|Year : 2016 | Volume
| Issue : 2 | Page : 79-90
Necrotizing enterocolitis - Some things old and some things new: A comprehensive review
Khalid N Haque
Reader in Neonatal Medicine (retired), University of London, UK, International Faculty Professor, Children's Hospital and Institute of Child Health, Lahore, Pakistan
|Date of Web Publication||8-Apr-2016|
Khalid N Haque
276, Club Drive, San Carlos, California 94070, USA
Source of Support: None, Conflict of Interest: None
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency encountered in particular the preterm infants and less often in term and near-term infants. Since its description in the late 1950's and early 1960's, its incidence along with its associated mortality and morbidity has remained unchanged. In babies born < 1500 g or before to 32 weeks of gestation, its incidence ranges between 3% and 12% and mortality is between 20 and 30%, with highest among those requiring surgery. With better understanding of etiology and pathophysiology, it is now being increasingly recognized that "NEC" as diagnosed by most clinicians in clinical practice may not be a single disease but a spectrum of diseases that present with similar signs and symptoms. NEC is currently thought to be due to dysbiosis of the intestinal microbiome and an uncontrolled exuberant inflammatory response to this microbial imbalance. This comprehensive review discusses the differences between NEC as seen in term and near-term babies as opposed to that seen classically in preterm infants. It also discusses the epidemiology, pathogenesis, and newer diagnostic modalities for the diagnosis of NEC in great depth, because it is only by understanding and appreciation of these dynamics that is likely to lead to the development of successful strategies for prevention, diagnosis, treatment, and improved outcome.
Keywords: Dysbiosis, gastrointestinal, microbiome
|How to cite this article:|
Haque KN. Necrotizing enterocolitis - Some things old and some things new: A comprehensive review. J Clin Neonatol 2016;5:79-90
| Introduction|| |
Necrotizing enterocolitis (NEC) by one name or the other has been recognized since 1828 when preterm infants started being housed in specialized hospital units. Schmidt and Quaiser reported its pathological characterization in 1952.  They called it "enterocolitis ulcerosa necroticans." Initially, the condition was reported to occur in "epidemics" thus suggesting an infectious etiology. However, it was gradually recognized that NEC is an "endemic" condition that is seen in almost all neonatal units worldwide.
NEC has a highly variable geographical incidence, e.g., the incidence in Japan is reported to be low (1.5%). The overall incidence reported from most parts of the world varies between 3 and 12%  with some countries reporting a falling incidence while others reporting an increasing trend.  Some reports have suggested seasonal variation in the incidence of NEC with higher incidence during the winter months. 
In USA and Canada, where the incidence of NEC is among the highest (7-12%) in babies weighing between 500 and 1500 g, approximately 20-30% of these babies die  and nearly quarter of survivors go on to develop microcephaly and are substantially at a greater risk of developing neurodevelopmental delay and other morbidities such as cholestasis and short gut syndrome. ,, It is estimated that in the USA, the cost of caring for infants with NEC is between 500 million and 1 billion dollars per year.  Length of stay in hospital is also increased by 20-60 days compared to infants without NEC. 
Though the precise etiology of NEC remains elusive and debatable, it has become clear that what has been classically defined as NEC is more than one disease. Initially, as NEC occurred in clusters, it was thought to have an infectious etiology by organisms such as Pseudomonas spp., Clostridium perfringens, various virus or infection by nosocomial organisms. Based on animal studies, Mizrahi  and others in the 60's postulated mesenteric hypoperfusion as the main etiological factor of NEC rather than infection. By the 70's, it became clear that NEC did not have a monocasual etiology but a multi-factorial one that included the immaturity of the preterm intestine, bowel ischemia, intestinal infection/infiltration with enterotoxin-producing pathogens, formula, and/or hyperosmolar feeding but till this date, the sequence or the interplay of these factors remains blurred. Till, we are able to clearly delineate the exact pathogenesis of NEC, strategies to prevent and manage NEC would remain a matter of continued and intense research. Hence, a review such as this is needed and will need to be updated from time to time.
| Epidemiology|| |
As indicated earlier, NEC is perhaps the most common gastrointestinal emergency in the preterm infant. Its true incidence is unknown as even now a number of gastrointestinal conditions are "lumped" under the term "NEC". Over decades, in countries where smaller and smaller babies of lower and lower gestation are surviving the incidence of NEC has increased while some other countries have reported a decrease in NEC.  Incidence in USA and Canada is reported to be between 7 and 12% of all preterm births. ,,,, Gordon et al.  have clearly shown that there is an inverse relationship between the onset of NEC and gestational age, i.e., the lower the gestational age, the later the onset of NEC. Using a very large national data set (Pediatrix), they also showed that the incidence of "classic NEC" peaked around the postgestational age of 29-31 weeks, whereas earlier presentation suggested a diagnosis of spontaneous intestinal perforation (SIP). Hunter et al.  suggest that the peak incidence of NEC occurs approximately 3 weeks after birth in infants born before 32 weeks of gestation, 2 weeks after birth in infants born between 32 and 36 weeks of gestation, and under a week in infants born after 36 weeks of gestation.
