Introduction

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Biological agents ("biotherapeutic agents" or "probiotics") have been used to treat a variety of infections, most notably infections of mucosal surfaces such as the gut and vagina (box). After the discovery and development of antibiotics, the value of these traditional treatments diminished. Now, however, we are being forced to look at alternatives to antibiotics to combat the ever increasing number of infections that occur because of excessive use of antibiotics.

The term "probiotic" was first used to describe "a live microbial supplement, which beneficially affects the host by improving its microbial balance."¹ Since then, research has looked at possible clinical uses for these agents and in 1995, when a greater understanding of their properties had developed, the term "biotherapeutic agents" was proposed to describe micro-organisms with specific therapeutic properties that also inhibit the growth of pathogenic bacteria.²

A number of agents have been isolated and studied with a view to clinical use. Streptococcus thermophilus and Lactobacillus bulgaricus, commonly used in the dairy food industry, were among the first to be studied. Other strains that have been used are Bifidobacterium bifidum, B longum, Enterococcus faecium, Saccharomyces boulardii, L acidophilus, L casei, and Lactobacillus GG. However, doctors are still reluctant to use these agents in clinical practice.

In this paper, we review the results from various trials carried out to study their benefits. We also look at the properties of biotherapeutic agents and options for further research.

	Materials and methods

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Literature search
We searched Medline between 1966 to 2000 with the terms "probiotics," "biotherapeutic agents," "lactobacilli," "antibiotic associated diarrhoea," and "Clostridium difficile." We also searched the Cochrane Controlled Trials Register and the Cochrane Database of Systematic Reviews. We restricted the search of Medline to published literature that had an English abstract; reviews identified by the searches of Medline and the Cochrane Database gave information about trials without an English abstract.^3-6 We included all randomised double blind trials that compared the effects of probiotic therapy and placebo (both given in combination with antibiotics). We independently assessed articles and abstracts, and we each put forward articles for inclusion.

Overall, we identified 38 relevant papers on the use of probiotics. We excluded 28 of the 38 papersthree single blind trials,^7-9 two letters on experimental use of probiotics, ^{10 11} three case series,^12-14 and three trials originally done for another indication.^15-17 We also excluded five trials that studied only traveller's diarrhoea ^{3 18-21} and 12 trials that studied infectious diarrhoea. ^{4-6 22-30} Of the latter, one trial was in normal subjects exposed to a bacterial challenge,⁴ one in patients infected with HIV,⁶ nine in children with diarrhoea of mixed causes, and one in adults with diarrhoea of mixed causes.²⁶ We excluded the trial in HIV patients because the results may not be generalised to other patients.⁶

Ten double blind placebo controlled trials were relevant to our area of interest (nine published in English and one in French).^31-40 Our meta-analysis included nine that looked at prevention of diarrhoea. We excluded the other trial, which looked at treatment of diarrhoea⁴⁰: this trial had two arms looking specifically at outcome of treatmentone of recurrent diarrhoea caused by Clostridium difficile and the other of non-recurrent disease caused by this organism.

Meta-analysis and data abstraction
The meta-analysis was carried out according to the recommendations of the QUOROM statement.⁴¹ The key outcome data taken for analysis included the sample size (table 1), treatment regimens, and numbers of patients in both arms of the study who had an absence of diarrhoea (table 2).

Quantitative data synthesis and validity assessment
We used the percentage of patients without diarrhoea in the probiotic and placebo groups as an outcome measure. We defined diarrhoea as "a change from the patient's normal bowel habit, with two or more loose or watery stools for at least two days." We performed three separate analyses: one for the four prevention trials using S boulardii, one for the five trials using lactobacilli or enterococci, and one on pooled data from all nine trials.

We ensured that the odds ratios were not heterogeneous across the trials by performing tests of homogeneity across all nine trials (P=0.246), across the four yeast trials (P=0.065), and across the five non-yeast trials (P=0.573). As these tests did not achieve significance, we combined the information in the tables, using the Mantel Haenszel method. We also calculated summary odds ratios and 95% confidence intervals for these analyses, and we plotted a graph by using the log of the odds ratios to determine the benefit of treatment over placebo.

