogurt containingL. acidophilus

Lacidofil containingLactobacillus acidophilus

(Reflor, Sanofi-Synthelabo Ilac A.S., Istanbul,

(Korea Yakult Company Limited; Chunan-Si,

‘each tablet containing 108L. reuteriATCC

‘8L. reuteriATCCeach tablet containing 10

250 mg sachets ofSaccharomyces boulardii

‘‘669 L.salivarius, 1109L. acidophilus,110

‘6Lactobacillus plantarum, 2109 L. reuterii,

8 cfu ofL. reuterieach pill containing 10

‘‘669L. caseisubsp.rhamnosus, 2109210

ATCC 55730(SD2112) (BioGaia, Sweden),

‘69Streptococcus termophilus, and510

250 mL commercial yogurt containing

69bLf 20 mg bid; Pb containing 510

Bifidobacterium infantisandB.longum,

7 cfu/mLBifidobacterium animalis10

Chungnam, S. Korea), qd, 3 weeks

‘61109 L.sporogenes, bid, 7 days

MethodsEradication TherapyProbiotics Supplementation

15 mg/kg qd, amoxicillin

(4–6 weeks later) lansoprazole 30 mg bid,

omeprazole 1 mg/kg/d,

bid, amoxicillin 1 g bid,

bid, amoxicillin 1 g bid,

500 mg bid, amoxicillin

500 mg bid, amoxicillin

500 mg bid, amoxicillin

50 mg/kg/d for 5 days;

later) omeprazole 1 mg/kg/d

plus amoxicillin 1 g, bid

clarithromycin 500 mg

clarithromycin 500 mg

tinidazole 20 mg/kg/d

(4 weeks later) rabeprazole 20 mg bid

for 5 days; rabeprazole

clarithromycin 500 mg

(8 weeks later) esomeprazole 20 mg

(4 weeks later) omeprazole 1 mg/kg

50 mg/kg qd, 7 days

(6 weeks later) pantoprazole 40 mg

(6 weeks later) lansoprazole 30 mg

for the next 5 days

bid, clarithromycin

bid, clarithromycin

bid, clarithromycin

500 mg bid for the

qd, clarithromycin

bid and tinidazole

(4 weeks later) standard PPI bid,

15 mg/kg/d and

1 g bid, 14 days

1 g bid, 10 days

plus amoxycillin

1 g bid, 7 days

clarithromycin

next 5 days

20 mg bid,

7 days

7 days

+HistologyUBT/UBT

UBT/UBT (20 days

++HistologyHpSA

++HistologyRUT

+UBT/HpSAUBT

+HpSAUBT/UBT

adults Histology/UBT

adults Histology/UBT

control)Patients Diagnositic

children UBT/UBT

adults UBT/UBT

Goldman[23]2006Argentina 65 (33/32)symptomatic

Zieminak[25]2006Poland245 (53/192)symptomatic

[28] 2008Italy40 (20/20)symptomatic

LB[30] 2008Italy33 (17/16)symptomatic

[27] 2007Italy206 (105/101)symptomatic

Kim[29]2008Korea347 (168/179)symptomatic

Lionetti[24]2006Italy40 (20/20)symptomatic

Cindoruk[26]2007Turkey124 (62/62)symptomatic

children

adults

adults

adults

(references)Year Area Case Number

(treatment/

Table 1.Cont.

De Bortoli N

Scaccianoce

Francavill

Authors

30.9% (13/42)

68.8% (22/32) 52.9% (18/34)

40.6% (13/32)

patients) (treatment/

66.7% (10/15) 20.0% (3/15)

25.0% (4/16)

71.2% (79/111) 5.2% (6/115)

3.5% (4/114)

75.6% (34/42) 8.3% (4/48)

control)

/

/

77.4% (24/31) /

/

89.1% (82/92) /

% Rradication

(237/331) PP

of Control

(79/114) PP

(18/36) PP

(20/38) PP

(24/31) PP

(34/42) PP

(10/16) PP

(22/32) PP

(82/95) PP

ITT 50.0%

ITT 52.6%

ITT 77.4%

ITT 71.6%

100% (45/45) ITT 80.9%

53.3% (8/15) ITT 62.5%

67.6% (23/34) ITT 68.8%

ITT 69.3%

77.2% (71/92) ITT 86.3%

(237/296)

