Prof Sanjay Patole, MD, DCH, FRACP, MSc, DrPH

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Probiotics for preterm neonates what lies ahead?

• Prof Sanjay Patole, MD, DCH, FRACP, MSc, DrPH
• Centre for Neonatal Research and Education
• KEM Hospital for Women, University of Western
  Australia, Perth

2
Routine probiotic supplementation (RPS)

• Reduced incidence of NEC associated with
  introduction of probiotics in a NICU Hoyos AH
  1999
• Cohort study of probiotics in a North American
  NICU Janvier et al. 2014
• Currently 15 tertiary NICUs in Australia provide
  RPS for preterm VLBW neonates

3
Probiotics prevent NEC in preterm neonates

• 30 Trials from 17 nations (n6655), 13
  Systematic reviews
• NEC Stage II RR 0.39 (95 CI0.27-0.56),
  plt0.00001
• All cause mortality RR 0.58 (95 CI0.46-0.75),
  plt0.0001
• NEC related mortality RR 0.38 (95 CI 0.18
  -0.82)
• Time to full feeds WMD -1.32 (95 CI -1.48 to
  -1.17)
• Probiotics for preterm neonates Enough is
  enough!!
• Barrington 2012

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• Probiotics fishing in the ocean. Vandenplas 2012
• The politics of probiotics probiotics, NEC and
  the ethics of neonatal research. Janvier 2013
• Probiotics to prevent NEC- Too cheap and easy?
  Taylor 2014
• Myth NEC probiotics will end the disease.
  Caplan 2011
• Probiotics strain for credibility.
  Hamilton-Miller 2000

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Probiotics for preterms- what lies ahead?

• Challenges and opportunities

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Extremely preterm neonates

• Extremely preterm neonates are most deserving of
  probiotic supplementation.
• Data on ELBW neonates from RCTs (N1500) and
  reports on routine use of probiotics is assuring.
• Probiotic sepsis is easy to treat compared with
  sepsis due to other organisms.

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• Benefits of probiotics in ELBW neonates may be
  suboptimal
• Frequent exposure to antibiotics
• Frequent stoppage of feeds
• Recurrent episodes of late onset sepsis by CONS
• Dependence on parenteral nutrition

8
Exposure to antibiotics

• Early postnatal exposure to Ampicillin and
  gentamicin had significant adverse effects on
  evolution of gut flora in infants.
• Antibiotic-treated infants had ?? Proteobacteria
  (p0.0049) and ?? Actinobacteria (p0.00001), ?
  Bifidobacterium (p0.0132) and ? Lactobacillus
  (p0.0182) compared with controls 4 weeks after
  stopping antibiotics.
• Proteobacteria levels significantly higher by
  week 8 in the treated infants (p0.0049).
• Fouhy Antimicrob Agents Chemother 2012

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Exposure to antibiotics

• Preterm neonates who received 5-7 days of empiric
  antibiotics in the 1st week had relative
  abundance of Enterobacter (p0.016) and lower
  bacterial diversity in week 2 and 3.
• Higher frequency of NEC, LOS, and death in those
  receiving early antibiotics vs those not exposed
  to antibiotics.
• Greenwood, J Pediatr 2014 Feb
• Association of prolonged exposure to antibiotics
  with LOS, NEC and death in preterm neonates.
• Cotten, Kuppala, Alexander, Shah

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Intrauterine growth restriction (IUGR)

• Dorling et al Meta analysis of independent case
  series
• 14 studies compared NEC rates in neonates who had
  fetal AREDF-UA with controls (forward fetal EDF).
  
