Premature birth remains one of the most challenging problems in pediatrics. The implementation of sophisticated technologies and neonatal specific interventions have contributed to an increase in survival among very low-birth-weight-infants from 15% to 75% over the past decade. However, with these gains in survival, morbidity related to immaturity of gastrointestinal function has increased significantly. Infants who previously would have succumbed to complications associated with prematurity and low birth weight are surviving, but often with impaired growth and neurodevelopment as a result of gastrointestinal complications such as necrotizing enterocolitis (NEC).
Necrotizing Enterocolitis in Premature Infants
Katherine Gregory, RN, PhD; Research Associate, Pediatric GI/Mucosal Immunology Laboratory, MassGeneral Hospital for Children; Haley Nurse Scientist, Brigham and Women’s Hospital; Associate Professor of Nursing, W. F. Connell School of Nursing, Boston College
NEC, an inflammatory gastrointestinal disease, is among the most significant complications of prematurity.It is both life-threatening and associated with significant long-term morbidity. Research on NEC has explored the inflammatory cascade and factors related to enteral feeding, yet, the pathogenesis remains elusive. NEC is especially problematic because the disease often presents in a sudden and rapidly progressive manner, making it a difficult disorder to anticipate and potentially prevent. For these reasons, our team is seeking to identify biomarkers of NEC that will help predict which premature infants are at greatest risk of disease, and in addition, to define the microbiome aspects of NEC so that effective prevention strategies may be developed. The use of urine for the identification of biomarkers and stool for microbiome analysis has the advantage of a noninvasive approach that will not deplete the premature infant of a limited blood volume.
Urinary Biomarkers of NEC
Several cytokines, specifically inflammatory mediators, have been implicated in the pathogenesis of NEC and have been studied as candidate biomarkers of this disease. These lines of inquiry have made a contribution to our understanding of the inflammatory response that is thought to play a role in NEC. However, up or down regulation of a specific cytokine or set of cytokines may not be fully attributed to NEC. A premature infant might exhibit these biologic events in response to any type of systemic inflammatory response (e.g., sepsis). Thus, the ideal biomarker predicting NEC would be one that is specific to the gastrointestinal tract. In our work, we have measured intestinal fatty acid binding protein (IFABP), a small cytoplasmic protein found within the cells at the tip of the intestinal villi, an area commonly injured as a result of inflammatory bowel diseases such as NEC.
We have conducted a case-control study of 140 infants born prior to 29 weeks gestation: 70 infants with NEC and gestational age-matched controls. The diagnosis of NEC, as defined by Bell Stages I-IIIB, was required for assignment to the case group. IFABP was measured in urine by ELISA. Infants were drawn from a prospective cohort study and had urine samples available within seven days prior to disease. Logistic regression was conducted to assess the relationship between iFABP and NEC. Analyses were also performed using a subset of subjects who had urine samples available within three days of diagnosis (n=98).
We found that median IFABP in controls was 4,470 pg/mL [1,792,12,010] and cases was 16,276pg/mL [7,767, 39,031] (p<.0001). Logistic regression analysis adjusted for 5 minute Apgar score, administration of maternal steroids, small for gestational age, mode of delivery, and breast milk exposure in logistic regression, revealed odds ratio for the association of IFABP and NEC of 1.947(p<.0001) [CI:1.465,2.587]. Within a subset of the sample for whom urine specimens were available within three days, median IFABP in controls was 4,110pg/mL [1,792, 10,116] and in cases was 19,301pg/mL [9,524, 54,325] (p<.0001). Adjusting for the same variables, this logistic regression resulted in an odds ratio of 2.505[CI:1.678,3.741] (p<.0001).
Our findings suggest that higher levels of IFABP in urine of premature infants obtained within a week of diagnosis are highly associated with NEC and might be more strongly predictive for the subset of urine samples obtained within three days prior to clinical evidence of NEC. If confirmed, urinary IFABP might prove a useful test of impending NEC, with the potential of ameliorating disease progression. This work was conducted in collaboration with Linda Van Marter, MD, MPH at Brigham and Women’s Hospital and with the support of the Clinical and Biostatistical Core for the Children’s Hospital Specialized Center of Research in Lung Biology (HL67669), The Harvard Newborn Medicine Clinical and Translational Research Consortium, and the American Nurses Foundation.
Microbiome Aspects of NEC
Study of the human microbiome has become one of the most rapidly growing fields in science. The microbiome has been explored in the context of obesity, inflammatory bowel disease, allergic response, cancer, late-onset autism, and premature birth. The studies conducted to date suggest that the composition of colonizing microbiota of the intestine may be an important determinant as to whether or not an individual develops a specific condition. Since premature infants have an abnormal pattern of colonization, tend to colonize with fewer bacteria, and are exposed to highly pathogenic institutional organisms, examining the microbiota in premature infants may be an important determinant in the pathogenesis of NEC.
Defining the intestinal microbiota in premature infants who are at risk for NEC is an innovative approach that will lead to a better understanding of the pathogenesis of the disease and thus, an improved ability to develop clinical interventions and strategies for disease prevention. The premature gut is poorly colonized with protective bacteria at birth and thus, is vulnerable to disease. Disease risk is amplified by the routine administration of antibiotics. We hypothesize that NEC may evolve because the protective effect of colonizing bacteria is absent, resulting in infants who are susceptible to gut pathogens and prone to acquire pathogens in the hospital environment.In this project, we aim to (i) conduct feasibility of microbiome analyses using fecal matter obtained from premature infants and (ii) study differences in the microbiome of premature infants who develop NEC from those who do not develop NEC. The central hypothesis is that the fecal elimination of specific human microbiota will differentiate a population of premature infants who develop NEC from those who do not develop this devastating gastrointestinal disease. To date, we have prospectively collected fecal material on over 600 low gestational age infants at risk for NEC. Analysis of the microbiota of infants who developed NEC as compared to unaffected infants is currently underway. In addition, we are studying differences in the microbiota associated with different nutritional exposures during the early neonatal period. We anticipate that our study findings will not only highlight differences in the diversity of bacterial species under conditions of health and disease, but will also provide specific insights on how we might intervene by introducing bacterial species that promote optimal intestinal colonization during the early neonatal period. This project is supported by the National Institute for Nursing Research and Harvard Catalyst.
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