(no. of

(18/36)

(20/38)

50.0%

52.6%

80.0%

% Eradication

of Treatment

(264/330) PP

(95/115) PP

(36/46) PP

(25/38) PP

(26/31) PP

(45/48) PP

(23/34) PP

(71/98) PP

patients)

BifidobacteriumDN-173 010, qd, 14 d ITT 65.8%

bid, 8 days ITT 83.9%

Korea), tid, 4 weeks ITT 80.0%

4 weeks ITT 93.8%

ITT 53.3%

.(10 cfu/g), bid, 4 weeks ITT 82.6%

boulardii, qd, 14 days ITT 72.4%

9 cfu/mLLactobacillus GG, bid, 7 daysITT 67.6%10

250 mL kefir, bid, 141dITT 78.3%

(264/309)

(8/15) PP

(95/106)

(no. of

(36/46)

(25/38)

(26/31)

78.3%

65.8%

83.9%

85.4%

89.6%

‘‘669),Bifidobacterium infantis(2109),(210

yogurt containing 112 gL. gasseriOLL2716

capsules containingsaccharomyces250 mg

Saccharomyces boulardii(Enterol; Biocodex,

6capsule containing 5109L. acidophilus,

Gentilly Cedex, France), 250 mg/day, bid,

Lacteol (BioSau´de laboratories, Portugal),

‘69), andStreptococcus termophilus(510

669),L. reuteri(2109),plantarum(510

‘63109 cfu/gSaccharomyces boulardii

‘10 cfu/g125 ml yogurt containing 10

a probiotic mixture withLactobacillus

Lactobacillus casei subsp.Rhamnosus

(Bioflor250, Kuhnil Pharmacy, Seoul,

69),Lactobacillus acidophilus(110

‘6Lactobacillus sporogenes(1109);

69),Bifidobacterium longum(210

6Lactobacillus salivarius(1109),

MethodsEradication TherapyProbiotics Supplementation

5 g/dose, bid, 7 days

10 mg/kg bid, amoxicillin

(4 weeks later) pantoprazole 40 mg bid,

esomeprazole 0.5 mg/kg

(4–6 weeks later) lansoprazole 30 mg bid,

later) lansoprazole 30 mg bid,

(5 weeks later) lansoprazole 30 mg bid,

bid, amoxicillin 1 g bid,

bid, amoxicillin 1 g bid,

bid, amoxicillin 1 g bid,

bid, amoxicillin 1 g bid,

bid, amoxicillin 1 g bid,

bid, amoxicillin 750 mg

500 mg bid, amoxicillin

(4 weeks later) omeprazole 20 mg bid,

later) rabeprazole 10 mg bid,

UBT (4–6 weeks later) omeprozole 0.5 mg/kg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 200 mg

25 mg/kg bid, 7 days

$UBT (6 weeks later) esomeprazole 20 mg

amoxicillin 25 mg/kg

bid, clarothromycin

bid, clarithromycin

bid, clarithromycin

1 g bid, 8 days

(4–6 weeks later) omeprozole or

7.5 mg/kg bid,

bid, 7–10 days

bid, 7 days

14 days

14 days

14 days

7 days

7 days

adults Histology-RUT-culture/

++HistologyRUTUBT/

+UBTHpSA (8 weeks

+Histologyculture/

adults UBT/UBT (45 days

adults Histology/HpSA

+HistologyRUT/

+HistologyRUT

adults Histology/UBT

children Histology/UBT

control)Patients Diagnositic

adults UBT/UBT

Szajewsk[32]2009Poland66 (34/32)asymptomatic

Madeiros[36]2011France62 (31/31)asymptomatic

Hurduc[31]2009Romania90 (48/42)symptomatic

MS[30] 2008Italy31 (15/16)symptomatic

Bekar[35]2011Turkey82 (46/36)symptomatic

Ozdil[37]2011Turkey193 (98/95)symptomatic

Yasar[34]2010Turkey76 (38/38)symptomatic

Song[33]2010Korea661 (330/331)symptomatic

Deguchi[38]2012Japan229 (115/114)symptomatic

children

children

adults

(references)Year Area Case Number

(treatment/

Table 1.Cont.