Earlier studies suggested that there were no geographical, seasonal, or racial differences in the incidence of NEC;  recent studies have challenged this view. There is evidence that Afro-American neonates have a higher incidence of NEC than their white counterparts and that NEC is slightly more frequent during the winter months. ,, Lower frequency of NEC is reported from Japan, Switzerland, Italy, and Austria whilst higher frequency of the disease is reported from Ireland, UK, USA, and Canada. ,,, It is unclear whether these differences are due to care strategies, environment, climate, ethnic background, or genetics. Unfortunately, robust data from developing countries is not available to give comparative figures.
Hull et al.  in a very large study from 55 neonatal units in the USA involving 215,057 very low birth weight infants found that the overall incidence of NEC was 9% of which approximately half were thought to have "medical NEC" and the other half to have "surgical NEC," i.e., those potentially requiring surgery. They reported an overall mortality of 28%. For "medical NEC," mortality in this cohort was 21%, which is much higher than the 6-10% reported from other studies. ,,, Mortality in babies with NEC who required surgery was 30%. 
In one of the largest studies of its kind, Ahle et al.  using the Swedish National Data Base studied the epidemiology and trends of NEC in Sweden between the years 1987 and 2009. They reported data from 2,381,318 live births and found the incidence of NEC to be 3.4/10,000 live births, boys having more NEC than girls (3.7 vs. 3.0/10,000 live births, P = 0.02, relative risk [RR] =1.2, 95% confidence interval [CI]: 1.06-1.40, P = 0.005) this difference remained when adjusted for birth weight, gestational age, birth year. They also observed a consistent seasonal variation with NEC peaking in November with a trough in May. They concluded by stating, "after an initial decrease, the incidence of NEC has increased in Sweden."
| Differential Diagnosis|| |
Prior to discussing the pathogenesis of NEC, it is important to "de-lump" the other conditions that hitherto have been included within the diagnosis of NEC. These conditions not only differ in their pathogenesis but also in strategies to prevent or treat them. They share NEC-like symptoms but usually occur in late preterm or term infants during the 1 st week of life as opposed to true NEC that is seen in very preterm (<32 weeks gestation) infants and occurs during the second or 3 rd week of life.
In term and near-term infants, the NEC-like symptoms may be due to perinatal stress (birth associated events like asphyxia) where mesenteric blood flow is affected, ,, cyanotic heart disease, aganglionosis, intestinal anomalies, maternal substance abuse (cocaine), formula feeding, rapid advancement of feeds, polycythemia with hyperviscosity or maternal chorioamnionitis. The pathogenesis in all these conditions is triggered by mesenteric hypoxia and ischemia that by activating platelet aggravating factor (PAF) and toll-like receptor TLR-4 expression in enterocytes initiates the cascade of apoptosis leading to intestinal mucosal necrosis.
Spontaneous intestinal perforation
This is the major differential diagnosis that has been confused with NEC. The main differentiating features between NEC and SIP are that SIP occurs within the first few days of life in premature infants and is not associated with inflammation or feeding, but a correlation has been reported with steroid and or Indomethacin therapy. ,, SIP has a nonischemic pathology  and histology showing thinned or segmental necrosis of the muscularis interna a common end point of NEC but SIP histology lacks coagulative necrosis and focal hemorrhage seen in NEC. Histologically and biochemically, there is no or minimal inflammation and activation of proinflammatory molecules (cytokines and chemokines) in SIP in contrast to NEC where inflammation and activation of pro-inflammatory molecules is massive. Clinically, it is difficult to differentiate between SIP and NEC though Gordon et al. have suggested a classification system to do this but that classification is not universally used.
Infants with severe sepsis sometimes develop intestinal ileus. The signs and symptoms are that of sepsis along with vomiting or abdominal distension with absence of bowel sound on auscultation. Diagnosis is confirmed by plain X-ray of abdomen. X-ray examination usually shows multiple fluid levels with distended bowel loops but no evidence of pneumatosis or biliary gas.
Viral enterocolitis is another condition that is often confused with NEC. It occurs at any gestation, at any age and is usually seen in clusters. Interestingly, it is characterized by early lymphocytosis something that is not seen with classical NEC.
