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

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Advances in necrotising enterocolitis

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

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Necrotising enterocolitis in preterm neonates

• Significant mortality and mortality including
  long term NDI
• Outcomes are worse, especially in ELBW neonates
  requiring surgical intervention for NEC.
• Health burden of Stage II NEC has not changed
  significantly despite the advances in neonatal
  intensive care, and extensive research over
  decades.
• Poorly understood pathogenesis is the main reason
  for the failure to develop strategies for
  prevention of NEC.

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Pathophysiology of NEC

• Excessive intestinal inflammation due to an
  immature innate immune response (TLR4)
• Lu et al Pathophysiology 2014
• Decreased diversity, complexity and stability of
  gut flora (dysbiosis) and delayed colonization
  by beneficial microbes
• Intestinal epithelial integrity is controlled by
  a tightly regulated balance between proliferation
  and differentiation of epithelium from ISC and
  cellular loss by apoptosis. It involves various
  signaling pathways.
• Kandasamy et al Pathophysiology. 2014

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Recent advances in NEC

• Pathogenesis TLR, gut microbiome, gut-liver
  axis, dysbiosis and fat intake, MDF 88, late
  onset infections,
• Early detection Biomarkers, imaging
• Monitoring Near-infrared spectroscopy, imaging
• Treatment ? Stem cells

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Recent advances in NEC

• Primary prevention Probiotics, prebiotics,
  lactoferrin, arginine, standardised slow feeding,
  oral surfactant, G-CSF, EPO, and relaxin, PGE2
• Secondary prevention ?? Probiotics,
  pentoxifylline
• Transfusion associated NEC Feeding, gut
  oxygenation, Hb

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TLR4

• Expression, localization and signaling of TLR4 in
  colonic epithelium may be developmentally
  regulated.
• Hydrocortisone may accelerate the TLR development
  towards an adult type.
• Meng et al Ped Res 2014
• Prevention of ER stress reduced TLR4-mediated
  intestinal stem cell (ISC) apoptosis and mucosal
  disruption. These changes suggest that increased
  ER stress within the premature bowel predisposes
  to NEC.
• Afrazi et al. J Biol Chem. 2014 Apr

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Gut dysbiosis and NEC

• Two fecal microbiota signatures (Clostridium and
  Klebsiella OTUs) and need for prolonged CPAP
  oxygen signal increased risk of NEC in
  pre-symptomatic infants.
• These biomarkers will assist development of a
  screening tool to allow very early diagnosis of
  NEC.
• Sim et al Clin Infect Dis. 2014 Oct
• NEC is associated with an abundance of strict
  anaerobes and a decrease in community diversity.
• Brower-Sinings PLOS ONE 2014

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DNA methylation

• Pre- and postnatal changes in intestinal DNA
  methylation may contribute to high NEC
  sensitivity in preterm neonates.
• Optimizing gene methylation changes via
  environmental stimuli (e.g. diet, nutrition, gut
  microbiota), may make immature infants more
  resistant to gut dysfunction.
• Gao et al BMC Genomics. 2014 Aug

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Genomics in NEC vs SIP

• The different genome-wide expression profiles
  suggest that NEC and SIP are likely two different
  diseases caused by distinct etiology and
  pathophysiology.
• This first comprehensive database of gene
  expression profiles could help in developing
  disease-specific diagnostic and prognostic
  biomarkers and new treatments.
• Chan et al Ann Surg. 2014 Dec

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Lysosomal enzymes New biomarkers?

• Gut ischemia is associated with ?plasma lysosomal
  enzymes
• Case-control study (15 neonates with NEC vs. 18
  controls)
• Plasma activities of ß-glucosidase (ABG),
  a-glucosidase (GAA), and galactocerebrosidase
  (GALC) significantly higher in NEC vs. controls
  (ABG, p0.009 GAA, plt0.001 GALC, plt0.001).
• GAA and GALC had highest diagnostic value with
  AUC of 0.91 and 0.87.
• Benkoe et al Clin Chim Acta. 2015

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Biomarkers IL8, serum and liver FABP

• Serum concentrations of I-FABP, L-FABP and IL-8
  were significantly higher in infants with NEC
  compared with controls.
• IL-8 had highest diagnostic value with an AUC of
  0.99, followed by L-FABP and I-FABP.
• Significant correlation between IL-8 and both
  FABPs in infants with NEC.
• Benkoe J Pediatr Surg. 2014

