Clinical trial NCT05335577: Role of Urinary Claudin-2, Caveolin-1, and EGF as Diagnostic Biomarkers of Necrotizing Enterocolitis in Preterm Neonates
Official title: Concentrations of Urinary Claudin-2, Caveolin-1, and Epidermal Growth Factor (EGF) as the Prototypes of Non-Invasive Biomarkers in the Diagnosis of Necrotizing Enterocolitis in Preterm Neonates
Study type: OBSERVATIONAL
Overall status: UNKNOWN
Conditions: Enterocolitis, Necrotizing
Infant, Premature
Keywords: Necrotizing enterocolitis
Preterm neonates
Claudin-2
Caveolin-1
Epidermal growth factor
Bell's Criteria
Lead sponsor: University of Brawijaya
Sponsor class: OTHER
First posted: 2022-04-19
Enrollment: 30
Sex: ALL
Minimum age (years): 1
Maximum age (years): 28
Standard ages: CHILD
Accepts healthy volunteers: yes
Observational model: COHORT
Time perspective: PROSPECTIVE
Sampling method: NON_PROBABILITY_SAMPLE
Study population: The target population of this study are neonates that would be categorized into three groups, i.e. preterm neonates with NEC, without NEC, and healthy term infants. The subjects are recruited from Perinatology Unit, Saiful Anwar General Hospital from March-May 2022.

Brief summary: Our study aims to determine the differences in the concentration of urinary claudin-2, caveolin-1, and epidermal growth factor (EGF) as non-invasive biomarkers in the diagnosis of Necrotizing Enterocolitis (NEC). We compare the concentration of urinary claudin-2, caveolin-1, and EGF between preterm neonates at risk of NEC and healthy term infants as the basis for determining NEC biomarkers with the most optimum sensitivity and specificity. This analytical observational study is based on biomolecular profiling with a prospective cohort design approach. The research subjects are a group of preterm neonates (gestational age of 28-34 weeks) who were admitted in Perinatology Unit, Department of Pediatrics, Saiful Anwar General Hospital, Malang and whom diagnosed with NEC using Bell's criteria and serum TGF-β levels. Subjects are selected by consecutive sampling and single-blind analysis was performed in the Laboratory of Bioscience and Biomedicine, Faculty of Medicine, University of Brawijaya.

During the research process, groups of preterm and term neonates would be observed and their clinical development followed. The collection of biologic samples would be taking 10 cc of urine and 40 mg of feces on day-5 (D5) and 7 (D7). The consecutive manner of urinary sampling was regarded to assess whether there was a time-related protein expression in the course of the NEC process. Faecal samples would be assessed for microbiota profile analysis described by the ratio of Proteobacteria: Firmicutes and Bacteroidetes to represent dysbiosis process in NEC. After 7 days, the subjects would be grouped into a group of preterm neonates with NEC, a group of healthy term neonates as a control, while a group of preterm infants at whom during the course of the study did not develop NEC, would be assigned to group of premature neonates without NEC.

Urinary protein concentrations from the three groups would then be analyzed and adjusted with normalized creatinine, so that the levels of these three proteins could be assessed quantitatively using the ELISA (Enzyme-Linked Immunosorbent Assay) method. The results would be compared with the microbiota profile as the golden standard for NEC cases. Through statistical tests, sensitivity, specificity and cut-off of selected protein levels would be assessed as diagnostic biomarkers of NEC.

Detailed description: Necrotizing enterocolitis (NEC) is a multifactorial syndrome of acute intestinal ischemic necrosis which is one of the acute intestinal emergencies in neonates with high morbidity and mortality rates were higher in preterm. Inflammation and ischemia are the main pathogenesis of NEC. The diagnosis of NEC is established based on the demographic condition and clinical presentations, and further confirmed by the presence of pneumatosis intestinalis on plain abdominal radiographs. However, the clinical symptoms of NEC were often overlapped with other cases, especially diseases related to inflammation and sepsis, and the limitations of diagnostic methods both clinically and radiologically in identifying the early phase of NEC, make biomarker studies continue to be carried out to find diagnostic methods that would be able to predict, diagnose and differentiate NEC from non-NEC cases in a timely manner. Thus, a diagnostic method with high sensitivity and specificity is needed in cases of NEC in premature infants.

Several studies have shown that the immaturity of the gastrointestinal tract is related to the condition of the gastrointestinal cell barrier in infants who are susceptible to disintegration. The disruption in the expression of various proteins that make up the gastrointestinal tight junction in NEC, makes the tight junction protein component suspected to be a potential biomarker in the early phase of NEC. This is closely related to the presence of the inflammatory process of the NEC. Inflammatory conditions disrupt intestinal microcirculation, which results in the emergence of intestinal ischemia, and trigger the degradation of the tight junction components of the gastrointestinal tract through urine. This degradation process occurs in the early phase of the NEC disease course.