Food-protein induced enterocolitis syndrome
Food-protein induced enterocolitis syndrome is an uncommon condition more likely to be seen in Asian and African infants who are more prone to have cow's milk intolerance.  The disease is not associated with a robust IgE response but increase in eosinophils into the intestine recruited by elevated interleukin (IL)-5.  Challenge with cow's milk antigen causes them to release their granules. Since preterm infants do not have the capacity to develop true allergy, they have low levels of mucosal necrosis when overchallenged by excessive antigens (cow's milk proteins). Hence, it is suggested that neonatal cow's milk allergy associated NEC is milder with low mortality.  Treatment here is total avoidance of not only cow's milk but also maternal milk as mother's milk may have sufficient cow antigen from their diet to sustain the allergic response in their babies. 
| Pathophysiology/Pathogenesis|| |
Neonatologists have known since the first descriptions of NEC that while prematurity was the major determinant but for NEC to develop it requires other factors such as decreased intestinal barrier function, or an event that compromises its integrity,  impairs intestinal immune defense plus the inflammatory propensity of the premature gut, , and the presence of food and bacteria in the gut [Figure 1].
Understanding 'that pathophysiology of NEC is evolving and is not yet fully understood. The main factor other than the immature motility and absorptive capacity of the newborn's intestine in the first few weeks of life after birth is its dependence on innate rather than adaptive immunity though the two are intertwined. The innate system that consists of cells and their receptors act as "first responders," i.e., they respond rapidly to microbes while the immune system activation requires prior exposure to antigenic stimuli. In the newborn, the adaptive system is significantly underdeveloped thereby increasing the risk of developing NEC. Immunologically, the vulnerability of the preterm infant to NEC is related to the transition from innate to adaptive immune function both systemically and locally in the gut itself. In the gut, once the mucosal barrier (which is poor in the preterm infant due to reduced mucin-producing goblet cells and weak "tight junctions" in between mucosal cells) is disrupted leading to invasion by organisms causing an imbalance between TLR-4 (proinflammatory) and TLR-9 (anti-inflammatory), which are thought to be the main immunological drivers in the development of NEC.
In the neonatal intestine, the TLR-4 gradually increases in expression with increasing gestational age till term when their number falls rapidly along with their surface expression. TLR-4 signaling regulates the balance between injury and repair in the newborn intestine. TLR-4 receptors are important because they sense macromolecules of pathogens, e.g., lipopolysaccharide (LPS)  and are an important regulatory factor for transcription of nuclear factor kappa-B (NFkB), which is underexpressed in the newborn. , Activated TLR-4 increases apoptosis and reduces enterocyte proliferation  and migration. Thus, TLR-4 activation within the intestinal epithelium exerts a deleterious effect on the intestinal mucosa by causing injury and reducing repair.
Enterocyte apoptosis is one of the first histopathological events in the development of NEC. Physiologically, TLR-4 expression gradually increases in the naοve newborn's gut with gestational age until term, when its abundance precipitously falls and its surface expression is also actively downregulated in few weeks after birth. In the preterm, the TLR-4 (numbers and expression) does not fall for a few weeks after birth and are activated secondary to colonization of the gut by both commensal and pathogenic organisms. This continued expression of TLR-4 maybe responsible for the timing of NEC in the preterm infant. The more the TLR-4 is expressed, the more vigorous the apoptosis and severity of the NEC. Unlike in the adults, the tendency of the preterm intestines toward inflammation is not only due to an abundance of TLR-4 receptors but also the intestinal epithelial cells expressing human leukocyte antigen (HLA1) and HLA-DR that accentuate local inflammatory response by acting as antigen presenting cells. , Fetal and neonatal intestinal epithelial cells also produce more cytokines such as IL-1, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) and PAF when triggered leading to the activation and migration of neutrophils and macrophages making the preterm infants intestine vulnerable to NEC. ,,,,,,
The role of adaptive immunity has not been well studied in NEC. However, we know that T-cells are present in the fetal gut and their numbers and activation is increased in chorioamnionitis… a risk factor for the development of NEC. A subset of T-cells is the T regulatory (Treg) cells are in abundance in the intestinal mucosa of preterm infants. They are responsible for preventing excessive activation of innate and adaptive immune systems in the intestinal tract.  Treg cells act through cytolysis of effector T-cells, secretion of inhibitory cytokines such as IL-10, IL-35, transforming growth factor-beta, and by other ways. , Thus, Treg cells regulate response to limit intestinal injury and inflammation by enhancing adaptive immunity in the gut. 