• 9 studies showed ?odds of NEC in those with fetal
  AREDF.
• OR 2.13 (95 CI 1.49-3.03)
• ADC Fetal Neonatal Ed 2005

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• Frequent signs of feed intolerance (e.g.
  abdominal distension, visible ropy bowel loops,
  large/coloured gastric residuals) and the fear of
  NEC means it often takes 2-3 weeks to reach
  120-150ml/kg/day feeds.
• Median (IQR) time to full feeds in IUGR vs AGA
  extremely preterm neonates 20 (15-34) vs. 16
  (12-24) days, p0.008
• Shah et al. JMF Neonatal Med 2014 Oct

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Kempley et al

• Post-hoc analysis of data on neonates lt29 weeks
  from a RCT (ADEPT) comparing benefits of starting
  feeds Early (D2) vs Late (D6) in preterm
  neonates (lt35 weeks) with IUGR.
• Feed increments as per the protocol should have
  achieved full feeds by D16 in the early and D20
  in the late group.
• ADC Fetal Neonatal Ed 2014

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• Neonates lt29 weeks achieved full feeds
  significantly later and had higher incidence of
  NEC vs those 29 weeks.
• Median (IQR) age 28 (22-40) vs 19 (17-23) days
• HR 0.35 (95 CI 0.3 to 0.5)
• NEC 32/83 (39) vs 32/312 (10)
• RR 3.7 (95 CI 2.4-5.7)

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NEC and feed intolerance in IUGR

• Fetal hypoxia and redistribution of the GI blood
  flow to spare the brain from hypoxic injury
• Hypoxic-ischaemic injury of the gut affects
  development of its motor, secretory, and mucosal
  functions, and increases its postnatal
  vulnerability to ileus, altered colonization, and
  bacterial invasion.
• Postprandial rise in SMA flow is compromised
• Pseudo-obstruction due to meconium plug, ? LOS

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IUGR

• Significantly decreased intestinal weight and
  length, ileal and colonic weight/cm, and villous
  sizes at birth in piglets with IUGR vs same-age
  controls.
• ? Markers of apoptosis and ? markers of
  proliferation
• DInca J Nutr 2010
• ?Bioavailability of butyrate in IUGR could
  adversely affect colonocyte proliferation,
  colonic homeostasis, and reduce mucin secretion.
• Gaudier 2004, Barcelo 2000

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IUGR

• IUGR impairs mucus barrier development and is
  associated with long-term alterations of mucin
  expression.
• Lack of an efficient colonic barrier induced by
  IUGR may predispose to colonic injury in neonatal
  as well as later life.
• Continuously impaired intestinal development in
  neonatal piglets with IUGR.
• Fanca-Berthon 2009, Wang 2010

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IUGR

• Effect of IUGR on cecocolonic microbiota from
  birth to adulthood in rats with vs without IUGR
• Bacterial density ? at D5 and ? at D12 in IUGR
• Adult rats with IUGR had fewer Bifidobacteria at
  D40 and more bacteria related to Roseburia
  intestinalis at D100
• Fanca-Berthon JPGN 2010

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Baseline fecal Bifidobacteria in IUGR

• No baseline differences in the proportion of
  detectable B. counts between extremely preterm
  IUGR and AGA neonates.
• Probiotic IUGR vs AGA 7(33) vs 22 (42),
  p0.603
• Control IUGR vs AGA 1(6) vs 1 (2), p0.429
• Patole et al. PLOS ONE 2014 March
• (Post-hoc analysis of data on lt28 week
  IUGR vs AGA)

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Response to probiotic suppl. in IUGR

• Response to probiotic did not differ between IUGR
  and non-IUGR neonates (p0.589), after adjusting
  for baseline counts and treatment allocation.
• IUGR neonates on probiotic (vs placebo) showed a
  non-significant trend towards a younger postnatal
  age at FEF (adjusted for age at start of MEF)
• Median (IQR) age 16 (12-26) vs 19 (11-25) days

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Probiotics can facilitate enteral nutrition

• Secreted products
• Products of fermentation (SCFA)
• Influence on intestinal neuroendocrine factors
• Gut mediators secreted as an immune reaction to
  probiotics
• Soret 2010, Barbara 2005, Cherbut 2003

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Opportunities for advancing knowledge

• Assessing nutritional benefits of probiotics is
  important.
• Jape-Athalye et al AJCN 2014 Nov.
• Colonisation depends on strain properties, and
  host related factors such as gestational and
  postnatal age
• Animal models Strain selection for clinical use
  (Wu 2013)
• Early vs Late Highest colonization rate when the
  suppl. was started between 24 and 48 hours after
  birth. (Yamasaki 2012)
• Single ve Multi-strain probiotic (Ishizeki 2013)
• Live vs Inactivated/killed probiotic (Awad 2010)

