Fardou H. Heida

A Necrotizing Enterocolitis-Associated Gut Microbiota Is Present in the Meconium: Results of a Prospective Study

BACKGROUND Anomalous intestinal microbiota development is supposedly associated with development of necrotizing enterocolitis (NEC). Our aim in this study was to identify the intestinal microbiota of patients at risk for NEC. METHODS In a prospective trial that investigated prognostic factors for development of NEC in high-risk neonates (NTR4153), 11 NEC cases were gestational age/birthweight matched with controls (ratio of 1:2). Feces were collected twice a week. We used the first feces sample of each patient (meconium), as well as the last 2 feces samples prior to development of NEC. DNA was extracted, and the bacterial 16S rRNA genes were analyzed on a MiSeq sequencer. RESULTS The presence and abundance of Clostridium perfringens (8.4%) and Bacteroides dorei (0.9%) in meconium were increased in neonates who developed NEC compared with controls (0.1% and 0.2%; both species, P < .001). In post-meconium samples, the abundance of staphylococci became negatively associated with NEC development (P = .1 and P = .01 for consecutive samples); Clostridium perfringens continued to be more prevalent in NEC cases. Early enteral feeding and, in particular, breast milk were correlated with an increase in lactate-producing bacilli in post-meconium samples (ρ = -0.45; P = .004). CONCLUSIONS A NEC-associated gut microbiota can be identified in meconium samples; C. perfringens continues to be associated with NEC from the first meconium till just before NEC onset. In contrast, in post-meconium, increased numbers of staphylococci were negatively associated with NEC. These findings suggest causality but this causality should be verified in trials of induced infection in animals, targeted antibiotics, and/or probiotics. CLINICAL TRIALS REGISTRATION CALIFORNIA trial, registered under trial number NTR4153 in the Dutch Trial Registry.

Paneth cells in the developing gut: when do they arise and when are they immune competent?

Background:Little is known about the perinatal development of Paneth cells (PCs) during gestation and the relation with necrotizing enterocolitis (NEC). We aimed to investigate when PCs arise and when they become immune competent during gestation.Methods:We included 57 samples of ileum tissue of fetuses/infants with a gestional age (GA) between 9 and 40 wk taken as part of a standard autopsy procedure. Hematoxylin-eosin staining and anti-human defensin 5 immunohistochemistry were performed. We performed a semi-quantitative assessment of (immune-competent) PC numbers per 10 crypts per tissue section per GA.Results:The number of PCs and the number of immune-competent PCs increased with increasing GA (Spearman’s ρ = 0.41, P = 0.002 and ρ = 0.61, P < 0.001, respectively). Whereas significantly higher PC numbers were observed after 37 wk gestation (median 7, range 0–12) compared to preterm infants (median 0, range 0–15; P = 0.002), we counted higher numbers of immune-competent PCs already in infants with GA above 29 wk (median 6, range 0–18) compared to infants with GA under 29 wk (median 2, range 0–9; P < 0.001).Conclusion:The significant increase of immune-competent PCs starting from a GA of 29 wk mimics the rise in incidence of NEC during a similar postmenstrual age in preterm infants.

Risk factors associated with postnecrotizing enterocolitis strictures in infants

INTRODUCTION Survivors of necrotizing enterocolitis (NEC) often develop a post-NEC intestinal stricture, causing severe and prolonged morbidity. OBJECTIVES We first aimed to determine the incidence of post-NEC strictures. Second, we aimed to determine risk factors associated with intestinal post-NEC strictures. MATERIALS AND METHODS A total of 441 patients diagnosed with NEC Bells stage ≥2 were retrospectively included in three academic pediatric surgical centers between January 2005 and January 2013. Clinical data were related to the occurrence of intestinal post-NEC strictures. Post-NEC strictures were defined as clinically relevant strictures with a radiological and/or surgical confirmation of this post-NEC stricture. RESULTS The median gestational age of the 337 survivors of the acute phase of NEC was 29weeks (range 24-41) and median birth weight was 1130g (range 410-4130). Of the survivors, 37 (17%) medically treated NEC patients developed a post-NEC strictures versus 27 surgically treated NEC patients (24%; p=0.001). Highest C-reactive protein (CRP) level measured during the NEC episode was associated with the development of post-NEC strictures (OR 1.20, 95% confidence interval 1.11-1.32; p=0.03). No post-NEC strictures were detected in patients with CRP levels <46mg/L. CONCLUSION This multicenter retrospective cohort study demonstrates an overall incidence of clinical relevant post-NEC strictures of 19%, with a higher rate (24%) in NEC cases treated surgically. Increased CRP levels during the NEC episode were associated with the development of post-NEC strictures.