In summary, NEC is a disease that is characterized by impaired signaling in response to colonization of the gut of the newborn. Prematurity, endotoxemia, and hypoxia all lead to persistent upregulation of intestinal TLR-4. NEC reflects the inability of the newborns intestine to downregulate TLR-4 signaling to become tolerant of intestinal microflora.
Role of intestinal microbiota in necrotizing enterocolitis
Microbial dysbiosis (alteration of intestinal microbial composition) and excessive intestinal inflammatory response associated with it is currently thought to be the most likely pathogenic mechanism for the development of NEC. Intestinal microbiota are involved in regulating the multiple pathways giving rise to an interactive host-microbiota metabolic, signaling, and immune inflammatory process between the gut and other organs including the brain leading to systemic and long-term effects of NEC.
Fetal and newborn infants intestine physiologically and rapidly acquire commensal microbiota during and soon after birth. The composition of this community of intestinal microbiota is derived from maternal colonic and vaginal flora (Enterobacteriae, Enterococci, Staphylococci).  This microbiota further develops in its diversity and is altered by factors such as feeding (breast milk vs. formula milk), use of antibiotics or histamine type 2 (H2) blockers, and the neonatal unit environment itself.
Physiologically, there is a physical separation between the intestinal epithelial cells and the microorganisms maintained by the gel-like mucus layer lining the intestinal epithelium. This layer inhibits bacterial contact with the mucosal surface by My88 dependent secretion of bactericidal C-peptide lectin RegIIIg from the Paneth cells.  In the preterm, this protective mechanism is inefficient and along with poor tight junctions allows the microbes to enter through the mucosal surface. Once in the mucosa, the intestinal (pattern recognition receptors, e.g., TLR-4) are able to specifically recognize (microbial-associated molecular patterns, e.g., LPS or peptoglycans) to setup inflammation. Two other elements of innate immunity that can induce NEC or increase its severity are (PAF, also an upregulator of TLR-4) that senses the metabolic stress caused by the microbial burden and eosinophil's that have the capacity to sense a bacterial specific metabolite N-formyl-methionyl-leucyl-phenylalanine , while some TLRs are proinflammatory that increase cytokine-mediated (IL-8) inflammatory response in the gut.  Nanthakumar et al.  and others , have suggested that the exaggerated inflammatory response in the preterm gut is due to deficient expression of inhibitors of NFkB inflammatory pathway. Furthermore, immature intestine may develop inflammatory responses as the microbial colonization increases with feeding and down-regulation of IL-1 receptor associated with kinase 1, which is an essential component of TLR-4 signaling.  In the intestine, there are other receptors that help maintain intestinal homeostasis by supporting cytoprotection. They secrete antimicrobial peptides and activate commensal (friendly) bacteria. ,, TLR-9 has the opposite effect to TLR-4 it is able to sense the CpG (CpG sites are relatively rare (~1%) on vertebrate genomes in comparison to bacterial genomes or viral DNA) repeats within the DNA of commensal bacteria helping them proliferate.  In brief, intestinal expression of TLR-4 and TLR-9 is reciprocally related during development. TLR-4 within the intestine is functionally active, and that TLR-9 activation with CpG-DNA can limit TLR-4 signaling in enterocytes via a mechanism that requires IRAK-M, thus preventing the development of NEC by limiting TLR-4 induced enterocyte apoptosis and bacterial translocation.
To summarize, the newborn intestine should be able to tolerate beneficial commensal bacteria and other microbes without causing inflammation and injury despite its hyperinflammatory bias. It does that by recognition of friendly commensal bacteria by TRLs or by balancing the proinflammatory and anti-inflammatory TRLs (TRL-4 versus TRL-9) and downregulating NFkB pathway. Other friendly (probiotic) bacteria also reduce inflammation by blocking the degradation of IkB, an inhibitor of NFkB.
Another family of proteins that is generating interest in NEC research is Heparin-binding epithelial growth factor (HB-EGF). It is a naturally occurring glycoprotein produced by monocytes and macrophages. It is also expressed by endothelial, epithelial, and muscle cells and is present in significant quantities in the amniotic fluid and breast milk. HB-EGF is a repair protein that has proliferative and anti-inflammatory properties that are upregulated in response to tissue damage.  HB-EGF acts via several pathways to cytoprotect, i.e., as a growth factor, it stimulates enterocytes to proliferate and migrate and repair intestinal injury.  It protects epithelial cells from TLR-4 induced apoptosis and decreases the production of injurious mediators such as nitric oxide and free radicals. , HB-EGF also decreases neutrophil-endothelial adhesion thus preventing tissue destruction and intestinal injury from activated neutrophils. ,,
| Who is at Risk of Getting Necrotizing Enterocolitis?|| |
While preterm infants < 31 weeks of gestation are at greatest risk of developing NEC, nearly 10% of term or near-term infants also develop NEC. Interestingly, the risk factors in the two groups are different; the very preterm are more likely to develop NEC due to intestinal immaturity, whereas term and near-term infants usually develop NEC secondary to multiple triggering factors.