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Opportunities

• Real life benefits of probiotics may not be as
  dramatic as reported in RCTs.
• Reporting outcomes and safety data on RPS is
  important to know real life benefits and
  uncommon/rare adverse effects.
• Strain specific population data for guiding
  clinical practice.
• Assessing the economic benefits of probiotics is
  important.
• Advances in technology Improve tolerance of
  probiotic strains to bile, acid, and oxygen for
  enhanced benefits.

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Challenges

• Cooperation between various stakeholders is
  urgently required for quality control and
  classification of probiotics.
• Field difficulties and priorities in resource
  limited set ups
• Politics of probiotics
• Probiotics will not be a panacea for NEC, an
  illness that is known to present at different
  postnatal ages with different triggers and
  different presentations.

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Challenges Probiotic bacteremia/sepsis

• Case series of Bifidobacterium longum bacteremia
  in three preterm infants on probiotic therapy.
  Zbinden et al. Neonatology. 2015
• Bifidobacterium longum bacteremia in preterm
  infants receiving probiotics. Bertelli et al.
  Clin Infect Dis. 2014
• Fatal gastrointestinal Mucormycosis in an infant
  following use of contaminated ABC Dophilus powder
  from Solgar Inc. http//www.cdc.gov/fungal/rhizopu
  s-investigation.html

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Resource limited set ups

• Probiotic issues
• Product/Strain selection, Cost, Cold storage?
• Import or locally available? Quality assurance
  and check?
• Microbiology back up on site? Baseline data?
• Priorities VLBW, ELBW, IUGR? Hospital vs
  Community?
• Strategies for prevention of NEC
• Antenatal glucocorticoids, Maternal/Donor breast
  milk
• Avoid formula, Standardised feeding protocol
• Avoid undue prolonged exposure to antibiotics

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Probiotics for preterm neonates

• All good?

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PIPs trial

• Multi-centre double blind randomised placebo
  controlled trial
• B. breve BBG-001 ( 2.1 to 5.3 108 cfu daily) in
  infants lt31weeks
• Randomised before 48 hrs.
• Primary outcomes NEC Bell Stage II, LOS,
  Death.
• ITT analysis adjusted for sex, gestation and
  randomisation within 24 hours and allowing for
  clustering of multiples.
• Costeloe et al. Arch Dis Child 201499
  A23-A24

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PIPs results

• 1310 infants randomised
• Median gestation 28.0 weeks, Birth weight1010g
• Age starting intervention 44 hours
• No adverse events related to the intervention
• No benefits in ANY of the outcomes of interest
• Conclusions
• B. breve BBG-001 did not have any advantage
• Highlight need to assess the efficacy of
  different strains
• Challenges the validity of combining trials using
  different probiotic interventions in meta-analyses

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• Thank you!!

30
Prebiotics in preterm neonates

• 7 RCTs (n417), NEC 5 trials (n345), LOS 3
  trials (n295)
• NEC RR 1.24 (96 CI 0.56-2.72)
• LOS RR0.81 (95 CI 0.57-1.15)
• TFF 3 RCTs (n295) no improvement
• Bifidobacteria growth ?? in prebiotic group
• WMD 0.53 (95 CI 0.33, 0.73) 106 colonies/g,
  p lt0.00001)
• Reduced stool viscosity and pH
• No significant adverse effects
• Srinivasjois Clin Nutr 2013 Dec

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Opportunities in the field of prebiotics

• Large RCTs of Prebiotics vs placebo, Pro vs
  Synbiotic
• Assess consumption of specific HMOs by different
  probiotic strains for developing optimal pre and
  probiotic combinations (Synbiotic)
  Garrido et al. Microbiology 2013
• Maternal vs donor breast milk HMO and secretor
  status

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Before vs After RPS lt33 weeks (n834 vs 990)