Scaccianoce

Authors

18.9% (17/90)

16.7% (15/90)

64.5% (20/31) 64.7% (22/34)

67.6% (23/34)

92.8% (65/70) 39.7% (29/73)

65.8% (50/76)

58.2% (32/55)

71.2% (37/52)

patients) (treatment/

61.5% (48/78) 1.3% (1/78)

0.0% (0/79)

control)

76.5% (26/34) /

/

69.7% (23/33) /

% Rradication

of Control

(73/106) PP

(26/34) PP

(23/33) PP

(48/79) PP

(73/90) PP

(20/34) PP

(65/76) PP

(44/52) PP

88.2% (30/34) ITT 76.5%

83% (83/100) ITT 68.9%

90.9% (30/33) ITT 69.7%

81.6% (62/76) ITT 60.7%

82.1% (69/84) ITT 81.1%

61.3% (19/31) ITT 58.8%

94.4% (67/71) ITT 85.5%

96.1% (49/51) ITT 84.6%

(73/106)

(no. of

(73/86)

(44/49)

68.9%

84.9%

89.8%

% Eradication

of Treatment

(83/100) PP

(30/34) PP

(30/33) PP

(62/78) PP

(69/90) PP

(19/34) PP

(67/73) PP

(49/55) PP

patients)

ITT 88.2%

ITT 90.9%

Lactobacillus acidophilus, qd, 14 d ITT 79.5%

ITT 76.7%

ITT 91.8%

ITT 89.1%

150 mg probiotic yogurt, bid, 7 daysITT 55.9%

‘69B. infantis2036, bid, 7 daysITT 83%310

(no. of

bulgaricus),Bifidobacteriumstrains (B. breveand

‘8 CFU/percapsule, bid,total viable count is 10

Balance capsue containingLactobacillusstrains

68Bifidobacterium bifidum,acidophilus, 510

‘69 CFUsStreptococcus faecium,and 1.2510

(L. casei, L. rhamnosus, L. acidophilus, andL.

B. longum), andStreptococcus thermophiles,

‘9LactobacillusMilano, Italy), containing 10

‘669L. salivarius, 1109L. acidophilus,110

‘6Lactobacillus plantarum, 2109L. reuterii,

‘669L.casei subsp.rhamnosus, 2109210

‘61.25109 CFUsLactobacillus acidophilus,

‘61.25109 CFUsLactobacillus rhamnosus,

thermophilus,Bifidobacterium infantisand

‘probiotic combination (restore, 109 cfu/

each sachet, Protexin Co, UK) containing

‘69 CFUsBifidobacterium bifidum1.2510

Lactogermine plus (Humana Italia s.p.a.,

‘6the PB supplement containing 5109

‘69Streptococcus termophilus, and510

Bifidobacterium infantisandB. longum,

109Streptococcus thermophilus, 109

Lactobacillus acidophilus,Lactobacillus

the probiotic compound containing

rhamnosus,Lactobacillus bulgaricus,

‘69L. sporogenes, qd, 7 days110

Bifidobacterium breve, qd, 7 days

Lactobacillus casei,Streptococcus

MethodsEradication TherapyProbiotics Supplementation

67probiotics, containing 310

Lactobacillus bulgaricus

bid, 30 days

14 days

bid, furazolidone 200 mg

20 mg bid, clarithromycin

(4 weeks later) omeprazole 1 mg/kg qd,

bid, tetracycline 500 mg

later) pantoprazole 40 mg qd,

(8–10 weeks later) esomeprazole 20 mg bid

for 5 days; esomeprazole

bid, amoxicillin 1 g bid,

bid, amoxicillin 1 g bid,

bid, amoxicillin 1 g bid,

240 mg bid, amoxicillin

UBT (4 weeks later) omeprazole 20 mg bid,

(4 weeks later) omeprazole 20 mg bid,

later) omeprazole 20 mg bid,

plus amoxicillin 1 g, bid

(4–8 weeks later) omeprazole 0.5 mg/kg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

