www.mdpi.com/journal/antibiotics Review

Department of Medicinal Chemistry, University of Washington, VA Puget Sound Healthcare System, 1660 S. Columbian Way, S-152, Seattle, WA 98108, USA; E-Mail: [email protected]; Tel.: +1-206-277-1780; Fax: +1-206-764-2935

Academic Editor: Yung-Fu Chang Received: 9 March 2015 / Accepted: 2 April 2015 / Published: 13 April 2015

Abstract: Clostridium difficile infections are a global clinical concern and are one of the leading causes of nosocomial outbreaks. Preventing these infections has benefited from multidisciplinary infection control strategies and new antibiotics, but the problem persists. Probiotics are effective in preventing antibiotic-associated diarrhea and may also be a beneficial strategy for C. difficile infections, but randomized controlled trials are scarce. This meta-analysis pools 21 randomized, controlled trials for primary prevention of C. difficile infections (CDI) and four trials for secondary prevention of C. difficile recurrences and assesses the efficacy of specific probiotic strains. Four probiotics significantly improved primary CDI prevention: (Saccharomyces boulardii, Lactobacillus casei DN114001, a mixture of L. acidophilus and Bifidobacterium bifidum, and a mixture of L. acidophilus, L. casei and L. rhamnosus). None of the tested probiotics significantly improved secondary prevention of CDI. More confirmatory randomized trials are needed to establish if probiotics are useful for preventing C. difficile infections.

Keywords: probiotics; clostridium difficile infections; diarrhea; meta-analysis

Clostridium difficile infections (CDI) have been a difficult clinical issue for over four decades, with a nearly one-half a million cases per year in the U.S., resulting in 29,000 deaths per year, increased costs of healthcare, outbreaks of CDI in hospitals and long-term care facilities and 83,000 cases of recurrent

CDI per year the U.S. [1]. Prevention of CDI has relied on multidisciplinary infection control practices, but guidelines have been found to be difficult to implement globally [2,3].

An innovative strategy to prevent CDI involves using probiotics at the same time antibiotics are given. One recent quasi-experimental study was done in Canada, which gave the mixture of L. acidophilus, L. casei and L. rhamnosus (BioK+) to all patients receiving antibiotics at two hospitals over time and found a significant reduction in the incidence of CDI cases and recurrences at these facilities [4]. Some probiotic strains have been found to be effective for prevention of antibiotic-associated diarrhea (AAD) and for the treatment of CDI [5]. Since CDI accounts for nearly one-third of all AAD cases, this strategy is worth evaluating since CDI persists in impacting our healthcare systems. However, studies of CDI prevention and probiotics have been largely limited to CDI being evaluated as a secondary outcome of AAD studies, leading to underpowerment for CDI outcomes [6]. The technique of meta-analysis allows the pooling of different trials to overcome the low power bias due to the small individual sample sizes. In this paper, randomized, controlled trials of good quality will be pooled to assess probiotic strains for primary and secondary prevention of CDI.

1. 2.1. Initial Screening of Data Search

The literature review yielded 474 abstracts relating to probiotics and CDI that were screened for inclusion. Of those, 323 were excluded after initial screening according to our exclusion criteria (Figure 1): reviews (n = 152), pre-clinical animal models or phase two studies for pharmacokinetics, formulation or safety (n = 81), no control group or case series (n = 54), commentaries (n = 20) or not randomized (n = 16).

1. 2.2. Secondary Screening of Full Articles
2. 2.3. Included Trials
3. 2.4. Study Design

Degree of blinding in primary prevention. Of the 21 trials, most (n = 18, 86%) were double-blinded (used placebos that were of identical appearance as the probiotic formulation), while three (14%) had open controls, which used either no treatments [20,25] or had an active control with another strain (L. casei Shirota) of probiotic as a comparator [31].

Degree of blinding in secondary prevention. All four trials had double-blinded, placebo controlled controls.

Attrition in primary prevention trials. As shown in Table 2, attrition ranged from 0%–43% in the 21 trials, drop-outs typically due to adverse events or loss to follow-up. Six trials (29%) reported no attrition, eight (38%) had low attrition frequencies from 1%–10%, four (19%) had moderate attrition from 12%–26%, while three (14%) reported high attrition frequencies (38%–43%). Of the 14 trials with attrition, only two (14%) used Intent-to-Treat (ITT) analysis [20,23], while most (86%) used as-perprotocol (APP) analysis.

