Circulating Mitochondrial and Bacterial DNA as Biomarkers of Sepsis

Abstract

Introduction: Sepsis is a leading cause of death in critically ill patients. Conventional methods of diagnostics in patients with suspected sepsis have limited sensitivity and long turnaround times. These factors contribute to indiscriminate use of broad-spectrum antibiotics and antimicrobial resistance. Our goal was to investigate where direct molecular analysis of circulating cell-free DNA in plasma could identify the microbial etiology of sepsis and improve assessment of prognosis. Methods: We conducted a prospective study of 30 consecutive patients suspected of sepsis in the Surgical Trauma ICU and compared with 22 healthy volunteer controls. Longitudinal plasma samples were collected at the time of workup for sepsis (day 0) and on days 7 and 14. Blood samples were collected in Streck Cell-Free DNA tubes and processed within 24 hours. DNA was extracted using QIAamp Circulating Nucleic Acid Kit. We measured mtDNA levels in plasma using real-time quantitative PCR targeting the mitochondrial NADH1 gene. Absolute mtDNA copies were calculated by comparing with known standards, pre-quantified using droplet digital PCR. Whole genome sequencing of cell-free DNA from plasma samples of three patients with positive blood cultures and 1 healthy volunteer control were performed to detect pathogen DNA. Results: We analyzed 72 serial plasma samples from 30 patients with suspected sepsis. Median mtDNA levels in controls were 602 ± 636 copies/µL of plasma (median ± IQR). In comparison, median mtDNA levels at day 0 in patients with SIRS were 3318 ± 1960 copies/µL (p<10⁻⁸, area under the ROC curve: 0.939). mtDNA levels were correlated with peripheral WBC count and respiratory rate (p=0.026 and 0.013 respectively) but not with temperature, heart rate or systolic BP. 3/30 patients died within the same hospital stay and last recorded mtDNA levels were higher as compared to survivors (p=0.028). Three out of thirty patients with a diagnosis of sepsis had positive blood cultures. Concordant results were found between conventional microbiology and next-generation sequencing of cell-free plasma DNA in 1 patient. Conclusions: Circulating mtDNA levels in patients with suspected sepsis are five-fold higher than healthy controls. Longitudinal changes in mtDNA are correlated with conventional markers of systemic inflammatory response and can be a biomarker for outcomes. Sequencing of cell-free DNA in plasma of sepsis patients can enable identification of bacteria and viral pathogens, although additional optimization of laboratory and informatics protocols is needed.