www.jpnim.com Open Access eISSN: 2281-0692 Journal of Pediatric and Neonatal Individualized Medicine 2014;3(2):e030245 doi: 10.7363/030245 Received: 2014 Sept 10; accepted: 2014 Sept 28; published online: 2014 Oct 21 Review From the “old NEC” to the “new NECs” Melania Puddu1, Maria Antonietta Marcialis1, Anna De Magistris1, Roberta Irmesi1, Elisabetta Coni1, Luigi Mascia2, Vassilios Fanos1 1Neonatal Intensive Care Unit, Neonatal Pathology, Puericulture Institute and Neonatal Section, AOU and University of Cagliari, Italy 2Pediatric Surgery, ASL 8, Cagliari, Italy Proceedings Proceedings of the International Course on Perinatal Pathology (part of the 10th International Workshop on Neonatology · October 22nd-25th, 2014) Cagliari (Italy) · October 25th, 2014 The role of the clinical pathological dialogue in problem solving Guest Editors: Gavino Faa, Vassilios Fanos, Peter Van Eyken Abstract Necrotizing enterocolitis (NEC) is an acute inflammatory disease of the neonatal intestine that strikes in 1 of 1,000 live births. Its etiology is unknown. This review describes in detail the new NECs especially those which affect preterm infants: contagion or lymphocytosis associated, transfusion associated and cow’s milk allergy associated. A wide repertory of images are presented, together with algorithms for differential diagnosis. Keywords Necrotizing enterocolitis, intestinal mucosa barrier, NEC sub-classes, prevention, gastrointestinal neonatal problems. Corresponding author Melania Puddu, Neonatal Intensive Care Unit, Neonatal Pathology, Puericulture Institute and Neonatal Section, AOU and University of Cagliari, Italy; email: [email protected]. How to cite Puddu M, Marcialis MA, De Magistris A, Irmesi R, Coni E, Mascia L, Fanos V. From the “old NEC” to the “new NECs”. J Pediatr Neonat Individual Med. 2014;3(2):e030245. doi: 10.7363/030245. 1/17 www.jpnim.com Open Access Journal of Pediatric and Neonatal Individualized Medicine • vol. 3 • n. 2 • 2014 Definition and incidence Necrotizing enterocolitis (NEC) is an acute inflammatory disease of the neonatal intestine. Its etiology is uncertain and it strikes in 1 of 1,000 live births. It is characterized by necrosis of the intestinal mucosa that extends to the deepest layers, mostly involving the proximal ileum and the colon. The most affected are premature infants with an incidence of 7% to 10% in very low birth weight (VLBW) infants. Incidence is inversely related to weight and gestational age (GA). Age at onset varies inversely with GA: those born at term develop the disease within a few days of birth while those of < 30 wks GA develop it after some weeks. The disease has a mortality from 20% to 50% depending on case histories [1-3]. Classification: from the “Old NEC” to the “New Figure 1. Bell’s classification, extensively used to define NECs” NEC (“Old NEC”), may be compared with a funnel where very different neonatal conditions caracterized by At the end of the 1970s, when NEC was described mild or severe gastrointestinal symptoms are collected. prevalently in term- or late preterm infants, Bell’s NEC: necrotizing enterocolitis; FIP (Feed Intolerance of classification [4] provided useful grounds for Prematurity); SIP (Spontaneus Intestinal Perforation); ANIDs (Acquired Neonatal Intestinal Diseases). grouping under a single denomination different forms that had in common a single characteristic: progression towards intestinal perforation. But with progress in neonatology and the ever-increasing with different etiologies. This new interpretation has onset of the disease in VLBW infants, given their several advantages: constant increase in survival, clinicians realized that • it introduces a separation between the NEC of the use of Bell’s classification, which is actually a term and late preterm neonates and that of very stadiation of the disease, led to the definition of NEC preterms; (“Old NEC” in Fig. 1) a series of acquired neonatal • within the NEC of preterms it provides a division intestinal diseases (ANIDs) with a different etiology into four subclasses with different etiologies that did not necessarily lead to intestinal perforation. according to risk factors, clinical and laboratory For example, Bell’s Stage I included non-specific data: abdominal symptoms (such as the broad range of 1. classic NEC; symptoms of Feed Intolerance of Prematurity [FIP]) 2. NEC associated with epidemics of viral and systemic signs of infection, while Bell’s stage gastroenteritis and/or lymphocytosis; IIIb could include Spontaneous Intestinal Perforation 3. NEC associated with cow’s milk proteins (SIP), a condition quite different from the etiological, intolerance; clinical and pathological standpoints [5]. 