A. Helps, E. Bell and R. Mactier. Glasgow Renal and Transplant Unit, Western Infirmary, Glasgow, Scotland, UK, G11 6NT

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1 International Journal of Probiotics and Prebiotics Vol. 10, No. 4, pp , 2015 ISSN print, Copyright 2015 by New Century Health Publishers, LLC All rights of reproduction in any form reserved PROSPECTIVE RANDOMIZED DOUBLE-BLIND STUDY OF EFFICACY OF PROBIOTIC MILK DRINK IN REDUCING THE INCIDENCE OF ANTIBIOTIC-ASSOCIATED DIARRHOEA AND CLOSTRIDIUM DIFFICILE DIARRHOEA A. Helps, E. Bell and R. Mactier Glasgow Renal and Transplant Unit, Western Infirmary, Glasgow, Scotland, UK, G11 6NT [Received June 20, 2015; Accepted October 23, 2015] [Communicated by Prof. Francesco Marotta] ABSTRACT: This prospective double-blind randomized placebo-controlled trial evaluated the effect of a probiotic fermented milk drink containing Lactobacillus casei Shirota on the incidence of antibiotic-associated diarrhoea (AAD) and Clostridium difficile-associated disease (CDAD) on renal unit inpatients. The probiotic drink or placebo was administered to 85 inpatients within 48 hours of commencing antibiotics. Incidence of AAD, CDAD and mortality was recorded. AAD occurred in 36.36% cases in the probiotic group and 34.15% in the placebo group. Two patients developed CDAD during the study period, both of whom were in the placebo group (p=0.15). Nausea was the most common side effect. The percentage of patients that died during the 12-week follow-up period was 13.6% and 12.2% in the probiotic and placebo groups respectively. Multivariate analysis showed that proton pump inhibitor use was associated with AAD, CDAD and death. There was no evidence of a reduction in the incidence of AAD or CDAD in renal inpatients administered probiotic within 48 hours of commencing antibiotics. The study was underpowered due to a reduction in the background rate of CDAD and under-recruitment during the study period this may have resulted in small differences in AAD between groups not being identified. KEY WORDS: antibiotic associated diarrhoea, Clostridium difficile associated diarrhoea, lactobacillus, nephrology, probiotic Corresponding Author: Dr A Helps, University Hospital Crosshouse, Kilmarnock, KA2 0BE. Telephone ; Fax ; aileen.helps@nhs.net INTRODUCTION Antibiotic-associated diarrhoea (AAD) and, in particular, Clostridium difficile-associated diarrhoea (CDAD) are major causes of morbidity and mortality in hospitalised patients who are administered broad-spectrum antibiotics (Hickson et al., 2007, Warny et al., 2005). Cephalosporins, co-amoxiclav and quinolones are the main culprit antibiotics, and the incidence of AAD may approach 25% of patients who are administered these antibiotics (Bartlett and Gerding, 2008, Bergogne- Berezin, 2000). The main predisposing factors to AAD in addition to exposure to antibiotics are: age over 65 years, prolonged hospital stay and proton pump inhibitor therapy (Aseeri et al., 2008, Khanna et al., 2012). AAD usually occurs within 5-10 days of starting antibiotics but may develop earlier than 5 days or later than 10 weeks after stopping antibiotics (McFarland, 1998, Bartlett and Gerding, 2008). Probiotics are defined as live micro-organisms which confer a health benefit to the host when administered in adequate numbers (Organisation, 2001). There is some evidence that probiotics may be helpful in reducing the incidence of AAD and C. difficile infections. A double-blind randomised study of 100g (97ml) Actimel administered twice daily to inpatients reported a 22% risk reduction in AAD and 17% reduction in C. difficile infection in the probiotic treated group (Hickson et al., 2007). The positive results of this study are supportive of the use of probiotic drinks to reduce AAD and CDAD but require confirmation. AIM This study was performed to evaluate the effect of a probiotic fermented milk drink containing Lactobacillus casei Shirota (Yakult) on the incidence of AAD and CDAD in a renal unit inpatient population. DESIGN This was a prospective double-blind randomised controlled study of the incidence of AAD and CDAD in renal unit inpatients taking either probiotic milk drink or placebo. Each