clarithromycin 500 mg

furazolidone 3 mg/kg

15 mg/kg bid, 7 days

tinidazole 500 mg bid

amoxicillin 50 mg/kg

UBT (8 weeks later) lansoprazole 30 mg

bismuth subcitrate

bid, clarithromycin

for the next 5 days

500 mg bid and

bid, amoxicillin

25 mg/kg bid,

1000 mg bid,

bid, 14 days

bid, 7 days

bid, 7 days

7 days

7 days

7 days

+HistologyHpSA/HpSA

HpSA/UBT (6–8 weeks

+++HistologyRUTUBT

+HistologyRUT/HpSA

++HistologyRUTUBT/

++HistologyRUTUBT/

+HistologyUBT/UBT

+HistologyRUT/UBT

adults UBT/UBT (4 weeks

control)Patients Diagnositic

Du YQ[39]2012China157 (78/79)symptomatic

Emirates 206 (100/106)symptomatic

Ahmad[43]2013Iran66 (33/33)symptomatic

Navarro[44]2013Brazil107 (55/52)symptomatic

Tolone[42]2012Italy68 (34/34)symptomatic

Manfredi[40]2012Italy149 (73/76)symptomatic

Mirzaee[41]2012Iran68 (34/34)symptomatic

Shavakhi[45]2013Iran180 (90/90)symptomatic

children

children

adults

adults

adults

adults

adults

(references)Year Area Case Number

(treatment/

Dajani[46]2013United

Arab

Table 1.Cont.

Authors

(no. of patients)

% Side Effects

(treatment/

control)

% Rradication

of Control

patients)

(no. of

% Eradication

of Treatment

patients)

(no. of

23.5% (12/51)

64.4% (29/45) 10.2% (5/49)

(29/51) PP

83.7% (26/43) ITT 56.9%

(36/49) PP

ITT 73.5%

each packs of probiotics containing 2 gL.

‘62 gB. bifidum-12(4.3108 cfu/100 g),

69 cfu/100 g) andacidophilus-5(4.710

,.5 years old, qd;5 years old, bid,

MethodsEradication TherapyProbiotics Supplementation

14 days

amoxicillin 30–50 mg/kg

bid, and metronidazole

(6 weeks later) PPI 0.6–0.8 mg/kg bid,

15–20 mg/kg bid for

10–15 mg/kg bid,

children, 14 days

penicillin allergic

clarithromycin

control)Patients Diagnositic

children UBT/UBT

Wang YH[47]2013China100 (49/51)symptomatic

(references)Year Area Case Number

(treatment/

doi:10.1371/journal.pone.0111030.t001

Table 1.Cont.

Authors

in an intervention group who received probiotics plus the identical antibiotic therapy; 4) there should be data on successful eradication rates available; 5) for the analysis of side effects data on occurrence of side effects during treatment were required as well. Exclusion criteria were as follows: 1) the design and the definition of the trials were obviously different from those specified above; 2) essential information was not provided; 3) papers were letters, commentaries, editorials, reviews and duplicate publications; 4) articles were written in a language other than English.

Data Extraction

Two authors extracted data independently from all eligible papers. For conflicting evaluations, another author was consulted to solve the dispute and a final decision was made by the majority of the votes. The data extracted included authors, year of publication, base characteristics of the patients, details of the H. pylori eradication therapy, details related to interventions, primary and secondary outcomes, and confirmation methods of H. pylori infection. Extracted information was entered into a database.

Assessment of Study Quality

We used the Cochrane Tool of Bias [15] to assess study quality. Two authors independently evaluated all studies. Results were then compared and discussed to form consensus. If consensus could not be reached, another author was consulted and a decision made by the majority of the votes.

