Reference intervals for stool calprotectin in preterm neonates and their utility for the diagnosis of necrotizing enterocolitis.

OBJECTIVE: Calprotectin is an antimicrobial protein found in stool when released by granulocytes. We sought to create stool calprotectin reference ranges in preterm neonates and to evaluate whether levels exceeding the upper reference interval are diagnostic for necrotizing enterocolitis (NEC). STUDY DESIGN: Stool calprotectin was measured in premature neonates without gastrointestinal pathology to create reference intervals. For comparison, levels from infants undergoing "rule out NEC" evaluations were plotted on these reference intervals. RESULTS: Stool calprotectin reference intervals were created according to gestational age at birth and corrected gestational age. Levels during "rule out NEC" evaluations were more often above the upper reference interval with NEC vs. those without NEC. CONCLUSIONS: Stools from preterm neonates have a higher range of calprotectin than stools from healthy term neonates. In evaluating preterm neonates for NEC with stool calprotectin, a calprotectin upper reference interval that incorporates corrected gestational age best predicts the diagnosis of NEC.

Elevated fecal calprotectin levels during necrotizing enterocolitis are associated with activated neutrophils extruding neutrophil extracellular traps.

OBJECTIVE: Neonates with necrotizing enterocolitis (NEC) have higher calprotectin levels in stool than do healthy neonates. However, it is not known whether high stool calprotectin at the onset of bowel symptoms identifies neonates who truly have NEC vs other bowel disorders. STUDY DESIGN: Neonates were eligible for this study when an x-ray was ordered to 'rule-out NEC'. Stool calprotectin was quantified at that time and in a follow-up stool. Each episode was later categorized as NEC or not NEC. The location of calprotectin in the bowel was determined by immunohistochemistry. RESULTS: Neonates with NEC had higher initial and follow-up stool calprotectin levels than did neonates without NEC. Calprotectin in bowel from neonates with NEC was within neutrophil extracellular traps (NETs). CONCLUSION: At the onset of signs concerning for NEC, fecal calprotectin is likely to be higher in neonates with NEC. Calprotectin in their stools is exported from neutrophils via NETs.

The complete blood cell count in a refined cohort of preterm NEC: the importance of gestational age and day of diagnosis when using the CBC to estimate mortality.

OBJECTIVE: The value of the white blood cell count (WBC) in necrotizing enterocolitis (NEC) is controversial. One reason for this confusion may be that the various WBC lineages change substantially with increasing gestational age and thereby age of NEC onset. This study postulated that if a data set was large enough and the diagnosis of NEC clean enough, absolute WBC counts would facilitate prediction of NEC mortality. The objective of this study was to determine whether absolute WBC counts enhance the prediction of NEC mortality. STUDY DESIGN: A de-identified data subset from the Pediatrix national data set specific to the diagnoses of NEC in patients who had a CBC drawn on the day of diagnosis (exclusive of the diagnoses of spontaneous intestinal perforations and congenital anomalies) was the target for analysis. Values of primary interest included: gestation, day of diagnosis, absolute WBC count, platelet count, hematocrit, mortality and the day of diagnosis. Stepwise regression analysis was used to predict mortality. RESULT: A total of 4059 (79%) survivors and 1107 (21%) infants who died with a diagnosis of medical or surgical NEC were included in the data set. Associations with mortality were found with low gestational age, low platelet count, low hematocrit, high band/segmented neutrophil ratio, earlier day of diagnosis, high birth weight z-score, non-white race, no antenatal steroids in gestations above 24 weeks, absolute lymphocyte count adjusted for gestational age, and absolute monocyte count high and low values. A stepwise regression analysis yielded a receiver-operator curve of 0.819 with a sensitivity of 65% and specificity of 84%. CONCLUSION: Absolute WBC values enhance prediction of NEC survival when used in combination with readily available data on the day of NEC diagnosis.

Can a national dataset generate a nomogram for necrotizing enterocolitis onset?