• NEC/All cause mortality 73 (9) vs 52 (5)
• OR 0.57 (0.38-0.85), p0.005
• NEC ( Stage II) 25 (3.0) vs 15 (1.5)
• OR 0.53 (0.27-1.01), p0.054
• All cause mortality 56 (7) vs 39 (4.0)
• OR 0.58 (0.37-0.91), p0.019
• Any gut perforation 31 (3.7) vs 15 (1.6)
• (Dec 2008-Nov 2010) vs (June 2012-May 2014) _at_
  KEM Perth

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Before vs After RPS lt28 weeks (n250 vs 250)

• (1) NEC/All cause mortality 52 (21) vs 34 (14)
• OR 0.62 (0.37-1.02), p0.05
• (2) NEC ( Stage II) 16 (6) vs 10 (4),
• OR 0.66 (0.29, 1.49), p0.31
• (3) All cause mortality 42 (17) vs 26 (10)
• OR 0.59 (0.33-1.03), p0.06
• (4) Any gut perforation 22 (8.8) vs 9 (4.1)

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• It can be argued that infection with
  lactobacilli is preferable over potential
  pathogens like Klebsiella, Enterobacter, or
  yeast.
• Kliegman and Willoughby. Pediatrics 2005
• The debate may be shifted from whether it is
  safe to give probiotics to whether it is safe not
  to give probiotics to premature neonates.
• Sanders et al, Gut Microbes 2010

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Single vs multistrain probiotics

• Colonisation of an ecosystem providing a niche
  for gt 400 species is anticipated to be more
  successful with multistrain rather than
  monostrain probiotics.
• Given the association of development of
  monoflora with impending NEC, probiotics may
  protect VLBW neonates by enforcing diversity of
  flora or by preventing colonization with
  pathogens.
• Kleigman et al. Pediatrics 2005

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• Based on the complexity of gut flora and the
  pathogenesis of NEC, and the multiple mechanisms
  of benefit of probiotic strains, multistrain
  probiotics may be more effective than
  single-strain probiotics.
• Combination of probiotic strains in a product
  does not necessarily add to the benefits of each
  strain. Consensus meeting report London,
  Nov 2009
• Strain combinations can be antagonistic,
  compatible or synergistic. Salminen et al.
  2009

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Dose

• There will be an optimal dose below which
  benefits may not occur, as survival and
  proliferation to adequate numbers after
  overcoming the barriers (e.g. gastric acid, bile,
  competing pathogens), is not ensured.
• Lewis et al. 1998, Martin et al. 2008
• To be functional, probiotics have to be viable
  and in sufficient dosage levels, typically 106 to
  107 cfu/g of the product. Galdeano et al.
  2004, Shah et al. 2000

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• No standardised number of probiotic bacteria that
  would ensure an effect.
• The effective quantity, for a given effect and a
  given strain, is the quantity which has
  demonstrated an effect in a clinical trial.
• Consensus meeting report- London, November
  2009.

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Scientific advances

• Microencapsulation, improving thermal tolerance
  of strains
• ? Gastric transit, GI persistence, and efficacy
  by cloning listerial betaine uptake system into
  the strain
• Evaluating bile salt hydrolase to increase BA
  tolerance
• Evaluating mucin degradation activity and
  translation ability
• Designer (Genetically modified) probiotic strains
• Metagenomics and metabonomics

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Can more trials help?

• A RCT of 2000 neonates and a baseline incidence
  of 8 would have to show a doubling of the
  incidence of NEC to overturn the benefits shown
  in the trials completed to date. Such a reversal
  of effects has never been demonstrated in
  clinical medicine.
• Barrington KJ, Arch Dis Child Educ Pract Ed
  2011
• A RCT of 4,500 neonates will have to show no
  effect (RR 1.0) in mortality after probiotic
  supplementation.

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Economic analyses (? NEC by 50)

• NEC expenses 10 to 15 million dollars/year in
  Australia
• Probiotic cost 30 to 70 per baby (5000/year)
• Dont forget the lifelong stress of parents
  caring for a child with NDI after severe NEC