4. NEC associated with red blood cell (RBC) This confusion was caused by the lack of a transfusions; distinction between NEC in the premature and NEC • it allows to establish incidence and epidemiology in term infants and, within these two large categories, of different subsets in the single NICU and to by a line of identification of possible causes and implement tailored preventive and therapeutic effects, also in consideration of recent studies that measures. show a connection with the ischemic, infective and allergic disorders, with nutrition and drugs. Pathophysiology In recent years a new approach [6] (NEC reductionism) has allowed to overcome the issue, at The events leading up to NEC are multifactorial least in part (“New NEC” in Fig. 2), by recognizing and complex, including a history of a hostile that NEC is the point of arrival of different affections intrauterine environment, a difficult perinatal 2/17 Puddu • Marcialis • De Magistris • Irmesi • Coni • Mascia • Fanos Journal of Pediatric and Neonatal Individualized Medicine • vol. 3 • n. 2 • 2014 www.jpnim.com Open Access Figure 2. Necrotizing enterocolitis (NEC) reductionism [6] (“New NEC” in the figure) introduces a separation between Term-NEC and Preterm-NEC and provides a division into four subclasses with different etiologies within the latter. transition and a complicated neonatal period. Three inherent in the phlogosis and being itself the cause conditions are required for the onset of necrosis of of further damage to the mucosa. the mucosa: a recently colonized intestine, as is that Some elements of vulnerability of the mucosa of the neonate, the presence of food in it, a triggering barrier may favor the bacterial invasion, especially event that damages the mucosa barrier [6]. in preterms. In the “New NEC” the first distinction between Fig. 3 schematically shows the process that NEC in the term neonate and NEC in the preterm seems to be more involved in the evolution of neonate allows us to divide them into two large the damage and subsequent necrosis: different groups on a pathophysiological basis: indeed, only underlying factors are discussed in the following in term newborns NEC (ischemic NEC) does the paragraphs. ischemia appear to be the initial event while in preterm newborns it is almost always the result of the Microbiota effect of different etiological factors. Some of these act earlier (prematurity, delayed feeding, altered The flora that normally populates the intestine intestinal colonization by antibiotics, mother’s (microbiota) plays an important role in maintaining milk lack, too-rapid progression in enteral feeding), the intestinal barrier: its microorganisms are while others have a later influence (viral epidemics, recognized as commensals and perform their allergy to cow’s milk proteins, transfusions) [7]. precious duties in the development of the mucosa Whatever the initial trigger, ischemic or of other and the intestinal immune system. Alteration of origin, the pathological process begins with the the microbiota (through exposure to nosocomial breaking down of the intestinal protective barrier, bacteria, antibiotics, lack of mother’s milk) or the invasion of the mucosa by bacteria and the its poverty due to factors that delay colonization triggering of an overreaction of the intestinal cells (i.e. delay in the beginning of enteral feeding) with consequent damage to the mucosa and final may thus play an important role in the genesis of necrosis. Ischemia is an integral part of the process NEC, selecting species that may direct the immune since it is caused by the endothelial dysfunction response towards inflammation [8]. From the “old NEC” to the “new NECs” 3/17 www.jpnim.com Open Access Journal of Pediatric and Neonatal Individualized Medicine • vol. 3 • n. 2 • 2014 Figure 3. Regardless of the risk factor involved, the pathological process begins with the breaking down of the intestinal protective barrier, the invasion of the mucosa by bacteria and the triggering of an overreaction of the intestinal cells with consequent damage to the mucosa and final necrosis. The process is carried out by different mediators and worsened by altered microbioma and by fragile points in the mucosa barrier (gel-like mucus barrier lack and low production of bactericidal peptides by Paneth cells). PAF: Platelet Activation Factor; TLR:Toll-Like