OBJECTIVE: Mother's own milk and donor human milk use is increasing as a means of necrotizing enterocolitis (NEC) prevention. Early onset of enteral feeding has been associated with improvement of many outcomes but has not been shown to reduce the incidence of NEC. Better definition of the window of risk for NEC by gestational strata should improve resource management with respect to donor human milk and enhance our understanding of NEC timing and pathogenesis. Our objective was to establish a NEC dataset of sufficient size and quality, then build a generalizable model of NEC onset from the dataset across gestational strata. STUDY DESIGN: We used de-identified data from the Pediatrix national dataset and filtered out all diagnostic confounders that could be identified by either specific diagnoses or logical exclusions (example dual diagnoses), with a specific focus on NEC and spontaneous intestinal perforation (SIP) as the outcomes of interest. The median day of onset was plotted against the gestational age for each of these diagnoses and analyzed for similarities and differences in the day of diagnosis. RESULT: Onset time of medical NEC was inversely proportional to gestation in a linear relationship across all gestational ages. We found the medical NEC dataset displayed characteristics most consistent with a homogeneous disease entity, whereas there was a skew towards early presentation in the youngest gestation groups of surgical NEC (suggesting probable SIP contamination). CONCLUSION: Our national dataset demonstrates that NEC onset occurs in an inverse stereotypic, linear relationship with gestational age at birth. Medical NEC is the most reliable sub-cohort for the purpose of determining the temporal window of NEC risk.

Fulminant necrotizing enterocolitis in a multihospital healthcare system.

OBJECTIVE: A subset of necrotizing enterocolitis (NEC) cases is fulminant, characterized by rapid progression to death with massive bowel necrosis found at laparotomy or autopsy. We sought to identify and report all such cases in a multihospital healthcare system during the past 9 years and to characterize this entity using case-control methodologies. STUDY DESIGN: This was a multicentered, cross-sectional, historic cohort study conducted using Intermountain Healthcare hospital patient data. All neonates who died of NEC within 48 h of onset, during 2001 to 2009, were compared with two matched control groups: (1) demographically matched controls who developed non-fulminant NEC, (2) demographically matched controls that did not develop NEC. RESULT: During this period, 2 71 327 live births occurred in the Intermountain Healthcare hospitals. Of these, 318 had a diagnosis of NEC (Bell stage ≥II). Also during this period, 205 other neonates were transferred into an Intermountain hospital for treatment of NEC. Of these 523 NEC cases, 35 (6.7%) had a fulminant course. Compared with the non-fulminant cases, the fulminant group were born at lower weight (1088±545 vs 1652±817 g, P=0.000) and earlier gestational age (27.5±3.3 vs 31.1±4.4 weeks, P=0.000), and were more likely to have: (1) radiographic evidence of portal venous air (P=0.000), (2) hematocrit <22% (P=0.000), (3) increase in feeding volume >20 ml/kg/day (P=0.003), (4) immature to total (I/T) neutrophil ratio >0.5 (P=0.005), (5) blood lymphocyte count <4000/µl (P=0.018), (6) an increase in concentration of human milk fortifier within 48 h before developing NEC (P=0.020). CONCLUSION: Portal venous air, anemia, rapid feeding escalation, a high I/T neutrophil ratio, a low lymphocyte count and recent increases in fortifier may all be associated with fulminant NEC.

Neonates presenting with bloody stools and eosinophilia can progress to two different types of necrotizing enterocolitis.

OBJECTIVE: We hypothesized that neonates with bloody stools and concomitant eosinophilia are likely to have atopic enteropathy rather than necrotizing enterocolitis (NEC). STUDY DESIGN: This was a retrospective cross-sectional study using electronic medical records and paper charts. Records of neonates admitted to any Intermountain Healthcare NICU between 1 January 2005 and 30 June 2010 were eligible if 'bloody stools' were listed in any archive. Qualifying records were divided into two groups depending on whether or not within 72 h of passing bloody stool eosinophil counts were above the 95th percentile reference range limit for age. RESULT: Bloody stools were identified in 275 predominantly Caucasian neonates. Fifty-four of these had eosinophilia and 221 had normal eosinophil counts. Those with eosinophilia were born at a slightly younger gestational age (31.3 ± 4.6 vs 32.6 ± 4.0 weeks, mean ± s.d., P=0.032). Contrary to our hypothesis, those with eosinophilia did not have a lower rate of pneumatosis or bowel resection, or death ascribed to NEC. Eosinophilia was more common among those who had a red blood cell (RBC) transfusion within 48 h before passing bloody stools (P<0.001). Those with a recent RBC transfusion were the only neonates to have NEC surgery or to die from NEC. Preceding the bloody stools, those with no antecedent transfusion had been fed a larger volume (P=0.014), and had trends toward receiving calorically enriched feedings (P=0.055) and recent addition of human milk fortifier (P=0.060). Eosinophil counts following RBC transfusion tended to increase for 3-6 days, but when bloody stools were not preceded by transfusion the eosinophil counts were more static over that period. CONCLUSION: In this predominantly Caucasian group of neonates with bloody stools, the presence of eosinophilia did not identify a benign condition distinct from NEC. A total of 44% of these neonates had transfusion-associated NEC. Eosinophils could have a previously unrecognized role in the pathogenesis of this NEC subtype.