Prenatal risk factors
Any maternal condition that stimulates the fetal intestinal inflammatory cascade such as maternal hypertension, pregnancy induced hypertension, maternal infection, problems related to placental blood flow or recreational drug use (cocaine) with resultant damage to the vasculature in the watershed areas may lead to mucosal damage and NEC. ,,, Recently, the association between HIV-positive mothers and NEC in their preterm infants has been described.  Histological chorioamnionitis with associated vasculitis increases the risk of infant developing NEC 2.5 fold (odds ratio [OR] 2.6, P = 0.02). 
Intrapartum risk factors
The major intrapartum risk factor is hypoxic-ischemic compromise.  Though unlikely to be a major cause of NEC in the very preterm infant, hypoxia, and ischemia are known to modulate the balance of microvascular tone and production of vascular regulators such as endothelin and epidermal growth factor  that play a role in the development of NEC.
Delivery by cesarean section where the opportunity for the baby to acquire colonization by friendly maternal vaginal commensal bacteria is lost,  which increases the chances of the infant developing NEC particularly if not breastfed. Gregory  have shown that babies who require bag-mask resuscitation (P ≤ 0.002), intubation (P ≤ 0.001), hemodynamic support (P ≤ 0.0001), require inotropic support (P ≤ 0.0001), or had hypotensive episode (P ≤ 0.0001) in the delivery room were between 6.4 and 28.6 times more likely to develop NEC than those who did not require any of the above. Gregory went on to conclude that the "infant most at risk for developing NEC is one weighing between 500 and 1500 g, is <28 weeks in gestation and who requires resuscitation in the delivery room.
Risk factors in the early neonatal period
Several factors in early neonatal care cause intestinal dysbiosis in the gut of the newborn. Antibiotic therapy soon after birth reduces microbial diversity and the growth of friendly commensal bacteria such as Bifidobacter and adversely affects the intestinal innate and adaptive immune systems thus increasing the risk of NEC. ,,,, There is good evidence that lack of antibiotic exposure or reduced duration of exposure to antibiotics reduces the risk of NEC  and conversely the longer the antimicrobial therapy, the greater the risk of NEC.  Chong et al.,  in a recent study, showed a tenfold reduction in the rate of NEC in two centers who changed to using piperacillin and tazobactam as their first-line empirical antibiotic therapy from ampicillin and gentamicin.
Hemodynamically, significant patent ductus arteriosus (PDA) and its treatment with indomethacin have been shown to increase the risk of NEC , while treatment of PDA with ibuprofen is associated with a risk reduction of 0.68% (95% CI: 0.47-0.99). 
Neonatologists often use H2 blockers. H2 blockers by reducing gastric acidity potentiate bacterial growth in the gut. Their use is associated with increasing the risk of infant developing NEC (OR = 1.71, 95% CI: 1.34-2.19, P ≤ 0.0001). ,
It is widespread practice that feeding is withheld once a diagnosis of NEC is considered. Recent thinking suggests that complete withholding of feeds may lead to intestinal atrophy, increased translocation of microbes through the intestinal mucosa increasing the risk of NEC and its severity and systemic infection. 
There is a total consensus that infants who are breastfed given breast milk fortified feeds are 6-10 times less likely to develop NEC compared to formula fed infants. , However, there is debate about the use of trophic feeds and the rate of increase of feeds. Cochrane systemic review by Bombell and McGuire  showed no effect of early trophic feeding versus no feeding on the incidence of NEC (RR 1.07; 95% CI: 0.067-1.70) and the risk difference of 0.01 (95% CI: 0.04-0.05). Similarly, Morgan et al.  in another Cochrane review found no difference in the risk of NEC when feeds were advanced slowly (15-20 ml/kg/day) versus when advanced at the rate of 30-35 ml/kg/day.
Currently, many units are using formula milk containing probiotics. Probiotics are live commensal bacteria that help colonize the gut with friendly bacteria, improve gut motility, quality of intestinal mucus, and control the production of inflammatory cytokines.  Meta-analysis of over two thousand infants has shown that probiotics reduce the risk of NEC by 65% (number to treat 25 infants to save 1 case of NEC).  Wang et al.  in a meta-analysis of 20 randomized controlled trials has shown that probiotics containing both Lactobaccillus and Bifidobacter reduced both the incidence and mortality from NEC whereas probiotic containing only Bifidobacter reduces the incidence of NEC but has no effect on mortality. Role of prebiotics in prevention or treatment has not been fully evaluated. Mother's milk with its many cellular and immunological benefits is also the best to colonize the gut with friendly bacteria. Therefore, it should be the first choice and if it is not available then pasteurized donor milk should be used and only if neither of these are available then feeding with cow's milk formula containing both Bifidobacter and Lactobacillus should be used.