Receptors; IGFβ2:Transforming Growth Factor β2; TREG: T regulatory cells. Fragile points in the mucosa barrier the TLRs (Toll-like receptors): the response, which initially is cytoprotective, may be destructive [10]. The weakness of the tight junctions, the scarcity The TLR4s induce apoptosis to trap the pathogen of the gel-like mucus layer that acts as an obstacle contained within the agonizing enterocyte; in to the entrance of bacteria towards the surface and particular, they note the presence of the bacterial the reduced secretion of bactericidal substances, in lipopolysaccaride (LPS) and its expression (which particular the bactericidal C-peptide lectin RegIIIγ increases with gestational age and is drastically produced by the Paneth cells, represent further reduced at term), appears to have an important elements of fragility of the intestinal mucosa [9]. responsibility in determining the severity of NEC. The TLR4s appear to be up-regulated in NEC to The TLR and PAF pathways the detriment of TLR9s, which have an antagonistic effect on apoptosis and are activated by commensals In the triggering of inflammatory mechanisms such as lactobacilli and bifidobacteria, the presence leading to necrosis of the intestinal mucosa, of which appears to reduce the risk of NEC by great importance is attributed to activation by the increasing the expression of TLR9 [10, 11]. intestinal epithelial cells of specific receptors for Moreover, a powerful lipid mediator, PAF the recognition of the bactericidal ligands (PPRs – (platelet activation factor), is produced in large pattern recognition receptors) and in particular of amounts by the epithelial cells in the NEC-affected 4/17 Puddu • Marcialis • De Magistris • Irmesi • Coni • Mascia • Fanos Journal of Pediatric and Neonatal Individualized Medicine • vol. 3 • n. 2 • 2014 www.jpnim.com Open Access intestine: it stimulates the new production of TLR4 Clinic and in turn is stimulated by the LPS, the receptor of TLR4, which facilitates the release of the The symptomatology is similar in the different neutrophils. There thus appears to be a correlation forms: it may begin with aspecific signs that progress between TLR4 and PAF, which may represent the insidiously for several days or have a fulminant two main pathways in NEC pathogenesis [12]. onset leading in a short time to multiorgan system The timing of NEC may actually be determined dysfunction and shock. by the TLR4s since their expression begins to be The most frequent symptoms when the terminal quite present (29-32 weeks) just when NEC arises a ileum (the typical seat in the VLBW infants) is few weeks after birth [6]. involved are abdominal distension, gastric residuals, In synthesis, the incapacity of the intestine to bilious vomiting and absence of stools. If the colon down-regulate the TLR4s hinders it from becoming is involved the first symptom may be only blood in tolerant of the luminal bacteria and the exaggerated the stool. TLR4 signal of the bacterial colonization results in a As the disease progresses abdominal distension damage to the mucosa by apoptosis and a reduced increases, the abdomen becomes firm and tender capacity to repair itself if damaged. and may assume an erythematose or bluish color when intestinal perforation takes place (Fig. 4). In The macrophages such a case, in male neonates even the scrotum may turn bluish due to the passage of peritoneal liquid Even the macrophages presence, starting from the perforated loop. Signs of overall distress, from the 11th or 12th week of gestation, appears to such as lethargy, apnea, bradycardia, temperature participate in the onset of this intolerance: in the instability, impairment of peripheral perfusion and mature intestine they are “tolerant” to the bacteria the need for artificial ventilation are quite frequent. of the nearby intestinal lumen and consequently Laboratory tests may show anemia, neutropenia, do not have an inflammatory response to them. left shift of neutrophils, metabolic acidosis and This tolerance, attributed to the down-regulation hyponatremia. In 40% to 60% of cases there may of TGF-β2 (transforming growth factor β2), is be positivity for blood cultures, commonly gram seemingly lacking in preterms and this facilitates negatives. the onset of the inflammatory process [13]. NEC may recur in 5% of cases, especially in neonates with congenital heart disease and in the The eosinophils forms with viral pathogenesis or caused by allergy to cow’s milk proteins [2, 15]. Finally, in some forms of NEC, such as that associated with