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Biomarkers Fecal Calprotectin, iFABPu

• Rapid Fecal Calprotectin (FC) Analysis Point of
  care testing for diagnosing early NEC.
• Bin-Nun et al Am J Perinatol 2014
• Five subjects developing NEC (Stage II 3, Stage
  III 2)
• The day before first clinical manifestation of
  NEC, the iFABPu/uCr gt10.2 pg/nmol predicted
  impending NEC with a sensitivity of 100 and a
  specificity of 95.6.
• iFABPu/uCr didnt predict NEC 2 days before first
  s/o NEC
• Gollin et al Neonatology. 2014

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Probiotics

• Infants Start early, single vs multi-strain,
  alternate days, Formula vs breast milk
• Maternal suppl. Temporary colonisation of the
  infant
• Significance of maternal secretor status,
  consumption of HMOs by probiotic strains (?
  Designer synbiotics)
• Continued suppl. during stage II may prevent
  progression to stage III NEC
• Nutritional benefits while reducing NEC and
  mortality
• Safety Probiotic bacteremia, sepsis,
  contamination

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Prebiotics

• (1) GOS-FOS reduces suspected NEC, time to full
  feeds, and hospital stay in preterm VLBW infants
  lt 34 weeks (n77)
• NEC 1 (4.0) vs. 11 (22.0), HR 0.49 (95 CI
  0.29-0.84) p0.002
• TFEF 11 (7-21) vs. 14 (8-36) days p 0.02
• Hospital stay 16 9-45 vs. 25 11-80 days p
  0.004
• Armanian 2014 Nov
• (2) Disialyllacto-N-tetraose prevents NEC in
  neonatal rats. Jantscher-Krenn et al. Gut.
  2012

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Arginine

• Two RCTs (N425) 235 included in the systematic
  review
• L-arginine reduced stage II and III NEC by 59
  compared with placebo (RR 0.41, 95 CI 0.20 to
  0.85, NNT9, p0.02).
• All stages of NEC reduced by 60 (RR 0.40, 95
  CI 0.23 to 0.69, NNT5, p 0.001).
• No significant difference in neurodevelopmental
  disability at 3 years (RR 0.65 95 CI
  0.23-1.83, p0.41)
• Mitchell et al 2014

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Lactoferrin Multicentre RCT (n743)

• NEC significantly lower with BLF and BLFLGG
• BLF 5/247 (2.0), BLFLGG 0/238 (0),
  Controls14/258 (5.4)
• BLF vs Control RR 0.37 (95 CI 0.136-1.005
  p0.055)
• BLFLGG vs Control RR 0.00 p lt0.001
• Death and/or NEC significantly lower with both
  treatments
• BLF 4.0, BLFLGG3.8, Control10.1
• BLF vs Control RR 0.39 (95 CI 0.19-0.80
  p0.008)
• BLFLGG vs Control RR 0.37 95 CI 0.18-0.77
  p 0.006)
• No adverse effects or intolerances to treatment
• Manzoni et al 2014

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Standardised slow-delayed feeding for ELBW

• 125 ELBW in SSEF group vs. 294 historical
  controls
• Longer time to start feeds, TFEF, TPN and CVL
  days in SSDF
• No significant difference in any NEC (5.6 vs
  11.2 p0.10) or surgical NEC (1.6 vs 4.8
  p.17) in SSDF vs. controls
• BW lt750 g NEC (2.1 vs 16.2 p lt.01) or
  combined NEC/death (12.8 vs 29.5 p.03)
  significantly less in the SSDF group
• No significant diff. in discharge weight or stay
  in adjusted analysis
• Viswanathan JPEN 2014

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Oral G-CSF and EPO

• RCT, N90, Gestation 33 weeks
• Four groups 20 each on rhG-CSF, rhEPO, or both,
  and 30 received distilled water as placebo
• Test solution given orally at the start of feeds
  and discontinued when reaching 100 mL/kg/day
  feeds or after a maximum of 7 days, whichever
  came first
• El-Ganzouri et al J Pediatr 2014

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G-CSF and EPO

• Neonates on oral rhG-CSF and/or rhEPO had better
  feed tolerance, and reached 75, 100, and
  150 mL/kg/day feeds earlier, with weight gain,
  and shorter stay (plt.05).
• NEC reduced from 10 to 0 in all treatment
  groups (plt.05)
• Shorter NBM duration of NBM for feed
  intolerance in both rhG-CSF and rhEPO compared
  with placebo group neonates (plt .05).
• Serum G-CSF and EPO levels at D0 and D7 did not
  differ
• No adverse effects