Gastric residuals suggest feeding intolerance; however, it only indicates compromise of intestinal integrity when either the residuals are bloody or in excess of 30-50% of the previous feed. ,,
Other risk factors that have been associated with increased risk of NEC include red blood cell transfusion. This is most often seen in premature infants who are fully formula fed and have AB blood group. , Posttransfusion NEC occurs usually 12-48 h after red blood cell transfusion and is often severe  with approximately 38% of infants with posttransfusion NEC requiring surgery.  The association with AB blood group suggest that AB blood group epitopes that are known to be expressed on the neonatal enterocytes  may be vulnerable to serum antibodies in blood products. Similarly, the use of high-dose immunoglobulins for hemolytic disease has been associated with NEC (OR = 31.66; 95% CI: 3.25-308.57).  This is possibly due to severe opsonization effect of using anti-AB immunoglobulins. Recently, NEC has been associated with cow's milk intolerance due to increased intestinal eosinophil's induced transmigration. ,
Though genetic factors have not been involved in the causation of NEC, a number of genes have been implicated in either affecting the outcome or modifying the disease severity.  Single nucleotide polymorphism in several genes, e.g., IL-4 receptor (+1902G) (protective), IL-8 (−607A) (severity), vascular EGF (+450C) (increased risk), and carbamoyl-phosphate synthetase 1 (T450N) (increased risk), and Sampath et al. have identified the NFkB1 (g-24519 del ATTG promoter polymorphism) variant in all cases of NEC. ,,,,, Bhandari et al.  have reported NEC in either one or both twins in 9 (14%) of 63 pairs of monozygotic twins and in 29 (15%) of 189 dizygotic twins. Thus, the genetic association needs to be further investigated, as does the racial factor NEC being more common in African American infants. 
| Clinical Presentation|| |
The initial presentation of NEC may not be very dissimilar to the presentation of neonatal sepsis. The infant is usually 31 weeks or less in gestation, is stable and feeding well on formula milk who insidiously over a few days starts showing nonspecific symptoms such as temperature instability (7-20%), apnea and bradycardia (32-60%), fluctuating blood pressure with hypotensive episodes (20-80%), increasing gastric residuals (50-90%), emesis (30-40%) abdominal wall redness, tenderness, and distension (10-70%), absent bowel sounds (20-40%), lower right quadrant mass (1-2.7%), and blood in stools (14-40%) (Figures quoted here are averages from a number of studies). Occasionally, the presentation may be acute with sudden collapse and shock-like symptoms with blood in stools; this presentation is more often seen in term or near-term infants who have endured a hypoxic-ischemic event.
Since Bell et al.  first published a staging system in 1978, it has been refined many times, ,,, but all classifications have similar shortcomings of the original Bells's system. NEC can develop without any radiological evidence of pneumatosis or intraluminal or portal gas (prerequisite for the diagnosis of NEC in the staging systems) thus; progression from one stage to another can be missed.  A more reliable staging system perhaps including specific biomarkers is needed. The author prefers to use a more clinical based user-friendly staging system as shown in [Table 1].
|Table 1: Staging of necrotizing enterocolitis based on clinical signs and symptoms |
Click here to view
Laboratory and radiological markers of necrotizing enterocolitis
Though radiology is considered the gold standard diagnostic tool for the diagnosis of NEC, quite often infants with NEC do not have any specific radiological findings. Often infants with NEC or those developing NEC develop nonspecific metabolic or respiratory acidosis, thrombocytopenia, and neutropenia  but if they show lymphocytosis, then rotavirus-associated NEC is very likely.  Blood culture is positive in less than a third of cases, and it is unclear whether this is as a result of bacterial translocation or systemic blood stream infection leading to NEC.
