Diagnostic and prognostic markers in sepsis

Sepsis is a life-threatening disease affecting millions of people globally. The more severe forms are considered to be a consequence of an unbalanced systemic inflammatory response to infection, causing organ dysfunction, vascular leakage and hypotension. An early diagnosis followed by appropriate antimicrobial therapy is critical for the outcome. Conversely, inappropriate antibiotic use will escalate antibiotic resistance. Therapeutic guidance from microbiological cultures is lacking in the early hospital course, and better tools are needed for prompt identification and severity stratification of sepsis patients. The aim of this thesis was to assess the clinical impact of severe sepsis and the diagnostic properties of clinical and biological markers in patients with a suspected or established serious infection. The diagnostic value of clinical and laboratory variables in predicting infections that require antibiotic treatment was evaluated in a prospective observational study of adult patients with suspected severe infections. We also analyzed the relations between severe sepsis, systemic inflammatory response syndrome (SIRS), and the clinical course. We concluded that increased C-reactive protein, white blood cell count, respiratory rate and a decreased hemoglobin level contributed independently to an accurate selection of patients for antibiotic therapy. Procalcitonin did not provide guidance on antibiotic decisions, but was associated with bacteremia and severe sepsis. In addition, severe sepsis was a common condition (42%), but mortality was low (5%), suggesting that severe sepsis is a more benign condition than earlier reported. SIRS did not exhibit discriminative ability in the classification of sepsis. We used the enzyme-linked immunospot (ELISpot) assay to study the spontaneous as well as the lipopolysaccharide (LPS)-induced secretion of a number of pro- and anti-inflammatory cytokines from leukocytes of septic patients and healthy controls. We concluded that circulating leukocytes did not appear to be the source of the increased plasma levels of cytokines observed in sepsis. A selective sepsis-induced downregulation of cytokine secretion in response to LPS was found: while the numbers of IL-6 and TNF-  secreting cells remained similar, significantly fewer IL-1 IL-10, IL-12p40 and GM-CSF secreting cells were seen in samples from septic patients as compared to healthy controls. The reduced number of cytokine secreting cells in response to LPS stimulation correlated with disease severity. LPS-induced cytokine secretion from polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) from healthy donors was analyzed by ELISpot. PMN were found to secrete the two chemokines IL-8 and MIP-1β in response to LPS. Also TNF was secreted but by significantly fewer cells. PBMC had a broader cytokine secreting repertoire and released considerably larger amounts of the investigated cytokines, with CD14+ monocytes being the primary source of production

Nuclease-assisted suppression of human DNA background in sepsis.

Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C0t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C0t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found

Schematic representation of the presented background suppression method.

<p>In septic blood, the amount of human DNA (black) exceeds pathogen DNA (green) amount (A). The extracted DNA is fragmented, denatured and re-hybridized, then dsDNA is degraded with nucleases (red) specific to double stranded DNA. Since rare DNA re-hybridizes slower, mostly abundant DNA will be degraded.</p

Standard curves and nuclease effect using amplicons to model excess human DNA.

<p>Human β-actin (a) and <i>E. coli</i> (b) primers provided the same R2 (>0.99) value and sensitivity (1–10 copies as the lower limit of detection) on amplicons in triplicate trials. ΔCt was 3.2 for human, 3.37 for <i>E. coli</i>. Error bars represent standard deviation.</p

Representative amplification curves of 10 experiments show background suppression on a septic blood sample, with blue lines representing the human DNA (β-actin), and pink lines show <i>E. coli</i> DNA amount.

<p>While the amount of human DNA has degraded, the <i>E. coli</i> DNA amount did not change.</p

Influenza-associated invasive aspergillosis in patients admitted to the intensive care unit in Sweden : a prospective multicentre cohort study

BACKGROUND: The purpose of this study was to prospectively investigate the incidence of influenza-associated pulmonary aspergillosis (IAPA) in influenza patients admitted to intensive care units in Sweden. METHODS: The study included consecutive adult patients with PCR-verified influenza A or B in 12 Swedish intensive care units (ICUs) over four influenza seasons (2019-2023). Patients were screened using serum galactomannan and β-d-glucan tests and fungal culture of a respiratory sample at inclusion and weekly during the ICU stay. Bronchoalveolar lavage was performed if clinically feasible. IAPA was classified according to recently proposed case definitions. RESULTS: The cohort included 55 patients; 42% were female, and the median age was 59 (IQR 48-71) years. All patients had at least one galactomannan test, β-d-glucan test and respiratory culture performed. Bronchoalveolar lavage was performed in 24 (44%) of the patients. Five (9%, 95% CI 3.8% - 20.4%) patients were classified as probable IAPA, of which four lacked classical risk factors. The overall ICU mortality was significantly higher among IAPA patients than non-IAPA patients (60% vs 8%, p = 0.01). CONCLUSIONS: The study represents the first prospective investigation of IAPA incidence. The 9% incidence of IAPA confirms the increased risk of invasive pulmonary aspergillosis among influenza patients admitted to the ICU. Therefore, it appears reasonable to implement a screening protocol for the early diagnosis and treatment of IAPA in influenza patients receiving intensive care. TRIAL REGISTRATION: ClinicalTrials.gov NCT04172610, registered November 21, 2019