Necrotizing enterocolitis during the first week of life: a multicentered case-control and cohort comparison study.

OBJECTIVE: Necrotizing enterocolitis (NEC) is rare during the first week of life; most cases occur after 2 to 4 weeks. We hypothesized that when NEC develops in the first week, certain predisposing factors and feeding practices are identifiable. To test this, we sought to identify every case of NEC diagnosed during the first week within the Intermountain Healthcare system during the most recent 6-year period. STUDY DESIGN: Data were collected from neonates admitted to any Intermountain Healthcare neonatal intensive care unit (NICU) with a date of birth from 1 January 2001 through 31 December 2006. Electronic and paper records were obtained for all with a diagnosis of NEC (Bell stage >or=II) within the first 168 h. X-rays, physician notes, nursing records, laboratory reports and operative reports were subjected to critical review to reexamine the diagnosis of NEC. Among those with confirmed NEC, we recorded underlying conditions and every feeding given prior to the diagnosis of NEC. Comparisons were made with patients that did not develop NEC, yet were cared for in the same NICUs, during the same period of time, and of the same gestational ages. RESULT: A total of 28 neonates were identified electronically as having NEC during the first week. Critical review confirmed this in 21, but 5 were determined at laparotomy to have had spontaneous intestinal perforation, and 2 others were found on surgical reports to have had a congenital infarction of the colon. Total 20 of the 21 confirmed cases developed NEC while in a NICU being treated for another condition. The exception was a small-for-gestational-age neonate in a well baby nursery. Compared to 6100 controls, the 21 with early NEC were more likely to have had a meconium-positive test for illicit drug exposure (P<0.005), early onset sepsis (P<0.034) and respiratory distress (P<0.039). They were less likely than case-controls to have been fed human milk (P=0.003) and were more likely to have been fed formula exclusively (P=0.019). None who were fed human milk exclusively developed early NEC. Twelve of the twenty-one were fed (by gavage or bottle) amounts exceeding the upper limit of volumes taken by breastfed neonates. CONCLUSION: We speculate that the prevalence of NEC during the first week could be reduced by identifying at-risk patients, feeding them human milk exclusively for the first week and using feeding volumes that do not exceed that taken by healthy breastfed neonates.

Emerging trends in acquired neonatal intestinal disease: is it time to abandon Bell's criteria?

In the last decade, it has become increasingly clear that necrotizing enterocolitis (NEC) is neither a uniform nor a well-defined disease entity. There are many factors that are forcing this unwelcome realization upon the neonatal and pediatric surgery communities. In the course of this manuscript we will review the history and the physical findings of the disparate etiologies of acquired neonatal intestinal diseases (ANIDs), some which do lead to the common final pathology of NEC and some which do not. New guidelines for distinguishing between ANIDs will also be suggested.

New insights into spontaneous intestinal perforation using a national data set (3): antenatal steroids have no adverse association with spontaneous intestinal perforation.

