This was an interventional clinical trial conducted on 55 preterm neonates (< 36 weeks’ gestation) admitted to the neonatal intensive care units (NICUs) of University Hospitals during the period from November 1, 2015 till July 31, 2017.
Patients
Forty preterm infants were diagnosed as having neonatal sepsis based on clinical signs and laboratory indicators including Rodwell’s hematological score ≥ 3 [9]. Fifteen healthy, growing, gender, and gestational age-matched preterm infants were studied as control after ruling out sepsis. These were included for the assessment of basal MDA levels in the healthy preterm.
A full history was taken for all neonates including maternal, obstetric, and perinatal history. Gestational age (GA) was calculated based on the date of the last menstrual period and confirmed by using the modified Ballard score [10].
Mode of delivery and Apgar score at 1 and 5 min were recorded. Birth anthropometric parameters were measured. All neonates received routine neonatal care according to our NICU protocol.
Preterm infants were excluded from the study if they had congenital anomalies, evidence of perinatal asphyxia, contraindication of enteral feeding, and/or oxygen needs with a fraction of inspired oxygen above 30%, either on invasive or non-invasive mechanical ventilation.
The study design was a parallel trial with an allocation ratio of 1:1. Simple randomization of septic neonates (n = 40) was done using odd- and even-numbered prewritten cards according to sequence of enrollment. We assigned odd numbers to melatonin treatment (MT group; n = 20); combined with conventional therapy of sepsis, and even numbers to lone conventional therapy (CT group; n = 20).
Intervention
Melatonin 10 mg rapid release capsules (Puritans pride®) were used. MT group was given melatonin at a total dose of 20 mg dissolved in 4 ml of distilled water via the enteral route in two doses of 10 mg (2 ml) each, with a 1-h interval
Laboratory investigations were done at the time of diagnosis of sepsis in patients and on day 1 in the control group and was repeated in patients on day 3. These included C-reactive protein (CRP) assay by latex agglutination test and complete blood count using Sysmex XT-1800i (Sysmex, Kobe, Japan).
Venous samples were withdrawn in gel tubes and were allowed to clot for 30 min before centrifugation at 2000×g for 15 minutes at 4 °C.
MDA detected by Colorimetric method using Lipid Peroxide (Malondialdehyde) Assay Kit supplied by BIODIAGNOSTIC® (cat no. MD 25 28). Serum MDA was reassessed in MT group 4 and 72 h after melatonin administration and in CT group, 72 h after the initial sample. Serum MDA concentrations were measured with an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s protocol (Biodiagnostics).
Blood cultures were done using BD BACTEC PEDS PLUS/F culture vials (Becton, Dickinson and Company Spark, Ireland).
Sample size calculation
Assuming an effect size for melatonin on CRP levels of one (1) a sample size of 20 would be enough to detect such effect if true at 0.05 alpha error and 0.8 power of the test.
Statistical methods
Data were collected, revised, coded, and entered into the Statistical Package for Social Science (IBM SPSS), version 23. Parametric quantitative data were presented as mean, standard deviation and range, while non-parametric data were presented as median and inter-quartile ranges (IQR). Qualitative variables were presented as numbers and percentages.
We used chi-square test and/or Fisher exact test for comparison of qualitative data within groups.
Independent t test was used to compare parametric quantitative data and Mann-Whitney for non-parametric data
The comparison between two paired groups with quantitative data and parametric distribution was done by using paired t test while data with non-parametric distribution were done by using Wilcoxon rank test. Spearman correlation coefficients were used to assess the correlation between two quantitative parameters in the same group.
The confidence interval was set to 95% and the margin of error accepted was set to 5%. So, the p value was considered significant if < 0.05 and highly significant if < 0.01.