Increased incidence of necrotizing enterocolitis in the Netherlands after implementation of the new Dutch guideline for active treatment in extremely preterm infants: Results from three academic referral centers.

INTRODUCTION Necrotizing enterocolitis (NEC) is a severe inflammatory disease, mostly occurring in preterm infants. The Dutch guidelines for active treatment of extremely preterm infants changed in 2006 from 26+0 to 25+0weeks of gestation, and in 2010 to 24+0 of gestation. We aimed to gain insight into the incidence, clinical outcomes and treatment strategies, in three academic referral centers in the Netherlands over the last nine years. METHODS We performed a multicenter retrospective cohort study of all patients with NEC (Bell stage ≥2a) in three academic referral centers diagnosed between 2005 and 2013. Outcome measures consisted of incidence, changes in clinical presentation, treatment strategies and mortality. RESULTS Between 2005 and 2013 14,161 children were admitted to the neonatal intensive care unit in the three centers. The overall percentage of children born at a gestational age of 24weeks and 25weeks increased with 1.7% after the introduction of the guidelines in 2006 and 2010. The incidence of NEC increased significantly (period 2005-2007: 2.1%; period 2008-2010 3.9%; period 2011-2013: 3.4%; P=0.001). We observed a significant decrease of peritoneal drainages (↓16%; P=0.001) and a decrease of laparotomies (↓24%; P=0.002). The mortality rate (33% in 2011-2013) remained unchanged. CONCLUSION The incidence of NEC significantly increased in the last nine years. The increase in incidence of NEC seemed to be related to an increase in infants born at a gestational age of 24 and 25weeks. The percentage of patients needing surgery decreased, while 30-day mortality did not change. LEVEL OF EVIDENCE Level IV.

The relation between splanchnic ischaemia and intestinal damage in necrotising enterocolitis

Objectives The underlying pathophysiology of necrotising enterocolitis (NEC) remains incompletely understood, particularly the role of intestinal perfusion. We aimed to determine the relation between cerebral and splanchnic fractional tissue oxygen extraction (FTOE), a marker for tissue underperfusion, with intestinal fatty acid-binding protein in plasma (I-FABPp), a marker for intestinal damage, in infants with NEC. Furthermore, we investigated the combined courses of cerebral and splanchnic FTOE values and I-FABPp levels in uncomplicated (conservative treatment) and complicated NEC (surgery or death). Design This study was part of a prospective observational cohort study. Patients We included 19 preterm infants with NEC (9 uncomplicated, 10 complicated). Interventions Using near-infrared spectroscopy, we measured regional cerebral and splanchnic tissue oxygen saturations continuously for 48 h after NEC onset. We measured I-FABPp levels simultaneously. Main outcome measures We used Spearman correlation tests to calculate correlation coefficients between FTOE values and I-FABPp levels in uncomplicated and complicated NEC. Results Median (range) gestational age was 28 (25–36) weeks and median (range) birth weight was 1290 (740–2400) g. Cerebral and splanchnic FTOE values correlated strongly with I-FABPp levels (rho between .745 and 0.900; p<0.001–0.037) during the first 16 h after NEC onset. Thereafter, in uncomplicated NEC, splanchnic FTOE values increased while I-FABPp levels decreased concomitantly. In complicated NEC both splanchnic FTOE values and I-FABPp levels decreased. Conclusions Combining cerebral and splanchnic FTOE values with I-FABPp levels, gives insight in the pathological chain of events resulting in progression or recovery of intestinal ischaemia in NEC. Trial registration number NTR3239.

Bloodstream infections during the onset of necrotizing enterocolitis and their relation with the pro-inflammatory response, gut wall integrity and severity of disease in NEC.

