Interplay between human STING genotype and bacterial NADase activity regulates inter-individual disease variability.

Variability in disease severity caused by a microbial pathogen is impacted by each infection representing a unique combination of host and pathogen genomes. Here, we show that the outcome of invasive Streptococcus pyogenes infection is regulated by an interplay between human STING genotype and bacterial NADase activity. S. pyogenes-derived c-di-AMP diffuses via streptolysin O pores into macrophages where it activates STING and the ensuing type I IFN response. However, the enzymatic activity of the NADase variants expressed by invasive strains suppresses STING-mediated type I IFN production. Analysis of patients with necrotizing S. pyogenes soft tissue infection indicates that a STING genotype associated with reduced c-di-AMP-binding capacity combined with high bacterial NADase activity promotes a 'perfect storm' manifested in poor outcome, whereas proficient and uninhibited STING-mediated type I IFN production correlates with protection against host-detrimental inflammation. These results reveal an immune-regulating function for bacterial NADase and provide insight regarding the host-pathogen genotype interplay underlying invasive infection and interindividual disease variability.

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase. METHODS: Bacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry. RESULTS: Here, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation. CONCLUSIONS: Our findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

Discriminatory plasma biomarkers predict specific clinical phenotypes of necrotizing soft-tissue infections.

BACKGROUNDNecrotizing soft-tissue infections (NSTIs) are rapidly progressing infections frequently complicated by septic shock and associated with high mortality. Early diagnosis is critical for patient outcome, but challenging due to vague initial symptoms. Here, we identified predictive biomarkers for NSTI clinical phenotypes and outcomes using a prospective multicenter NSTI patient cohort.METHODSLuminex multiplex assays were used to assess 36 soluble factors in plasma from NSTI patients with positive microbiological cultures (n = 251 and n = 60 in the discovery and validation cohorts, respectively). Control groups for comparative analyses included surgical controls (n = 20), non-NSTI controls (i.e., suspected NSTI with no necrosis detected upon exploratory surgery, n = 20), and sepsis patients (n = 24).RESULTSThrombomodulin was identified as a unique biomarker for detection of NSTI (AUC, 0.95). A distinct profile discriminating mono- (type II) versus polymicrobial (type I) NSTI types was identified based on differential expression of IL-2, IL-10, IL-22, CXCL10, Fas-ligand, and MMP9 (AUC >0.7). While each NSTI type displayed a distinct array of biomarkers predicting septic shock, granulocyte CSF (G-CSF), S100A8, and IL-6 were shared by both types (AUC >0.78). Finally, differential connectivity analysis revealed distinctive networks associated with specific clinical phenotypes.CONCLUSIONSThis study identifies predictive biomarkers for NSTI clinical phenotypes of potential value for diagnostic, prognostic, and therapeutic approaches in NSTIs.TRIAL REGISTRATIONClinicalTrials.gov NCT01790698.FUNDINGCenter for Innovative Medicine (CIMED); Region Stockholm; Swedish Research Council; European Union; Vinnova; Innovation Fund Denmark; Research Council of Norway; Netherlands Organisation for Health Research and Development; DLR Federal Ministry of Education and Research; and Swedish Children's Cancer Foundation.

Performance of a closed-loop glucose control system, comprising a continuous glucose monitoring system and an AI-based controller in swine during severe hypo- and hyperglycemic provocations.

Intensive care unit (ICU) patients develop stress induced insulin resistance causing hyperglycemia, large glucose variability and hypoglycemia. These glucose metrics have all been associated with increased rates of morbidity and mortality. The only way to achieve safe glucose control at a lower glucose range (e.g., 4.4-6.6 mmol/L) will be through use of an autonomous closed loop glucose control system (artificial pancreas). Our goal with the present study was to assess the safety and performance of an artificial pancreas system, composed of the EIRUS (Maquet Critical Care AB) continuous glucose monitor (CGM) and novel artificial intelligence-based glucose control software, in a swine model using unannounced hypo- and hyperglycemia challenges. Fourteen piglets (6 control, 8 treated) underwent sequential unannounced hypoglycemic and hyperglycemic challenges with 3 IU of NovoRapid and a glucose infusion at 17 mg/kg/min over the course of 5 h. In the Control animals an experienced ICU physician used every 30-min blood glucose values to maintain control to a range of 4.4-9 mmol/L. In the Treated group the artificial pancreas system attempted to maintain blood glucose control to a range of 4.4-6.6 mmol/L. Five of six Control animals and none of eight Treated animals experienced severe hypoglycemia (< 2.22 mmol/L). The area under the curve 3.5 mmol/L was 28.9 (21.1-54.2) for Control and 4.8 (3.1-5.2) for the Treated animals. The total percent time within tight glucose control range, 4.4-6.6 mmol/L, was 32.8% (32.4-47.1) for Controls and 55.4% (52.9-59.4) for Treated (p < 0.034). Data are median and quartiles. The artificial pancreas system abolished severe hypoglycemia and outperformed the experienced ICU physician in avoiding clinically significant hypoglycemic excursions.