2 146 Probiotic milk drink and antibiotic-associated diarrhoea 65 ml bottle of probiotic milk drink contained 6.5 x 10 9 live organisms of the probiotic bacterial strain Lactobacillus casei Shirota. The study was conducted according to the guidelines laid down in the Declaration of Helsinki and was healthcare related research covered by NHS indemnity. It was approved by the local Research and Development department and by NHS Greater Glasgow and Clyde research and ethics committee, (reference 08/S0704/4), on the 11 th July METHODS Patient demographics and diagnoses on admission were recorded and used as baseline characteristics for patient randomisation and study analyses. Inclusion criteria 1. inpatient in a renal ward and prescribed a course of antibiotic within the past 48 hours with the exception of vancomycin alone or metronidazole (alone or in combination with another antibiotic) 2. aged 18 years or greater Exclusion criteria 1. diarrhoea on randomisation or within the preceding week 2. lactose intolerance or intolerance to dairy products 3. regular probiotic use in the 4 weeks prior to randomisation 4. antibiotic use in the 4 weeks prior to randomisation 5. patients unable to give written consent within 48 hours of starting antibiotics 6. patients on induction dose immunosuppression (e.g. recent transplant or induction therapy of vasculitis) 7. active inflammatory bowel disease or bowel surgery less than 6 months prior to admission Eligible patients commencing antibiotics were asked for informed consent within 48 hours of starting antibiotics. Patients were randomised to either probiotic milk drink twice daily or placebo drink twice daily within 48 hours of starting one or more of the above antibiotics and continued until 7 days after stopping antibiotics. Patients, nursing staff and investigators were blinded to which study product had been assigned to the patient. Randomization was stratified by age via an online portal designed by the Robertson Centre of Biostatistics, University of Glasgow. The renal unit medical and nursing staff supervised compliance with the probiotc milk drink or placebo. All patients were managed as per the hospital infection control recommendations to minimise the risk of developing or transferring infectious diarrhoea. Stool samples were sent for culture to attempt to identify a specific cause in all patients who developed diarrhoea as per usual practice. The frequency and duration of diarrhoea, identified aetiology, and antimicrobial treatment of diarrhoea, were recorded. Patients were followed up for 12 weeks after recruitment with AAD, CDAD, further hospital admission and mortality documented. The primary outcome was the incidence of AAD. AAD was assessed by daily monitoring of frequency of loose or semi-formed stools by renal nursing staff and development of abdominal pain. Diarrhoea was defined as 3 or more loose bowel motions in 24 hours. Stool samples were sent following the diagnosis of AAD. The main secondary outcome was the incidence of C. difficile diarrhoea. C. difficile cell culture and stool cultures were performed on all patients with undiagnosed diarrhoea to identify if enteric pathogens were present. The other secondary outcomes were: Total days until discharge from hospital or patient death Total treatment days on oral metronidazole or vancomycin for CDAD treatment Adverse events attributable to probiotic drink or control drink Significant differences in routine laboratory tests such as serum albumin Data were analysed using an intention to treat analysis. The Chi squared test, Mann Whitney U test and Student s unpaired T test were used where appropriate. Univariate and multivariate binomial regression were used to determine factors associated with the development of AAD and CDAD. With α=0.05 and a power of 90% to detect an absolute difference of 20% between the proportion of patients with AAD in the placebo (assumed at 30%) and probiotic (assumed at 