1. Probiotics – Do They Work? Prevention/Treatment of GI disease in pediatrics?

2. DISCLAIMER I would like to thank Prof. Hania Szajewska, Dept. Pediatrics, University Warsaw, Poland, who provided me with charts from her presentation at the Nestle Nutrition Institute Course in Singapore 2013.

3. 10:1

4. There are 10 times more microorganisms in and on us than we have cells that make up our body (mainly in the gut)  gut microbiota

5. Gut microbiota 1000 – Up to 1000 different species of bacteria 170 – Each individual harbors some 170 bacterial species out of a total of about 1000 that are predominant in the gut 150 – The gut microbiota encode 150 times as many genes as our own genome Qin et al. Nature 2010;464:59-65.

6. IBD Diabetes NEC Johnson & Versalovic. Pediatrics 2012;129:950-60. Differences in gut microbiota between healthy controls and disease states

7. Differences in gut microbiota between healthy controls and obese subjects Obese subjects have less variability in their microbiota than healthy non-obese subjects.

8. Gut microbiota might be an essential factor in certain pathological disorders Efforts to optimize the intestinal microbial milieu have increased the interest in probiotics (and/or prebiotics) Manipulation of the human microbiome

9. What are probiotics? Definition  Live microorganisms which when administered in adequate amounts confer a health benefit on the host Examples  Lactobacilli  Bifidobacteria  S boulardii Joint FAO/WHO Expert Consultation 2001

10. Probiotics Genera, species, and strains Why the stain- not just probiotics? GenusSpeciesStrain LactobacillusrhamnosusATCC 53103 (GG) LactobacilluscaseiDN-114 001 LactobacillusreuteriDSM 17938 Bifidobacteriumanimalis subsp. lactisHN019 WHO Global Guideline. Probiotics and prebiotics. 2011. All probiotics are not created equal Supplement companies make unproven claims !!!!!!

11. What are the implications of the strain-specifity? Documentation No extrapolation Dosage WHO Global Guideline. Probiotics and prebiotics. 2011.

12. Mechanisms of action Non-immunologic Immunologic O'Toole PW, Cooney JC. Interdiscip Perspect Infect Dis. 2008;2008:175285 WHO Global Guideline. Probiotics and prebiotics. 2011.

13. Effectiveness of interventions. Published RCTs & systematic reviews/ meta-analyses on probiotics RCTs 810 Meta-analyses 101 Cochrane Collaboration (search date: Jan 2013)

14. For infants and children: Supplementation of infant formula with probiotics Provide clinically proven supplements

15. Administration of probiotic-supplemented formula beyond early infancy B lactis Bb12 3 RCTs RR 0.5 (0.36-0.8) NNT 7 There is evidence from the trials that supplementation of infant formula with B lactis Bb 12 is associated with a reduction in the risk of nonspecific GI infections. J Pediatr Gastroenterol Nutr 2011;52:238-50. Prevention of Gastrointestinal Infections

16. ESPGHAN Committee on Nutrition J Pediatr Gastroenterol Nutr 2011;52:238-50.

17. Administration≤4 moBeyond early infancy GrowthNo safety concerns Limited evidence No safety concerns Limited evidence Clinical outcomes GI infections Too much uncertainty to draw reliable conclusions from the results Reduction in the risk of non-specific GI infections Other clinical outcomes Limited evidence Some clinical benefits Adverse effectsNS J Pediatr Gastroenterol Nutr 2011;52:238-50. Supplementation of infant formula with probiotics – term infants ESPGHAN Committee on Nutrition

18. Treatment - Acute Gastroenteritis What is the evidence that probiotics work?

19. Acute gastroenteritis Duration of diarrhoea MetaanalysisProbioticRCT (n)WMD (95% CI) Szajewska et al. J Pediatr Gastr Nutr 2001 Various8 (773)-20 h (-26 to –14) Van Niel et al. Pediatrics 2002 Various7 (675)-17 h (-29 to –7) Huang et al. Dig Dis Sci 2002 Various18 (1917)-19 h (-26 to –14) Allen et al. Cochrane Review 2010 Various35 (4555)-25 h (-16 to -34) Reduced duration of diarrhoea

20. A common criticism Mixing apples & oranges

21. Lactobacillus GG 11 RCTs, n=2483 Saccharomyces boulardii 8 RCTs, n=1052 Update: Szajewska et al. Aliment Pharmacol Therap 2007;25:257-64 Szajewska et al. Aliment Pharmacol Ther 2009;30:960-1 Reduced duration of diarrhoea Acute gastroenteritis Duration of diarrhoea

22. S boulardii Diarrhea lasting ≤4 days Cochrane review 2010 6 RCTs (n=606) RR 0.37 (0.21 to 0.65) NNT 3 (2 to 3)

23. Treatment of acute gastroenteritis Current recommendations ESPGHAN/ESPID 2008AAP 2010 Yes Probiotics may be an effective adjunct to the management of AGE. Because there is no evidence of efficacy for many preparations, we suggest the use of probiotic strains with proven efficacy and in appropriate doses There is some evidence in otherwise healthy infants and young children to support the use of probiotics early in the course of diarrhea from acute viral gastroenteritis. Examples Lactobacillus GG, Saccharomyces boulardii JPGN 2008;46:619-21Pediatrics 2010;126:1217-31.

24. Proportion of patients with watery diarrhoea L reuteri 4 × 10 8 CFU Duration of diarrhoea L reuteri 2.1 ± 1.7 d Placebo 3.3 ± 2.1 d Mean difference -1.2 d (-2 to -0.3) Other probiotics also may be used provided their efficacy is documented in high quality RCTs (or in meta-analyses). ESPGHAN 2008

25. Antibiotic-associated diarrhoea

26. Prevention of AAD Hempel at al. JAMA 2012;307:1959-1969 Total 63 RCTs N= 11 811 RR 0.58 (0.5 to 0.68) Children 16 RCTs RR 0.55 (0.38 to 0.8)

27. 45% reduction in the risk of AAD Number needed to treat 11 i.e. you have to treat 11 patients to prevent 1 from AAD

28. Number needed to treat InterventionNNT Statins for myocardial infarction for one year Vitamin D for hip fractures Aspirin for cardiovascular protection Slide from Dan Merenstein

29. Number needed to treat InterventionNNT Statins for myocardial infarction for one year 100-427 Vitamin D for hip fractures Aspirin for cardiovascular protection Slide from Dan Merenstein

30. Number needed to treat InterventionNNT Statins for myocardial infarction for one year 100-427 Vitamin D for hip fractures 50 Aspirin for cardiovascular protection Slide from Dan Merenstein

31. Number needed to treat InterventionNNT Statins for myocardial infarction for one year 100-427 Vitamin D for hip fractures 50 Aspirin for cardiovascular protection 40 Slide from Dan Merenstein

32. Number needed to treat InterventionNNT Statins for myocardial infarction for one year 100-427 Vitamin D for hip fractures 50 Aspirin for cardiovascular protection 40 Probiotics for prevention of AAD 11 Slide from Dan Merenstein

33. AAP recommendation Prevention of AAD – There is some evidence to support the use of probiotics to prevent antibiotic-associated diarrhoea Thomas et al. Pediatrics 2010;126:1217-31. All probiotics are not created equal

34. Prevention of Clostridium difficile- associated diarrhea Johnston et al. Ann Intern Med. 2012

35. Probiotics (as a group)  reduced risk of C. difficile-diarrhea 20 RCTs N= 3821 RR 0.34 (0.24-0.49) Johnston et al. Ann Intern Med. 2012 RCT, badanie z randomizacją

36. Johnston et al. Ann Intern Med. 2012 Effect size (example) S. boulardii (n=1232) 1.4% vs. 3.7% RR 0.39 (0.19-0.82)  risk 61% November 2012 November 2012 All probiotics are not created equal

37. TREATMENT OF CLOSTRIDIUM DIFFICILE DIARRHEA Hot topic: Fecal microbiota transplantation (enema, colonoscopy, nasogastric tube) Traditional Chinese medicine (4th cent.) Transplant «healthy» microbiota Seems to be safe and works Randomized clinical trial: van Nood et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. N Engl J Med 2013 Questions: dosage, timing, standardized «healthy» microbiota?

38. IBD – probiotic supplements fecal microbiota transplantation No effect in Crohn`s diease VLS#3 probiotic combination + concomindant therapy. Remission rate in ped. UC significantly higher (93 vs 36%) Microbiota transplantation: mild-to-moderate UC: N=10 (pediatric); Enema daily for 5 days; Family or close relation. Clinical remission at 1 week (3); clinical response at 1 mo (6); Kunde et al, JPGN, 2013

39. Nosocomial diarrhoea Meta-analysis of 20 surveillance studies (Pediatrics 2012;129:e1011) Incidence per 100 hospitalisations Overall nRV2.9 (1.6 – 4.4.) nRV in children <2 y, hospitalized during the epidemic months 8.1 (6.4 – 9.9)

40. B. bifidum+Str. thermRR (95% CI)NNT (95% CI) Saavedra Lancet 1994 0.2 (0.06-0.8)5 (3-20) L. delbrueckii H2B20 Penna Pediatria (São Paulo) 2009 1.6 (0.6-4.0)NS Lactobacillus GGRR (95% CI)NNT (95% CI) Szajewska J Pediatr 2000 0.2 (0.06-0.6)4 (2-10) Mastretta JPGN 2002 0.8 (0.6-1.3)NS Hojsak Pediatrics 2010 0.4 (0.25-0.7)15 (9-34) Prevention of nosocomial diarrhoea What is known on this topic?

