2. Plan  Background  The evidence so far  Gaps in our knowledge  PiPS

3.  As survival increases so the proportion of deaths attributed to NEC and infection rises The problem

4. National data: trends

5. US ‘linked’ data : singletons Lucaks SL et al 2004. Peds Inf Dis J.

6. Primary cause of death 19952006pRR (95% CI) Infection8%16%<0.0011.4 (1.2 – 1.6) NEC4%12%<0.00011.7 (1.5 – 1.9)  As survival increases so the proportion of deaths attributed to NEC and infection rises Source: The EPICure studies

7. Why probiotics?

8. Normal development of bowel flora  Sterile at birth …..  Healthy babies colonised rapidly by maternal faecal flora  Flora of breast fed babies dominated by Bifidobacteria

9. Benefits of the microbial-host interaction BenefitMechanism Maintain mucosal barrier integrity Reduce mucosal permeability Increase mucus production Strengthen tight junctions Inhibit bacterial translocation Regulate appropriate bacterial colonisation Modulate microflora growth and adherence Produce toxins to aerobic bacteria Reduce intraluminal pH Compete with pathogens for binding sites Activate intestinal immune defences Increase faecal IgA and enhance IgA response Produce short chain fatty acids Increase leucocyte phagocytosis Modulate intestinal inflammation Increase T-cell &macrophage cytokine production Decrease production of anti-inflammatory cytokines Decrease proinflammatory cytokines Promote Th1 cytokine profile Martin CR & Walker WA: Seminars in Perinat 2008;

10. Gut flora in preterm babies  Slower acquisition, further delayed by antibiotics  Less diversity  Fewer commensals  More potentially pathogenic strains Gewolb IH et al Arch Dis Childh 1999

11. Probiotic  A live micro-organism which when administered in adequate amounts confers a health benefit on the host (World Health Organisation, 2010)

12. Systematic reviews:  Barclay AR et al., JPGN 2007  Deshpande G et al., Lancet 2007  AlFaleh KM & Bassler D, Cochrane 2008  Mihatsch., 2008  Guthmann., 2010  Deshpande G et al., Pediatrics 2010  AlFaleh et al., Cochrane 2011

13. Systematic reviews:  Barclay AR et al., JPGN 2007  Deshpande G et al., Lancet 2007  AlFaleh KM & Bassler D, Cochrane 2008  Mihatsch., 2008  Guthmann., 2010  Deshpande G et al., Pediatrics 2010  AlFaleh et al., Cochrane 2011

14. Outcome: ‘Definite’ NEC Deshpande et al. 2010

15. Outcome: Blood culture positive sepsis Deshpande et al. 2010

16. Outcome: Death Deshpande et al. 2010

17. Summary of outcomes  Significant reduction of ‘definite’ NEC  No effect on blood culture positive sepsis  Significant reduction ‘all cause’ mortality  No reported problems with safety

18. A closer look at those trials designed to study clinical outcomes  Dani C et al 2002  Lin H-C et al 2005  Bin-Nun A et al 2005  Samanta M et al 2008  Lin H-C et al 2008 These studies account for 85% of the babies included in the updated systematic review

19. Characteristics of clinical studies <2008 Authors Year PopulationExclusions (other than anomalies & refusals) InterventionPrimary outcome Incidence in comparator group Dani et al 2002 Multicentre <33w or<1500g n=585 Mean wt >1300g Death <14dLactobacillus GG To discharge UTI NEC ≥stage2 Sepsis all after 7d 5.2% 2.8% 4.1% Lin et al 2005 Single centre 7d Lines out>24h n=367 Mean wt c.1100g Deaths or NEC in 1 st week L acidophilus B infantis To discharge Death or NEC ≥stage 2 12.8% (powered at 23%) Bin-Nun et al 2005 Single centre <1500g starting milk on weekday n=147 (3 not analysed) Mean wt >1100g No information B infantis Str thermophilus B bifidus To 36w Any NEC16.4%

20. Characteristics of clinical studies <2008 Authors Year PopulationExclusions (other than anomalies & refusals) InterventionPrimary outcome Incidence in comparator group Dani et al 2002 Multicentre <33w or<1500g n=585 Mean wt >1300g Death <14d 100/685 Lactobacillus GG To discharge UTI NEC ≥stage2 Sepsis all after 7d 5.2% 2.8% 4.1% Lin et al 2005 Single centre 7d Lines out>24h n=367 Mean wt c.1100g Deaths or NEC in 1 st week 48/417 L acidophilus B infantis To discharge Death or NEC ≥stage 2 12.8% (powered at 23%) Bin-Nun et al 2005 Single centre <1500g starting milk on weekday n=147 (3 not analysed) Mean wt >1100g No information B infantis Str thermophilus B bifidus To 36w Any NEC16.4%

21. Characteristics of 2 recent studies Authors Year PopulationExclusions (other than anomalies & refusals) InterventionPrimary outcome Incidence in comparator group Samanta et al 2008 Single centre <32w &<1500g Survived 48h Mean wt >1100g Deaths due to ‘other neonatal illness’: B infantis B bifidum B longum L acidophilus To discharge NEC ≥2 Sepsis Death due to NEC or sepsis 15.8% 29.5% 14.7% Lin et al 2008 Multicentre <34w &<1500g & enterally fed Asphyxia Formula fed Comg anolmaly NBM >3w L acidophilus B bifidum For 6w Death or NEC≥2 in the 6w study period 9.2% (was powered at 25%)