allergy to cow’s milk proteins and Diagnostics perhaps also that associated with transfusions, the eosinophils apparently participate in the destructive The radiographic signs are those most useful in process of the intestinal mucosa through recognition diagnosing the disease. of a specific bacterial metabolite: fMLP (N-formyl- Pneumatosis (Fig. 5) and the presence of gas in methionyl-leucyl-phenylalamine) [14]. the portal vein are pathognomonic: gas, produced by the bacteria, may generate a linear image if it is The advent of adaptive immunity in the intestinal wall, circular if subserous and in the shape of small bubbles if submucosal. Pneumatosis is With the increase in gestational age and the found more frequently in the lower quadrants but may beginning of proper enteral feeding, the innate involve the entire intestine. immunity, which includes all the processes described Other non-specific radiographic aspects are the above, gradually gives way to the adaptive immunity increase in thickness of the loops walls, the dilation of mediated by the B and T cells: so the production of some of them and the presence of “fixed loops” (bowel superficial IgA increases, the expression of TLR4 without peristalsis) (Fig. 6). The series of loops struck decreases, that of TGF-β2 increases with a later by the pathological process may form a mass distinct reduction of activation of the macrophages, the from the rest of the intestine due to the considerable thickness of the gel-like mucus layer increases and, thickening of the wall and loss of normal shape. The in absence of exaggerated bacterial growth, the risk persisting radiographic finding of a fixed loop is an of NEC is reduced [10, 11, 13]. indication of a necrotic bowel loop. The localization of From the “old NEC” to the “new NECs” 5/17 www.jpnim.com Open Access Journal of Pediatric and Neonatal Individualized Medicine • vol. 3 • n. 2 • 2014 A. B. Figure 4. A. 26 wks infant with shiny, distended, erythematous abdomen (advanced necrotizing enterocolitis [NEC]) treated with ileal resection at 15 days of life. B. Necrotic patches on the wall of the ileum. A. B. Figure 5. Supine (A) and lateral (B) radiographs of the abdomen obtained in a neonate with necrotizing enterocolitis (NEC) showing bowel wall thickening, dilatation of the loops with gas and extensive intramural gas. 6/17 Puddu • Marcialis • De Magistris • Irmesi • Coni • Mascia • Fanos Journal of Pediatric and Neonatal Individualized Medicine • vol. 3 • n. 2 • 2014 www.jpnim.com Open Access dilated loops at the center of the abdomen may indicate Pneumoperitoneum (Fig. 7) is a sign of perfo- the presence of a peritoneal effusion that causes them ration and in its initial phase it is easier to see with a to “float”. In cases in which main clinical features are left lateral decubitus radiography [2, 15]. minor abdominal distension and ileus, we see loops Auxiliary to the x-ray diagnosis, abdominal ultra- with no or quite limited amounts of gas inside them. sound has come into wide use when NEC is suspected. Figure 6. Images obtained at 12-hour intervals show dilated and “fixed” loops. A. B. Figure 7. Supine (A) and lateral (B) radiographs of the abdomen showing dilatation of the loops with gas and free intraperitoneal gas (pneumoperitoneum). From the “old NEC” to the “new NECs” 7/17 www.jpnim.com Open Access Journal of Pediatric and Neonatal Individualized Medicine • vol. 3 • n. 2 • 2014 It allows the revealing of the presence of slight Inside different NECs effusions as well as small amounts of free gas in the abdomen. It is also possible to assess peristalsis, the In the ambit of NEC subclasses, below are thickness of the intestinal loops and the presence of examined the physiopathological and clinical pneumatosis and portal gas (Fig. 8). The Doppler characteristics of each of them (Tab. 1), together ultrasound examination allows assessment of with possible specific preventive and therapeutic perfusion of the loops [16]. measures that can be applied. Term and late preterm NEC This appears in approximately 1 out of 20,000 term neonates with a mortality of 25% to 30%. Its onset is generally in the first week of life in neonates in NICUs for other reasons. Certain diagnoses (congenital cyanogen heart diseases, in particular the single ventricle, perinatal asphyxia, hypotension, especially when associated with late- onset sepsis, polycythemia) have been found to be more common in these neonates as is the association with the use of formula milk and the administration of food volumes