Neutrophil Dysfunction, Immature Granulocytes, and Cell-free DNA are Early Biomarkers of Sepsis in Burn-injured Patients: A Prospective Observational Cohort Study.

OBJECTIVE: The aim of this study was to measure neutrophil function longitudinally following burn injury and to examine the relationship between neutrophil dysfunction and sepsis. BACKGROUND: Sepsis prevalence and its associated mortality is high following burn injury, and sepsis diagnosis is complicated by the ongoing inflammatory response. Previous studies have suggested that neutrophil dysfunction may underlie high infection rates and sepsis postburn; however, neutrophil dysfunction has not been thoroughly characterized over time in burns patients. METHODS: Neutrophil phagocytosis, oxidative burst capacity, and neutrophil extracellular trap (NET) generation (NETosis) were measured from 1 day to up to 1 year postburn injury in 63 patients with major burns (≥15% total body surface area). In addition, immature granulocyte (IG) count, plasma cell-free DNA (cfDNA), and plasma citrullinated histone H3 (Cit H3) levels were measured. RESULTS: Neutrophil function was reduced for 28 days postburn injury and to a greater degree in patients who developed sepsis, which was also characterized by elevated IG counts. Plasma cfDNA and Cit-H3, a specific marker of NETosis, were elevated during septic episodes. The combination of neutrophil phagocytic capacity, plasma cfDNA levels, and IG count at day 1 postinjury gave good discriminatory power for the identification of septic patients. CONCLUSION: Neutrophil function, IG count, and plasma cfDNA levels show potential as biomarkers for the prediction/early diagnosis of sepsis postburn injury and neutrophil dysfunction may actively contribute to the development of sepsis. Targeting neutrophil dysfunction and IG release may be a viable therapeutic intervention to help reduce the incidence of nosocomial infections and sepsis postburn.

Heparin resistance in severe thermal injury: A prospective cohort study.

BACKGROUND: Low molecular-weight heparin (LMWH) is routinely administered to burn patients for thromboprophylaxis. Some studies have reported heparin resistance, yet the mechanism(s) and prevalence have not been systematically studied. We hypothesized that nucleosomes, composed of histone structures with associated DNA released from injured tissue and activated immune cells in the form of neutrophil extracellular traps (NETs or NETosis), neutralize LMWH resulting in suboptimal anticoagulation, assessed by reduction in anti-factor Xa activity. METHODS: Blood was sampled from >15% total body surface area (TBSA) burn patients receiving LMWH on days 5, 10 and 14. Peak anti-factor Xa (AFXa) activity, anti-thrombin (ATIII) activity, cell-free DNA (cfDNA) levels and nucleosome levels were measured. Mixed effects regression was adjusted for multiple confounders, including injury severity and ATIII activity, and was used to test the association between nucleosomes and AFXa. RESULTS: A total of 30 patients with severe burns were included. Mean TBSA 43% (SD 17). Twenty-three (77%) patients were affected by heparin resistance (defined by AFXa activity <0.2 IU/mL). Mean peak AFXa activity across samples was 0.18 IU/mL (SD 0.11). Mean ATIII was 81.9% activity (SD 20.4). Samples taken at higher LWMH doses were found to have significantly increased AFXa activity, though the effect was not observed at all doses, at 8000 IU no samples were heparin resistant. Nucleosome levels were negatively correlated with AFXa (r = -0.29, p = 0.050) consistent with the hypothesis. The final model, with peak AFXa as the response variable, was adjusted for nucleosome levels (p = 0.0453), ATIII activity (p = 0.0053), LMWH dose pre-sample (p = 0.0049), drug given (enoxaparin or tinzaparin) (p = 0.03), and other confounders including severity of injury, age, gender, time point of sample. CONCLUSIONS: Heparin resistance is a prevalent issue in severe burns. Nucleosome levels were increased post-burn, and showed an inverse association with AFXa consistent with the hypothesis that they may interfere with the anticoagulant effect of heparin in vivo and contribute to heparin resistance. Accurate monitoring of AFXa activity with appropriate therapy escalation plans are recommended with dose adjustment following severe burn injury.