Statistical Analysis

Statistical heterogeneity was analyzed with Chi-squared distributed Chochran’s Q and the I-squared statistics (I2=100%6 (Qdf)/Q). I-square indicates the percentage of variation across studies due to hetereogeneity as opposed to chance. We assumed sufficient homogeneity to apply a fixed-effects model (Mantel-Haenszel) for the meta-analysis if I-square was under 40% and/or Q was not significant at p,0.05. Otherwise, we opted for a random-effects model (DerSimonian and Laird). The jacknife was used to assess the influence of individual studies, i.e. estimation of the overall effect was repeated while omitting one study at each time. Funnel plots and Harbord’s modified test for small study effects were used to assess publication bias [16]. The influence of the probiotic strain applied (multi-strain interventions were excluded from this analysis), blinded vs. non-blinded trials, and in the case of eradication rates, the effectiveness of antibiotic therapy in the control group (eradication rate .80% vs. ,80%) was assessed with sub-group analysis. Concerning eradication rates, metaregression was used to determine the influence of studies in pediatric vs. adult as well as symptomatic and asymptomatic populations. All statistical analysis was performed with STATA 12.0 (Stata Corporation, Texas, USA).

Results Study Characteristics

The bibliographic search yielded 1114 articles, 40 of which were reviewed in full text (Figure 1) [17–56]. Of these studies, 33 RCTs [17–49] met the inclusion criteria for the analysis of eradication rates and 20 were eligible for analyzing side effects. These trials randomized a total of 4459 patients, 4261 of which were followed up. Nine studies enrolled only children [24– 26,33,34,36,44,45,49] and 24 were undertaken exclusively with adults [17–23,27–32,35,37–43,46–48]. From all the included trials, 29 used PPI-triple therapy [17–25,27–29,31–41,43– 46,48,49], three used sequential therapy [26,30,42] and one used bismuth-quadruple therapy [47]. These studies were undertaken

1. Figure 2. Risk of bias graph (A): review authors’ judgements about each risk of bias item presented as percentages across all included studies. (B) Risk of bias summary: review authors’ judgements about each risk of bias item for each included study. (+) = low risk of bias, (?) = unclear, (-) = high risk of bias.

1. doi:10.1371/journal.pone.0111030.g002

in Argentina [25], Brazil [46], mainland China [41,49], China (Taiwan) [21], Finland [23], France [38], Iran [43,45,47], Italy [17–20,22,26,29,30,32,42,44], Japan [40], Korea [31,35], Poland [27,34], Romania [33], Turkey [28,36,37,39], United Kingdom [24] and the United Arabian Emirates [48].(Table 1)

Trial Quality Assessment

Figure 2A shows authors’ judgements for each Cochrane risk of bias item and Figure 2B presents the Cochrane risk of bias score for each citation included.

Eradication Rates

Eradication risk ratios (RRs) were available for 4459 patients (2189 in the probiotics supplementation group and 2270 in the control group). Heterogeneity was found to be low for the overall incidence of eradication rates in ITT analysis (x2=42.97, p=0.167, I2=18.5%) but higher for PP analysis (x2=58.55, p=0.006, I2=41.9%). Therefore, a fixed effects model based on Mantel-Haenszel’s estimation method was used in the case of ITT and a random effects model in the case of PP analysis. Nonetheless, the pooled RRs from intention-to-treat (ITT) and from pre-protocol (PP) analysis for the probiotics supplementation group over controls were very similar with 1.122 (95% CI 1.086– 1.159) and 1.114 (95% CI 1.070–1.159) respectively (Figure 3A&B).

Omitting individual studies from the meta-analysis did not change occurrence of RRs being significantly higher than 1. Funnel-plot and Harbord’s modified test showed no evidence for publication bias (p =0.64). Neither the age of the population (pediatric vs. adult, p=0.127) nor symptom status (p=0.314) played a role according to the meta-regression.

Studies used different strains of probiotics with 13 applying Lactobacillus [17–20,24,26,27,30,32,34,38,40,41], two using Bifidobacterium [36,48], one using Bacillus clausii [22], five using Saccharomyces [20,28,33,35,39] and 15 using multistrain [20,21,23,26,29,31,32,37,42–47,49]. Among those studies applying individual probiotic supplementation, four specific species were effective: Lactobacillus acidophilus (pooled RR=1.235, 95% CI 1.090–1.400), Lactobacillus casei DN-114001 (pooled RR=1.473, 95% CI 1.13–1.949), Lactobacillus gasseri (pooled RR=1.192, 95% CI 1.028–1.382), Bifidobacterium infantis 2036 (pooled RR=1.205, 95% CI 1.031–1.408). While double-blinded (pooled RR=1.118, 1.045–1.196) and non-blinded trials (pooled RR=1.120, 95% CI 1.080–1.162) basically arrived at the same results, sub-group analysis by effectiveness of the antibiotic therapy in the control group revealed that supplementation with probiotics solely increased eradication rates in relatively ineffective therapies.

