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Necrotizing enterocolitis (NEC) is a neonatal disease with its pathogenesis still not well understood, although it is hypothesized to be related to decreased perfusion of the intestinal wall. The current study aimed to evaluate the plasma lactate levels and assess the validity of plasma and urinary intestinal fatty acid binding protein (I-FABPp and I-FABPu/Cru respectively) in NEC.
The study included 55 neonates with variable Bell's stages who were comparable with 23 matched controls. Colorimetric assays of plasma lactate and ELISA assays of I-FABP in both serum and urine of the included neonates have been performed.
There were significantly higher median levels of I-FABPp, I-FABPu and lactate among cases (2.84 ng/ml, 1.74 ng/g creat. and 32.34 mg/dl, respectively) compared with controls (0.16 ng/ml, 0.60 ng/g creat. and 15.33 mg/dl, respectively) with p ˂ 0.05 for all. I-FABPp at cut-off point >3.24 ng/ml showed 90% sensitivity, 72% specificity, PPV = 52.6%, NPP = 94.7%, while for I-FABPu (at cut-off point > 2.93 ng/g creat.) those values were 90%, 92%, 81.8% and 95.8% respectively, in discriminating stage IIIA from stage II with p = 0.001. In predicting surgical NEC, I-FABPp at the cut-off point of 6.95 ng/ml revealed 75% sensitivity, 100% specificity, PPV = 100%, NPP = 95%, while for I-FABPu (cut-off point>4.13 ng/g creat.) they were 100%, 76.19%, 44.4 %and 100%, p = 0.04.
s: In addition to clinical judgment, sonographic data and plasma lactate, I-FABPp was shown to be a specific marker for early identification of surgical NEC, while I-FABPu could be more useful for differentiating Bell's stage II from stage III.
Its cause is still not well understood and though it is related to decreased intestinal wall perfusion and ischemia especially in immature guts with disruption of the intestinal barrier, activation of the inflammatory mediators, and enabling of bacterial passage.
Multiple risk factors have been implicated in its development, such as preterm birth, umbilical catheterization, perinatal asphyxia, early enteral feeding in preterm newborns, and congenital heart defects.
Plasma lactate level is an indicator for the normal metabolism of the intestinal flora. Progressive ischemic injury to the mesentery with mucosal damage and the changes in the homeostasis of the intestinal flora lead to bacterial translocation and may cause an increase in the serum lactate levels.
Prognostic value of intestinal fatty acid binding protein, L-lactate and D-dimer levels, and their combination in the early stage of acute mesenteric ischemia confirmed with histological outcomes: an experimental study.
The incidence of surgical necrotizing enterocolitis (NEC) has not decreased significantly over the past decade. Pneumoperitoneum and clinical deterioration (despite maximal medical therapy) are the most common indications for operative treatment.
Therefore, the current study aimed to evaluate the role of I-FABPp and I-FABPu as biomarkers for early diagnosis of necrotizing enterocolitis in neonates, in addition to serum lactate levels and ultrasonographic findings. Also, the study aimed to compare and correlate the plasma and urinary I-FABP2 levels with various ultrasonographic findings among neonates with NEC.
2. Patients and methods
2.1 Study design and participants
The current prospective cohort study has been conducted with 55 neonates (preterm or full-term) of both sexes with various NEC stages admitted to the Neonatal Intensive Care Unit, Pediatrics Department, South Valley University Hospitals, Qena, Egypt, in addition to 23 healthy neonates attending the NICU for routine clinical follow-up, age- and sex-matched with included cases, who were selected as a control group. The study was approved by the Ethics committee of the Faculty of Medicine, South Valley University, Qena, Egypt, and it was conducted in accordance with the Declaration of Helsinki. Informed written consent has been obtained from the parents of every included neonate. The study duration was one year from January 1st, 2018 to December 30th, 2018.
2.2 Patients' selection criteria
Neonates with NEC were categorized according to Modified Bell's system which was first proposed by Bell et al. as stage I (suspected), II (proven) or III (advanced),
In this modified scheme, stages are further subdivided into A or B depending on the radiographic findings: stage IA or B (normal or intestinal dilation), stage IIA (ileus, pneumatosis intestinalis) or IIB (portal venous gas), stage IIIA (ascites) or IIIB (pneumoperitoneum).