Platelet count: A predictor of sepsis and mortality in severe burns.

BACKGROUND: Platelet cells, or thrombocytes, have additional roles to haemostasis. After burn injury, platelet counts drop to a nadir at days 2-5 then rise to a peak between days 10-18. The nadir has previously been associated with mortality but there is currently no thorough investigation of its potential to predict sepsis in adults. The primary objective of this study is to assess whether platelet count can predict survival and sepsis in adults with severe burn injuries. METHODS AND FINDINGS: A retrospective cohort analysis of platelet count and other blood parameters in 145 burn patients with a TBSA greater than 20%. AUROC analysis revealed that the platelet count and rBaux score together produce moderate discrimination for survival at less than 24h after injury (AUROC=0.848, 95%CI 0.765-0.930). Platelet count at day 3 combined with TBSA has a modest association with sepsis (AUROC=0.779, 95%CI 0.697-0.862). Multivariable Cox regression analysis revealed platelet peak was the strongest predictor of mortality. CONCLUSIONS: A reduced peak platelet count is a strong predictor of 50-day mortality. Platelet count nadir may have some association with sepsis.

Prospective comparative evaluation study of Laser Doppler Imaging and thermal imaging in the assessment of burn depth.

INTRODUCTION: The accurate assessment of burn depth is challenging but crucial for surgical excision and tissue preservation. Laser Doppler Imaging (LDI) has gained increasing acceptance as a tool to aid depth assessment but its adoption is hampered by high costs, long scan times and limited portability. Thermal imaging is touted as a suitable alternative however few comparison studies have been done. METHODS: Sixteen burn patients with 52 regions of interests were analysed. Burn depth was determined using four methods LDI, thermal imaging, photographic and real-time clinical evaluation at day 1 and day 3. LDI flux and Delta T values were used for the prediction of outcomes (wound closure in <21 days). Photographic clinical evaluation of burn depth was performed by 4 blinded burn surgeons. RESULTS: Accuracy of assessment methods were greater on post burn day 3 compared to day 0. Accuracies of LDI on post burn day 0 and 3 were 80.8% and 92.3% compared to 55.8% and 71.2% for thermal imaging and 62.5% and 71.6% for photographic clinical assessment. Real-time clinical examination had an accuracy of 88.5%. Thermal imaging scan times were significantly faster compared to LDI. DISCUSSION: LDI outperforms thermal imaging in terms of diagnostic accuracy of burn depth likely due to the susceptibility of thermal imaging to environmental factors.

Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI.

Soluble glycoprotein VI (sGPVI) is shed from the platelet surface and is a marker of platelet activation in thrombotic conditions. We assessed sGPVI levels together with patient and clinical parameters in acute and chronic inflammatory conditions, including patients with thermal injury and inflammatory bowel disease and patients admitted to the intensive care unit (ICU) for elective cardiac surgery, trauma, acute brain injury, or prolonged ventilation. Plasma sGPVI was measured by enzyme-linked immunosorbent assay and was elevated on day 14 after thermal injury, and was higher in patients who developed sepsis. sGPVI levels were associated with sepsis, and the value for predicting sepsis was increased in combination with platelet count and Abbreviated Burn Severity Index. sGPVI levels positively correlated with levels of D-dimer (a fibrin degradation product) in ICU patients and patients with thermal injury. sGPVI levels in ICU patients at admission were significantly associated with 28- and 90-day mortality independent of platelet count. sGPVI levels in patients with thermal injury were associated with 28-day mortality at days 1, 14, and 21 when adjusting for platelet count. In both cohorts, sGPVI associations with mortality were stronger than D-dimer levels. Mechanistically, release of GPVI was triggered by exposure of platelets to polymerized fibrin, but not by engagement of G protein-coupled receptors by thrombin, adenosine 5'-diphosphate, or thromboxane mimetics. Enhanced fibrin production in these patients may therefore contribute to the observed elevated sGPVI levels. sGPVI is an important platelet-specific marker for platelet activation that predicts sepsis progression and mortality in injured patients.