Radiological findings of an ileus or thickened (fixed loop) are nonspecific but finding of intraluminal (Pneumatosis intestinalis) or portal gas which is present in 50-75% of cases is considered diagnostic. Shebrya et al.  have shown that abdominal ultrasound is more sensitive than plain X-ray abdomen. Till date, serial plain X-ray abdomen and cross table lateral X-ray with a horizontal beam are the most often used technologies to detect subtle early collection of free air in the abdomen. , Urboniene et al.  have evaluated the RI and the PI of the superior mesenteric artery in preterm infants and found 96.3% of infants with NEC had an RI of >0.75 (sensitivity 96.3%, specificity 90.9%, OR = 260) whereas 88.9% of infants with NEC had a PI > 1.85 (sensitivity 88.9%, specificity 78.7%, OR = 29) suggesting Doppler measurements of intestinal blood flow as a useful bedside tool for predicting and diagnosing NEC.
| Other Tests|| |
Breath hydrogen sampling has been used for early detection of NEC  but has not gained popularity due to technical difficulties of doing the test in preterm infants and its poor predictive value.
Given the genetic predisposition toward NEC explained earlier, researchers have searched for genetic markers that could predict the diagnosis of NEC. Single-neucleotide polymorphism of CD-14, TLR-4 or caspase-recruitment domain 15, gene that encodes carbamoyl-phosphate synthase 1 and IL-18 AA genotype have been found useful, ,,,, but they lack sensitivity and specificity.
Infants who develop NEC have microbial dysbiosis compared to those who do not. Culture-based studies have clearly demonstrated these differences in fecal bacteria up to 72 h before the onset of NEC.  These changes include increase in Enterobacter and Escherichia More Details Coli with concomitant decrease in Streptococcus faecalis and other Staphylococcus species. Recent nonculture molecular 16S ribosomal RNA-based studies have shown changes in the prevalence of C. perfringens, Firmicutes, Proteobacteria, and Enterobacter prior to the onset of NEC , and decrease in Bifidobacter and changes in unidentified strains similar to Klebsiella.  These changes occur rapidly within the first few weeks of postnatal age , explaining the timeline for the development of NEC in preterm infant. Availability of nonculture molecular 16S ribosomal RNA-based studies offer a great potential for predicting NEC.
As described earlier, the pathogenesis of NEC involves a pathway that includes endogenous inflammatory mediators that cause intestinal injury, e.g., TNF-α, IL-8, PAF, and TLR-4 plus many cytokines and chemokines. Measurements of these mediators on their own have not achieved high sensitivity or specificity. Acute-phase reactants such as C-reactive protein or procalcitonin are raised in stage II and III of NEC but do not offer much in the way of sensitivity and specificity.
Fecal calprotectin is established screening test for the diagnosis of inflammatory bowel disease  in adults. Houston and Morgan  have shown that in the newborn, particularly the preterm infant, it overestimates the risk of NEC and thus should not be used to diagnose or predict NEC.
Intestinal fatty acid binding protein (I-FABP) is a specific indicator of early enterocyte death and is liberated into the blood stream as soon as the integrity of the enterocyte cell membrane is compromised.  Guthmann et al.  measured both I-FABP and liver FABP (L-FABP) and found that though L-FABP was more sensitive of intestinal injury, urinary I-FABP greater than 2 pg/nmol to creatinine ratio clearly distinguishes NEC from other conditions. Also associated with NEC are low levels of arginine and glutamine, but their measurement has not been found useful.
Interestingly, salivary derived epidermal growth factor which is physiologically low in preterm infants shows a very rapid rise during the 1 st week of life in infants who go on to develop NEC. 
New molecular techniques such as mass spectrometry, transcriptomics, and proteomics have emerged in developing new and novel biomarkers. Ng et al.  using proteomic techniques have found that proapolipoprotein CII and des-arginine variant of serum amyloid A as the most promising biomarkers for the diagnosis of NEC (sensitivity 90%, specificity 95%) but their study has to be validated from other institutions using larger cohort of patients.
Thus, advances in molecular diagnostic technologies in particular gene sequencing, mass spectrometry, and proteomics offer greater hope for diagnostic test for NEC. However, until these tests are made available, we have to contend with nonspecific tests with poor sensitivity and specificity.
| Management/Treatment Of Necrotizing Enterocolitis|| |
Management of a case of NEC and treatment has not changed over last three decades. It is based on the principles of resting the bowel by stopping feeds (there is no consensus for how long to stop feeds for), gastric decompression by placing an oral-gastric tube, management to support nutritional needs by parenteral nutrition and supporting the metabolic, cardiovascular, and respiratory effects of systemic disease.  Antibiotics (there is no consensus on which ones to use or for how long) are always given for possible infective etiology (though <10-15% of cases have positive blood culture) or complications. Often antianaerobics or antifungals are added if perforation is suspected or confirmed. , Small studies have shown enteral aminoglycoside therapy may prevent NEC, but risk of resistance development has prevented the adoption of this strategy.
Frequent radiological and laboratory assessments (there is again no consensus on how frequent) are done to monitor the progress of the disease.