Risk Factors and Predictors of Mortality in Streptococcal Necrotizing Soft-tissue Infections: A Multicenter Prospective Study.

BACKGROUND: Necrotizing soft-tissue infections (NSTI) are life-threatening conditions often caused by ß-hemolytic streptococci, group A Streptococcus (GAS) in particular. Optimal treatment is contentious. The INFECT cohort includes the largest set of prospectively enrolled streptococcal NSTI cases to date. METHODS: From the INFECT cohort of 409 adults admitted with NSTI to 5 clinical centers in Scandinavia, patients culture-positive for GAS or Streptococcus dysgalactiae (SD) were selected. Risk factors were identified by comparison with a cohort of nonnecrotizing streptococcal cellulitis. The impact of baseline factors and treatment on 90-day mortality was explored using Lasso regression. Whole-genome sequencing of bacterial isolates was used for emm typing and virulence gene profiling. RESULTS: The 126 GAS NSTI cases and 27 cases caused by SD constituted 31% and 7% of the whole NSTI cohort, respectively. When comparing to nonnecrotizing streptococcal cellulitis, streptococcal NSTI was associated to blunt trauma, absence of preexisting skin lesions, and a lower body mass index. Septic shock was significantly more frequent in GAS (65%) compared to SD (41%) and polymicrobial, nonstreptococcal NSTI (46%). Age, male sex, septic shock, and no administration of intravenous immunoglobulin (IVIG) were among factors associated with 90-day mortality. Predominant emm types were emm1, emm3, and emm28 in GAS and stG62647 in SD. CONCLUSIONS: Streptococcal NSTI was associated with several risk factors, including blunt trauma. Septic shock was more frequent in NSTI caused by GAS than in cases due to SD. Factors associated with mortality in GAS NSTI included age, septic shock, and no administration of IVIG.

Patient's characteristics and outcomes in necrotising soft-tissue infections: results from a Scandinavian, multicentre, prospective cohort study.

PURPOSE: Necrotising soft-tissue infections (NSTI) are characterised by necrosis, fast progression, and high rates of morbidity and mortality, but our knowledge is primarily derived from small prospective studies and retrospective studies. METHODS: We performed an international, multicentre, prospective cohort study of adults with NSTI describing patient's characteristics and associations between baseline variables and microbiological findings, amputation, and 90-day mortality. RESULTS: We included 409 patients with NSTI; 402 were admitted to the ICU. Cardiovascular disease [169 patients (41%)] and diabetes [98 (24%)] were the most common comorbidities; 122 patients (30%) had no comorbidity. Before surgery, bruising of the skin [210 patients (51%)] and pain requiring opioids [172 (42%)] were common. The sites most commonly affected were the abdomen/ano-genital area [140 patients (34%)] and lower extremities [126 (31%)]. Monomicrobial infection was seen in 179 patients (44%). NSTI of the upper or lower extremities was associated with monomicrobial group A streptococcus (GAS) infection, and NSTI located to the abdomen/ano-genital area was associated with polymicrobial infection. Septic shock [202 patients (50%)] and acute kidney injury [82 (20%)] were common. Amputation occurred in 22% of patients with NSTI of an extremity and was associated with higher lactate level. All-cause 90-day mortality was 18% (95% CI 14-22); age and higher lactate levels were associated with increased mortality and GAS aetiology with decreased mortality. CONCLUSIONS: Patients with NSTI were heterogeneous regarding co-morbidities, initial symptoms, infectious localisation, and microbiological findings. Higher age and lactate levels were associated with increased mortality, and GAS infection with decreased mortality.

Pivotal Role of Preexisting Pathogen-Specific Antibodies in the Development of Necrotizing Soft-Tissue Infections.