Attrition in secondary prevention trials. Of the four trials, three reported attrition rates from no attrition [39,41], to 16% attrition [38], but attrition was not reported in one trial [40].

Eligible Antibiotic Exposures

Daily Dose of Probiotic (cfu/day)

Duration of Probiotic Treatment

Duration Follow-up

CDI in Probiotic Group (%)

CDI in Control Group (%)

Probiotic

Reference

Primary prevention of CDI L. casei Shirota nr 6.5 ×109 duration + 1 week 4 weeks 9/76 (0%) ns 1/82 (1.2%) Wong 2014 [7]

L. acidophilus mixed, 77% beta-lactams 6 ×1010 2 weeks 0 0/23 (0%) ns 1/16 (6.2%) Safdar 2008 [8] L. plantarum 299v mixed 1 ×1010 duration + 1 week 1 week 1/74 (1.3%) ns 0/76 (0%) Lonnermark 2010 [9] Bacillus clausii mixed, beta-lactams 4 ×109 duration 6 weeks 0/162 (0%) ns 1/160 (0.6%) Destura 2008 [10] C. butyricum 588 mixed, 87% beta-lactams 1–4 ×107 6 days 0 0/83 (0%) ns 0/27 (0%) Seki 2003 [11]

L rhamnosus (strains E/N, Oxy, Pen) mixed, mostly pen and ceph 4 ×1010 duration (x = 8 day) 2 weeks 3/120 (2.5%) ns 7/120 (5.8%) Ruszczynski 2008 [12] L. rhamnosus GG +L. acido. La5 + Bifido. lactis Bb12 mixed, nr types 5 ×1010 2 weeks 0 0/34 (0%) ns 1/29 (3.4%) Wenus 2008 [13]

L. acidophilus (CUL 60 and CUL 21) + Bifido. bifidum CUL20 +Bifido. lactis CUL34

mixed, 21% single, 70% pen 6 ×1010 3 weeks 10 weeks 12/1470 (0.8%) ns 17/1471 (1.2%) Allen 2013 [14]

VSL#3 mixed, 75% pen 9 ×1011 duration + 1 week 3 weeks 0/117 (0%) ns 0/112 (0%) Selinger 2013 [15] Secondary prevention of CDI

4/11 (36%) recurred

L. plantarum 299v mixed 5 ×1010 5.4 weeks 4.5 weeks

6/9 (67%) Wullt 2003 [16]

Abbreviations: Bifido., Bifidobacterium; C., Clostridium; cfu, colony-forming unit; L., Lactobacillus; ns, not significant; VSL#3, contains Bifido. breve, Bifido. longum, Bifido. infantis, L. acidophilus, L. plantarum, L. casei, L. bulgaricus, Strept. thermophilus; x, mean.

Single or Multiple Types of Inciting Antibiotics

Most Common Type of Antibiotic

Enrolled population % Attrition

Type(s) of Infections Reference

adults, I 43 59% multiple 36% cepha mixed, nr Surawicz 1989 [17] adults, I 38 82% multiple beta-lactams mixed, nr McFarland 1995 [18] elderly, I 4.2 nr nr nr Lewis 1998 [19] adult, O 3.3 100% multiple amox and clarithromycin H. pylori infections Duman 2005 [20]

pediatric, I&O 8.5 nr 41% cepha 68% resp, 29% otitis media Kotowska 2005 [21] adults, I 0 nr 83% beta-lactams nr Can 2006 [22] adults, O 4.6 100% single 100% amox 88% resp Bravo 2008 [23] adults, I 26 69% single mixed, nr nr Pozzoni 2012 [24]

pediatric, I 15 nr 52% cepha resp Shan 2014 [25]

pediatric, O 28.7 nr 66% amox 74% otitis media, 26% resp Arvola 1999 [26] adults, I 11.6 nr 69% beta-lactams nr Thomas 2001 [27] adults, I 0 nr cepha nr Miller 2008a [28] adults, I 0 69% single 50% cepha nr Miller 2008b [28] adults, I 5.5 only 34% with VAP on abx nr pneumonia Morrow 2010 [29] adults, I 19 61% single 66% amox or cepha 49% resp Hickson 2007 [30] adults, I 0 nr 60% amp or cepha 80% resp or GU Dietrich 2014 [31] elderly, I 8 nr nr nr Plummer 2004 [32] adults, I 0 nr mixed nr Rafiq 2007 [33] adults, I 0 nr 48% ceph nr Stein 2007 [34] adults, I 0 nr 59% quinolones 92% resp Beausoleil 2007 [35]

adults, I&O 7.4 nr 78% beta-lactams 39% resp Sampalis 2010 [36] adults, I 9 nr 41% cepha 47% resp Gao 2010a [37] adults, I 7 nr 37% cepha 47% resp Gao 2010b [37]

