Search

Bloodstream infections (BSIs) require an accurate and fast identification of causative pathogens. Molecular diagnostics, in particular polymerase chain reaction (PCR)-based approaches for BSI diagnostics directly from whole blood, suffer from limitations such as inhibition leading to invalid results. In this retrospective study, we analyzed 23 parameters for their potential interference with LightCycler SeptiFast PCR tests (n = 2167) routinely performed at our institution. The overall inhibition rate was 9.1%. Test date, type of ward, procalcitonin levels, high leukocyte counts, and absolute neutrophil count were significantly associated with inhibition. For a subset (n = 448), cut-off values for leukocyte counts of < 5700 cells/μL and ≥ 26,900 cells/μL were significantly associated with a low (5%) and high (67%) inhibition risk. For patients with a moderate to high leukocyte count (5700-26,900 cells/μL), the additional administration of hydrocortisone significantly increased the inhibition risk. Furthermore, freezing of blood samples prior to DNA extraction and SF testing appeared to neutralize inhibitory factors. It remains to be investigated whether other molecular diagnostic tests are susceptible to similar inhibiting parameters.

There is increasing evidence that C-reactive protein (CRP) can mediate inflammatory reactions following the transformation of functionally inert pentameric CRP (pCRP) into its structural isoform pCRP* and into monomeric CRP (mCRP). This conversion can occur on the membranes of apoptotic or activated cells or on extracellular vesicles (EVs) shed from the cell surface. Here, we characterized the association of CRP with EVs in plasma from sepsis patients using flow cytometry, and found highly elevated levels of total EV counts and CRP + EVs as compared to healthy individuals. We further assessed the ability of PentraSorb CRP, an extracorporeal device for the adsorption of CRP, to deplete free CRP and CRP + EVs. Treatment of septic plasma with the adsorbent in vitro resulted in almost complete removal of both, free CRP and CRP + EVs, while total EV counts remained largely unaffected, indicating the detachment of CRP from the EV surface. EVs from septic plasma elicited a release of interleukin-8 from cultured human monocytes, which was significantly reduced by adsorbent treatment prior to EV isolation. Our findings provide evidence that CRP + EVs exhibit pro-inflammatory characteristics and can contribute to the spreading of inflammation throughout the circulation on top of their pro-coagulant activity.

The timely recognition of sepsis and the prediction of its clinical course are challenging due to the complex molecular mechanisms leading to organ failure and to the heterogeneity of sepsis patients. Treatment strategies relying on a "one-fits-all" approach have failed to reduce mortality, suggesting that therapeutic targets differ between patient subgroups and highlighting the need for accurate analysis of the molecular cascades to assess the highly variable host response. Here, we characterized a panel of 44 inflammatory mediators, including cytokines, chemokines, damage-associated molecular patterns, and coagulation-related factors, as well as markers of endothelial activation in 30 patients suffering from renal failure in the course of sepsis. All patients received continuous veno-venous hemodialysis with either high cut-off filters or with standard filters, and mediators were quantified for all patients at the initiation of dialysis and after 24 h and 48 h. Mediator concentrations in individual patients ranged widely, demonstrating the heterogeneity of sepsis patients. None of the mediators correlated with SAPS III or TISS scores. The overall in-hospital mortality of the study population was 56.7% (57.1% vs. 56.3% for high cut-off vs. standard filter). The two filter groups differed regarding most of the mediator levels at baseline, prohibiting conclusions regarding the effect of standard filters versus high cut-off filters on mediator depletion. The elevation and correlation of damage-associated molecular patterns and markers of endothelial activation gave evidence of severe tissue damage. In particular, extracellular histones were strongly increased and were almost 30-fold higher in nonsurvivors as compared to survivors, indicating their diagnostic and prognostic potential., Competing Interests: The authors have no conflict of interest to declare., (Copyright © 2021 Tanja Eichhorn et al.)

Blood culture represents the current reference method for the detection of bacteria or fungi in the circulation. To accelerate pathogen identification, molecular diagnostic methods, mainly based on polymerase chain reaction (PCR), have been introduced to ensure early and targeted antibiotic treatment of patients suffering from bloodstream infection. Still, these approaches suffer from a lack of sensitivity and from inhibition of PCR in a number of clinical samples, leading to false negative results. To overcome these limitations, various approaches aiming at the enrichment of pathogens from larger blood volumes prior to the extraction of pathogen DNA, thereby also depleting factors interfering with PCR, have been developed. Here, we provide an overview of current systems for diagnosing bloodstream infection, with a focus on approaches for pre-analytical pathogen enrichment, and highlight emerging applications of pathogen depletion for therapeutic purposes as a potential adjunctive treatment of sepsis patients., (Copyright © 2018. Published by Elsevier Inc.)

Sepsis is currently viewed as a fundamental disintegration of control functions from intracellular signalling to immunoregulatory and neuroendocrine mechanisms. The immediate threat in sepsis is invasive infection, and the need to activate immune defense mechanisms to clear the pathogen before irreparable damage occurs. In the process of pathogen elimination, however, the systemic host response to infection may cause collateral damage to the endothelium and may lead to the destruction of host tissues.A number of experimental models have been developed to monitor endothelial activation and to study endothelial dysfunction under septic conditions. Here, we review the application of these models to assess the highly variable host response in sepsis and to investigate the efficacy of adsorbent-based extracorporeal therapies. We also highlight the need for efficient diagnostic tools, which are indispensable to select patients who are likely to benefit from distinct adjunctive therapies.