41. 3 RCT, n=1043 RR 0.5 (0.4 – 0.7) NNT 13 (95% CI 9 – 28) 3 RCT, n=1043 RR 0.5 (0.4 – 0.7) NNT 13 (95% CI 9 – 28) Szajewska et al. Aliment Pharmacol Therap 2011 What is new on this topic? LGG in the prevention of nosocomial diarrhoea Meta-analysis

42. L reuteri DSM 17938 in the prevention of nosocomial diarrhoea Wanke & Szajewska. J Pediatr 2012;161:40-43.e1 In hospitalized children, the administration of L reuteri DSM 17938 compared with placebo had no effect on the overall incidence of nosocomial diarrhea, including rotavirus infection

43. RR 0.4 (0.25-0.7) NNT 15 (9-34) RR 0.4 (0.25-0.7) NNT 15 (9-34) RR 0.4 (0.18-0.85) NNT 30 (16-159) RR 0.4 (0.18-0.85) NNT 30 (16-159) Hojsak I, Abdovic S, Szajewska H, Kolacek S. Pediatrics 2010;125:e1171-7 LGG in the prevention of nosocomial gastrointestinal and respiratory tract infections

44. To use or not to use probiotics for preventing nosocomial diarrhoea?

45. Lactobacillus GG Study designNNT (95% CI) Szajewska APT 2011 Meta-analysis of 3 RCTs (n=1043) 13 (9-28) B. bifidum+Str. therm Saavedra Lancet 1994 RCT (n=55) 5 (3-20) L. delbrueckii H2B20 Penna Pediatria (São Paulo) 2009 RCT (n=139) NS L reuteri DSM 17938 Wanke & Szajewska J Pediatr 2012 RCT (n=106) NS Prevention of nosocomial diarrhoea Summary 2013

46. Infantile colic Prevalence – 3 to 40% of infants Rationale for the use of probiotics – An aberrant gut microbiota in colicky infants Lower counts of intestinal lactobacilli Increased concentration of coliformis Savino & Tarasco. Curr Opin Pediatr 2010;22:791-7.

47. Could something as simple as a probiotic supplement stop a colicky baby from crying so much? Newsweek, January 2011

48. Infantile colic L reuteri DSM 17938 RR (95% CI)NNT (95% CI) Dzień 71.6 (1.1-2.2)3 (2-8) Dzień 142.1 (1.2-3.8)3 (2-9) Dzień 211.3 (1.01-1.8)5 (3-25) Savino et al. Pediatrics 2010;126:e526-33. L. reuteri DSM 17 938 at a dose of 10 8 CFU/d in breastfed infants improved symptoms of infantile colic and was well tolerated and safe AAP 2010 There may be benefit for treating infantile colic with probiotics, but further studies are necessary. Day 0 Day 21 0 Day 7 Day 14 Day 21 Day 7 Day 14 Day 21 Day 7 Day 14 Day 21

49.  Treatment success Reduction on the daily average crying time ≥50% RR (95% CI)NNT (95% CI) P value Day 7 -7 (4 to 19) 0.026 Day 14 4.3 (2.3 to 8.7)2 (2 to 3) <0.001 Day 21 3.2 (1.8 to 4.0)2 (2 to 3) <0.001 Day 28* 1.6 (1.3 to 2.1)3 (2 to 5) <0.001 * Follow-up visit 1 week after the termination of the intervention Szajewska, Gyrczuk, Horvath. J Pediatr 2013; 162:257-262

50.  Duration of crying Throughout the study period, the crying time was significantly reduced in the probiotic group compared with the placebo group Szajewska, Gyrczuk, Horvath. J Pediatr 2013; 162:257-262

51.  Parental percetion of colic severity  Family quality of life Throughout the study period, in the probiotic group compared with the placebo group: reduction in the parental perception of colic severity improved parental/family quality of life throughout the study