10%) groups we estimated that we needed a sample size of 164 (82 in each group). RESULTS Eighty-five patients, who were admitted to the renal unit and met the inclusion and exclusion criteria, were recruited to this study within 2 days of starting antibiotics during a 44 month period (01/03/ /11/2012). Forty-four of the patients were randomised to probiotic milk drink and 41 to placebo. The main reasons why 716 additional screened patients were not recruited were: Recent antibiotic use (n=220) Current metronidazole use (n=75) Declined to take part in the study (n=68) Antibiotic administration for >48 hours (n=65) Unable to give informed consent (n=59) Vancomycin prescribed as a single agent (n=51) Too unwell to participate (n=43) Diarrhea (n=43) Long term antibiotic use (n=37) High dose immunosuppression (n=36) Unknown (n=19) There were no significant differences in patient baseline characteristics between the probiotic milk drink and placebo groups at the time of randomisation (Table 1). There was no

3 Probiotic milk drink and antibiotic-associated diarrhoea 147 significant difference in primary and secondary outcomes between those taking probiotic milk drink and placebo (Table 2). The only exception to this was improved compliance in the placebo group at the end of the antibiotic course (85.4% v 65.9%; p=0.03). This difference was not maintained at completion of the study (p=0.12). AAD occurred in 36.4% of cases in the probiotic milk drink group and 34.1% in the placebo group. There was no significant difference in the incidence of AAD when non-compliant patients were excluded from the analysis. Only 2 patients developed CDAD, both of whom were in the placebo group. Neither patient developed toxic megacolon or required colectomy. Nausea was the most common side effect in both groups and was the predominant reason given for non-compliance with the drinks. There was no significant difference in the proportion of patients taking 2 or more antibiotics at the time of recruitment or in the site of infection at recruitment. There was no significant change in laboratory parameters between probiotic and placebo groups. Readmission to the renal unit occurred in 47.7% patients in the probiotic group and 36.6% patients in the placebo group during the 3 month follow up period (p=0.29). The percentage of patients that died during the 3 month follow-up period was 13.3% and 12.2% in the probiotic and placebo groups respectively (p=0.843). There was no significant difference in length of admission between the two groups. TABLE 1. Summary of patient characteristics at recruitment. *Interquartile range, **Chronic obstructive pulmonary disease Characteristic Probiotic milk drink (n=44) Age (mean, IQR*) (52.02, 74.43) Placebo (n=41) (50.69, 76.40) P value Sex (% male) 27 (61.4) 26 (63.4) Renal replacement therapy emergency hemodialysis Long-term hemodialysis peritoneal dialysis renal transplant Antibiotic duration (mean, IQR) (7.0, 14.0) (6.0, 10.75) Comorbidity diabetes mellitus 15 (34.09) 15 (36.59) ischemic heart disease 16 (36.36) 16 (39.02) cerebrovascular disease 7 (15.91) 7 (17.07) hypertension 29 (65.91) 32 (78.05) asthma/copd** 5 (11.36) 8 (19.51) malignancy 2 (4.55) 4 (9.76) vasculitis 0 2 (4.88) Previous CDAD 1 (2.27) 2 (4.88) Medications proton pump inhibitor 27 (61.36) 20 (48.78) antidiarrheal agents 0 1 (2.44) immunosuppression 8 (18.18) 6 (14.63) Laboratory parameters Hemoglobin (mean, IQR) 10.5 (9.1, 11.9) 10.3 (8.7, 12.0) white cell count (mean, IQR) 9.36 (6.79, 10.99) (8.05, 12.45) platelet count (mean, IQR) 233 (154, 296) 233 (182, 280) C reactive protein (mean, IQR) 109 (40, 163) 98 (17, 170) Serum albumin 28.8 (26.3, 33.0) 28.0 (23.5, 32.0) 0.550