OBJECTIVE: To examine whether antenatal steroids (ANS), alone or with early indomethacin, are associated with spontaneous intestinal perforation (SIP). SIP is a known complication of concurrent post-natal administration of glucocorticoid and indomethacin in extremely low birth weight (ELBW) infants. STUDY DESIGN: A large de-identified national data set was retrospectively examined for infants with SIP without any report of other malformation or necrotizing entrocolitis. A control group was then derived matching for gender and birth weight (+/- 20 g). Pre- and post-natal variables were tested by both univariate and multivariate analysis to identify associations with SIP. RESULTS: From January 1996 to June 2004, there were 2 27 711 discharges from Pediatrix neonatal intensive care unit sites. From this population 388 infants with SIP associated with ELBW were compared to matched controls. Infants with SIP were more likely to have received early indomethacin and to have received a combination of early indomethacin with post-natal glucocorticoids (P < 0.05 for both). When used alone (without subsequent indomethacin), ANS showed no association with SIP. When used in conjunction with indomethacin, ANS did not increase the rate of SIP beyond indomethacin alone. CONCLUSION: ELBW Infants that acquire SIP were more likely to have been exposed to early indomethacin and post-natal glucocorticoids. However, no association was found between SIP and ANS within a well-powered cohort.

New insights into spontaneous intestinal perforation using a national data set: (2) two populations of patients with perforations.

BACKGROUND: Spontaneous intestinal perforation (SIP) is increasingly common in the premature infant and has been demonstrated to be associated with early postnatal administration of glucocorticoids and indomethacin. Before survival of the extremely low birth weight (ELBW) infant, SIP was thought to be a rare, congenitally acquired disease sporadically affecting the muscularis of the distal intestine. These disparate views of etiology have not been previously reconciled in the literature. OBJECTIVES: (1) To establish a cohort of SIP patients in a national data set. (2) To use timing of diagnosis as a unique data element and thereby differentiate between SIP cases which are susceptible to postnatal risk factors versus those occurring at or immediately after birth (and therefore not exposed to postnatal risk factors). METHODS: A large identified national data set was used to retrospectively look at timing of diagnosis and then the cohort was divided into postnatal treatment groups for further subanalyses. This analysis resulted in the division of the cohort into early and late diagnosis SIP subcohorts. These were then queried retrospectively by univariate analysis to look for differences in demographics between the two (using a P-value < 0.05). RESULTS: There were 633 patients with SIP evaluated in this data set. The early SIP cohort (0-3 days) was made up of 116 infants with a median BW of 1.401 kg, whereas the late cohort (4-14 days) held 386 infants with a median BW of 775 g. Infants with early SIP were significantly more likely to: be male, have higher Apgar scores, have not received antenatal steroids, surfactant or required mechanical ventilation. CONCLUSIONS: Two populations of infants acquire SIP: ELBW infants acquire SIP on average between 7 and 10 days of life after exposure to indomethacin and glucocorticoids (either endogenous or exogenous), and infants with early SIP (0-3 days) who are significantly less likely to have been exposed to postnatal risk factors and are less premature.

New insights into spontaneous intestinal perforation using a national data set: (1) SIP is associated with early indomethacin exposure.

BACKGROUND: Spontaneous intestinal perforation (SIP) is increasingly common in the premature infant and is associated with significant morbidity. Indomethacin use has been implicated as a co-risk factor for SIP when combined with glucocorticoids, but previous evidence argued against indomethacin being an independent risk factor when used prophylactically. OBJECTIVES: (1) To establish a homogeneous cohort of SIP patients in a national data set and to contrast them to patients with surgical necrotizing enterocolitis (NEC). (2) To test the hypothesis that early post-natal indomethacin is independently associated with SIP. METHODS: A large de-identified data set was retrospectively queried by diagnosis, and then multiple antenatal and post-natal variables were tested by both univariate and multivariate analysis to identify associations with SIP. Sub-analyses were also performed to look at the timing of drug administration. RESULTS: There were 2105 patients evaluated in the data set. Patients were divided into matched controls (n = 581), those with SIP without report of NEC (n = 633) and those with NEC requiring surgery (n = 891). Infants with SIP were more likely to have a patent ductus arteriosus and more likely to be treated with vasopressors than either control or NEC patients. Compared to infants with NEC, patients with SIP were smaller, less mature and required more support. SIP was also diagnosed earlier than NEC (median of 7 vs 15 days). Patients with SIP were more likely to be treated with indomethacin, hydrocortisone or both on days of life 0-3 than controls. CONCLUSIONS: (1) Surgical NEC and SIP have significant differences in presentation, demographics and morbidity. (2) A detailed look at drug timing revealed that early post-natal indomethacin is independently associated with SIP.

