Transcript Probiotics for prevention of necrotizing enterocolitis in

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
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.
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
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
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
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
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
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
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
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 β-glucosidase (ABG), α-glucosidase
(GAA), and galactocerebrosidase (GALC) significantly higher
in NEC vs. controls (ABG, p=0.009; GAA, p<0.001; GALC,
p<0.001).
 GAA and GALC had highest diagnostic value with AUC of
0.91 and 0.87.
Benkoe et al Clin Chim Acta. 2015
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
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 >10.2 pg/nmol predicted impending NEC with a
sensitivity of 100% and a specificity of 95.6%.
 iFABPu/uCr didn’t predict NEC 2 days before first s/o NEC
Gollin et al Neonatology. 2014
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
Prebiotics
(1) GOS-FOS reduces suspected NEC, time to full feeds, and
hospital stay in preterm VLBW infants < 34 weeks (n=77)



NEC: 1 (4.0%) vs. 11 (22.0%), HR: 0.49 (95% CI: 0.290.84); p=0.002
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
Arginine
 Two RCTs (N=425); 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, NNT=9,
p=0.02).
 All stages of NEC reduced by 60% (RR: 0.40, 95% CI: 0.23 to
0.69, NNT=5, p= 0.001).
 No significant difference in neurodevelopmental disability at 3
years (RR: 0.65; 95% CI: 0.23-1.83, p=0.41)
Mitchell et al 2014
Lactoferrin: Multicentre RCT (n=743)
NEC significantly lower with BLF and BLF+LGG
 BLF: 5/247 (2.0%), BLF+LGG: 0/238 (0%), Controls:14/258 (5.4%)
 BLF vs Control: RR: 0.37 (95% CI: 0.136-1.005; p=0.055)
 BLF+LGG vs Control: RR: 0.00; p <0.001
Death and/or NEC significantly lower with both treatments
 BLF: 4.0%, BLF+LGG:3.8%, Control:10.1%
 BLF vs Control: RR: 0.39 (95% CI: 0.19-0.80; p=0.008)
 BLF+LGG 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
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%; p=0.10)
or surgical NEC (1.6% vs 4.8%; p=.17) in SSDF vs. controls
 BW <750 g: NEC (2.1% vs 16.2%; p <.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
Oral G-CSF and EPO
 RCT, N=90, 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
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 (p<.05).
 NEC reduced from 10% to 0% in all treatment groups (p<.05)
 Shorter NBM duration of NBM for ‘’feed intolerance’’ in both
rhG-CSF and rhEPO compared with placebo group neonates
(p< .05).
 Serum G-CSF and EPO levels at D0 and D7 did not differ
 No adverse effects
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
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
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-1β, IFN-γ and
TNF-α) assessed by ELISA;TLR4 levels assessed by western
analysis
Oral SP-A
 Treatment with SP-A significantly reduced mortality and
assessment of NEC
 SP-A significantly reduced IL-1β and TNF-α levels, but had
little effect on elevated levels of IFN-γ
 SP-A treatment significantly reduced expression of intestinal
TLR4, key in NEC pathogenesis
Quintanilla et al JPGN 2014
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
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
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
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
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
Hepatic blood flow in NEC
Aim: To evaluate portal and hepatic vein flow in NEC
Methods
 Patient (suspected/definite NEC, n=24) vs. controls (n=25)
 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
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
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 (< 32 weeks) VLBW infants
Patel et al Ped Crit Care Med 2014
Abdominal NIS: Results
 Mean AStO2 in normal preterm infants (n=78) during the first
week of life was significantly higher than in those (n=14) 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; p=0.01).
 Infants with NEC had significantly more variation in AStO2
during and after feeding in the first 2 weeks of life.
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
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, 1.212.9), 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 (OR=11.8; IC, 1.595.8) with intestinal stenosis following NEC.
 None of the x-ray findings were associated with complications.
Conclusion: Sonography reliable for assessing prognosis in NEC
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
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
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
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
Thank you!!