Background: Necrotizing soft-tissue infections (NSTI) are the most severe form of bacterial-induced tissue pathology. Their unpredictable onset and rapid development into life-threatening conditions considerably complicate patient treatment. Understanding the risk factors for NSTI in individual patients is necessary for selecting the appropriate therapeutic option. Methods: We investigated the role of pathogen-specific antibodies in the manifestation of NSTI by performing a comparative serologic approach, using plasma samples and bacterial isolates from patients with clinical NSTIs or nonnecrotizing STIs caused by Streptococcus pyogenes. We also evaluated the potential beneficial effect of intravenous immunoglobulin (IVIG) treatment. Results: We identified a hitherto overlooked state of serologic susceptibility in patients with NSTIs during the earliest stages of the infection that is potentially linked to disease progression. Thus, all patients with NSTIs included in this study exhibited a deficiency in specific antibodies directed against the causative S. pyogenes strains and the majority of their exotoxins during the initial stage of the infection. We also showed that the clinical use of IVIG during the course of infection compensates the observed antibody deficiency but is unable to halt the disease progression, once tissue necrosis has developed. Conclusion: These observations emphasize the requirement of preexisting pathogen-specific antibodies to prevent the irreversible progression of tissue infections into severely spreading NSTIs and urge further investigations on the beneficial effect of IVIG-based early phase intervention strategies to prevent the severe effects of this devastating bacterial infection.

Hyperbaric Oxygen Therapy as Adjuvant Treatment for Hardware-Related Infections in Neuromodulation.

BACKGROUND: In neuromodulation therapies, hardware-related infections are a major challenge often leading to hardware removal. OBJECTIVE: To investigate the role of adjuvant hyperbaric oxygen therapy (HBOT) in hardware-related infections. METHODS: Fourteen hardware-related infection events in 12 consecutive patients between 2002 and 2015 were treated with antibiotics and adjuvant HBOT at the Karolinska University Hospital (Stockholm, Sweden). Two time-independent infection events related to hardware replacements occurred in 2 patients. Infection resolution and the need for hardware removal were assessed. RESULTS: Twelve out of 14 events of hardware-related infection were successfully treated without hardware removal (86%). The 2 patients treated twice with HBOT on 2 time-independent occasions could retain their hardware in both cases. Hardware was removed following HBOT failure in 2 infection events, with long-term infection control achieved in all patients. Further, an intrathecal pump malfunction caused by HBOT at 2.8 bars was observed, leading to a change in the manufacturer's guidelines. CONCLUSIONS: This study indicates a potential benefit of adjuvant HBOT in the treatment of hardware-related infections in neuromodulation, diminishing the need for hardware removal and treatment interruption. Prospective studies are warranted to establish the role of adjuvant HBOT in the treatment of hardware-related infections in neuromodulation.

Assessment of Platelet Function in Traumatic Brain Injury-A Retrospective Observational Study in the Neuro-Critical Care Setting.

BACKGROUND: Despite seemingly functional coagulation, hemorrhagic lesion progression is a common and devastating condition following traumatic brain injury (TBI), stressing the need for new diagnostic techniques. Multiple electrode aggregometry (MEA) measures platelet function and could aid in coagulopathy assessment following TBI. The aims of this study were to evaluate MEA temporal dynamics, influence of concomitant therapy, and its capabilities to predict lesion progression and clinical outcome in a TBI cohort. MATERIAL AND METHODS: Adult TBI patients in a neurointensive care unit that underwent MEA sampling were retrospectively included. MEA was sampled if the patient was treated with antiplatelet therapy, bled heavily during surgery, or had abnormal baseline coagulation values. We assessed platelet activation pathways involving the arachidonic acid receptor (ASPI), P2Y12 receptor, and thrombin receptor (TRAP). ASPI was the primary focus of analysis. If several samples were obtained, they were included. Retrospective data were extracted from hospital charts. Outcome variables were radiologic hemorrhagic progression and Glasgow Outcome Scale assessed prospectively at 12 months posttrauma. MEA levels were compared between patients on antiplatelet therapy. Linear mixed effect models and uni-/multivariable regression models were used to study longitudinal dynamics, hemorrhagic progression and outcome, respectively. RESULTS: In total, 178 patients were included (48% unfavorable outcome). ASPI levels increased from initially low values in a time-dependent fashion (p < 0.001). Patients on cyclooxygenase inhibitors demonstrated low ASPI levels (p < 0.001), while platelet transfusion increased them (p < 0.001). The first ASPI (p = 0.039) and TRAP (p = 0.009) were significant predictors of outcome, but not lesion progression, in univariate analyses. In multivariable analysis, MEA values were not independently correlated with outcome. CONCLUSION: A general longitudinal trend of MEA is identified in this TBI cohort, even in patients without known antiplatelet therapies. Values appear also affected by platelet inhibitory treatment and by platelet transfusions. While significant in univariate models to predict outcome, MEA values did not independently correlate to outcome or lesion progression in multivariable analyses. Further prospective studies to monitor coagulation in TBI patients are warranted, in particular the interpretation of pathological MEA values in patients without antiplatelet therapies.