far above those that would be ingested with breastfeeding. Relatively recently another condition associated with term NEC has been described: neonatal abstinence from oppioids. The food factor is important also in this case: breastfeeding is usually not recommended in such neonates and their extreme irritability, often interpreted as hunger, leads to a Figure 8. Supine abdominal radiograph shows bubbles of rapid increase in the amount of food administered. portal venous gas projected over the liver (arrow). Table 1. Pathophysiological and main clinical characteristics of different NECs. NEC with NEC with cow’s NEC with RBC Classic NEC Term NEC contagion milk allergy transfusion Ileocecal junction, Where Terminal ileum Descending colon Not specific Terminal ileum ascending colon When 3-4 wks Variable > 6 wks 5-6 wks 1 wk Prematurity, IUGR, congenital preeclampsia, ELBW, exposure to cardiopathy, Formula milk and PROM, feeding at least 1 previous policythemia, Why Viral epidemics fortifiers made with malpractice, transfusion donor, sepsis, hypoxia, cow’s milk proteins antibiotics, formula severe anemia ischemia, formula feeding feeding Ischemic and/ Eosinofilic Abnormal response or hemorrhagic Mesenteric Coagulative phlogosis, edema of mesenteric How necrosis, edema ischemia, necrosis and necrosis, blood flow velocity, and phogosis, colic coagulative necrosis ascitis, pneumatosis coagulative necrosis pneumatosis, ascitis Clinics Ileus Yes Delayed Yes Yes Variable Rectal bleeding Rare Yes Yes Yes Variable Pneumatosis Yes Yes Yes Yes Yes NEC: necrotizing enterocolitis; PROM: premature rupture of membranes; ELBW: extremely low birth weight; IUGR: intrauterine growth restriction. 8/17 Puddu • Marcialis • De Magistris • Irmesi • Coni • Mascia • Fanos Journal of Pediatric and Neonatal Individualized Medicine • vol. 3 • n. 2 • 2014 www.jpnim.com Open Access In all conditions described the impairment of near term with a positive anamnesis for hypoxic- mesenteric perfusion is supposedly to be the first ischemic events or in the syndrome of abstinence hit while the food factor would act in triggering the from oppioids [3, 17, 18]. symptoms [3, 17, 18]. Even in the term neonate activation of the two “Classic” NEC in the VLBW preterm TLR4-PAF pathways appears to play a preponderant role: the expression of TLR4s, which is running This affects approximatly 7% of neonates in this out at the end of gestation, may be re-activated by weight category, with a mortality of about 20% to the PAF released by the enterocytes in response to 40% [2]. hypoxia [6]. Symptoms usually appear after the first two Since in term baby there is no slowing of weeks of life. Risk factors, other than preterm birth, progression along the ileum as in VLBW infant, are represented by pre-eclampsia, PROM (premature the site of the mucosa damage in most cases is rupture of membranes), chorionamnionitis, poorly distal (ileocecal valve and ascending colon) and the managed feeding regimen, antibiotics and the use of symptoms of intestinal obstruction may be lacking formula milk [19]. while the emission of bloody stools may be the Since it is mostly the distal ileum that is affected, initial peculiar symptom (Fig. 9). the main symptoms are those related to obstruction, The use of mother’s milk is, as in all forms while the presence of bloody stools is infrequent [2]. of NEC, the gold standard for prevention. The The onset generally does not take place until food activation of a feeding regimen that includes a volumes reach about 80 ml/kg or if enteral feeding slow increase in food intake is presumably to is well under way and tolerated with volumes of > be taken into consideration even in babies born 120 ml/kg [5]. A. B. C. D. E. Figure 9. Necrotizing enterocolitis (NEC) in a term baby formula milk fed began with emission of bloody stools: at 4 days severe abdominal distension and tenderness. A. The newborn underwent surgery for total colectomy and terminal ileostomy. B. On exploration there were multiple gangrenous and necrotic patches on the wall of the colon. C. Widespread colonic pneumatosis. D. Radiographic evidence of ileus. E. Abdominal ultrasound evidence of portal gas (arrows). From the “old NEC” to the “new NECs” 9/17 www.jpnim.com Open Access Journal of Pediatric and Neonatal Individualized Medicine • vol. 3 • n. 2 • 2014 In a certain number of neonates the progression assumed in relation to the volume of total milk is particularly serious and death takes place rapidly (mother’s milk + formula milk). The incidence of in