Neonates who presented with pneumoperitoneum by x-ray as in Bell's scoring IIIB or who had any pervious GIT operation or proved to have another diagnosis. Those who had incomplete data, or with congenital anomalies or metabolic error disorders, or neonates whose parents refused to participate in the study, were excluded from the study.
2.3 Clinical evaluation of the included neonates
Detailed medical history was taken focusing on the following: postnatal age of the patients, gestational age, age of onset of the symptoms, obstetric history as pre-eclampsia, DM and chorioamnionitis, and time and type of first feeding. Full clinical examination was done for the included patients with special concern with the abdominal examination. The examination included general examination, anthropometric measurements (weight, length, head circumference), Down score, and cardiac, chest, and neurological examination.
2.4 Investigatory battery
2.4.1 Imaging tools
Plain x-ray abdomen AP view in the supine position, using Philips medical systems (D-22335, Hamburg, Germany) was used for all included patients on admission to assess the presence of abnormal bowel dilatation, intramural gas, portal venous gas, separation of bowel loops and fixed appearance of bowel loops on serial films.
Abdominal ultrasonography including gray scale and color Doppler examination using GE LOGIQ P6 ultrasound machine, USA; using convex and linear transducers (3.4 and 11.8 MHz) was administered to all study patients. Gray-scale abdominal US was used to assess the presence of peritoneal fluids, peritoneal air, portal venous gas, bowel wall thickening (2.7 mm or greater), bowel wall thinning (1.0 or lesser), increased bowel wall echogenicity, peristalsis, and pneumatosis intestinalis (echogenic spots). Color Doppler US examination was done to evaluate the perfusion state of the intestinal wall and mesenteric vessels, indicating early increased vascularity (hyperperfusion) giving (Y) shaped pattern and late hypoperfusion of the necrotic bowel loops or even complete loss of perfusion, Fig. 1.
The final decision regarding the presence or absence of intestinal necrosis was based on the US and Doppler findings (portal venous gas, ascites, hypoperfusion) of which the surgeon was aware when making decisions on the possible need for surgery. Neonates having necrotic intestinal segment upon laparotomy underwent resection and diversion.
2.4.2 Laboratory workup
In addition to the routine laboratory complete blood counts (using Cell Dyn 1800-Abbott diagnostics, Germany), C-reactive protein (using the semi-quantitative latex agglutination test, AVITEX CRP kits; Catalog No. OD023; supplied by Omega Diagnostics, UK) and serum electrolytes (sodium, potassium and ionized calcium, using Dimension® EXL™ 200 Integrated Chemistry System, Germany), 4-ml venous blood samples and 2 ml of urine were collected from every included neonate at time of diagnosis of suspected or definite NEC stage after clinical and imaging evaluation. Two ml of blood samples were placed on fluorinated tubes and the remaining 2 ml were evacuated into EDTA containing tubes; both samples were then centrifuged at 3500 rpm for 10 min, and the separated plasma was transferred into 1 ml cryotubes and stored at −80 °C for later biochemical analysis. Urine samples were collected using urine collecting bags, evacuated into 1 ml cryotubes and stored at −80 °C for later biochemical analysis of I-FABPu which was expressed as ratio to urinary creatinine (ng/g creat.). At the time of assay of I-FABPu, urine samples were incubated at 37 °C for 15 min and were then centrifuged at 3500 rpm for 5 min and supernatants were used for the biochemical assays.
Biochemical assays included: A) plasma lactate level, which was measured using commercially available colorimetric assay kit supplied by Spectrum Diagnostics, Cairo, Egypt, Catalog No: 274001, using spectrophotometer (Chem-7, Erba Diagnostics Mannheim GmbH, Germany); and plasma and urinary I-FABP levels, which were measured using commercially available ELISA assay kit supplied by Chongqing Biospes Co. Ltd, China with catalog number BYEK2440, using microplate ELISA reader (EMR-500, USA).
2.5 Statistical analysis
Data entry and data analysis were done using SPSS version 19 (Statistical Package for Social Science). Data were presented as a number, percentage, the mean and standard deviation for parametric data, and the median and inter-quartile range for non-parametric data. Chi-square test and Fisher exact test were used to compare qualitative variables. Mann–Whitney U test was used to compare between two quantitative variables and Kruskal-Wallis test was used to compare more than two quantitative variables for non-parametric data. Spearman correlation test was done to measure the correlation between quantitative variables in case of non-parametric data. Medcalc Program was used to calculate sensitivity, specificity, positive and negative predictive values. P-value was considered statistically significant when <0.05.
