Lipocalin-2 and calprotectin as stool biomarkers for predicting necrotizing enterocolitis in premature neonates

Abstract

Background: Necrotizing enterocolitis (NEC) is a major challenge for premature infants in neonatal intensive care units and efforts toward the search for indicators that could be used to predict the development of the disease have given limited results until now.

Methods: In this study, stools from 132 very low birth weight infants were collected daily in the context of a multi-center prospective study aimed at investigating the potential of fecal biomarkers for NEC prediction. Eight infants (~6%) received a stage 3 NEC diagnosis. Their stools collected up to 10 days before diagnosis were included and matched with 14 non-NEC controls and tested by ELISA for the quantitation of eight biomarkers.

Results: Biomarkers were evaluated in all available stool samples leading to the identification of lipocalin-2 and calprotectin as the two most reliable predicting markers over the 10-day period prior to NEC development. Pooling the data for each infant confirmed the significance of lipocalin-2 and calprotectin, individually and in combination 1 week in advance of the NEC clinical diagnosis.

Conclusions: The lipocalin-2 and calprotectin tandem represents a significant biomarker signature for predicting NEC development. Although not yet fulfilling the "perfect biomarker" criteria, it represents a first step toward it.

Impact: Stool biomarkers can be used to predict NEC development in very low birth weight infants more than a week before the diagnosis. LCN2 was identified as a new robust biomarker for predicting NEC development, which used in conjunction with CALPRO, allows the identification of more than half of the cases that will develop NEC in very low birth weight infants. Combining more stool markers with the LCN2/CALPRO tandem such as PGE2 can further improve the algorithm for the prediction of NEC development.

Fig. 1. Detection of CALPRO, LCN2, and HAPTO in the stools of NEC and non-NEC premature infants by ELISA.

Stool samples analyzed were those collected over a 10-day period preceding NEC diagnosis (round symbols) and in matched non-NEC infants (square symbols). Simple linear regression for the levels of biomarkers in the stools were calculated for both NEC and non-NEC with R² values varying between 0.02 and 0.0003 while the slopes were considered nonsignificant for all. For simplicity, only the regression line (black line, R²) for NEC samples are shown (left panels). The biomarker data were then subjected to ROC curve analyses (right panels) showing sensitivity and specificity values and cut-off evaluation (dotted lines *, left panels).

Fig. 2. Relative levels of biomarkers in the stools of NEC infants for distinct periods preceding diagnosis.

For each NEC infant, the stool samples were pooled for three periods relative to the diagnosis: group A, −10 to −7 days; group B, −6 to −3 days; and group C, −2 to 0 days while non-NEC samples (NN) were pooled for the 10-day period. The means ± SEM are illustrated. *P < 0.05 (^#P < 0.07) using the Wilcoxon matched pairs signed-rank test by comparison with NN.

Fig. 3. Relative levels of various combinations of biomarkers in the stools of NEC infants for distinct periods preceding diagnosis.

For each NEC infant, the stool samples were pooled for three periods relative to the diagnosis: group A, −10 to −7 days; group B, −6 to −3 days; and group C, −2 to 0 days while non-NEC samples (NN) were pooled for the 10-day period and the data for combining the biomarkers were processed as indicated. The means ± SEM are illustrated. *P < 0.05 (^#P < 0.07) using the Wilcoxon matched pairs signed-rank test by comparison with NN.

Fig. 4. Detection of CALPRO in the stools of NEC and non-NEC infants according to their gestation ages.

Stool samples analyzed were those collected in the period preceding NEC diagnosis and in matched non-NEC infants according to their gestational age. Simple linear regression was calculated with R² squared = 0.023 and a slope considered nonsignificant.