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Relaxin

• Relaxin (RLXN), a hormone in breast milk but
  absent from formula, is a potent vasodilator
• Hypothesis Relaxin-supplemented feeds would
  decrease NEC severity and increase intestinal
  blood flow in a rat pup model of the illness
• Matheson J Ped Surg 2014

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Relaxin

• Addition of relaxin to NEC group feeds improved
  the degree of ileal injury
• Ileal blood flow was decreased in NEC pups vs.
  controls but the addition of relaxin to ONE feed
  increased baseline ileal blood flow in the NEC
  group compared to NEC alone
• Addition of relaxin to ALL feeds significantly
  increased baseline ileal blood flow
• Matheson J Ped Surg 2014

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Oral surfactant protein-A (SP-A)

• Experimental NEC in newborn Sprague-Dawley rat
  pups by daily formula feeds and intermittent
  hypoxia.
• Purified human SP-A (5?µg/day) administered by
  oral gavage. After 4 days, surviving pups were
  sacrificed, and histological examination of
  distal terminal ileal sections was conducted.
• Intestinal inflammatory cytokine levels (IL-1ß,
  IFN-? and TNF-a) assessed by ELISATLR4 levels
  assessed by western analysis

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Oral SP-A

• Treatment with SP-A significantly reduced
  mortality and assessment of NEC
• SP-A significantly reduced IL-1ß and TNF-a
  levels, but had little effect on elevated levels
  of IFN-?
• SP-A treatment significantly reduced expression
  of intestinal TLR4, key in NEC pathogenesis
• Quintanilla et al JPGN 2014

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PGE2

• Indomethacin, a non-selective PG inhibitor for
  closing PDA, is associated with intestinal
  perforation inducing an NEC-like illness.
• Aim Define the contribution of PGE2 and its
  receptor EP4 to intestinal blood flow regulation
  in preterm neonates with NEC.
• Methods Rat pup model of NEC. At 48hours of age,
  intestinal laser Doppler blood flow was assessed
  at baseline and after IP indomethacin, PGE2, EP4
  antagonist, or EP4 agonist.
• K Walker J Pediatr Surg 2014

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PGE2

• At baseline, NEC pups had lower intestinal blood
  flow than controls
• Indomethacin, PG E2 and EP4 agonist increased
  ileal blood flow, but PGE2 and EP4 agonist
  increased blood flow the most in NEC pups
• EP4 antagonist decreased intestinal perfusion in
  both groups
• K Walker J Pediatr Surg 2014

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Fluroscopy for detecting stricture

• 56 patients, 51 UGI-SBFT and 85 CE, 25 strictures
  detected
• Small bowel (SB) strictures CE vs. UGI-SBFT had
  higher sensitivity (0.667 vs 0.00) and similar
  specificity (0.857 vs 0.833).
• SB and/or colonic strictures CE had a
  sensitivity of 0.667 and a specificity of 0.951.
• Strictures more likely in symptomatic vs.
  asymptomatic infants (28 vs 8, p??0.002)
• Contrast enema (CE) is the investigation of
  choice
• Wiland et al JPGN 2014

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Sonography in NEC

• Prospective study 26 consecutive NEC Stage
  II/III infants
• At least one abdominal US performed in each
  patient
• Surgery at the discretion of the surgeon
• US showed signs of intestinal necrosis in 5/26
  patients, all 5 had laparotomy.
• The sensitivity, specificity, positive and
  negative predictive values of US for the
  detection of bowel necrosis were 100, 95.4, 80.0,
  and 100, respectively.
• Yikilmaz et al. Pediatr Surg Int. 2014

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Sonography in NEC

• Abdominal US can identify those infants with NEC
  who may need surgery by detecting bowel necrosis
  (prior to the development of perforation or
  medical deterioration) with high sensitivity and
  specificity.
• Early surgical intervention may improve outcomes
• Yikilmaz et al. Pediatr Surg Int. 2014

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Hepatic blood flow in NEC

• Aim To evaluate portal and hepatic vein flow in
  NEC
• Methods
• Patient (suspected/definite NEC, n24) vs.
  controls (n25)
• Daily serial DUS performed after suspecting NEC
  and continued until the initial day of enteral
  feeding
• Portal blood flow (PBF) and "hepatic blood flow
  ratio" (RHBF) were calculated
• Akin et al JMFNM 2014