Abbreviations: amox, amoxicillin; amp, ampicillin; cepha, cephalosporin; GU, genital-urinary infections; I, inpatient; nr, not reported; O, outpatient; resp, respiratory infections; VAP, ventilator-associated pneumonia.

1. 2.5. Patient Population
2. 2.6. Antibiotic Exposure
3. 2.7. Interventions

Probiotics in primary CDI prevention trials. Details of the intervention for the 21 RCT (23 treatment arms) for the primary prevention of CDI are given in Table 3. Five different types of probiotics were described in the 21 trials: three single-strain probiotics (Saccharomyces boulardii CNCM I-745 (S. boulardii), Lactobacillus rhamnosus GG, L. casei DN114001)) and two types of probiotic mixtures: (L. acidophilus and Bifidobacterium bifidum) and (L. acidophilus CL1285 and L. casei LBC80R and L. rhamnosus CLR2 (La+Lc+Lr)). Newer strain designations for several probiotics and the retrospective review of older studies may have used different strain designations, but were, in fact, the same strain. The most recent strain designations are used in this study. The most current strain designation for S. boulardii is CNCM I-745, the registration number at the Pasteur Institute [42], but older studies also refer to this strain as S. boulardii lyo, or S. boulardii, with no strain designation or by the brand name “Florastor”. L. casei DN114001 is also cited as the brand name “Actimel”. The mixture of L. acidophilus CL1285 and L. casei LBC80R and L. rhamnosus CLR2 is also cited as the brand name “Bio K+” [43].

1. S. boulardii 2 ×1010 capsules duration + 2 weeks 0 3 (2.6%) 5 (7.8%) 26.5% Surawicz 1989 [17]
2. S. boulardii 3 ×1010 capsules duration + 3 days 7 3 (3.1%) 4 (4.2%) 2.6% McFarland 1995 [18] S. boulardii 4.5 ×109 capsules duration (x = 7 days) 0 5 (15%) 3 (8.3%) 7.2% Lewis 1998 [19] S. boulardii 1 ×1010 capsules duration (x = 2 weeks) 4 days 0 (0%) 1 (0.5%) 3.3% Duman 2005 [20] S. boulardii 1 ×1010 wafers duration (x = 1 week) 0 3 (2.5%) 10 (7.9%) 35.6% Kotowska 2005 [21] S. boulardii 1 ×1010 capsules duration 4 0 (0%) 2 (2.6%) 9.1% Can 2006 [22] S. boulardii 1 ×1010 capsules 12 days 9 days 0 (0%) 0 (0%) -- Bravo 2008 [23] S. boulardii 1 ×1010 capsules duration + 7 days 12 3 (2.8%) 2 (2%) 3% Pozzoni 2012 [24] S. boulardii 1 ×1010 powder duration (x = 2 weeks) 2 1 (0.7%) 8 (5.6%) 51.9% Shan 2014 [25]

L. rhamnosus GG 4 ×1010 capsules duration (x = 7–10 day) 12 1 (1.6%) 1 (1.7%) 10% Arvola 1999 [26] L. rhamnosus GG 2 ×1010 capsules 2 weeks 1 2 (1.5%) 3 (2.2%) 2.7% Thomas 2001 [27] L. rhamnosus GG 4 ×1010 capsules duration (x = 2 weeks) 4 4 (4.2%) 7 (7.4%) 9.2% Miller 2008a [28] L. rhamnosus GG 1.2 ×1011 capsules duration (x = 2 weeks) 4 2 (1.3%) 0 11.2% Miller 2008b [28] L. rhamnosus GG 4 ×109 capsules duration (x = 15 day) 0 4 (5.8%) 13 (18.6%) 52.9% Morrow 2010 [29]

