Necrotizing enterocolitis (NEC) usually occurs in very low birth weight infants (VLBWI, birth weight <1500 g) (1) and it is the most common gastrointestinal emergency in the neonatal intensive care unit (NICU) (2), with a high mortality. The pathophysiology of NEC is poorly understood but is surely multifactorial. Prematurity is the single most important risk factor, inasmuch as a sharp decrease in incidence of NEC is observed at approximately 35 to 36 weeks of postconceptional age (3). Associated risk factors are enteral feeding (4), no breast-feeding (5), intrauterine growth restriction, bowel ischemia (6), and infection (3,7,8). As for ischemic risk factors, preterm infants commonly have a patent ductus arteriosus (PDA) with a left-to-right shunt, diminishing blood flow to the mesenteric vascular bed. Inasmuch as NEC mortality and morbidity are extremely high, early diagnosis becomes essential. Residual volume increase is one of the gastrointestinal criteria to define NEC (9), but studies on qualitative and quantitative gastric residual features as markers of NEC risk are still lacking, to our knowledge. The main aim of this analysis was to evaluate gastric residuals as a means of identifying patients at risk for NEC. This study was also designed to investigate risk factors, besides prematurity and birth weight, in a population of VLBWI born in a tertiary center.
PATIENTS AND METHODS
We performed a case-control study among VLBWI with a gestational age (GA) of more than 23 weeks who were admitted to the NICU, Department of Pediatrics, Turin University, Italy, between January 1994 and March 2005. Patients who were moved to another unit within the first week of life or infants with severe congenital anomalies (10) were excluded. During hospitalization, nutritional practices were standardized for all babies according to guidelines for preterm infants receiving either enteral or parenteral nutrition (11). Both enteral and parenteral nutrition were begun simultaneously on day 1. Fluids were started at 60 to 80 mL · kg−1 · day−1 on day 1 and increased to 130 to 160 mL · kg−1 · day−1 by day 7. Carbohydrates were begun at 6 to 8 g · kg−1 · day−1 on day 1 and increased progressively to a maximum of 15 g · kg−1 · day−1. Protein intake was started at 0.5 g · kg−1 · day−1 on day 1 and increased to 3.5 g · kg−1 · day−1 by day 7. Lipids were introduced at 0.5 g · kg−1 · day−1 on day 1 and increased to 3 g · kg−1 · day−1 by day 7. Enteral nutrition was started with human milk, 10 to 20 mL · kg−1 · day−1, and increased daily by 10 to 20 mL · kg−1 · day−1. Maternal milk (MM) was used, or, if the supply of MM was inadequate, human bank pasteurized donor milk (DM). Human milk fortifier was added to human milk when the intake reached 100 mL · kg−1 · day−1 and continued until the infant attained a weight of 1.8 kg or was discharged. If no MM was available, then feeding was carried on with fortified DM until 32 weeks of GA and then progressively replaced by preterm formula until the infant attained a weight of 1.8 kg or was discharged. Infants were fed by tube every 3 hours or by continuous orogastric feeding if signs of feeding intolerance were present. Feeding tolerance was evaluated by use of a specific schedule and took into consideration abdominal distention, gastric residual volumes, emesis, stool frequency, bradycardia, and apnea. When gastric residual volumes were greater than 50% of feed volume, feeding was reduced or withheld, at the discretion of the care provider, with simultaneous increase of parenteral nutrition.
All babies received a daily feeding supplementation with probiotics (12–14). Probiotics have routinely been used since 1995; 1 case infant and its control infant did not receive any supplementation. Probiotics given were Lactobacillus GG (3*109 lactobacilli/day) or Lactobacillus casei (8*109 lactobacilli/day). When feeding was withheld at the onset of NEC, probiotics were also withheld. Cases were defined as occurring in babies who experienced proven NEC (Bell stage ≥2) (9) before death or hospital discharge. In all of the cases, NEC diagnosis was confirmed by abdominal radiography. All of the cases were reviewed and confirmed by a team (E.B., A.C., F.G.) because most of these cases were evaluated retrospectively by chart review. Once NEC was diagnosed, enteral feeding was withdrawn and an orogastric tube was positioned immediately to decompress the stomach. Total parenteral nutrition was always started and continued for 7 to 10 days because enteral feedings are withheld in our nursery for this period of time. Broad-spectrum antibiotics were initiated, and abdominal radiographs were taken for correct NEC staging and for monitoring the occurrence of intestinal perforation. Infants with advanced NEC (stage 3 or greater) received pediatric surgical evaluation. Each NEC case infant was matched with a control infant by GA (in complete weeks without consideration of extra days) and birth weight (±50 g). GA was assessed by first trimester ultrasound scan. When more than 1 infant was identified as a close match, the infant with the closest birth date was chosen. The mean period from birth date of case infants and matched control infants was 1.0 year (SD 1.6 years).