3.1 Baseline characteristics of the included neonates
The current study was conducted on 55 neonates with necrotizing enterocolitis (35 males and 20 females). Their mean post-natal age was 16.85 ± 6.34 days and their mean gestational age was 33.38 ± 3.39 weeks. For 23 healthy controls (15 males and 8 females) matched with the study patients for gestational age, post-natal age and sex, with mean gestational age was 35.30 ± 3.86 weeks and post-natal age was 14.30 ± 6.41 days. The included neonates with NEC were consecutive neonates with suspected NEC (55); after clinical and imaging investigations, 35 of them were proved definite NEC on admission, while the remaining 20 neonates recovered and did not progress into definite NEC.
3.2 Possible associated risk factors for NEC among the included cases
Among the neonates with NEC, male-to-female ratio was 1.75. Forty cases (72.7%) were preterm and 15 (27.3%) were full term. Forty-five cases (81.8%) were delivered by cesarean section and the remaining 18.2% (10 cases) were delivered via normal vaginal delivery. Birth anoxia was present in 5 cases (9.1%). Maternal history of pre-eclampsia was present in 15 cases (27.3%), diabetes mellitus in 11 cases (20%), with chorioamnionitis and irradiation in 6 cases (10.9%) each. Regarding the feeding type, breast feeding was present in 9.1% (5 neonates), while 31 neonates (56.4%) were artificially fed and the remaining 19 neonates (34.5%) had history of mixed feeding (both breast and artificial milk).
3.3 Clinical and imaging data of the included neonates with NEC
According to the Modified Bell's system, the study neonates with NEC were classified into the following subgroups; stage Ia, Ib, IIa, IIb and IIIa (each included 10 cases except for stage IIb with 15 patients).
All cases (100%) exhibited signs of feeding intolerance in the form of vomiting, gastric residuals and abdominal distension. Eight patients (14.5%) had audible intestinal sounds, 18 patients (32.7%) had abdominal tenderness and 8 patients (14.5%) had abdominal cellulitis. Constipation was present in 39 cases (70.9%). Twenty-five (45.4%) cases had lethargy and weak reflexes. The study cases showed significantly lower mean ± SD diastolic blood pressure and significantly higher Down score (36.24 mmHg ±4.82,2.24 ± 0.98 respectively) in comparison to the controls (41.30 mmHg ±5.19, 0.70 ± 0.67, respectively), (p ˂0.05 for both).
As regards the ultrasonographic and Doppler findings of the study cases, intestinal wall thickening was present in all cases (100%), intramural gas was frequent in 27 cases (49.1%), 10 cases (18.2%) had good bowel perfusion and 15cases (27.3%) revealed presence of peristalsis. Portal venous gas was detected in 19 cases (34.5%) and ascites was present in 16 cases (29.1%) (Fig. 1).
3.4 Routine laboratory investigations of study groups
There were statistically significant lower total calcium, hemoglobin levels and platelet counts (9.00 mg/dl ± 1.22, 13.73 g/dl ± 2.60 and 174.04 ˟103/μl ± 141.81, respectively) with significantly higher white blood cell counts (10.01 ˟103/μl ± 3.61) among cases when compared with the controls (10.14 mg/dl ± 0.82, 16.20 g/dl ± 1.48, 429.40 ˟103/μl ± 76.67 and 6.90˟103/μl ± 2.23, respectively), with p <0.05 for all. CRP was positive in 25.5% (14) of cases.
3.5 Plasma lactate, I-FABPp and I-FABPu levels among the studied groups
There were significantly higher median levels of I-FABPp, I-FABPu and lactate among cases (2.84 ng/ml, 1.74 ng/g creat. and 32.34 mg/dl, respectively) when compared with the controls (0.16 ng/ml, 0.60 ng/g creat. and 15.33 mg/dl, respectively) with p ˂0.05 for all (Table 1).
Table 1Comparison of the plasma I-FABP, urinary I-FABP and blood lactate levels among the included cases versus controls.