Publication bias
A funnel plot (fig 1)⁴² did not show any publication bias; it showed that the larger studies found benefit and the smaller studies gave results varying from good to no benefit. Two further tests were carried out to investigate possible publication biasthe Begg and Mazumdar adjusted rank correlation test for publication bias and the Egger et al regression asymmetry test for publication bias.

View larger version (6K): [in this window] [in a new window]   Fig 1.   Funnel plot of odds ratio against sample size

View larger version (11K): [in this window] [in a new window]   Fig 2.   Funnel plot using data from Begg and Mazumdar's adjusted rank correlation test58

View larger version (8K): [in this window] [in a new window]   Fig 3.   Plot of publication bias using data from Egger et al's regression asymmetry test; 95% confidence interval is given between markers at x=0

	Results

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Nine trials were included in the final analysis (fig 4). The study regimens used probiotics combined with one antibiotic or a variety of antibiotics (table 2). All trials studied the efficacy of a probiotic in the prevention of antibiotic associated diarrhoea. The numbers of patients and the duration of follow up varied greatly from study to study, but the patients' characteristics were similar for the active treatment and placebo groups within each study.

We calculated the odds ratio on the basis of the proportion of patients free of diarrhoea on treatment compared with that in control groups. After tests of homogeneity, summary odds ratios and 95% confidence interval limits were provided for the combined data of the four trials that used S boulardii (yeast trials), the five non-yeast trials, and all nine trials together. The combined odds ratios for the four yeast trials and for the five non-yeast trials were similar (0.39 (95% confidence interval 0.25 to 0.62) and 0.34 (0.19 to 0.61), respectively); both favoured active treatment over placebo in the prevention of antibiotic associated diarrhoea. The odds ratio for pooled data from all nine trials was in favour of active treatment over placebo in the prevention of antibiotic associated diarrhoea (0.37; 0.26 to 0.53). Six studies showed a significant benefit of probiotic treatment compared with placebo (P<0.05) (fig 5). ^{31 33 34 36 37 39} One study showed benefit for only a subgroup of patients who did not receive non-antibiotic drugs likely to induce diarrhoea, such as magnesium hydroxide (for constipation), lactulose, and bisacodyl (for hepatic encephalopathy).³²

View larger version (34K): [in this window] [in a new window]   Fig 4.   Meta-analysis profile summarising flow trials. RCT=randomised controlled trial

Findings
McFarland et al showed that S boulardii lessened diarrhoea associated with antibiotics that contain a  lactam ring and prevented recurrent C difficile infection when given in combination with standard antibiotics. ^{37 40} There was no benefit, however, when S boulardii was used to treat primary infection with C difficile.⁴⁰

Schellenberg et al reported success with brewer's yeast, S cerevisiae, for the treatment of C difficile colitis.¹¹ This finding was criticised in one paper,⁴⁵ but was supported in another.⁴⁶

Lactobacillus GG was successfully used to treat a group of patients with recurrent diarrhoea caused by C difficile.¹⁰ Vanderhoof et al showed a significant reduction in antibiotic associated diarrhoea, using this agent in children.³⁹ The other trials in our meta-analysis used different strains of lactobacilli, two trials used two different strains of lactobacilli in combination. ^{32 35} Wunderlich et al showed that the strain of enterococcus strain SF68 that produced lactic acid was useful.³⁴ In the Orrhage study, a combination of L acidophilus and Bifidobacterium survived in the human gut and reduced the faecal counts of clostridia.³⁶

View larger version (29K): [in this window] [in a new window]   Fig 5.   Plot of the log of odds ratios for the proportion of patients free of diarrhoea in treatment groups compared with control groups

	Discussion

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Our meta-analysis of trials that used live organisms to prevent diarrhoea associated with antibiotics shows that probiotics may be effective in preventing antibiotic associated diarrhoea. We had only a small number of trials in our meta-analysis, and it should be noted that the different antibiotics used in the trials may have altered the risk of patients getting diarrhoea and their response to the probiotics. Although probiotics have been used to prevent or treat diarrhoea of other causesnamely traveller's diarrhoea and infantile infectious diarrhoeawe did not include trials that investigated probiotics in these indications; however, most of these studies showed positive results, and some reviews have been encouraging.⁴³

The way in which probiotics affect the gut has drawn much interest. To combat the problems of gastrointestinal infection, a probiotic must be non-pathogenic and must act against pathogens by different mechanisms from antibioticsfor example, by competition. More importantly, they should have a fairly rapid onset of action and survive the challenges of gastric acid, bile, or concurrent antibiotics. It is also desirable that they modify immune processes to destroy the invading organism. Saccharomyces boulardii and lactobacilli display these common properties.