Demographic variables were recorded for all of the infants. Information on all of the study infants was obtained from the NICU database and the medical record by use of a specific data collection form. In the VLBWI population, GA, birth weight, sex, being born small-for-GA (defined as birth weight below the 10th centile of the reference for northwest Italy) (15), Apgar score, PDA (defined with direct visualization of ductus diameter >1.5 mm associated with a left atrium-to-aorta ratio >1.5 (16), at echographic screening of all VLBWI, between 24 and 48 hours from birth) were studied. Infants with severe congenital heart disease were identified by cardiac ultrasound and excluded from analysis. All of the case infants with hemodynamically significant PDA were treated with intravenous ibuprofen with a first dose of 10 mg/kg, followed at intervals of 24 hours by 2 doses of 5 mg/kg. Only when pharmacological therapy failed was surgical ligature performed.
Maternal age, hypertensive disease of pregnancy, maternal drug use, and single or twin pregnancies were also recorded. The day of NEC diagnosis was recorded as day 0, and the corresponding age, expressed in postnatal days, was also recorded. For each case-control pair, we used day 0 as the same postnatal day at which NEC developed in the case infant. Data were collected for all infants from day of birth to day 0. Data recorded daily included weight, type and volume of feed, day and type of first enteral feed, enteral feeding rate of advancement, and age in days at the beginning of human milk fortification. Feeding tolerance was assessed by the following characteristics: maximum gastric residual volume detected (comparison was made considering the residual as percentage of previous feeding), total amount of residuals (calculated as percentage of total amount of feeding given during the previous 24 hours), and aspect of residuals (assessed as clear, bilious, blood-stained, or hemorrhagic). Gastric residual volume was determined by aspiration of gastric contents from the indwelling orogastric tube every 3 hours in all infants before the next feeding. Gastric residuals up to 2 mL in infants weighing <750 g and up to 3 mL in infants from 750 to 1500 g were excluded from statistics, as suggested by Mihatsch et al (17) in their study. The risk factors evaluated were race, sex, Apgar score, respiratory distress syndrome (PaO2 < 50 mmHg or cyanosis without O2 supplementation and consistent chest radiographs), sepsis (clinical syndrome characterized by systemic signs of infection with increased C-reactive protein or positive blood culture), number of days with an umbilical vein catheter (for a maximum of 4 days), and antibiotics given during the week before day 0. Umbilical arterial catheterization was never used. The mean GA at the onset of NEC was also recorded. Maternal and pregnancy risk factors included single or twin pregnancies, prolonged rupture of membranes (defined as >24 hours), mode of delivery, and hypertensive disease of pregnancy. An evaluation was carried out on overall mortality, mortality within 7 days of postnatal age, mortality between 7 and 28 days of postnatal age, and mortality due to NEC.
Odds ratio with 95% confidence intervals was used for categorical variables. Independent sample t test, or Mann-Whitney test if the distribution was not normal, was used for continuous variables to report mean difference and standard deviation. All of the statistical computations were performed by use of SPSS for Windows, version 12.0 (SPSS, Chicago, IL). P < 0.05 was used to define statistical significance.
In all, 844 VLBWI, 414 male and 430 female, were admitted to the NICU within the study period; the overall mortality before discharge was 123/844 (14.6%). Mortality within 7 days of postnatal age was 84/844 (10%); mortality between 7 days and 28 days of postnatal age was 31/760 (4%). The mean GA was 29.2 weeks (SD 2.8, range 23–39); the mean birth weight was 1065 g (SD 276, range 280–1495). The frequency of NEC was 2% (17/844) of VLBWI.