Discharge outcomes of extremely low birth weight infants with spontaneous intestinal perforations.

OBJECTIVE: To examine discharge outcomes of extremely low birth weight infants (ELBW) with spontaneous intestinal perforation (SIP). STUDY DESIGN: A single-center retrospective cohort study of all ELBW infants admitted to the University of Virginia neonatal intensive care unit between July 1996 and June 2004. RESULTS: We found 35 patients with SIP (incidence 8.4%). The median gestational age was 25 weeks, median birth weight was 722 g, and 71% of the infants were male. Most infants (n=28) with SIP were diagnosed secondary to pneumoperitoneum; however, one-third (7) of infants<25 weeks had occult presentations without pneumoperitoneum. When controlled for gestational age, gender, multiple gestation, indomethacin, and glucocorticoid exposure, infants with SIP have a higher risk of PVL and death than infants without perforation. SUMMARY: Periventricular leukomalacia and death are significantly associated with SIP in ELBW after adjusting for gestational age, multiple gestation, indomethacin, and glucocorticoid exposure.

The cellular repressor of E1A-stimulated genes mediates glucocorticoid-induced loss of the type-2 IGF receptor in ileal epithelial cells.

Glucocorticoids induce hypertrophy of the neonatal ileal mucosa but the molecular mechanisms behind this growth induction remain poorly understood. Ileal epithelial cells (IECs) are dependent upon IGF-II for proliferation both in vivo and in culture. The type-2 IGF receptor (IGFR-2) is a lysosomal transport protein that attenuates IGF-II-driven growth and is highly abundant in the ileum. The cellular repressor of E1A-stimulated genes (CREG) is a secreted phosphoglycoprotein that affects cell fate via ligand binding with IGFR-2, although the mechanism by which it does so is unknown. We hypothesized that glucocorticoids might facilitate IGF-mediated hypertrophy through CREG-mediated degradation of IGFR-2. To test this hypothesis, confluent rat IECs (IEC-18) were cultured for 72 h with or without dexamethasone (DEX) and harvested for Western blot, immunocytochemistry, gene array and CREG immunoneutralization experiments. IGFR-2 and CREG immunohistochemistry were also performed in archived ileal specimens from control and DEX-exposed newborn mice and extremely premature infants to investigate in vivo and clinical relevance. DEX exposure was found to diminish IGFR-2 immunolocalization in cultured rat IECs, newborn mouse ileal mucosa and human neonatal ileal mucosa. Gene array data indicated that IGFR-2 expression was unchanged with DEX treatment, suggesting a mechanism of protein degradation. CREG immunolocalization and abundance was found to be increased by DEX and immunoneutralization of CREG resulted in the abolition of IGFR-2 degradation. We have concluded that CREG is a secreted mediator by which DEX induces degradation of IGFR-2 and speculate that this is a fundamental mechanism of mucosal growth induction.

Focal small bowel perforation: an adverse effect of early postnatal dexamethasone therapy in extremely low birth weight infants.

OBJECTIVE: We tested the hypothesis that early postnatal dexamethasone (EPD) increases the risk of focal small bowel perforation (FSBP) in extremely low birth weight (ELBW) infants. STUDY DESIGN: The techniques of meta-analysis were applied to studies evaluating EPD, which we identified through a systematic literature search. Studies were included if they were randomized, placebo-controlled trials of EPD, enrolled infants with birth weights < or =1000 g, and reported FSBP as an outcome variable. The Breslow-Day test was used to assess for homogeneity and a summary odds ratio was calculated using the Mantel-Haenszel exact method. RESULTS: Four studies, with a pooled sample of 1383 infants, were included in the primary analysis. The Breslow-Day test showed a p-value of 0.61, indicating homogeneity among the studies. FSBP was significantly higher in EPD treated infants [odds ratio 1.91, 95% confidence interval (CI) 1.21, 3.07; p=0.004]. CONCLUSION: EPD increases the risk of FSBP in ELBW infants.