a context of systemic sepsis. This most serious NEC decreases with the increase in the amount of NEC, also known as NEC totalis since necrosis mother’s milk [27]. involves the entire intestine, has not yet received a There is strong evidence in favor of donated pathogenetic explanation, although the hypothesis human milk compared to artificial milk in the of a genetic predisposition is plausible [20]. prevention of NEC, but there are no randomized studies comparing mother’s milk and donated milk. NEC and FIP The administration of donated milk is thus strongly recommended when mother’s milk is unavailable, NEC is frequently preceded by symptoms of keeping in mind that pasteurization and origin from FIP, a common problem in NICUs when the first mothers with term deliveries can reduce its content attempts at enteral feeding are made. It is caused of T and B cells, macrophages, neutrophils, IgA, by an intestinal dysmotility (responsibility for IgG and lactoferrin, thus making it less effective which is probably immaturity of the peristalsis in than the milk of one’s own mother [26-28]. the distal ileum) and by retarded gastric voiding The early beginning of enteral feeding, owing to [5, 21, 22]. The result is gastric retention, abdomi- its positive effect on the trophism of the intestinal nal distension, absence of stools, sometimes mucosa and the development of the microbiota, bilious vomiting, apnea occurrence. FIP is a has been shown to be effective in reducing the paraphysiological situation that is overcome in incidence of NEC (and its mortality) [29]. Instead, most cases but, given the greater risk of bacterial there is no evidence in this sense in favor of minimal invasion caused by food retention, it may develop enteral feeding, but since this practice, now applied into a true NEC if not treated properly: enemas in many NICUs, has no negative effects, it can be for failure to stool and appropriate diet protocol considered a sure alternative to the rapid increase in with a cautious increase in food volumes are useful enteral feeding [26]. measures for overcoming it. As concerns probiotics, their use has been shown to be promising in preventing NEC [30, 31] but NEC associated with delayed feeding the several studies performed have not provided suitable information on what probiotic or probiotics In the lowest gestational ages, NEC may appear to use, their dosage, which neonates to administer in neonates in whom, following a prolonged FIP or them to and when and for how long the treatment for other causes, progression in enteral feeding is should begin and last. The possibility that their quite slow. In such cases, at the time of onset not use may expose the premature intestine, already even pertinent volumes have been reached with lacking adequate defenses and with a tendency to minimal enteral feeding [6]. Atrophy of the mucosa inflammation leading to sepsis, has hindered their appears to be the preponderant pathogenetic factor: widespread use in NICUs: although almost no studies it is known that prolonged parenteral feeding have detected the onset of sepsis in connection with is associated with this condition which, when probiotics, clinical practice requires caution in this trophism is not re-established, leads to failure of sense, especially in cases of ELBW (extremely low the barrier function and the onset of the necrotic birth weight) infants [26]. process [23-25]. There is universal agreement on the need to take into account the epidemiology of the disease in their Prevention own NICU as the greater the incidence of NEC the greater would be the effectiveness of probiotics [30]. Attention of clinicians and researchers In a recent editorial in response to an article concerning the prevention of preterm NEC focuses by Shlomai et al. [32], in which the authors prevalently on three strategies: the use of mother’s recommend the administration of probiotics to all or donated breast milk, the early beginning of enteral ELBW infants regardless of the kind of milk used feeding, minimal (or trophic) enteral feeding, the (mother’s milk, formula milk or both together), use of probiotics. Moodi [33] expresses the following perplexity: is The efficacy of mother’s milk has now been it logical to administer a supplement of probiotics, widely acknowledged [26]. Protection against (so long as we know which ones to administer) to NEC depends on the amount of mother’s milk neonates who assume colostrum from the beginning 10/17 Puddu • Marcialis • De Magistris • Irmesi • Coni • Mascia • Fanos