Fecal calprotectin concentration in neonatal necrotizing enterocolitis

Abstract

Purpose

Among the many factors associated with acute intestinal mucosal infection, numerous studies have proposed the usefulness of fecal calprotectin. The aim of this study was to evaluate the usefulness of fecal calprotectin in the diagnosis of necrotizing enterocolitis (NEC).

Methods

We collected 154 stool samples from 16 very low birth weight and premature newborns at the Konyang University Hospital neonatal intensive care unit or neonatal nursery. The stool samples were collected using the Calprest device, and the fecal calprotectin level was measured with the BÜHLMANN Calprotectin enzyme-linked immunosorbent assay kit.

Results

Fecal calprotectin levels were significantly higher in the NEC group than in the non-NEC group (P=0.02). There was a significant positive linear relationship between the fecal calprotectin level and number of days after birth (P=0.00) in the gestational age <26 weeks group. There was a significant negative linear relationship between the calprotectin level and number of days after birth (P=0.03) in the gestational age ≥26 weeks and <30 weeks group. There was no difference in the calprotectin levels according to the type and method of feeding between the NEC and non-NEC groups.

Conclusion

Fecal calprotectin levels were significantly increased in premature infants with NEC. The fecal calprotectin test is a noninvasive, easy, and useful tool for the diagnosis of NEC.

The participants of this study were newborns in the Konyang University Hospital neonatal intensive care unit or newborn unit. All participants had gestational ages of [less than]37 weeks. We collected stools from these newborns over two separate periods. The first collection was from February to April 2012 and the second was from May to June 2013. We collected stools from premature infants with early symptoms and signs of NEC and normal premature infants with parental consent. We excluded newborns with congenital anomaly of intestine, acute gastroenteritis, or milk allergy.

Stool specimens were collected using a commercial kit (Calprest; Eurospital, Trieste, Italy). Using this kit, specimens were collected within 3 hours. Specimens were mixed with a roller shaker for over 20 minutes and centrifuged at 3,000 rpm for 5 minutes. Then, the top layer of the centrifuged specimen was transferred to a microcentrifuge and stored at -20℃. The longest period of storage was 3 months. Next, a calprotectin enzyme-linked immunosorbent assay [ELISA] kit (Calprotectin ELISA kit, Eurospital, Trieste, Italy) was used to measure the concentration of calprotectin in the specimens.

The medical records were checked and we interviewed their mothers to obtain information about the mothers' medical records; birth method; gestational age; birth weight; the age of the newborn at stool collection; and method, amount, and type of feeding. The blood tests were performed on the same day as the stools collection or within 3 days before or after stool collection.

Clinical symptoms observed at stool collection, radiological finding, blood test, and other test results of newborns with NEC were also recorded. We determined the level of disease severity using the modified version of Bell's staging criteria.

Total 154 stool samples were obtained from 16 premature infants. Twelve stool samples were obtained from 4 premature infants with NEC and 142 stool samples were obtained from 12 newborns without NEC (Fig. 1). The Modified Bell's stages were stage IA, IB, IIA, and IIB in 4 premature infants with NEC.

1. Statistical analyses

Statistical processing was carried out with SPSS ver. 10.0 (SPSS Inc., Chicago, IL, USA). The Mann-Whitney U test, chi square analysis, and Fisher exact test were used to compare NEC newborns with the non-NEC group. Logistic regression was performed for data that displayed statistical significance. Pearson's correlation coefficient and Spearman's rho coefficient were used for correlation analysis. The Mann-Whitney U test and independent t test were used to compare calprotectin concentrations in the normal newborn group. A P[less than]0.05 was considered significant.

2. Ethical issues

Before carrying out the study, we obtained approval following review by the Konyang University Hospital Medical Ethics Committee. The parents of the participants were briefed on the study and provided with explanations on how the gathered medical data could be used. We obtained parental consent before performing the study.

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