In the NEC group, 5 infants required surgical treatment. The study included 17 NEC case infants and 17 control infants for a total of 34 study participants. Only 1 control infant was chosen for each case infant because it was impossible to find in our case series 2 control infants matched closely with each case infant. The mean GA was 28.47 weeks (SD 1.9) in case infants and 28.47 weeks (SD 1.9) in control infants (P = 1); the mean birth weight was 923.53 g (SD 166) in case infants and 928.53 g (SD 166) in control infants (P = 0.93), as we had expected after matching. Mortality in the NEC group was significantly higher than in the control group, with 4 deaths among the NEC case infants (23.5%) and none among the control infants (P = 0.03). Risk factors were similar, as shown in Table 1. The mean age at onset of NEC was 25.7 days (SD 15.3; range 6–60; median value 19 days), and the mean weight reached on day 0 was 1096 g (SD 286) in the NEC group and 1160 g (SD 417) in the control group (P = 0.61). The mean GA at the onset of NEC was 31.3 weeks (SD 0.89, range 30–33, median value 31.0). The prevalence of sepsis until 24 hours before NEC diagnosis was 5 (29.4%) among NEC case infants and 1 (5.9%) among control infants (OR 6.7; CI 0.7–64.8). Twelve (70.6%) case infants received antibiotic treatment during the 2 weeks before NEC diagnosis, and so did 10 (58.8%) control infants (OR 1.7; CI 0.4–6.9).
Table 1 shows, together with other conditions, factors that may be associated with gut ischemia (maternal hypertension, 1-minute Apgar score ≤3, 5-minute Apgar score ≤3, umbilical vein catheter, respiratory distress syndrome, PDA). PDA was significantly associated with NEC: 58.8% of case infants and 23.5% of control infants had PDA (OR 4.6; CI 1.1–20.4) diagnosed during the first 48 hours of life. Surgical ligature was performed in 2 case infants and in 1 control infant (P = 0.54). In the case group, 1 mother used cannabinoids; in the control group, 1 mother used heroin.
In the first 2 weeks of life, both case infants and control infants received either MM or DM. Preterm formula was never adopted in that period. Enteral feeding was started at a mean age of 1.3 days (SD 1.2) in case infants and 1.5 days (SD 0.9) in control infants (P = 0.63). Nine (52.9%) case infants and 7 (41.2%) control infants were fed with more than 50% MM; no significant difference was noted in MM or DM volume intake. Fortification of MM or DM was made for 10 (58.8%) case infants and 9 (52.9%) control infants. The mean postnatal age at the beginning of human milk fortification was 15.90 days (SD 3.2) in the NEC group and 19.78 days (SD 8.3) in the control group (P = 0.19). The average daily increase in enteral feeding volumes was similar in both groups.
Gastric residuals were compared from birth to the onset of NEC between both groups. The mean maximum residual was 7.46 mL (SD 4.9, median 6) in NEC case infants and 4 mL (SD 3.5, median 3) in control infants (P = 0.04). The mean period between maximum residual day and day 0 in case infants was 17.33 days (SD 10.13, median 14.5). The mean daily feeding volume at the time the mean maximum residual occurred was 72.1 mL · kg−1 · day−1 (SD 42.3) in case infants and 68.3 mL · kg−1 · day−1 (SD 48.9) in control infants (P = 0.84). The mean value of maximum residual as percentage of the previous feed (Fig. 1) was 113.24% (SD 90.7) in case infants and 42.87% (SD 31) in control infants (P = 0.018). The 24-hour residual volume as percentage of the whole volume of daily feeds was 33.07 (SD 27.3) in the NEC group and 30.17 (SD 38) in the control group (P = 0.81). The percentage of infants with hemorrhagic residuals was significantly higher in the NEC group than in the control infants, whereas the difference was not significant for bilious residuals (Table 2). Blood-stained residuals were not a risk factor by themselves. The mean postnatal age at which NEC case infants showed hemorrhagic residuals was 3.1 days (SD 2.8, median 2), and the mean period between hemorrhagic residual and onset of NEC was 19.1 days (SD 13.8, median 15).
The 2.2% frequency of NEC among VLBWI observed in this study is low in comparison with a reported incidence of 7% of VLBWI (National Institute of Child Health and Human Development Neonatal Research Network, reporting incidence of 14 centers, 2001) (3), 3.8% of infants born at 24 to 31 weeks of GA in New South Wales and Australian Capital Territory (18), 7% of VLBWI observed by the Canadian neonatal network (19).