Cervical necrotizing fasciitis: descriptive, retrospective analysis of 59 cases treated at a single center.

To provide retrospective, descriptive information on patients with cervical necrotizing fasciitis treated at a single center during the years 1998-2014, and to evaluate the outcome of a newly introduced treatment strategy. Retrospective analysis of clinical data obtained from medical records. Mortality, pre-morbidity, severity of illness, primary site of infection, type of bacteria, time parameters. The observed 3-month mortality was 6/59 (10 %). The most common initial foci of the infection were pharyngeal, dental or hypopharyngeal. The most common pathogen was Streptococcus milleri bacteria within the Streptococcus anginosus group (66 % of the cases). Using a combined treatment with early surgical debridement combined with hyperbaric oxygen treatment, it is possible to reduce the mortality rate among patients suffering from cervical necrotizing fasciitis, compared to the expected mortality rate and to previous historical reports. Data indicated that early onset of hyperbaric oxygen treatment may have a positive impact on survival rate, but no identifiable factor was found to prognosticate outcome.

Biochemical response to hyperbaric oxygen treatment of a transhemispheric penetrating cerebral gunshot injury.

Hyperbaric oxygen (HBO) therapy has been suggested a treatment option in order to reduce the development of secondary insults succeeding traumatic brain injury. This case report studied the course of a 23-year-old gentleman with a close range transhemispheric gunshot wound. The biochemical parameters, using a multi-modal monitoring in the neuro-intensive care unit, improved following HBO treatment.

Formation of microparticles in the injured brain of patients with severe isolated traumatic brain injury.

The potential pathophysiological role of circulating microparticles (MPs) has been recognized in various conditions, such as cardiovascular and thrombotic diseases. Traumatic brain injury (TBI) has a complex pathophysiology that involves coagulopathy and inflammation. We investigated endothelial-, platelet-, and leukocyte-derived microparticles (EMPs, PMPs, and LMPs, respectively) in 16 patients with severe isolated TBI. Arterial and cerebrovenous samples were taken repeatedly, during 1-72 h after injury. Subpopulations of MPs, exposing tissue factor (TF) and P-selection, were also studied. MP counts in cerebrovenous samples, irrespective of cellular origin, were higher in TBI cases, compared to healthy controls (peak levels of EMPs were approximately 7 times higher, PMPs 1.4 times higher, and LMPs 2 times higher, respectively; p<0.001 for all). MP counts declined sharply from high levels shortly after the trauma toward slightly elevated levels 72 h later. EMPs and PMPs exposing TF, as well as PMPs exposing P-selection, showed a transcranial gradient with higher concentration in cerebrovenous, compared to arterial, samples. In contrast, LMPs exposing TF were higher in arterial samples, suggesting accumulation of LMPs in the brain. We conclude that the pattern of circulating MPs is altered after TBI. PMPs exposing P-selection and EMPs exposing TF seem to be generated in the injured brain, whereas LMPs exposing TF are accumulated. The pathophysiological significance of these changes in MP pattern in TBI should be further investigated. Including MPs exposing brain-specific antigens in the assessment of brain injury would give further information of origin and likely give additional information of the size of the injury, given that the MP phenotypes investigated in the present study are not brain-specific markers.

No protective role for hypoxic pulmonary vasoconstriction in severe hypergravity-induced arterial hypoxemia.

Supine subjects exposed to hypergravity show a marked arterial desaturation. Previous work from our laboratory has also shown a paradoxical reduction of lung perfusion in dependent lung regions in supine subjects exposed to hypergravity. We reasoned that the increased lung weight during hypergravity caused either direct compression of the blood vessels in the dependent lung tissue or that poor regional ventilation caused reduced perfusion through hypoxic pulmonary vasoconstriction (HPV). The objective of this study was to evaluate the importance of HPV through measurements of arterial oxygenation during exposure to hypergravity with normal and attenuated HPV. A further increased arterial desaturation during hypergravity with attenuated HPV would support the hypothesis that HPV contributes to the paradoxical redistribution of regional perfusion. In a two-phased randomized study we first exposed 12 healthy subjects to 5 G while supine during two single-blinded conditions; control and after 50 mg sildenafil p.o.. In a second phase, 12 supine subjects were exposed to 5 G during three single-blinded conditions; control, after 100 mg sildenafil p.o. and after inhalation of 10 µg iloprost. There was a substantial arterial desaturation by 5-30% units in all subjects with no or only minor differences between conditions. The results speak against HPV as a principal mechanism for the hypergravity-induced reduction of lung perfusion in dependent lung regions in supine humans.

Neuropsychological outcome following near-drowning in ice water: two adult case studies.