Background: High cutoff hemofilters might support the restoration of immune homeostasis in systemic inflammation by depleting inflammatory mediators from the circulation., Methods: Interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor-alpha depletion was assessed in 30 sepsis patients with acute renal failure using continuous veno-venous hemodialysis with high cutoff versus standard filters (CVVHD-HCO vs. CVVHD-STD) over 48 h., Results: The transfer of IL-6 and IL-8 was significantly higher for CVVHD-HCO, as shown by increased IL-6 and IL-8 effluent concentrations. The mean plasma cytokine concentrations decreased over time for all cytokines without detectable differences for the treatment modalities. No transfer of albumin was observed for either of the filters. C-reactive protein remained stable over time and did not differ between CVVHD-HCO and CVVHD-STD, while procalcitonin decreased significantly over 48 h for both treatment modalities., Conclusion: CVVHD-HCO achieved enhanced removal of IL-6 and IL-8 as compared to CVVHD-STD, without differentially reducing plasma cytokine levels., (© 2017 S. Karger AG, Basel.)

Endothelial activation with excessive recruitment and adhesion of immune cells plays a central role in the progression of sepsis. We established a microfluidic system to study the activation of human umbilical vein endothelial cells by conditioned medium containing plasma from lipopolysaccharide-stimulated whole blood or from septic blood and to investigate the effect of adsorption of inflammatory mediators on endothelial activation. Treatment of stimulated whole blood with polystyrene-divinylbenzene-based cytokine adsorbents (average pore sizes 15 or 30 nm) prior to passage over the endothelial layer resulted in significantly reduced endothelial cytokine and chemokine release, plasminogen activator inhibitor-1 secretion, adhesion molecule expression, and in diminished monocyte adhesion. Plasma samples from sepsis patients differed substantially in their potential to induce endothelial activation and monocyte adhesion despite their almost identical interleukin-6 and tumor necrosis factor-alpha levels. Pre-incubation of the plasma samples with a polystyrene-divinylbenzene-based adsorbent (30 nm average pore size) reduced endothelial intercellular adhesion molecule-1 expression to baseline levels, resulting in significantly diminished monocyte adhesion. Our data support the potential of porous polystyrene-divinylbenzene-based adsorbents to reduce endothelial activation under septic conditions by depletion of a broad range of inflammatory mediators., Competing Interests: Compliance with Ethical Standards All procedures performed in this study involving human samples were in accordance with the ethical standards of the institutional review boards as laid down in the Declaration of Helsinki. Conflict of Interest The authors declare that they have no conflict of interest.

The aim of this work was to establish and characterize a cell culture model for lipopolysaccharide (LPS)-induced activation of human endothelial cells. Monocytic THP-1 cells were stimulated for 4 h with 10 ng/ml LPS from Pseudomonas aeruginosa in media containing 10% human plasma. Culture supernatants containing LPS and factors secreted by THP-1 in response to stimulation were applied to human umbilical vein endothelial cells (HUVECs). Nuclear factor-κB (NF-κB) activity, expression of adhesion molecules, and cytokine secretion were quantified. In addition, the effect of adsorptive removal of tumour necrosis factor-α (TNF-α) from the THP-1 culture supernatant on HUVEC activation was assessed. After 4 h of stimulation, THP-1 cells secreted various mediators including TNF-α (854 ± 472 pg/ml), interleukin (IL)-8 (2069 ± 710 pg/ml), IL-18 (305 ± 124 pg/ml), IL-10 (14 ± 5 pg/ml), and IL-1β (24 ± 11 pg/ml). Stimulated HUVECs showed significantly increased NF-κB activity and secreted high amounts of IL-6 and IL-8. Additionally, adhesion molecules ICAM-1 and E-selectin were increased both in the culture supernatant and at the cell surface. Removal of TNF-α from the THP-1 culture supernatant prior to HUVEC stimulation resulted in a decrease in NF-κB activity, expression of adhesion molecules, as well as IL-6 secretion. The cell culture model established in this study permits the monitoring of LPS-induced endothelial activation, which plays a central role in sepsis and may serve to assess the effect of mediator modulation by methods such as extracorporeal blood purification.

Introduction: A pro-apoptotic effect of circulating mediators on renal tubular epithelial cells has been involved in the pathogenesis of sepsis-associated acute kidney injury (AKI). Adsorption techniques have been showed to efficiently remove inflammatory cytokines from plasma. The aim of this study was to evaluate the efficiency of the hydrophobic resin Amberchrom CG161 M to adsorb from septic plasma soluble mediators involved in tubular injury., Methods: We enrolled in the study 10 critically ill patients with sepsis-associated AKI and we evaluated the effects of their plasma on granulocyte adhesion, apoptosis and functional alterations of cultured human kidney tubular epithelial cells. We established an in vitro model of plasma adsorption and we studied the protective effect of unselective removal of soluble mediators by the Amberchrom CG161 M resin on septic plasma-induced tubular cell injury., Results: Plasma from septic patients induced granulocyte adhesion, apoptosis and altered polarity in tubular cells. Plasma adsorption significantly decreased these effects and abated the concentrations of several soluble mediators. The inhibition of granulocyte adhesion to tubular cells was associated with the down-regulation of ICAM-1 and CD40. Resin adsorption inhibited tubular cell apoptosis induced by septic plasma by down-regulating the activation of caspase-3, 8, 9 and of Fas/death receptor-mediated signalling pathways. The alteration of cell polarity, morphogenesis, protein reabsorption and the down-regulation of the tight junction molecule ZO-1, of the sodium transporter NHE3, of the glucose transporter GLUT-2 and of the endocytic receptor megalin all induced by septic plasma were significantly reduced by resin adsorption., Conclusions: Septic plasma induced a direct injury of tubular cells by favouring granulocyte adhesion, by inducing cell apoptosis and by altering cell polarity and function. All these biological effects are related to the presence of circulating inflammatory mediators that can be efficiently removed by resin adsorption with a consequent limitation of tubular cell injury.