4 148 Probiotic milk drink and antibiotic-associated diarrhoea TABLE 2. Comparison of outcomes in the probiotic milk drink and placebo groups Probiotic milk drink n=44 (%) Placebo n=41 (%) P value Antibiotic associated diarrhoea 16 (36.36) 14 (34.15) C. difficile diarrhoea 0 2 (4.88) Compliance 2 days after starting 32 (72.73) 36 (87.80) at end of antibiotic course 29 (65.91) 35 (85.37) week after stopping antibiotic 28 (63.64) 33 (80.49) Reasons for non compliance/ side effect nausea 7 (15.91) 3 (7.32) change in immunosuppression levels 0 1 (2.44) disruption in supply of drink 6 (13.64) 3 (7.32) deterioration in patient condition 2 (4.55) 1 (2.44) patient changed their mind regarding taking part in study 2 (4.55) 1 (2.44) Antibiotic associated diarrhoea in fully compliant patients 9/26 (34.6%) 11/33 (33.3%) Change in laboratory parameters between randomisation and discharge (death censored) Hb (g/dl) (mean, IQR) (-1.23, 0.13) (-1.18, 0.48) WCC (mean, IQR) (-3.15, 0.85) (-3.93, 0.06) Platelet count (mean, IQR) 37 (18, 75) 37 (-3, 64) C reactive protein (mean, IQR) -59 (-126, 0) -55 (-131, 2) Serum albumin (mean, IQR) -1.9 (-4.0, 0.25) -1.8, (-5.0, 1.0) Length of hospital admission (mean, IQR) (3, 12) 13.8 (6, 19) Readmitted during 3 month follow-up 21 (47.7) 15 (36.6) Died during hospital admission 2 (4.5) 1 (2.4) Died during 3 month follow-up 6 (13.6) 5 (12.2) Composite of AAD/ CDAD/ death 20 (45.5) 15 (36.6) Univariate binomial regression analysis was performed in order to determine factors associated with the development of AAD. No variable gave a statistically significant result. This was repeated using a composite end point of AAD, CDAD and death. Proton pump inhibitor (PPI) use was significantly associated with an increased risk of AAD, CDAD and death with an odds ratio of 2.79 (p=0.023). Total number of days on antibiotics was also associated with increased risk with odds ratio 1.05 (p=0.017). Requiring long-term renal replacement therapy (RRT) appeared to be protective although this did not reach statistical significance (OR 0.42; p= 0.066). Multivariate binomial regression analysis for a composite of AAD, CDAD and death was performed using a backwards selection method for those with p values < 0.2. Patients requiring RRT appear to have reduced risk of AAD, CDAD and death as a composite end point (OR 0.33; p= 0.054). PPI use was associated with significantly increased risk of developing the composite end point (OR 2.64; p= 0.003). DISCUSSION This double-blind, randomised placebo controlled study of high risk renal inpatients has shown no evidence of a decreased incidence of AAD or CDAD when prescribed a commercially available probiotic milk drink twice daily within 2 days of starting a course of antibiotics (Table 2). There was a high incidence of AAD in both groups, while only 2 (2.4%) of the 84 patients recruited to this study developed symptomatic CDAD. This is a lower incidence of CDAD than in previously reported studies in the Cochrane Systematic Review (Goldenberg et al., 2013). There was no increase in adverse events in the probiotic group. C. difficile cases in Scotland were falling consistently throughout the duration of the study from 1.29 per 1000

5 Probiotic milk drink and antibiotic-associated diarrhoea 149 occupied bed days in to per 1000 occupied bed days in The study therefore was performed during a time period when a bundle of measures was introduced to reduce the incidence of CDAD. Recruitment to this study did not meet the total number required from the power calculation but there was no trend towards a reduction in AAD in the probiotic group. The Cochrane Systematic Review identified 23 randomized controlled trials reporting on the effect of probiotics on prevention of CDAD (n=4213 patients) (Goldenberg et al., 2013). It found that the incidence of CDAD was 2.0% in the probiotic group compared to 5.5% in the placebo or no treatment control group with a relative risk of 0.36 (95% CI 0.26 to 0.51). A systematic review of the role of probiotics in AAD in over 10,000 patients in 82 randomised controlled trials, of which 57 involved a lactobacillus-based probiotic, found that using probiotics as adjunct therapy reduced the risk of AAD with an RR of 0.58 equating to a NNT of 13 (Hempel et al., 2012). The PLACIDE trial is the largest trial of probiotic use in AAD to date. It is a multicentre trial of probiotic against placebo in patients aged 65 years or older (Allen et al., 2013) and recruited 2974 patients from patients screened. AAD occurred in 12.9% of the probiotic group and 11.7% of the placebo group. Patients with acute pancreatitis or a history of mesenteric ischemia were excluded from the PLACIDE study as the PROPATRIA trial(besselink et al., 2008) found that probiotic administration was associated with a worse outcome in those with severe acute pancreatitis. No patients with pancreatitis were included in our trial. Research into the effects of probiotic on AAD has increased dramatically since this study began recruitment and there have been eleven double-blind randomised controlled trials published in the past 7 years (Bravo et al., 2008, Safdar et al., 2008, Wenus et al., 2008, Gao et al., 2010, Lonnermark et al., 2010, Sampalis et al., 2010, Pozzoni et al., 2012, Allen et al., 2013, Wong et al., 2013, Chatterjee et al., 2013, Selinger et al., 2013). Agents used and duration of probiotic use was variable and therefore it is difficult to generalise the studies as a whole to potential strain specific effects of L. casei Shirota used in the current study. The incidence of AAD in the control groups varied widely from 6.0% to 54.9% (Lonnermark et al., 2010, Wong et al., 2013, Chatterjee et al., 2013, Selinger et al., 2013). There were also highly variable background CDAD rates in the study populations with the incidence of CDAD in control groups ranging from 0 to 23.8% (Lonnermark et al., 2010). The range of antibiotics used may also have had implications on the transit of L. casei Shirota through the gut. The minimum inhibitory concentrations of lactobacilli have been studied with L. casei Shirota being susceptible to aminoglycosides, quinolones, beta-lactam antibiotics and macrolides, while resistant to vancomycin (Yuki et al., 1999, Kiwaki and Sato, 2009). There is evidence that L. casei Shirota survives in the gastrointestinal tract (Yuki et al., 1999) and patients with chronic kidney disease may have alterations in gut flora compared with the general population (Anders et al., 2013, Vaziri et al., 2013a). This results in differences in gut barrier function and possibly in bacterial translocation (Vaziri et al., 2013b, Simenhoff et al., 1978) and makes data from a general population difficult to extrapolate to renal patients. Use of proton pump inhibitors is now widely recognised as a risk factor for C. difficile (Aseeri et al., 2008). Whilst gastric acid does not kill spores of this pathogen, it has been suggested that survival and germination of the spores may be higher in reduced acid conditions. Raised ph levels in the stomach have also been associated with greater bacteria cell counts in the small and large intestines. CONCLUSION The role of probiotics in AAD is not fully understood. This double-blind randomised placebo-controlled study was not able to demonstrate any reduction in AAD or CDAD in patients with kidney disease who have commenced antibiotics within the past 2 days. The study was underpowered which may have resulted in small differences in AAD between the probiotic and placebo groups not being identified in this population. Larger, multicentre studies are required in renal patients. Such studies should be at low risk to the study population as the current pilot study showed that there were no significant adverse events despite the high co-morbidity and relative immunosuppression of the renal inpatient population. ACKNOWLEDGMENTS AND DECLARATION OF CONFLICT OF INTEREST This study was designed in conjunction with Dr Linda Thomas (Yakult UK Limited) with involvement from Dr Kaori Suzuki (Yakult Europe BV). Yakult Europe BV provided study drinks free of charge in addition to storage facilities for the drinks. They were not involved in patient recruitment or randomisation, data collection or statistical analysis, and did not have access to patient identifiable information. REFERENCES Allen, S. J., Wareham, K., Wang, D., Bradley, C., Hutchings, H., Harris, W., Dhar, A., Brown, H., Foden, A., Gravenor, M. B. and Mack, D. (2013). 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