52. Conclusion Exclusively or predominantly breastfed infants with infantile colic benefit from the administration of L reuteri DSM 17938 compared with placebo. Infantile colic L reuteri DSM 17938 Feb 2013 Feb 2013 Szajewska, Gyrczuk, Horvath. J Pediatr 2013; 162:257-262

53. How this intervention might work? ? The effect of L reuteri on – gut motility – colonic sensory nerves – colon contractile activity – pain perception Additional mechanisms include – anti-inflammatory effects documented both in vitro and in vivo – interaction with altered gut microbiota. Kunze et al. J Cell Mol Med. 2009;13(8B):2261-70. Wang et al. Neurogastroenterol Motil 2010;22:98-107, e33. Ma et al. Am J Physiol Gastrointest Liver Physiol 2009;296:G868-75 Indrio et al. J Physiol Pharmacol 2009;60(suppl 6):27-31. The use of L reuteri could be discussed with caregivers

54. Prevention of NEC

55. Controversy ‘It is time to change practice’ ‘Probiotics: are we ready for routine use?’

56. A common criticism Mixing apples & oranges

57. Current recommendations ESPGHAN 2009AAP 2010ASPEN 2012 No Efficacy and safety should be established for each product. Further studies are needed. There is some evidence that probiotics prevent NEC in VLBW infants (birth weight between 1000 and 1500 g), but more studies are needed. There are insufficient data to recommend the use of probiotics in infants at risk for NEC. Further research needed. JPGN 2009;49:1-9.Pediatrics 2010;126:1217-31.JPEN 2012;36:506-23.

58. Some researchers consider that the current evidence justifies the routine use of probiotics Deshpande et al. BMC Med. 2011;9:92.

59. Take home message Can probiotics prevent/treat disease in pediatrics? Yes, but all probiotics are not created equal.

61. Respiratory tract infections >5 y – 5 episodes/year <5 y – 3 episodes/year

62. Probiotics for preventing acute upper respiratory tract infections Cochrane review 2011 Probiotics were better than placebo in reducing  the number of participants experiencing episodes of acute URTIs  the rate ratio of episodes of acute URTI  antibiotic use

63. 2012 2001 2009 Lactobacillus GG for preventing acute respiratory tract infections

64. PopulationDoseDurationEffect Hatakka BMJ 2001 N=571 1-6 y ≈10 8 CFU7 mo (winter) LGG may reduce RTI and their severity Kumpu EJCN 2012 N=523 2-6 y ≈10 8 CFU7 mo (Oct-Apr) LGG reduced RTI in the completed cases (in terms of recovery of LGG in fecal samples), but not in the total population Hojsak Clin Nutr 2009 N=281 1-7 y 10 9 CFU3 mo (Nov-Feb) LGG can be recommended for decreasing the risk of upper RTI Total1375 Lactobacillus GG for preventing acute respiratory tract infections

65. PopulationDoseDurationEffect Hatakka BMJ 2001 N=571 1-6 y ≈10 8 CFU7 mo (winter) LGG may reduce RTI and their severity Kumpu EJCN 2012 N=523 2-6 y ≈10 8 CFU7 mo (Oct-Apr) LGG reduced RTI in the completed cases (in terms of recovery of LGG in fecal samples), but not in the total population Hojsak Clin Nutr 2009 N=281 1-7 y 10 9 CFU3 mo (Nov-Feb) LGG can be recommended for decreasing the risk of upper RTI Total1375 Hojsak Clin Nutr 2009 N=742 hospital 10 9 CFU  URTI RR 0.38 (0.2-0.85) Lactobacillus GG for preventing acute respiratory tract infections

66. Upper RTI (n=794) RR 0.7 (0.55 to 0.9) NNT 8 (5 to 15) All RTI (n=1295) RR 0.8 (0.5 to 1.3) NS Szajewska 2012 (unpublished)

67. Lactobacillus GG for preventing acute respiratory tract infections Use of antibiotics RR 0.89 (0.78 to 1.02) Szajewska 2012 (unpublished)

68. How the intervention might work Forsythe P. Chect 2011;139:901-908. Following immune challenge in the airway, cells activated in GALT traffic to the respiratory mucosa where they promote protective and antiinflammatory responses. Microbes in the intestine are sampled by DC directly or following translocation through M cells to the GALT. Phenotypic changes in the DC and the production of Th1 type and/or regulatory mediators Activation of IDO (indolamine 2,3 dioxygenase) and subsequent production of KYN (kynurenine) promotes Tregs and depletes Th2 cells.

