# Untargeted 1H-NMR Urine Metabolomic Analysis of Preterm Infants with Neonatal Sepsis
## 2 Materials and Methods  
### Study Population  
The current study was conducted in the Neonatal Ward and the NICU of the General University Hospital, and the Department of Pharmacy at the University of Patras. Seventy-one (n = 71) neonates were recruited in the study, only after informed consent and parental permission having been provided. The neonates were separated into the following four groups: Group A: Septic neonates diagnosed with EOS (n = 23); Group B: Septic neonates diagnosed with LOS (n = 11); Group C: Preterm neonates without any clinical sign or symptom of sepsis or other serious morbidity (n = 14) hospitalized in the NICU; Group D: Healthy preterm neonates (n = 23) not separated from their mother after birth. All NICU neonates were premature, with GA ranging from 25 to 36 GA weeks, while all healthy non NICU preterm neonates ranged from 35 to 36 GA weeks. For each neonate, the following were recorded: (a) perinatal data such as gestational age (GA), birth weight (BW), sex, delivery mode, premature rupture of membranes and Apgar score; (b) clinical data such as mechanical ventilation, treatment with antibiotics, positive blood cultures, C reactive protein (CRP), and breast feeding. All clinical characteristics and laboratory findings of the neonates participated in this study are listed in Table 1. The biological fluid under study was urine and the samples were collected in the first 24 h after birth for EOS neonates and in the third day of the extrauterine life for LOS neonates. The collection was carried out with the use of adhesive pediatric urine collection bags and 1.5 mL of each collected urine sample was transferred to sterile vials and remained at \(-80\)\({}^{\circ}\)C until the analysis.  
\begin{table}
\begin{tabular}{c c c c c} \hline  & **Group A** & **Group B** & **Group C** & **Group D** \\  & **EOS** & **LOS** & **Control NICU** & **Control Non NICU** \\  & **(n = 23)** & **(n = 11)** & **(n = 14)** & **(n = 23)** \\ \hline Male sex (n, \%) & 8 (35) & 5 (45) & 9 (64) & 18 (75) \\ GA (weeks) & 34 (26–36) & 34 (25–36) & 35 (31–36) & 36 (35–36) \\ BW (gr) & 2150 (770–4060) & 1820 (690–2900) & 2085 (1630–3540) & 2740 (2100–3700) \\ Small for GA (n, \%) & 5 (22) & 4 (36) & 2 (14) & 0 (00) \\ Cesarian Section (n, \%) & 8 (73) & 8 (73) & 11 (79) & 13 (57) \\ Apgar Score 1 st min & 8 (3–9) & 8 (3–9) & 8 (7–9) & 9 (5–9) \\ Apgar Score 10 th min & 9 (8–10) & 9 (8–10) & 9.5 (8–10) & 9 (7–10) \\ Antibiotics (n, \%) & 23 (100) & 11 (100) & 1 (7) & 11 (48) \\ premature Rupture of Membranes \textgreater{}18 h (n, \%) & 1 (4.3) & 0 (0) & 3 (21.4) & 0 (0) \\ Mechanical Ventilation (n, \%) & 17 (71) & 7 (64) & 4 (14) & 0 (0) \\ Nutrition & No & No & No & Breast milk \\ Laboratory findings & B.C negative, CRP positive findings (n, \%) & 17 (74) & 5 (45) & 0 (0) & 0 (0) \\ B.C positive, gram (+) (n, \%) & 4 (17) & 5 (45) & 0 (0) & 0 (0) \\ B.C positive, gram ( –) (n, \%) & 1 (4) & 1 (10) & 0 (0) & 0 (0) \\ B.C positive, & 1 (4) & 0 (0) & 0 (0) & 0 (0) \\ Fungi (n, \%) & & & & \\ \hline \end{tabular}
\end{table}
Table 1: Clinical characteristics and laboratory findings for septic and control neonates. Median and (minimum-maximum) values of the GA, BW, Apgar Score for the first and tenth minute of life, the number (percentage) of the male sex, the delivery mode (cesarian section), the small for gestational age (SGA) infants, the newborns with premature rupture of membranes (>18 h), treatment with mechanical ventilation and/or antibiotics, blood culture positive (Gram-positive, Gram-negative and fungi) and c reactive protein (CRP).