Several animal studies have shown that there is a correlation between abnormal expression of the proteins claudin-2, caveolin-1, and EGF and tight junction damage in NEC. Meanwhile, studies with a small number of neonates showed that the protein component is expressed in urine as a biomarker of tight junction damage in NEC and is independent of other inflammatory components, so that it is considered as a potential diagnostic biomarker in NEC cases.

This study aims to determine the differences in the expression of urinary claudin-2, caveolin-1, and EGF with NEC in preterm neonates at risk of NEC compared to healthy term infants as a basis for determining NEC biomarkers with optimum sensitivity and specificity, which then would be used as biomarker. The study was conducted using a cohort prospective method. The research subjects are a group of preterm neonates (gestational age of 28-34 weeks) who were admitted in Perinatology Unit, Department of Pediatrics, Saiful Anwar General Hospital, Malang and whom diagnosed with NEC using Bell's criteria and serum TGF-β levels obtained at Day-1 (D1). Subjects are selected by consecutive sampling and single-blind analysis was performed in the Laboratory of Bioscience and Biomedicine, Faculty of Medicine, University of Brawijaya.

During the research process, groups of preterm and term neonates would be observed and their clinical development followed. The collection of biologic samples would be taking 10 cc of urine and 40 mg of feces on day-5 (D5) and 7 (D7). The consecutive manner of urinary sampling was regarded to assess whether there was a time-related protein expression in the course of the NEC process. Faecal samples would be assessed for microbiota profile analysis described by the ratio of Proteobacteria: Firmicutes and Bacteroidetes to represent dysbiosis process in NEC. After 7 days, the subjects would be grouped into a group of preterm neonates with NEC, a group of healthy term neonates as a control, while a group of preterm infants at whom during the course of the study did not develop NEC, would be assigned to group of premature neonates without NEC.

Urinary protein concentrations from the three groups would then be analyzed and adjusted with normalized creatinine, so that the levels of these three proteins could be assessed quantitatively using the ELISA (Enzyme-Linked Immunosorbent Assay) method. The results would be compared with the microbiota profile as the golden standard for NEC cases. Through statistical tests, sensitivity, specificity and cut-off of selected protein levels would be assessed as prototype of diagnostic biomarkers in establishing NEC.

Sample size in this study was calculated by ANOVA repeated measure statistical analysis with a significance level of 0.05 and study power of 0.8. From the calculation, the subject needed is 10 patients for each group (total 30 subjects). The basic characteristics of the patient will be analyzed descriptively. All parameter data will be analyzed statistically by Statistical Product and Service Solution (SPSS) 20, preceded by normality test and homogeneity test. The study analysis will be sensitivity, specificity and cut-off of selected protein levels would be assessed as prototype of diagnostic biomarkers in establishing NEC. Further bivariate and multivariate analysis would be performed. Data transformation will be performed if needed in case of outlier data or out-of-range results.

Eligibility criteria: Inclusion Criteria of Subjects:

* Premature neonates with 28-34 weeks' gestational age
* Admitted in Perinatology Unit, Saiful Anwar General Hospital, Malang
* Parents/guardians have agreed and signed the informed consent of the study
* Neonates receive nutrition from breast milk or breast milk predominance
* NEC was diagnosed using Bell's modification criteria.

Inclusion Criteria of Control:

* Premature neonates with 28-34 weeks' gestational age, admitted in Perinatology Unit, Saiful Anwar General Hospital, Malang. Parents/guardians have agreed and signed the informed consent of the study. Neonates receive nutrition from breast milk or breast milk predominance.
* Term neonates with 37-42 weeks' gestational age, admitted in Perinatology Unit, Saiful Anwar General Hospital, Malang. Parents/guardians have agreed and signed the informed consent of the study. Neonates receive nutrition from breast milk or breast milk predominance.

Exclusion Criteria:

* Treated neonates who died during the study before the diagnosis of NEC was established
* Neonates whom require surgery during the study
* Parents/guardians stated that they were not willing to participate in the study.

Primary outcomes:
measure: Change of Urinary Claudin-2 Concentration
timeFrame: Day-5 and Day-7 of age
measure: Change of Urinary Caveolin-1 Concentration
timeFrame: Day-5 and Day-7 of age
measure: Change of Urinary EGF Concentration
timeFrame: Day-5 and Day-7 of age
measure: Bell's stage of NEC
timeFrame: Day-1 (Baseline)
Secondary outcomes:
measure: Change of fecal microbiota profile
timeFrame: Day-5 and Day-7 of age
measure: Change of Urinary Normalized Creatinine Level
timeFrame: Day-5 and Day-7 of age
measure: Serum TGF-β Concentration
timeFrame: Day-1 (Baseline)
Other outcomes:
Locations:
city: Malang
country: Indonesia
facility: Saiful Anwar General Hospital
state: East Java
Study officials:
affiliation: Saiful Anwar General Hospital
name: Brigitta IRV Corebime, M.D.(Paed)
role: PRINCIPAL_INVESTIGATOR
References: