Study design, setting, and population
This observational study was conducted in the ED of Shanghai South Campus, Renji Hospital, which is an urban university tertiary-care hospital with approximately120,000 ED visitors per year. From June 2018 to January 2019, 116 consecutive non-traumatic patients who fulfilled the sepsis-3 criteria defined by the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) were enrolled in the study. The exclusion criteria were as follows: younger than 18 years old, terminal stage of disease (malignant cancer of any type), end-stage renal disease, and patients who declined to participate in the study themselves or via their relatives. Ultimately, 107 consecutive patients were enrolled, and nine patients were excluded. All procedures performed in studies involving human participants were in accordance with the ethical standards of the Shanghai Jiaotong University School of Medicine, Renji Hospital Ethics Committee (NO.2016-109k) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards . All of the patients provided written informed consent.
Definitions and Grouping
The patients were divided into a sepsis group and a septic shock group according to the sepsis-3 criteria. That is, sepsis was clinically identified as an infection with a SOFA score ≥ 2, and patients with septic shock were clinically identified according to vasopressor requirement to maintain a mean arterial pressure of 65 mm Hg or greater and a serum lactate level >2 mmol/L (>18 mg/dL) in the absence of hypovolemia.
Septic cardiomyopathy (SCM) was defined as an acute syndrome of cardiac dysfunction that is unrelated to cardiac ischemia in patients with sepsis, B-type natriuretic peptide (BNP) and troponin elevations appear to reflect SCM.
Preparation and Quantification of Plasma DNA
Blood samples were drawn from patients in the two groups within 24 h after admission, transferred into ethylenediaminetetraacetic acid-coated blood collection tubes, and processed within 2h after venipuncture. The samples were left to rest for 30 min and then centrifuged immediately at 1914 ×g for 10 min to separate the plasma from the cellular components. The plasma samples were stored at −80°C for batch analysis.
DNA was isolated from the plasma using the QIAamp Blood Mini Kit (#51106; Qiagen GmbH, Hilden, Germany) according to the manufacturer’s manual. All samples were thawed on ice, and the level of the mtDNA gene cytochrome b was measured by a SYBR Green dye-based quantitative real-time polymerase chain reaction (qPCR) assay (TaKaRa, Japan) using an ABI Prism7900HT detection system with the following primers: forward, ATGACCCCAATACGCAAAAT; reverse, CGAAGTTTCATCATGCGGAG. The PCR mixture was set up in a reaction volume of 10 μL using 5 μL of 2× SYBR green Master Mix (2×), 0.5 μL forward primer (1 μM), 0.5 μL reverse primer (1 μM), 3 μL of nuclease-free H2O, and 1 μL of plasma extract. The following thermocycler conditions were used: 3-min incubation at 95°C, followed by 40 cycles of initial denaturation at 95°C for 30 s, annealing at 54°C for 45 s, and elongation at 68°C for 1 min. The concentration of mtDNA was determined using a standard curve generated by qPCR with construct plasmids (pMD® 18-T Vector; Sangon Biotech, Shanghai) containing the human mitochondrial cytochrome B gene sequences described above. Calibrators were prepared by serial 10-times dilution of the stock solution and contained 102 to 108 mtDNA copies/μL.
Clinical and laboratory data are expressed as number (percent) or median [interquartile range (IQR), i.e., 25th–75th percentile], as appropriate. Statistical calculations were performed with IBM SPSS software version 26.0 (SPSS Inc., Chicago, IL, USA). Bivariate comparisons were conducted with the Mann-Whitney U test for continuous variables, and with the chi-squared test or Fisher’s exact tests for categorical variables.Bonferroni adjustment was adopted to select variables(treatment process and sepsis indused organ injury werec excluded).The influence of clinical and baseline laboratory data, including respiratory tract infection(RSTI) ,coronary heart disease (CHD), in addition to the log mtDNA concentration and lactate levels（which adjusted P ＜0.002）, on 28-day mortality was evaluated in bivariate logistic regression models to determine independent predictors. Receiver operating characteristic (ROC) curve analyses were used to assess the predictive value of mtDNA on mortality by Sigma Plot 14.0, and cut-off values were calculated according to Youden’s index. The sensitivities and specificities were further used to calculate the positive and negative likelihood ratios. The Hosmer-Lemeshow goodness of fit test was used for verifying model calibration. All statistical tests were two-tailed, and P < 0.05 was considered to indicate a statistically significant difference.