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Hepatic blood flow in NEC

• Results
• PBF and RHBF significantly ? in patients vs.
  controls
• Clinical improvement in NEC associated with ?PBF
  and RHBF. Cut-off RHBF level for diagnosis of
  NEC 0.66.
• Conclusion
• DUS useful for diagnosis and follow-up of NEC by
  providing quantitative information on hepatic
  blood flow.
• Daily PBF and RoHBF measurements in neonates with
  NEC may be beneficial to make the decision of
  starting enteral feeding. Akin et al JMFNM
  2014

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Abdominal near-infrared spectroscopy (NIS)

• Background NIS is a noninvasive method of
  measuring local tissue oxygenation (StO2).
  Abdominal StO2 (AStO2) measurements in preterm
  piglets are directly correlated with changes in
  intestinal BF and markedly reduced by NEC.
• Aim To use NIS to establish normal values for
  abdominal StO2 in preterm infants and test
  whether they are reduced in infants who develop
  NEC
• Methods 100 preterm (lt 32 weeks) VLBW infants
• Patel et al Ped Crit Care Med 2014

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Abdominal NIS Results

• Mean AStO2 in normal preterm infants (n78)
  during the first week of life was significantly
  higher than in those (n14) who later developed
  NEC 77.3 14.4 vs 70.7 19.1, p 0.002
• AStO2 56 identified those progressing to NEC
• Sensitivity 86, Specificity 64, NPV 96,
  PPV 30
• AStO2 56 independently associated with a
  significantly increased risk of NEC (OR 14.1
  p0.01).
• Infants with NEC had significantly more variation
  in AStO2 during and after feeding in the first 2
  weeks of life.

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Abdominal sonography in NEC

• A retrospective study of 95 preterm infants (mean
  gestation 28.6 weeks), presenting with NEC and
  who underwent plain abdominal radiography and
  sonography, was performed.
• In uni- and multivariate analyses, radiographic
  and sonographic findings were correlated with
  complications ('surgery and/or death' and
  'stenosis')
• Garbi-Goutel et al J Pediatr Surg 2014

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Abdominal sonography in NEC

• Free intraperitoneal air (OR 8.0 IC, 1.4-44.2),
  free abdominal fluid (OR 3.5 IC 1.3-9.4), portal
  venous gas (OR 3.9 IC, 1.2-12.9), and bowel wall
  thickening (OR 2.8 IC,1.1-7.2) were
  significantly associated with surgery and/or
  death.
• Intramural gas was significantly correlated
  (OR11.8 IC, 1.5-95.8) with intestinal stenosis
  following NEC.
• None of the x-ray findings were associated with
  complications.
• Conclusion Sonography reliable for assessing
  prognosis in NEC

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Portal venous blood flow and gut ischemia

• Aim Evaluate the utility of portal blood flow
  and other hemodynamic measurements for early
  diagnosis of ischemia that may cause NEC
• Methods Measured neonatal PBF and hemodynamics
  in 75 neonates without congenital anomalies.
• All neonates followed for 1?month after birth.
  The average gestation and birth weight was 30.5
  weeks, and 1,172 g.
• Kobayashi et al. Eur J Pediatr Surg. 2014

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PVBF and gut ischemia

• PV cross-sectional area and BFV changed over time
  to maintain a fixed PVBF volume.
• Seven infants demonstrated a reduction in PVBF
  before development of abdominal symptoms.
• Both the cross-sectional area and BFV decreased
  over time before onset of NEC symptoms.
• Conclusion A significant decline in PBF volume
  may be useful for the early diagnosis of NEC.
• Kobayashi et al. Eur J Pediatr Surg. 2014

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Magnetic resonance imaging (MRI)

• The correlation of MRI results with histologic
  images of the excised ileal tissue samples
  strongly suggests that MRI can noninvasively
  identify NEC and assess intestinal injury prior
  to clinical symptoms in a physiologic rat pup
  model of NEC.
• Mustafi et al. NMR Biomed. 2014 Mar

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Stem cell therapy

• Amniotic fluid stem cells prevent development of
  ascites in a neonatal rat model of NEC.
• Zani et al. Eur J Pediatr Surg. 2014
• Amniotic fluid stem cells improve survival and
  enhance repair of damaged intestine in NEC via a
  COX-2 dependent mechanism in rats.
• Zani et al Gut 2014
• Review Yang et al. Methods Mol Biol. 2014

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• Thank you!!