As soon as the diagnosis of NEC is considered, most clinicians would inform their surgical team. Surgeons either use peritoneal drainage or laparotomy with bowel resection to decompress the gut and/or remove necrotic bowel. Infants who have peritoneal drainage often go on to require laparotomy.  Systematic reviews suggest that mortality may be 50% higher with peritoneal drainage compared with laparotomy.  Despite this, there is still debate whether primary peritoneal drainage has a role in critically ill preterm with NEC.  In babies who develop pancolitis (NEC totalis), then jejunostomy has shown some benefit compared to total bowel resection. 
| Prevention Strategies|| |
The major advance that has taken place in the management of NEC has been in the development of strategies to prevent or reduce the incidence of NEC. These include the following:
- Exclusive breastfeeding
- Promoting, preserving, and restoring healthy gut flora
- Use of standardized feeding protocols (SFPs)
- Limiting the use of anti-microbial therapy in the early neonatal period.
As stated earlier that the benefits of feeding the preterm infants, his/her own mother's milk have been shown over and over again to protect the baby from developing NEC. ,, For every 10 babies fed on mother's breast milk, one case of NEC is prevented.  If mother's milk is unavailable, then feeding donor breast milk will reduce the risk of NEC by 80% compared with formula milk feeding (RR = 0.21; 95% CI: 0.06-0.76).  If neither mother's milk nor donor breast milk is available, then preterm infants should be fed on preterm formula containing probiotics (Bifidobacter plus Lactobacillus).
Despite nine randomized controlled trials showing inconclusive evidence for trophic feeding,  this practice appears to be safe  and should be started as early as possible  keeping in mind that the premature intestine starts showing signs of atrophy if no feeds are given for 3 or more days.
Promoting, preserving, and restoring health gut flora
This can be achieved to a great extent by using probiotics in the feeds, i.e., using milk that contains Bifidobacter or Lactobacillus or both. In a large meta-analysis, Yang et al.  analyzed a total of 6655 preterm infants and found that probiotic supplementation reduced the risk of Bell Stage I and Stage II NEC (RR = 0.35, 95%, CI: 0.27-0.44, P ≤ 0.00001) and (RR = 0.34, 95% CI: 0.25-0.48, P ≤ 0.00001), respectively. The risk was also reduced for mortality from NEC without any increase in the risk for sepsis. There is not enough robust evidence for the benefits or otherwise of using prebiotics.
Use of standardized feeding protocol
Christensen et al.  in their article "Can we cut the incidence of NEC in half today?" suggested that one of the measures that can reduce the incidence of NEC is the institution of "SFPs." Patole and de Klerk  in a meta-analysis of 3762 infants weighing <1500 g described an approximate reduction in NEC up to 85% when SFP was used. A number of other researchers have shown that regardless of the protocol used the advantages of using SFPs outweighs using no SFP's in the prevention of NEC. 
Limiting the use of antimicrobial therapy in early neonatal period
Avoiding or limiting the use of empirical antibiotic therapy improves the healthy balance, quantity, and diversity of commensal bacteria in the neonatal gut offering the immunological advantages eluded to earlier in this review. In USA, the median duration of empiric antimicrobial treatment in neonates ranges from 3 to 9.5 days.  This study showed that the odds for developing NEC was 40% higher for infants receiving 7 versus 2 days empiric antibiotic therapy for culture negative or suspected early onset sepsis. ,
| Future|| |
With the use of "omic" technologies, it is hoped that sensitive and specific markers of NEC would be developed.
Lately, with evidence of HB-EGF having a role in cytoprotection and growth activity of the enterocytes and intestinal repair, Phase I and Phase II clinical trials are either underway or planned to establish the therapeutic use of HB-EGF to prevent and reduce the incidence of NEC.
| Conclusion|| |
A challenge that a comprehensive review of this magnitude poses is in providing a large amount of information and at the same time to provide the jobbing clinician advice/knowledge that is helpful in their daily practice.
It is now beyond doubt that NEC as clinically diagnosed today is not a single disease but an amalgam of conditions whose end point is injury to the premature gut. Thus, attempts should be made to clearly differentiate true NEC from other conditions masquerading as NEC. While the current diagnostic and management strategies and the outcome have not changed over many years, there have been advances made or are being made in understanding the pathogenesis of NEC and developing newer and unique biomarkers to predict or make early diagnosis so that robust treatment strategies could be adopted.
The greatest challenge is to bring this understanding of the pathogenesis and diagnostic tools together so that the clinician can diagnose and treat NEC better than before.
I am very grateful to Dr. Irfan Waheed and Dr. Talal Waqar for reviewing the manuscript and for their helpful suggestions.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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