INTRODUCTION Bacterial involvement is believed to play a pivotal role in the development and disease outcome of NEC. However, whether a bloodstream infection (BSI) predisposes to NEC (e.g. by activating the pro-inflammatory response) or result from the loss of gut wall integrity during NEC development is a longstanding question. OBJECTIVE We hypothesize that the occurrence of a BSI plays a complementary role in the pathogenesis of NEC. The first aim of the study was to correlate the occurrence of a BSI during the early phase of NEC with intestinal fatty acid-binding protein (I-FABP) levels, as a marker for loss of gut wall integrity owing to mucosal damage, and Interleukin (IL)-8 levels, as a biomarker for the pro-inflammatory cascade in NEC. The second aim of the study was to investigate the relation between the occurrence of a BSI and disease outcome. MATERIAL AND METHODS We combined data from prospective trials from two large academic pediatric surgical centers. Thirty-eight neonates with NEC, 5 neonates with bacterial sepsis, and 14 controls were included. RESULTS BSIs occurred in 10/38 (26%) neonates at NEC onset. No association between the occurrence of BSIs and I-FABP levels in plasma (cohort 1: median 11ng/mL (range 0.8-298), cohort 2: median 6.8ng/mL (range 1.3-15)) was found in NEC patients (cohort 1: p=0.41; cohort 2: p=0.90). In addition, the occurrence of BSIs did not correlate with IL-8 (median 1562pg/mL (range 150-7,500); p=0.99). While the occurrence of a BSI was not correlated with Bells stage (p=0.85), mortality was higher in patients with a BSI (p=0.005). CONCLUSION The low incidence of BSIs and the absent association of both the markers for loss of gut wall integrity and the pro-inflammatory response during the early phase of NEC, support the hypothesis that the presence of a BSI does not precede NEC.

Identification of bacterial invasion in necrotizing enterocolitis specimens using fluorescent in situ hybridization

Objective:Investigation of bacterial invasion into the intestinal wall in necrotizing enterocolitis (NEC) specimens.Study Design:We compared 43 surgical NEC specimens with 43 age-matched controls. We used fluorescent in situ hybridization (FISH), a universal bacterial probe together with species-specific probes for Clostridium spp., Enterobacteriaceae, bacteroides and enterococci/lactobacilli. We used a FISH scoring system to reveal invasion of the intestinal wall, in which 1 represented no colonies and 4 invasion of the intestinal wall.Results:We observed invasion of the intestinal wall in 22/43 of the most affected NEC tissue samples as compared with 16/43 in the least affected NEC tissue samples (P=0.03). A FISH score of 4 was reached in 7/43 control cases. Enterobacteriaceae dominated the NEC specimens. Clostridium spp. were detected occasionally in NEC samples.Conclusion:Bacterial invasion of the intestinal wall is more present in most affected NEC tissue samples compared with least affected NEC tissue samples or controls. Enterobacteriaceae are prevalent in advanced NEC.

PS-185 Intestinal Oxygen Extraction Strongly Correlates With I-fabp Levels, A Marker For Intestinal Damage

Background and aim It remains unknown whether near-infrared spectroscopy (NIRS) can be used to assess intestinal perfusion. Intestinal fatty acid binding proteins in plasma (I-FABPp) and urine (I-FABPu) are a direct measure of intestinal epithelial cell damage that may occur after intestinal hypoperfusion. We measured splanchnic fractional tissue oxygen extraction (FTOE) and correlated these FTOE values with I-FABP levels in preterm infants in the first 16 h after onset of necrotizing enterocolitis (NEC). Methods Preterm infants born between October 2010 and November 2012 were prospectively included when NEC was diagnosed (Bell stage ≥2). Regional tissue oxygen saturation of the liver (rlivSO2) and infra-umbilical (rintSO2) region were measured continuously by NIRS. Mean 8-hour FTOE values were calculated: FTOE = (SpO2-rSO2)/SpO2. Plasma and urine samples collected in the first 16 h after onset of symptoms were used for analysis. Spearman’s correlation test was used to calculate correlation coefficients. Results Twenty-one preterm infants were included (median [range] gestational age 28 [25–36] weeks, birth weight 1290 [740–2400] grams). Median [range] liver FTOE (livFTOE) was 0.33 [0.07–0.81], infra-umbilical FTOE (intFTOE) 0.48 [0.13–0.82], I-FABPp 16.3 [0.54–3748] ng/mL, and I-FABPu 89.9 [3.2–23,336] ng/mL. Table 1 shows strong positive correlations between FTOE and I-FABP levels. Abstract PS-185 Table 1 Correlation coefficients between FTOE and I-FABP Conclusion High intFTOE values, suggestive of an impaired intestinal blood flow, correlated strongly with I-FABP, i.e. with the extent of intestinal epithelial cell damage. These results indicate that intestinal NIRS monitoring can be used to assess intestinal perfusion in preterm infants with an impaired intestinal blood flow such as occurs in NEC.