Another sub-group analysis was done according to the effectiveness of eradication regimens. The less effective antibiotic therapies were, the more useful probiotic supplementation was: when eradication rate ,60%, pooled RR=1.28, 1.12–1.45; when eradication rate within 60%–69%, pooled RR=1.18, 1.10–1.27; when eradication rate within 70%–79%, pooled RR=1.11, 1.06– 1.17; while if eradication rate over 80%, the supplementation was useless (pooled RR=1.01, 0.96–1.77). (Figure 4)

Side Effects

There were 20 trials (2487 patients, 1269 in the probiotics supplementation group and 1218 in the control group) which

provided data on the overall incidence of side effects. Significant heterogeneity was found for the overall occurrence of side effects (I2=72.2%, P,0.001). Therefore, the random effects model was used. The pooled RR in the probiotics supplementation over control was 0.735 (95% CI 0.598–0.902) (Figure 5). While Harbord’s modified test for publication bias was insignificant (p =0.17), the Funnel Plot did reveal some asymmetry (Figure 6): Small studies showing a considerable reduction in side-effects occur more often than those only showing a small reduction. When stratified by probiotics, only the pooled RR for Saccharomyces boulardii indicates a significant reduction in side effects (0.335, 95% CI 0.220–0.510; LB: 0.892, 95% CI 0.632–1.259; MS: 0.760, 95% CI 0.568–1.017; BB: 0.895: 0.737–1.087).

Only eight studies reporting the overall incidence of side effects were blinded and solely results from non-blinded trials provided evidence for the reduction of side-effects (pooled RR=0.589, 95% CI: 0.412–0.842), while double blinded studies did not (pooled RR=0.889, 95% CI: 0.728–1.085) (Figure 7).

Discussion

This is the largest meta-analysis on the effects of probiotics on H. pylori eradication and side effects conducted to date. The quality of the trials was medium to low and no study had a low risk of bias across all Cochrane criteria. Overall, this analysis of 33 RCTs suggests that supplementation of antibiotic therapy with probiotics increases eradication rates compared to a placebo or no intervention. However, regarding individual probiotic strains, this could only be confirmed for several strains of Lactobacilus and one strain of Bifidobacterium. Moreover, probiotics only demonstrated additional effects if the eradication rates in the control groups were relatively low, i.e. cases where the antibiotic therapy was relatively ineffective. Although, we found an overall decrease of total side effects, this held true only for Saccharomyces boulardii and nonblinded RCTs.

In comparison with previously published meta-analysis

1. [8,9,10,11,12,13,14], our updated study represents the most comprehensive analysis. Various potential influence factors were taken into consideration such as age, symptom status, eradication therapy regime, eradication rate, bacterial strain and blinding method. Moreover, from the previous studies, six
2. [8,10,11,12,13,14] excluded trials using Quadruple therapy as the co-intervention and none performed subgroup analysis with regard to the efficacy of antibiotic therapy. Moreover, sensitivity analysis for side effects had not been conducted so far. Our results confirm previous meta-analyses only to some extent. At a first glance, probiotics seem effective in increasing eradication rates and decreasing side effects. At a closer look, however, evidence only supports these claims for specific probiotic strains, ineffective antibiotic therapies and low-quality trials (i.e. non-blinded studies) as far as side effects are concerned.

Before Marshall found H. pylori in 1984 [57], the stomach was considered to be a sterile organ due to its low pH level. In the aftermath, ‘H. pylori has been intensively studied and recent sequencing analysis of other gastric microbiota shows that H. pylori is not alone’ [58]. Although there are only 10

2-4 cfu/g in the gastric mucosa [59], these commensal bacteria can play an important role in maintaining human health. Probiotic supple-

‘

1. Figure 3. The effect of probiotics supplementation vs. without probiotics on eradication rates by intention-to-treat analysis in (A). RR, risk ratio; CI, confidence interval. The triangles represent individual studies and the size of the triangle represents the weight given to each study in the meta-analysis. The diamond represents the combined results. (B) The effect of probiotics supplementation vs. without probiotics on eradication rates by pre-protocol analysis. RR, risk ratio; CI, confidence interval. The triangles represent individual studies and the size of the triangle represents the weight given to each study in the meta-analysis. The diamond represents the combined results.

mentation as an approach to manipulate gastrointestinal flora has been intensively studied.