Sls1p is a membrane-bound regulator of transcription-coupled processes involved in Saccharomyces cerevisiae mitochondrial gene expression.

Mitochondrial translation is largely membrane-associated in S. cerevisiae. Recently, we discovered that the matrix protein Nam1p binds the amino-terminal domain of yeast mtRNA polymerase to couple translation and/or RNA-processing events to transcription. To gain additional insight into these transcription-coupled processes, we performed a genetic screen for genes that suppress the petite phenotype of a point mutation in mtRNA polymerase (rpo41-R129D) when overexpressed. One suppressor identified in this screen was SLS1, which encodes a mitochondrial membrane protein required for assembly of respiratory-chain enzyme complexes III and IV. The mtRNA-processing defects associated with the rpo41-R129D mutation were corrected in the suppressed strain, linking Sls1p to a pathway that includes mtRNA polymerase and Nam1p. This was supported by the observation that SLS1 overexpression rescued the petite phenotype of a NAM1 null mutation. In contrast, overexpression of Nam1p did not rescue the petite phenotype of a SLS1 null mutation, indicating that Nam1p and Sls1p are not functionally redundant but rather exist in an ordered pathway. On the basis of these data, a model in which Nam1p coordinates the delivery of newly synthesized transcripts to the membrane, where Sls1p directs or regulates their subsequent handling by membrane-bound factors involved in translation, is proposed.

Outcomes of burns in the elderly: revised estimates from the Birmingham Burn Centre.

Outcomes after burn have continued to improve over the last 70 years in all age groups including the elderly. However, concerns have been raised that survival gains have not been to the same magnitude in elderly patients compared to younger age groups. The aims of this study were to analyze the recent outcomes of elderly burn injured patients admitted to the Birmingham Burn Centre, compare data with a historical cohort and published data from other burn centres worldwide. A retrospective review was conducted of all patients ≥65 years of age, admitted to our centre with cutaneous burns, between 2004 and 2012. Data was compared to a previously published historical cohort (1999-2003). 228 patients were included. The observed mortality for the study group was 14.9%. The median age of the study group was 79 years, the male to female ratio was 1:1 and median Total Body Surface Area (TBSA) burned was 5%. The incidence of inhalation injury was 13%. Median length of stay per TBSA burned for survivors was 2.4 days/% TBSA. Mortality has improved in all burn size groups, but differences were highly statistically significant in the medium burn size group (10-20% TBSA, p≤0.001). Burn outcomes in the elderly have improved over the last decade. This reduction has been impacted by a reduction in overall injury severity but is also likely due to general improvements in burn care, improved infrastructure, implementation of clinical guidelines and increased multi-disciplinary support, including Geriatric physicians.

The Antibacterial Activity of Acetic Acid against Biofilm-Producing Pathogens of Relevance to Burns Patients.

INTRODUCTION: Localised infections, and burn wound sepsis are key concerns in the treatment of burns patients, and prevention of colonisation largely relies on biocides. Acetic acid has been shown to have good antibacterial activity against various planktonic organisms, however data is limited on efficacy, and few studies have been performed on biofilms. OBJECTIVES: We sought to investigate the antibacterial activity of acetic acid against important burn wound colonising organisms growing planktonically and as biofilms. METHODS: Laboratory experiments were performed to test the ability of acetic acid to inhibit growth of pathogens, inhibit the formation of biofilms, and eradicate pre-formed biofilms. RESULTS: Twenty-nine isolates of common wound-infecting pathogens were tested. Acetic acid was antibacterial against planktonic growth, with an minimum inhibitory concentration of 0.16-0.31% for all isolates, and was also able to prevent formation of biofilms (at 0.31%). Eradication of mature biofilms was observed for all isolates after three hours of exposure. CONCLUSIONS: This study provides evidence that acetic acid can inhibit growth of key burn wound pathogens when used at very dilute concentrations. Owing to current concerns of the reducing efficacy of systemic antibiotics, this novel biocide application offers great promise as a cheap and effective measure to treat infections in burns patients.