L. casei DN 114001 2 ×1010 drink duration + 1 week 4 0 (0%) 9 (17%) 81% Hickson 2007 [30] L. casei DN 114001 2 ×1010 drink duration (x = 6 days) 0 0 (0%) 3 (10%) 21.3% Dietrich 2014 [31]

L acidophilus +Bifido. bifidum 2 ×1010 capsules 20 d 0 2 (2.9%) 5 (7.2%) 11.5% Plummer 2004 [32] L acidophilus +Bifido. bifidum cfu nr (3g/day) capsules duration or LOS 0 5 (11%) 22 (40%) 88.0% Rafiq 2007 [33] L acidophilus +Bifido. bifidum 6 ×109 capsules 3 weeks 0 3 (14.3%) 1 (4.8%) 7.2% Stein 2007 [34]

L. acidophilus CL1285 + L. casei LBC80R + L. rhamnosus CLR2

5 ×1010 milk duration (x = 7–8 day) 3 1 (2.3%) 7 (15.6%) 44.2% Beausoleil 2007 [35]

L. acidophilus CL1285 + L. casei LBC80R + L. rhamnosus CLR2

L. acidophilus CL1285 + L. casei LBC80R + L. rhamnosus CLR2

5 ×1010 milk duration + 5 days 3 1 (0.5%) 4 (1.8%) 12.5% Sampalis 2010 [36]

5 ×1010 capsules duration + 5 days 3 8 (9.4%) 20 (23.8%) 64% Gao 2010a [37]

L. acidophilus CL1285 + L. casei LBC80R + L. rhamnosus CLR2

1 ×1011 capsules duration + 5 days 3 1 (1.2%) 20 (23.8%) 99.2% Gao 2010b [37]

Abbreviation: Bifido., Bifidobacterium; CDI, C. difficile infections; cfu, colony-forming units; L., Lactobacillus; LOS, length of stay; nr, not reported; S., Saccharomyces; x, mean.

The daily dose of probiotics varied widely from a lower daily dose in three treatment arms (4–6 ×109) [19,29,34] to higher doses ranging from 1–12 ×1010 colony-forming units (cfu) per day, while one study did not report their daily dose by cfu/d [33].

Most of the 23 treatment arms used a capsule formulation (74%), while four (17%) were given in milk or other drinks, or as powder (4%) or in wafers (4%).

Probiotics were given in conjunction with the antibiotics (usually started within 48–72 h of the antibiotic) and continued for either the duration of the antibiotic (12 treatment arms, 52%) or continued for 3–14 days after antibiotics were discontinued (7 arms, 30%). Four treatment arms gave the probiotic for a prescribed period (ranging from 14–21 days), regardless of the duration of antibiotics [23,27,32,34].

The duration of follow-up post-antibiotic and probiotic intervention ranged from 0–90 days. Eight (35%) of the treatment arms did not follow patients after the intervention had been discontinued. Most trial arms followed patients for 2–4 weeks (9 arms, 39%), or 1 week (2 arms, 9%) or for only four days (1, arm, 4%), while three (13%) had prolonged follow-up periods from seven to 12 weeks [18,24,26].

As CDI was usually a secondary outcome, not all enrolled trial participants were assayed for C. difficile, most trials tested for C. difficile when participants developed diarrheal symptoms, but not all trials successfully assayed all participants with diarrhea, nor provided data on the number of participants tested for C. difficile. One trial planned a priori to assay for C. difficile at enrollment, at the end of the intervention and end of follow-up, and successfully assessed 133 (69%) of trial participants, regardless of diarrheal symptoms [18]. Only three other trials reported the frequency of testing for C. difficile (done only if diarrhea developed), which was in a limited number of participants: n = 16 [20] or n = 46 [36], but one study only tested 50% (4/8) participants with diarrhea [23].

Probiotics in secondary CDI prevention trials. As shown in Table 4, four of six treatment arms tested a single strain of yeast (S. boulardii) [38,39] and two treatment arms tested a single strain of bacteria (L. rhamnosus GG) [40,41]. The three treatment arms in one trial combined S. boulardii or placebo in three separate antibiotic adjunctive treatments [either low dose vancomycin (500 mg/day), high dose vancomycin (2 g/day) or metronidazole (1 g/day)] [39]. The doses of vancomycin or metronidazole adjuncts were not controlled in the other three trials and were under the discretion of the patient’s primary provider. The daily dose of the probiotic varied from 2–3 ×1010/day [38,39] to 3 ×1011 [41], but daily dose was not provided in one trial [40]. Five of the treatment arms had a capsule formulation, while one used a probiotic yogurt [40]. The duration of probiotic intervention varied from 3–4 weeks, except in one trial that gave the intervention during adjunctive antibiotic therapy (typically 10–14 days), then extended the intervention for another three weeks [41]. The duration of follow-up was usually four weeks post-intervention, except for one trial that followed patients for 8.6 weeks [41].