While all previously conducted meta-analysis concluded that supplementation of antibiotic therapy with probiotics is effective in increasing eradication rates [8,9,10,11,12], our study identified only four effective individual strain: Lactobacillus acidophilus (Pubmed ID: 190198), Lactobacillus casei DN-114001 (Pubmed ID: 35628), Lactobacillus gasseri (Pubmed ID: 35528), Bifidobac-

terium infantis 2036 (Pubmed ID: 41468). However, more than one trial was conducted only for Lactobacillus acidophilus so far, limiting the generalizability of the findings regarding the other three strains. While Lactobacillus GG had previously been reported to be an effective strain [8], we do reach the same conclusion here.

Moreover, our results suggest that probiotic supplementation is only useful in less effective (eradication rate ,80%) antibiotic

1. Figure 5. The effect of probiotics supplementation vs. without probiotics on total side effects. RR, risk ratio; CI, confidence interval. The triangles represent individual studies and the size of the triangle represents the weight given to each study in the meta-analysis. The diamond represents the combined results.

therapies. Acceptable success rates have often been defined as 80% or more on an ITT basis [2]. In those effective regimens, probiotic supplementation may not be needed. On the other hand, however, increasing resistance to antibiotics, for example clarithromycin, has been noted [2]. Probiotics supplementation may be a potential approach for eliminating resistant strains. However, no study included in this meta-analysis reported the detection of antibiotic resistance. Further research is warranted to clarify this issue.

The occurrence of adverse effects is one of the major drawbacks of antibiotic treatment. Although antibiotics may modify the composition of intestinal bacteria, broad spectrum antibiotics also often lead to gastrointestinal side effects [20,60]. Results regarding the effectiveness of probiotics in reducing side effects from previous meta-analyses have been inconclusive [8,9,10,11,12]. While we found that probiotics reduce the overall occurrence of side effects in the pooled data, this result must be taken with caution. Only one specific strain significantly reduced side effects and this overall result was only confirmed for non-blinded trials. While eradication rates can be determined with objective measures, assessment of side effects must rely on patients’ subjective reporting. When patients are not blinded, their reports are likely to be biased due to awareness of a respective intervention.

1. Figure 7. Meta-analysis of side effects by double-blinded and nondouble-blinded trials. RR, risk ratio; CI, confidence interval. The triangles represent individual studies and the size of the triangle represents the weight given to each study in the meta-analysis. The diamond represents the combined results.

1. doi:10.1371/journal.pone.0111030.g007

Limitations of this meta-analysis include a limited number of high quality trials that could be analyzed, particularly regarding individual probiotic strains and side effects. Only one controlled trial was conducted for the following specific species Bacillus clausii, Bifidobacterium DN-173 010, Lactobacillus casei DN114001, Lactobacillus gasseri, Bifidobacterium infantis 2036. Accordingly, results regarding those strains need to be interpreted with caution. Thirteen of the analyzed trials did not provide data on overall occurrence of side effects. Moreover, tools used for measuring side effects and reporting individual side effects largely differed across studies. From 20 studies reporting on the overall incidence of side effects, only eight were blinded.

Conclusions

The pooled data suggests that supplementation with specific strains of probiotics compared with eradication therapy may be considered as an option for increasing eradication rates of H.

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Supporting Information

Checklist S1 PRISMA Checklist. (DOC)

Acknowledgments

We acknowledge the help of Dr. Andrew Pennycott in correcting the manuscript for grammar and style.

Author Contributions

Conceived and designed the experiments: YD GZ. Performed the experiments: YD XZ. Analyzed the data: YD JDR. Contributed reagents/materials/analysis tools: XZ. Wrote the paper: YD JDR GZ.

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