22. Characteristics of 2 recent studies Authors Year PopulationExclusions (other than anomalies & refusals) InterventionPrimary outcome Incidence in comparator group Samanta et al 2008 Single centre <32w &<1500g Survived 48h Mean wt >1100g Deaths due to ‘other neonatal illness’: 88/274 B infantis B bifidum B longum L acidophilus To discharge NEC ≥2 Sepsis Death due to NEC or sepsis 15.8% 29.5% 14.7% Lin et al 2008 Multicentre <34w &<1500g & enterally fed Asphyxia Formula fed Comg anolmaly NBM >3w 137/580, 98 of whom died L acidophilus B bifidum For 6w Death or NEC≥2 in the 6w study period 9.2% (was powered at 25%)

23. Published studies: general comments  Only 5 of 11 trials were designed to study clinical outcomes  Have only recruited babies receiving milk feeds  Not easy to separate out the smaller more immature babies  No study has undertaken stool microbiology to determine whether the babies are successfully colonised and how much cross-contamination is occurring  All use different products none of which is quality controlled

24. Safety  None of the published studies reports any complications  Published reports of lactobacilli and one case report of B breve septicaemia  Increased mortality associated with probiotic use in adults with acute pancreatitis, Besselink MGH et al., Lancet 2008.  The babies at highest risk of NEC and possibly of adverse effects have been excluded from the studies.

25. Major outstanding questions  Will probiotics reduce NEC and death in an unselected group of very preterm babies?  Why are probiotics apparently ineffective at preventing sepsis?  Is it necessary to give a multi-strain preparation to gain benefit?  Is it safe to give probiotics to the babies at highest risk of NEC?

27. Objective To determine whether early administration of Bifidobacterium breve strain BBG to preterm infants reduces the incidence of infection, necrotising enterocolitis and death.

28. Why is PiPS different?  It is statistically powered to study all three outcomes  Uses a more stringent definition of sepsis for the primary outcome  It has few exclusions and includes babies at high risk of NEC  The intervention is started early whether or not milk feeds have been started  The intervention is a single bacterial strain manufactured to high specification and with a CTA  Stool colonisation with the probiotic bacterium and the effect on intestinal flora will be studied

29. What will PiPS deliver?  Clarity as to whether B breve BBG prevents death, NEC and blood culture positive sepsis and whether it is safe in a high risk population of babies  Unique insight into the importance of ‘colonisation’ as opposed to simple ‘administration’ of probiotic on efficacy  Unique insight into the implications of probiotic usage on a Neonatal Unit for those babies for whom it is not prescribed

30. Trial design Double blind placebo controlled randomised trial

31. Primary outcomes  Any baby with an episode of blood stream infection, with any organism other than a skin commensal, diagnosed on a sample drawn more than 72h after birth and before death or discharge.  NEC, Bell stage II or III  Death before discharge

32. Secondary outcomes  A range of infective outcomes including CONs and numbers of septic screens and stool colonisation  Use of antimicrobials  Time to full feeds and growth  BPD, ROP, IVH etc.  Length of stay  Economic evaluation

33. Eligibility criteria  ≤30w +6d at birth  <48h after birth  Written informed consent Exclusion criteria  Lethal congenital malformation known at trial entry  Any known g-i malformation  No realistic chance of survival  Babies on antibiotics for suspected or proven infection are eligible for recruitment

34. Interventions Active  Bifidobacterium breve strain BBG freeze dried with corn starch re-suspended in 3ml 1/8 th strength Neocate: Iml (2.7± 0.5x10 9 cfu) to be given once a day starting ASAP after randomisation and continued to 36w post- menstrual age. Placebo  Freeze dried corn starch alone prepared and administered in the same way.

35.  Participating staff receive training about the trial, making up the intervention and completing the data collection forms  There is emphasis on avoiding cross colonisation of the placebo group with the administered probiotic strain

36. Clinical management  Feeding and all aspects of management, including the decision to omit a dose of the intervention, are at the discretion of the local clinicians

37. Data collection  Data collection is paper based and continues till discharge from hospital  Details of microbiology samples, positive cultures and details of antibiotic sensitivities are collected directly from laboratories

38. Sample collection  2 stool samples; as close as possible to 2w post-natal and 36w post-menstrual age  These samples are posted in a special container to the microbiology laboratory at the Royal London Hospital  No other additional samples are required

39. Power calculations and statistical significance  The incidence of all 3 primary outcome measures is estimated at around 15%  A trial of 1,300 babies will have 90% power to detect a 40% reduction in relative risk from 15 to 9%. Likewise if the incidence is nearer 12% we will still have 90% power to detect a 44% RR reduction from 12 to 6.7% and from 10 to 5.6%  For the primary outcomes a 95% Confidence Interval will be calculated; because there are a number of secondary outcomes 99% will be used

40. Analyses  Comparative analyses will be by Intention to Treat  The following pre-specified sub-groups will be analysed: gestation as per minimisation, male v. female, whether randomised in the first or second 24h  A secondary analysis of all clinical and microbiological outcomes will be conducted by whether the baby was colonised with B breve BBG  Logistic regression analysis will be used to study determinants of successful colonisation with B breve BBG in the active intervention group

41. Time-line  The aim is to recruit 1300 babies over a 2.5 year period

42. Practicalities of achieving target recruitment rates  NHS R&D approval  Adequate numbers of staff on the ground with GCP training who can take consent  Approvals in ‘networked’ hospitals so that babies can continue the intervention until 36w pma after returning to the referring hospital

43. Summary  Probiotics are the current best bet for a preventive intervention against NEC and are probably safe  We do not know which organism to use and which babies to give it to  PiPS is designed to fill some of the important gaps in our current knowledge

44. Thank you