In comparison of the plasma I-FABP, urinary I-FABP and blood lactate levels among the study neonates with necrotizing enterocolitis, increased with the progress of the NEC stages in a statistically significant manner with the lowest median levels at stage IA and the highest median levels at stage III-A, with p <0.05 for all (Table 2).
Table 2Comparison of the plasma I-FABP, urinary I-FABP and blood lactate levels among the included neonates with necrotizing enterocolitis according to Modified Bell's system stages and the control group.
Neonates with NEC n = 55
Controls n = 23
Stage I-A n = 10
Stage I–B n = 10
Stage II-A n = 10
Stage II-B n = 15
Stage III-A n = 10
I-FABPu (ng/g creat)
N.B: P value > 0.05 insignificant, ∗P < 0.05 significant. I-FABPp: plasma intestinal fatty acid binding protein; I-FABPu: urinary intestinal fatty acid binding protein. Kruskal-Wallis test was used.
Additionally, there were significantly positive correlations between I-FABPp and I-FABPu(r = 0.913, p = 0.000), I-FABPp and lactate (r = 0.611, p = 0.000) and between I-FABPu and lactate (r 0.682, p = 0.000) (Table 3). Non-significant correlations could be found between both I-FABPp and I-FABPu with gestational age, birth body weight or weight at onset of symptoms among the study cases, p <0.05 for all (Table 4).
Table 3Correlations between I-FABPp, I-FABPu and plasma lactate levels among the included neonates with necrotizing enterocolitis.
There were non-significant differences in the median values of I-FABPp and I-FABPu levels among the study cases with positive CRP vs. those who were negative for CRP, with p <0.05 for all, Table .5. The median levels of I-FABPp and I-FABPu were significantly higher among cases with positive ultrasonographic findings (portal venous gas 6.50 ng/ml and 2.93 ng/g creat., respectively) and ascites (4.50 ng/ml and 3.63 ng/g creat., respectively) when compared with cases who did not exhibit positive ultrasonographic findings (portal venous gas 2.90 ng/ml and 1.74 ng/g creat., respectively) and ascites (3.10 ng/ml and 2.93 ng/g creat., respectively), with p ˂0.05 for all, with non-significant differences as regards plasma lactate levels with p ˃0.05 for all (Table 6).
Table 5Comparison of I-FABPp and I-FABPp levels among the included neonates with necrotizing enterocolitis in terms of CRP.
Neonates with NEC (n = 55)
Negative CRP (n = 41)
Positive CRP (n = 14)
N.B: P value > 0.05 insignificant, I-FABPp: plasma intestinal fatty acid binding protein; I-FABPu: urinary intestinal fatty acid binding protein.
As regards the performance characteristics of I-FABPp and I-FABPu in discriminating stage IIIA from stage II, I-FABPp at cut-off point >3.24 ng/ml showed 90% sensitivity, 72% specificity, positive predictive value (PPV) = 52.6%, negative predictive value (NPP) = 94.7% with AUC = 0.768, while for I-FABPu, performance characteristics at cut-off point> 2.93 ng/g creat. were 90%, 92%, 81.8 and 95.8, respectively with AUC = 0.864 (Fig. 2).Regarding the performance characteristics of I-FABPp and I-FABPu in predicting NEC with impending perforation (negative outcome), I-FABPp at cut-off point 6.95 ng/ml revealed 75% sensitivity, 100% specificity, PPV = 100%, NPP = 95% with AUC = 0.881, while for I-FABPu at cut-off point>4.13 ng/g creat., results were 100%, 76.19%, 44.4% and 100 %with AUC = 0.821 (Fig. 2).
Eight cases were surgically explored who exhibited positive ultrasonographic findings in addition to the clinical judgment; 2 cases were stage II-B (one of them revealed full bowel wall necrotic intestinal segment without actual perforation, i.e. impending perforation); and 6 cases were stage–III–A (five of them confirmed to have impending intestinal perforation).There were higher ranges for both I-FABPp and I-FABPu levels among NEC with intestinal necrosis after exploration (5.71–8.22 ng/ml and 4.41–6.72 ng/g creat., respectively) than NEC without intestinal necrosis after exploration (1.23–4.51 ng/ml and 0.81–5.12 ng/g creat., respectively).
Necrotizing enterocolitis (NEC) is a common gastrointestinal disorder of newborns with high morbidity and mortality.
