The present study was a cross-sectional study that was carried out over a period of six months at the PICU of Cairo University Specialized Children Hospital, Egypt. The study protocol was approved by our Institutional Review Board and Ethical Committee.
We included children who fulfilled the following criteria: children aged from 1 month to 12 years with strong clinical diagnosis of septic shock at time of admission. We defined pediatric septic shock based on the American college of critical care medicine and international pediatric Society of Critical Care definitions for sepsis.[10] We excluded patients with known congenital heart disease or cardiomyopathy, or whose duration of stay in PICU was less than 48 hours. Children with known metabolic disorders or adrenal insufficiency were also excluded.
The Electrical Cardiometery (EC, manufactured by Oskypa Medical Data) parameters were measured in the emergency department after fluid boluses. Cold septic shock was defined as cardiac index <3.3 L/min/m2 and systemic vascular resistance index>1600 (dyne s/cm5/m²) while warm septic shock was defined as cardiac index >5.5 L/min/m2 and systemic vascular resistance index < 800 (dyne s/cm5/ m²).[11]
Sepsis profile was withdrawn before antibiotics administration in the hospital, including: complete blood count, C-reactive protein and blood culture (BACTEC Plus aerobic/F and BACTEC Plus anaerobic/F blood culture bottles; Becton, Dickinson and Company, Spain). Subcultures were done on blood, MacConkey’s and chocolate agars incubated in 5 % CO2 at 35 °C for 48 hours and on Sabouraud’s dextrose agar incubated at 25 °C for up to 14 days and examined every 48 hours for any growth. Further growth identification was done by Vitek 2 compact system (Biomerieux, France). Additionally, a broad-range bacterial and fungal PCR amplification for all culture negative samples was done [12, 13]
Amplified DNA fragments were separated by agarose gel electrophoresis and stained with ethidium bromide and visualized under UV light, using pan bacterial 536f 5′CAGCAGCCGCGGTAATAC[14] and the pan fungal ITS–1 5′TCCGTAGGTGAACCTGCGGG. The molecular sizes of fragments generated by electrophoresis were judged by comparison with the molecular weight standards as previously described [15]. An extracted DNA of E coli strain was used as a positive control, whereas the negative control to detect reagent contamination was included in each PCR reaction, containing all components except the DNA extract, which was replaced by 2 µl of sterile distilled H2O.
Sequencing analysis
Culture negative samples were negative for ITS–1 pan fungal gene, whereas positive for pan bacterial 16srRNA gene and were subjected to sequencing. All procedures for sequencing were conducted by Macrogen (Seoul, South Korea) using ABI PRISM BigDye Terminator Cycle Sequencing Kit with AmpliTaq DNA polymerase (FS enzyme; Applied Biosystems) according to manufacturer instruction. All fluorescent-labeled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. The samples were resuspended in distilled water and subjected to electrophoresis in an ABI 3730x1 sequencer (Applied Biosystem, USA). Nucleotide sequence similarities were determined using other known sequences found in the GenBank database using BLAST program of National Center for Biotechnology Information (NCBI) databases (http://www.ncbi.nlm.nih.gov/blast).
Statistical Analysis:
An Excel spreadsheet was established for the entry of data. We used validation checks on numerical variables and option-based data entry method for categorical variables to reduce potential errors. The analyses were carried with SPSS software (Statistical Package for the Social Sciences, version 24, SSPS Inc, Chicago, IL, USA). Frequency tables with percentages were used for categorical variables and descriptive statistics (median and interquartile range [IQR]) were used for numerical variables. Independent Student t-test, paired t-test, or Mann-Whitney tests were used to compare quantitative variables, while Chi-square test or McNemar-Bowker tests were used to analyze categorical variables. A p-value < 0.05 is considered statistically significant.