Probiotic daily dose (cfu/day)

Frequency CDI recurrences in probiotic

History of CDI

Adjunctive therapy (daily dose)

Duration treated (follow-up)

Frequency CDI recurrences in controls

Pop-ulation Type of controls

Probiotic

Power (%) Reference

124 adults, In & Out

placebo V or M (varied) S. boulardii 3 ×1010 4 weeks (4 weeks) 15/57 (26.3%)* 30/67 (44.8%) 49.5 McFarland 1994 [38]

I/R

R

83 adults, In & Out

placebo V (500 mg) S. boulardii 2 ×1010 4 weeks (4 weeks) 23/45 (51%) 17/38 (44.7%) 5.3 Surawicz 2000a [39]

R

32 adults, In & Out

placebo V (2 g) S. boulardii 2 ×1010 4 weeks (4 weeks) 3/18 (17%)* 7/14 (50%) 35.9 Surawicz 2000b [39]

R

53 adults, In & Out

placebo M (1g) S. boulardii 2 ×1010 4 weeks (4 weeks) 13/27 (48%) 13/26 (50%) 3.3 Surawicz 2000c [39]

I/R

25 adults, In & Out

placebo V (nr) M (nr)

L rhamnosus GG

nr 3 weeks (4 weeks) 4/11 (36.4%) 5/14 (35.7%) 5.7 Pochapin 2000 [40]

20% V (nr) 80% M (nr)

L rhamnosus GG + inulin

duration abx + 21 days (8.6)

3 ×1011

R 15 adults placebo

3/8 (37.5%) 1/7 (14.3%) 5.3 Lawrence 2005 [41]

* p < 0.05, Abbreviations: abx, antibiotics; CDI, Clostridium difficile infection; I, initial CDI episode; In, inpatient; L., Lactobacillus; M, metronidazole; Md, median; nr, not reported in paper/abstract; Out, outpatient; R, recurrent CDI; S., Saccharomyces; V, vancomycin.

1. 2.8. Pooled Efficacy of Probiotics for Primary CDI Prevention

Meta-analysis. A meta-analysis of the 23 treatment arms of probiotic versus controls was performed and the pooled results indicated a low degree of heterogeneity (I2 = 17.2%, p = 0.23), so a fixed-effect

1. model was used. As shown by the forest plot in Figure 2, when trials were pooled by similar types of probiotic species, four of five types of tested probiotic types were significantly effective for primary CDI prevention: S. boulardii (pRR = 0.50, 95% C.I. 0.29, 0.85), L. casei DN114001 (pRR = 0.07, 95% C.I.

1. 0.01, 0.55), the mixture of L. acidophilus and Bifido. bifidum (pRR = 0.41, 95% C.I. 0.21, 0.80), and the mixture of L. acidophilus and L. casei and L. rhamnosus (pRR = 0.21, 95% C.I. 0.11, 0.40). The pooled results for L. rhamnosus GG did not reach statistical significance. A funnel plot (data not shown) and Egger’s text for publication bias did not show significant publication bias (p = 0.17).

Study

ID

S boulardii

Surawicz_1989

McFarland_1995

Lewis_1998

Duman_2005

Kotowska_2005

Can_2006

Pozzoni_2012

Shan_2014

Bravo_2008

Subtotal (I-squared = 11.8%, p = 0.338)

.

L rhamnosus GG

Arvola_1999

Thomas_2001

Miller_2008a

Miller_2008b

Morrow_2010

Subtotal (I-squared = 0.0%, p = 0.509)

.

L casei DN114001

Hickson_2007

Dietrich_2014

Subtotal (I-squared = 0.0%, p = 0.604)

.

L acid + Bifid bifidum

Plummer_2004

Rafiq_2007

Stein_2007

Subtotal (I-squared = 49.2%, p = 0.140)

.

L acid + L casei + L rham

Beausoleil_2007

Psaradellis_2010

Gao_2010a

Gao_2010b

Subtotal (I-squared = 37.1%, p = 0.189)

.

Overall (I-squared = 17.2%, p = 0.232)

%

Weight

0.30 (0.01, 7.38)

1. 0.32 (0.09, 1.14)

1. 0.14 (0.01, 2.65)
2. 1.39 (0.24, 8.13)

0.13 (0.02, 1.02)

(Excluded)

0.50 (0.29, 0.85)

0.95 (0.06, 14.85)

0.32 (0.11, 0.92)

4.25 2.65

1.89

1.04

6.38

1.60

1.37

5.18

0.00

24.37

0.68

1.97

4.64

0.33

8.45

16.07

6.44

2.31

8.75

0.28 (0.11, 0.67)

3.00 (0.34, 26.56)

0.41 (0.21, 0.80)

3.30

13.06

0.66

17.02

0.15 (0.02, 1.14)

0.26 (0.03, 2.27)

0.40 (0.18, 0.85)

0.05 (0.01, 0.36)

0.36 (0.27, 0.48)

4.57

2.61

13.27

13.35

33.80

100.00

.00297 1 336

Figure 2. Forest plot of fixed effects model of meta-analysis of primary prevention of C. difficile disease by sub-group of probiotic type, x-axis indicates relative risk.

Sub-group analysis. Results from the meta-regression analysis for the adjunctive use of probiotics primary prevention of CDI did not find significant differences in associations between the study population (adult versus pediatric, p = 0.68), or daily dose of probiotic (>1010 cfu/day versus <1010 cfu/day, p = 0.18). Only the probiotic strain group showed significance, confirming the validity of analyzing efficacy by strain type.

1. 2.9. Pooled Efficacy of Probiotics for Secondary CDI Prevention

Meta-analysis. A meta-analysis of the six treatment arms of probiotic versus controls was performed and the pooled results indicated a moderate degree of heterogeneity (I2 = 35.4%, p = 0.17), so a fixed-effect

1. model was used. As shown by the forest plot in Figure 3, when trials were pooled by similar types of probiotic species, neither S. boulardii nor L. rhamnosus GG was significantly efficious for secondary CDI prevention. Publication bias was not assessed due to the limited number of available trials.

Figure 3. Forest plot of fixed effects model of meta-analysis of secondary prevention of C. difficile disease by sub-group of probiotic type, x-axis indicates relative risk.

Clinical recommendations for the use of probiotics in CDI disease has been limited by the scarcity of well-done, randomized controlled trials using CDI as their powered, primary outcome. Most of the evidence results from prevention of AAD trials, which include CDI only as a secondary outcome and did not consider this outcome when calculating the needed study size for their trials (52% had <10% power). As a consequence, most individual trials have not found statistically significant efficacy for

probiotics and the prevention of CDI. This meta-analysis pooled the results of these trials, resulting in a significant increase in power and detected some (but not all) probiotic types were effective in preventing primary cases of CDI. The evidence for probiotics and the secondary prevention of CDI recurrences remains hampered by a lack of randomized, controlled trials.

As research on probiotics has evolved, the efficacy and mechanisms-of-action has been found to be highly strain-specific, requiring that dissimilar types of probiotics to be analyzed as separate subgroups [44]. Previous meta-analyses on probiotics for the prevention of CDI done before these guidelines were established pooled dissimilar types of probiotic species in their analysis [5,45]. A recent metaanalysis chose to pool their main outcomes across probiotic species, based on the hypothesis that the efficacy should be similar, as the mechanisms-of-action is similar for different probiotics [46]. I would disagree with this hypothesis, as different probiotic strains can have different mechanisms-of-action and resulting degrees of efficacies [47]. Another recent meta-analysis did not separate the different types of probiotics in their nine included trials [48]. More recent meta-analyses have presented their results by probiotic sub-groups, but were not as comprehensive as this meta-analysis: One meta-analysis included 11 trials [6] and another was only done in five pediatric trials [49]. Another meta-analysis included 20 trials and did present pooled results by sub-groups, but the data was not presented within specific pooled probiotic groups [50].

The strengths of this meta-analysis include the extensive literature search of both established literature databases, use of grey literature and correspondence with experts in the field, inclusion of a large number of high to moderate quality randomized, controlled clinical trials, the analysis of the efficacy for both primary CDI prevention and secondary CDI prevention by probiotic type sub-groups and the use of standardized methods adhering to current meta-analytic guidelines. The result is a comprehensive evaluation of the types of probiotics that are effective in preventing CDI, allowing clinicians to evaluate whether the use of probiotics may be effective in their patients. Limitations of this meta-analysis are inherent in the reporting of published trials with missing data (for example, not all reported the types of antibiotics or the number of participants tested for C. difficile) and the limited number of confirmatory trials tested for each type of probiotic. Of the 15 different types of probiotics with randomized trials for the prevention of CDI, only five (33%) had multiple trials, allowing pooling of their results. More well-done trials need to be done testing the same types of probiotics.

1. 4.1. Aims

The two aims of this review were to assess the use of specific probiotics for: (1) primary prevention of C. difficile disease (CDI) and (2) secondary prevention of C. difficile recurrences. Primary prevention of CDI is defined as people without diarrhea symptoms who are exposed to antibiotics and are given the intervention who do not develop diarrhea associated with a positive C. difficile assay (culture, immune assay, cytotoxin test or other assay) within two months exposure to the inciting antibiotic. Secondary prevention of CDI (prevention of CDI recurrences) is defined as people who have recovered from at least one prior episode of CDI, are asymptomatic (no diarrhea) at the time of the intervention and do not develop a recurrence of CDI within 1–2 months of follow-up.

1. 4.2. Search Strategy
2. 4.3. Inclusion and Exclusion Criteria
3. 4.4. Data Extraction

The data was extracted from a database from a previous meta-analysis on primary prevention and updated with recent publications, while secondary prevention articles were added [6]. For articles published in abstract form only or for any missing significant data in full articles, further information was sought by contacting authors or by the company manufacturing the probiotic product. Using a standardized data extraction form, the following data was systematically collected: authors, year of publication and journal, population data (age range, setting, types of antibiotic exposures, types of inciting diseases), study aims and outcomes, study methods (study design, eligibility criteria, sample size calculations, interim analysis, statistical methods used, recruitment methods, subgroup analysis done), randomization (method of randomization allocation, randomization method), degree of blinding (open, single or double), intervention data (probiotic strains used, daily dose, duration of treatment, duration of follow-up, type of control used, treatment concealment), types of C. difficile assays done, results (balanced randomization achieved, attrition rate and reasons, comparison of treatment groups by

demographics, etc., CONSORT flow-chart provided), outcome data [by group, intent-to-treat (ITT) or as-per-protocol (APP) analysis], safety data (adverse events reported by group), discussion points (limitations, generalizability and comparison of study results to published papers), clinical trial registration, location of protocol, and source of funding.

1. 4.5. Interventions
2. 4.6. Statistical Analysis
3. 4.7. Publication Bias

To assess for publication bias, a funnel plot, as well as a weighted regression (Egger’s test) and a rank correlation test (Begg’s test for small study effects) were conducted [52,55]. Funnel plots show graphically that as sample sizes of trials increase, the precision is estimating the underlying treatment effect increases, which results in the effect estimates (relative risks) from small trials scattering more widely at the bottom of the graph and narrower scattering among larger studies. In the absence of

publication bias, the funnel plot resembles a symmetrical inverted funnel. Reporting bias (smaller studies showing no protective effect) often are not published, and are indicated by an asymmetrical appearance with a gap in the bottom left of a funnel plot [56].

Four different types of probiotics were found to be effective for primary prevention of CDI (S. boulardii, L. casei DN114001, the mixture of L. acidophilus and Bifido. bifidum and the mixture of L. acidophilus, L. casei and L. rhamnosus). L. rhamnosus GG was not significantly efficious for the primary prevention of CDI and the other 10 types of probiotics lacked a second trial, so pooling of their outcomes was not possible. More clinical experience with these four probiotics might be recommended to confirm if they are effective in larger populations of patients.

Only two types of probiotics (S. boulardii and L. rhamnosus GG) had sufficient numbers of trials for to assess secondary prevention of CDI by meta-analysis, but none of the pooled results reached statistical significance. It may be that neither of these strains were effective in this analysis for preventing CDI recurrences, but based on prior experience and use of these probiotics (mechanism of action studies, case series, etc.), there are indications that these probiotic strains may be effective if an effective combination of probiotic and anti-C. difficile antibiotics can be determined [57,58].