Two men, 56 and 33 years old, (case 1 and case 2) were examined neuropsychologically after successful resuscitation from circulatory arrest following extreme accidental hypothermia and near drowning. After submersion in ice water for at least 20 minutes they received CPR for 45 to 60 minutes. Body-core temperature at start of CPB was 24 degrees C and 22 degrees C, respectively. A neuropsychological examination was performed within two months after the accident and 1 year later. An additional follow-up interview was made 3 years after the accidents. Both had severe problems with memory, visuospatial performance, executive function, and verbal fluency. The follow-up demonstrated improvement in the visuospatial test in both and in the verbal learning, recall, and logical reasoning tests in case 2. Both still had problems with executive function, and case 2 also in verbal fluency. Case 1 also had problems with flexibility, planning and abstract ability. Despite the protective effects of hypothermia and gradual improvement of symptoms over time, some of the deficits were permanent. A thorough neuropsychological examination of patients suffered from anoxia is advisable, because gross neurological examination and MRI scans may not always reveal underlying brain dysfunction.

Platelet dysfunction in patients with severe traumatic brain injury.

Coagulopathy is a common phenomenon in traumatic brain injury (TBI) and a major contributor to a poor outcome. Thrombocytopenia is a strong negative prognostic factor in TBI, but bleeding tendency can be present even with a normal platelet count. We investigated platelet function in patients with TBI by means of modified thromboelastography (i.e., platelet mapping [TEG-PM]). Four groups were studied: (1) patients with severe isolated TBI (n = 20), (2) patients with general trauma without TBI (the ICU group, n = 10), (3) patients with chronic alcohol abuse (n = 7; as alcohol abuse is common in patients with TBI), and (4) healthy volunteers (n = 10). We measured platelet counts in venous blood (Plt), Ivy bleeding time, standard TEG parameters, and platelet responses to arachidonic acid (AA) and adenosindiphosphate (ADP), using TEG-PM. TBI patients had a lower Plt (180 +/- 68 x 10(9) ; mean +/- SD) and a longer bleeding time (674 +/- 230 sec) than healthy controls, (256 +/- 43 x 10(9), p < 0.01) and (320 +/- 95 sec, p < 0.005), respectively. TBI patients had dramatically lower platelet responses to AA (0-86%, mean 22%) compared to healthy controls (57-89%, mean 73%), the ICU group (4-75%, mean 49%), and the alcohol abusers (17-88%, mean 64%; p < 0.001). Responses to ADP did not differ significantly between the groups. Patients with low responsiveness to AA at admittance to the hospital were likely to develop bleeding complications later. Patients with TBI develop platelet dysfunction, which most likely contributes to bleeding complications. The observed platelet dysfunction appears to involve the cyclooxygenase pathway. TEG-PM analysis can be used to identify patients with a high risk of bleeding complications.

Coagulation abnormalities associated with severe isolated traumatic brain injury: cerebral arterio-venous differences in coagulation and inflammatory markers.

Although coagulopathy is known to be the major contributor to a poor outcome of traumatic brain injury (TBI), the mechanisms that trigger coagulation abnormalities have not been studied in detail. We undertook a prospective observational study at a neurosurgical ICU (NICU) in a university hospital. We examined 11 patients with severe isolated TBI, at admittance to the hospital and during the next 3 days. We collected cerebrovenous blood samples from a jugular bulb catheter, arterial blood, and cerebrospinal fluid (CSF) samples. We measured concentrations of thrombin-antithrombin complex (TAT), fibrin D-dimer (DD), prothrombin fragment 1 + 2 (F1 + 2), interleukin-6 (IL-6), and complement complex (C5b-9). All patients had some degree of consumption coagulopathy at the study start and a tendency to thrombocytopenia during the next few days. Levels of DD (3.6 +/- 2.7 mg/L), TAT (86 +/- 72 microg/L) and F1 + 2 (5.9 +/- 6.8 nmol/L) were significantly increased shortly after the trauma compared to reference values, with considerable transcranial gradients for TAT (49 microg/L) and F1 + 2 (3.2 nmol/L). Compared to controls, IL-6 levels were increased more than a hundredfold in both blood (283 +/- 192 ng/L) and CSF (424 +/- 355 ng/L) samples, with a transcranial gradient at the study start (107 ng/L). C5b-9 levels were moderately increased in blood samples, 270 +/- 114 microg/L, versus controls, 184 +/- 39 (p < 0.05). We conclude that activation of the coagulation system takes place during the passage of blood through the damaged brain, and is already evident hours after the trauma. IL-6 and activation of the complement system (C5b-9) co-vary with hemostatic parameters in TBI patients.