Antimicrobial dressings: Comparison of the ability of a panel of dressings to prevent biofilm formation by key burn wound pathogens.

UNLABELLED: Antimicrobial medicated dressings (AMD) are often used to reduce bacterial infection of burns and other wounds. However, there is limited literature regarding comparative efficacies to inform effective clinical decision making. OBJECTIVES: Following on from a previous study where we demonstrated good antibiofilm properties of acetic acid (AA), we assessed and compared the in vitro anti-biofilm activity of a range of AMDs and non-AMDs to AA. METHODS: Laboratory experiments determined the ability of a range of eleven commercial AMD, two nAMD, and AA, to prevent the formation of biofilms of a panel of four isolates of Pseudomonas aeruginosa and Acinetobacter baumannii. RESULTS: There is a large variation in ability of different dressings to inhibit biofilm formation, seen between dressings that contain the same, and those that contain other antimicrobial agents. The best performing AMD were Mepilex(®) Ag and Acticoat. AA consistently prevented biofilm formation. CONCLUSIONS: Large variation exists in the ability of AMD to prevent biofilm formation and colonisation of wounds. A standardised in vitro methodology should be developed for external parties to examine and compare the efficacies of commercially available AMDs, along with robust clinical randomised controlled trials. This is essential for informed clinical decision-making and optimal patient management.

Hydrotherapy in burn care: a survey of hydrotherapy practices in the UK and Ireland and literature review.

INTRODUCTION: Hydrotherapy is widely used in burns management however there are risks associated with its use, in particular cross-infection. Data regarding indications and techniques in common use is deficient. This study aimed to investigate hydrotherapy practices in the UK and Ireland. METHODS: A survey of the hydrotherapy practice of major burn care providers was performed by e mail and where necessary, follow up telephone contact. RESULTS: The survey included 28 burn care providers. 27 reported using hydrotherapy. Only 11 (41%) had defined indication criteria with 4 (15%) implementing a specific protocol. Variations in hydrotherapy practice were seen. CONCLUSION: Hydrotherapy is used nationwide, however considerable variation in practice exists. One area worthy of further consideration is the need for appropriate standards of infection control.

Seeking the source of Pseudomonas aeruginosa infections in a recently opened hospital: an observational study using whole-genome sequencing.

OBJECTIVES: Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition. STUDY DESIGN: An observational prospective cohort study. SETTING: Burns care ward and critical care ward in the UK. PARTICIPANTS: Patients with >7% total burns by surface area were recruited into the study. METHODS: All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates. RESULTS: WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment. CONCLUSIONS: This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a hospital ward.

Expression of the rDNA-encoded mitochondrial protein Tar1p is stringently controlled and responds differentially to mitochondrial respiratory demand and dysfunction.

The novel yeast protein Tar1p is encoded on the anti-sense strand of the multi-copy nuclear 25S rRNA gene, localizes to mitochondria, and partially suppresses the mitochondrial RNA polymerase mutant, rpo41-R129D. However, the function of Tar1p in mitochondria and how its expression is regulated are currently unknown. Here we report that Tar1p is subject to glucose repression and is up-regulated during post-diauxic shift in glucose medium and in glycerol medium, conditions requiring elevated mitochondrial respiration. However, Tar1p expression is down-regulated in response to mitochondrial dysfunction caused by the rpo41-R129D mutation or in strains lacking respiration. Furthermore, in contrast to the previously reported beneficial effects of moderate over-expression of Tar1p in the rpo41-R129D strain, higher-level over-expression exacerbates the ROS-derived phenotypes of this mutant, including decreased respiration and life span. Finally, two-hybrid screening and in vitro-binding studies revealed a physical interaction between Tar1p and Coq5p, an enzyme involved in synthesizing the mitochondrial electron carrier and antioxidant, coenzyme Q. We propose that Tar1p expression is induced under respiratory conditions to maintain oxidative phosphorylation capacity, but that its levels in mitochondria are typically low and stringently controlled. Furthermore, we speculate that Tar1p is down-regulated when respiration is defective to prevent deleterious ROS-dependent consequences of mitochondrial dysfunction.