69. Probiotics for preventing allergy Data from (some) RCTs Probiotic(s)Before delivery After delivery Outcome (AD) Effect LGG 4 wk6 mo2,4,7 y Yes LGG 6 wk6 mo2 yNo LGG From 36 wk of gestation Until delivery 1 yNo L acidophilus LAVRI A1 -6 mo2.5 yNo L reuteri ATCC 55730 6 wk12 mo2 y No Yes (IgE eczema) BL999 + LPR -6 mo1 yNo L rhamnosus HN001 From 35 wk of gestation 6 mo2 y/4 y Yes/Yes B animalis subsp. lactis No/No B bifidum & B lactis & L acidophilus 4-8 wk6 mo1 y Yes BL999 +LPR 8 wk2 mo2 y Yes BL999 + ST11 Yes

70. Probiotics for preventing allergy Meta-analysis Cochrane review 2007 Allergic disease or food hypersensitivity Insufficient evidence to recommend probiotics Lee JACI 2008 Atopic dermatitis ✔ Betsi Am J Clin Dermatol 2008 Atopic dermatitis ✔ (especially LGG) Doege Br J Nutr 2012 Atopic eczema ✔ Pelucchi Epidemiology 2012 Atopic dermatitis ✔

71. Probiotics supplementation during pregnancy or infancy for the prevention of atopic dermatitis Pelucchi et al. Epidemiology 2012:23:402-14. 18 publications based on 14 RCTs RR 0.79 (0.71 to 0.88)

72. Probiotics supplementation during pregnancy or infancy for the prevention of atopic dermatitis Moderate role of probiotics in the prevention of AD and IgE- associated AD in infants. The favorable effect was similar regardless of the time of probiotic use (pregnancy or early life) or the subject(s) receiving probiotics (mother, child, or both)

73. Would you recommend use of probiotics to prevent/treat allergic disease in your patients?

74. AAP recommendation The results of some studies support the prophylactic use of probiotics during pregnancy and lactation and during the first 6 mo of life in infants who are at risk of atopic disorders Further confirmatiory evidence is necessary before a recommendation for routine use can be made. Thomas et al. Pediatrics 2010;126:1217-31. All probiotics are not created equal

75. What is new? Design RCT, double-blind Participants Mothers & infants at high risk of allergy Intervention L rhamnosus HN001 B animalis subsp. lactis HN019 From 35 wk gestation until 6 mo if brestfeeding and infant supplementation until 2 y Comparison Placebo Primary outcome Allergic disease & sensitisation at 2 & 4 y (90% participated in follow-up) 2012

76. L rhamnosus HN001, but not B animalis subsp lactis HN019, reduced the cumulative prevalence of eczema, but not atopy, by 2 & 4 years. At 2 yearsAt 4 years Intervention until 2 y One of a very few studies to separately evaluate 2 different probiotics Wickens et al. Clin Exp Allergy 2012;42:1071-9. 2012

77. Maternal supplementation with LPR and BL999 or ST11 and BL999 during pregnancy and breastfeeding reduces the risk of eczema in the infant Rautava et al. JACI 2012;130:1355-60. 2012

78. Mechanisms of action Non-immunologic Probiotics – Digest food and compete for nutrients with pathogens – Alter local pH to create an unfavorable local environment for pathogens – Produce bacteriocins to inhibit pathogens – Stimulate epithelial mucin production – Enhance intestinal barrier function – Complete for adhesion with pathogens – Modify pathogen-derived toxins O'Toole PW, Cooney JC. Interdiscip Perspect Infect Dis. 2008;2008:175285 WHO Global Guideline. Probiotics and prebiotics. 2011.

79. Mechanisms of action Immunologic Probotics Increased phagocytosis capacity Increased NK cell activity Stimulation of IgA production Supression of lymphocyte proliferation Induction of apoptosis Increased cell-mediate immunity Delcenserie et al. Curr Issues Mol Biol. 2008;10(1-2):37-54 All probiotics are not created equal

80. Cytokines produced following the interaction of probiotics with the intestinal epithelium Delcenserie et al. Curr Issues Mol Biol. 2008;10(1-2):37-54 All probiotics are not created equal Mechanisms of action Immunologic

81. Some probiotics and their effects on dendritic cells maturation Delcenserie et al. Curr Issues Mol Biol. 2008;10(1-2):37-54 All probiotics are not created equal

82. RCT Cohort studies Case-control studies Case series studies Expert opinion, theories bases on physiology or plausibility, bench top research & animal studies Weak Strong Hierarchy of evidence for questions about the effectiveness of an intervention Guyatt, Rennie. User’s guides to the medical literature. 2002 Systematic review/meta-analysis