Our policy of exclusive use of MM or DM for the first 14 days of postnatal age without the addition of preterm formula did not allow us to study the role of preterm formula in the pathogenesis of NEC, but it may explain the low observed incidence of NEC (4,20–25). The use of standardized feeding schedules may also have contributed to this low incidence (26–29). By contrast, the incidence of NEC was so low that analysis was limited to a small number of pairs, and our study evaluated experience over a prolonged span of time. To our knowledge, there are no reported data of a higher risk for NEC related to MM fortification (30); the only result of fortification in our study concerned the initiation of fortification begun later in control infants than in case infants, even if the result was not statistically significant. Various risk factors have been implicated in the pathogenesis of NEC in recent studies, such as intrauterine growth restriction (31), low Apgar scores (7), maternal hypertension, and prolonged rupture of membranes (32), but in this study we found no significant difference between the 2 groups (Table 1).
A relation between PDA and NEC has been demonstrated in many studies (18), and in our study NEC was also significantly associated with PDA: 58.8% of NEC case infants versus 23.5% of control infants (OR 4.6; CI 1.1–20.4) had PDA. As long as all hemodynamically significant PDA were treated, in this study we were not able to determine separately the effect of PDA and of ibuprofen treatment on the incidence of NEC. Further studies are needed to confirm the observed association between PDA and NEC and particularly to investigate, by Doppler studies, the role of PDA in the pathogenesis of NEC.
Inasmuch as the onset of NEC is often overwhelming, intervention strategies rarely succeed after the presentation of clinical signs and symptoms. By contrast, a preventive approach, such as early detection of feeding intolerance, could be more successful. Gastric residuals are a significant marker of feeding intolerance (17,33). In our study we observed that maximum residual from birth to day 0 and maximum residual as a percentage of the previous feed (Fig. 1) were significantly higher in the NEC group. In their study, Mihatsch et al (17) tolerated gastric residuals up to 2 mL in infants weighing <750 g and up to 3 mL in infants from 750 to 1000 g. In a case-control study in a population of VLBWI, Cobb et al (33) found that gastric residual volume higher than 3.5 mL could be associated with a higher risk of NEC. Although larger gastric residual volumes were associated with NEC in our study, we were not able to assess a tolerance threshold before withdrawing feeds. A relation between higher maximum residual volumes as a percentage of the previous feed and NEC was observed in the case-control study by Cobb et al (33). However, in their study, only the 6 days before NEC onset were considered, whereas we considered the whole period from birth to NEC onset. Thus, we were able to observe that feeding intolerance was also present in NEC case infants in an earlier period, inasmuch as the mean period between maximum residual day and NEC diagnosis in case infants was 17.33 days (SD 10.13, median 14.5). In these cases, increased gastric residuals could have been an effect of gastrointestinal tract immaturity and poor gastric motility.
The most relevant result in our study seems to be that the number of infants with early blood-stained or hemorrhagic residuals was significantly higher in the NEC group than in the control group, whereas bilious residuals did not seem to be associated with NEC onset. The presence of bloody residuals seemed to be the best predictor for NEC in those infants. Inasmuch as bloody residuals were rarely detected a few days before NEC diagnosis and they were more often present in the first days of life, they cannot be considered an early sign of NEC but rather as an early sign of minimal loss of mucosal integrity, following prematurity, abnormal bacterial colonization, and compromised intestinal blood flow regulation. Such mucosal injury has a pivotal role in NEC pathogenesis of intestinal immaturity and compromise, predisposing to NEC. Therefore, the presence of bloody residuals requires the greatest caution in approaching enteral feeding.
Which factor induces or triggers feeding intolerance and ensuing NEC is still unknown or is at least multifactorial. Some patients may show presenting symptoms such as temperature lability, apnea, and bradycardia, but residuals may be the initial symptom in some or most patients because most patients are being fed at the time a diagnosis of NEC is first considered.
We conclude that for early detection of VLBWI at risk for NEC, both gastric residual volumes and bloody residuals may represent early relevant markers. Further studies are required to assess a residual volume threshold and to better understand the association between early bloody residuals and later onset of NEC.
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