A few live organisms have been used in many trials. S boulardii, a non-pathogenic yeast, is one such organism. It has a growth temperature of 37°C, rapidly colonises the bowel, does not alter the normal gut flora, and is cleared from the colon after treatment is discontinued.⁴⁴ Of the four yeast trials, two studies individually showed significant benefit, ^{31 33 37} but one did not³⁸; differences in the dose and duration of treatment with S boulardii and variations in the period of follow up may explain this disparity. Interestingly, S boulardii can also destroy the receptor site for C difficile toxin A and B by producing a protease⁴⁷; this could explain how S boulardii was noted to reduce the frequency of toxin B positivity.⁴⁰ This finding was criticised,⁴⁵ but it was also supported.⁴⁶

The other probiotic agent used widely in clinical trials is the Lactobacillus species. The mechanism of action of lactobacilli may be through multiple means: Lactobacillus GG has shown beneficial effects on intestinal immunity, it increases the numbers of cells that secrete immunoglobulin G and other immunoglobulins in the intestinal mucosa, and it stimulates the local release of interferon.⁴⁸ It also facilitates antigen transport to underlying lymphoid cells, and shows increased uptake in Peyer's patches.⁴⁸ Lactobacillus GG has also been shown to produce an antimicrobial substance that inhibits the growth of Escherichia coli, streptococci, C difficile, Bacteroides fragilis, and Salmonella.⁴⁹ L casei shirota also showed good survival in the gut in separate studies, and mucosal antibody titres (specific to lactobacilli) were increased in the presence of this agent. ^{50 51} Although there was no discernible change to the numbers of clostridia or enterococci, there was an increase in the numbers of excreted bifidobacteriaa normal bowel anaerobe. ^{50 51} It is possible that this increase in bifidobacteria interferes with the pathogenic potential of C difficile.

Advantages of S boulardii over current clinical practice include its ready availability in the form of brewer's yeast, its easy administration, and the remarkable cost effectiveness of its use compared with vancomycin when infection occurs.¹¹ However, there is a risk of fungaemia in immunocompromised patients⁵² and further large trials to document safety are needed before use of this agent will be accepted widely. Some papers report the development of septicaemia in immunocompromised patients and of endocarditis in those with damaged or artificial heart valves who have been treated with lactobacilli ^{53 54} ; it would seem prudent to avoid using lactobacilli in such patients.

Probiotics are a possible solution in the prevention of antibiotic associated diarrhoea. Clostridium difficile infection is increasingly prevalent in today's hospital setting, particularly in elderly patients, in whom 10-20% of such cases occur.⁵⁵ The incidence of antibiotic associated diarrhoea depends on the antibiotic used and each individual patient's risk factors. The standard regimens to treat colitis associated with Clostridium difficile are metronidazole and vancomycin; although these drugs are successful in 80% of cases, about 20% of patients suffer from recurrence.⁵⁶ In light of the need to control costs in these days of managed health care, we must re-examine the benefits of using live organisms. Whether the use of probiotics can actually reduce the length of hospital stay by reducing the incidence of infection with C difficile and the need to use antibiotics such as metronidazole and vancomycin are issues that need to be addressed in a clinical trial.

Conclusion
Our meta-analysis of nine trials shows that biotherapeutic agents may be useful in preventing antibiotic associated diarrhoea, but it provides little support for a role of probiotics in the treatment of such diarrhoea.

The increasing availability, lower costs, and relative lack of side effects of probiotics contrast with the problems associated with current antibiotic regimens. Commercially available strains are being marketed in capsules and yoghurt based drinks, but their potential benefit needs further investigation. It would be wrong to credit the proved benefits of one strain to an untested but closely related strain.⁵⁷ Data from trials have provided us with clear evidence on the efficacy of some strains in the gut, but we still need to see confirmation of their clinical benefit.