Abdominal ultrasound is more sensitive for NEC diagnosis than conventional X-ray in identification of free fluid in the abdominal cavity and in determination of the thickness and the perfusion of the intestinal loops.
investigated the role and accuracy of abdominal US in diagnosing NEC and disease severity, they reported that abdominal US showed significantly higher detection rates of portal venous gas and dilatation of the intestine than plain abdominal X-ray film, and the surgery/death group had significantly higher detection rates of dilatation of intestine, bowel wall thickening, peritoneal effusion and free intraperitoneal air compared with the medically treated group.
Plasma lactate and intestinal IFABP are two surrogate markers of evaluating intestinal mucosal integrity and barrier function, especially in cases with intestinal ischemia.
Acute intestinal ischemia, reperfusion, and bacterial colonization were associated with failure of the mucosal barrier, increasing the permeability of the intestinal wall and allowing lactate to enter the portal circulation with increased plasma lactate levels in both portal and systemic blood.
examined levels of plasma lactate in premature infants affected by NEC, finding them to increase significantly early in direct proportion to the overall extent of intestinal disease, but sensitivity and specificity were not reported. In the current study, plasma lactate was found to be elevated among the included neonates with NEC when compared with the controls.
I-FABP constitutes up to 2% of the cytoplasmic protein content of the mature enterocyte.
As a consequence, plasma and urinary i-FABP levels may reflect the extent of intestinal epithelial cell damage. Therefore, the plasma I-FABP (I-FABPp) and urinary I-FABP (I-FABPu) are promising biomarkers in early diagnosis of NEC.
We also noticed that I-FABPp and I-FABPu levels increased according to Bell's staging at the time of diagnosis of NEC. These data were in accordance with other studies.26,29Also, there were significant positive correlations between IFABPp and IFABPu and between lactate with both IFABPp and IFABPu. In agreement with our findings, Schurink et al.
emphasized the importance of such biomarkers as indicators of enterocyte damage and compromised intestinal barrier function (increased permeability) that were induced by ischemia and hypoperfusion.
Radiological findings were the most powerful predictors of the need for surgical intervention including the following; persistent dilation of the bowel loops, evidence of portal venous gas, bowel wall thickening, absent peristalsis and echogenic-free fluid or focal fluid collection. Many authors stated that air in the portal system is one of the indicators of surgical necessity as this is associated with the degree of intestinal necrosis.
Our findings revealed significantly higher I-FABPp and I-FABPu among neonates with NEC who exhibited positive sonographic findings (portal venous gas and/or ascites) than those who did not, indicating the informative value of such biomarkers regarding diagnosis and severity of NEC.
Various studies revealed that the levels of IFABP were a helpful marker of evaluating intestinal necrosis.
We plotted a ROC curve for both serum and urinary I-FABP as a marker for diagnosis of NEC, discriminating stage- II from stage-III with equal sensitivity (90% for each), but with higher specificity for I-FABPu (92%) than I-FABPp. The characteristic performances of both assays in the prediction of surgical NEC revealed that I-FABPp (100%) was more specific than I-FABPu (76.19%). Several studies found plasmatic concentrations of I-FABP to be a specific marker for early identification of severe NEC (Bell's stage III), they were less useful for differentiating initial Bell's stages.
demonstrated that both plasma and urine I-FABP levels were strongly associated with the length of bowel resection in newborns with surgical NEC, supporting the hypothesis that increased I-FABP levels correspond with the extent of necrotic tissue. Additionally, Evennett et al.
concluded that I-FABPu concentration could predict the extent of disease in necrotizing enterocolitis.
The current study provides additional evidence regarding the utility of serum and urinary I-FABP levels, when combined with clinical and imaging data, as a useful informative marker for early diagnosis and prediction of disease severity in NEC and in early recognition of impending perforation, thus decreasing the mortality associated with perforated NEC among neonates.
6. Study limitations
Its small sample size and lack of serial measurements of the studied biomarkers among the included cases were the main study limitations.
The current research has been funded by the authors themselves.
Declaration of Competing Interest
No potential conflict of interest was reported by the authors.
Prognostic value of intestinal fatty acid binding protein, L-lactate and D-dimer levels, and their combination in the early stage of acute mesenteric ischemia confirmed with histological outcomes: an experimental study.