Pancreatic stone protein as a novel marker for neonatal sepsis.

PURPOSE: Early-onset sepsis (EOS) is one of the main causes for the admission of newborns to the neonatal intensive care unit. However, traditional infection markers are poor diagnostic markers of EOS. Pancreatic stone protein (PSP) is a promising sepsis marker in adults. The aim of this study was to investigate whether determining PSP improves the diagnosis of EOS in comparison with other infection markers. METHODS: This was a prospective multicentre study involving 137 infants with a gestational age of >34 weeks who were admitted with suspected EOS. PSP, procalcitonin (PCT), soluble human triggering receptor expressed on myeloid cells-1 (sTREM-1), macrophage migration inhibitory factor (MIF) and C-reactive protein (CRP) were measured at admission. Receiver-operating characteristic (ROC) curve analysis was performed. RESULTS: The level of PSP in infected infants was significantly higher than that in uninfected ones (median 11.3 vs. 7.5 ng/ml, respectively; p = 0.001). The ROC area under the curve was 0.69 [95 % confidence interval (CI) 0.59-0.80; p < 0.001] for PSP, 0.77 (95 % CI 0.66-0.87; p < 0.001) for PCT, 0.66 (95 % CI 0.55-0.77; p = 0.006) for CRP, 0.62 (0.51-0.73; p = 0.055) for sTREM-1 and 0.54 (0.41-0.67; p = 0.54) for MIF. PSP independently of PCT predicted EOS (p < 0.001), and the use of both markers concomitantly significantly increased the ability to diagnose EOS. A bioscore combining PSP (>9 ng/ml) and PCT (>2 ng/ml) was the best predictor of EOS (0.83; 95 % CI 0.74-0.93; p < 0.001) and resulted in a negative predictive value of 100 % and a positive predictive value of 71 %. CONCLUSIONS: In this prospective study, the diagnostic performance of PSP and PCT was superior to that of traditional markers and a combination bioscore improved the diagnosis of sepsis. Our findings suggest that PSP is a valuable biomarker in combination with PCT in EOS.

Umbilical venous concentrations of estradiol in infants with early-onset neonatal sepsis and chorioamnionitis.

Objective: Fetuses are exposed to high concentrations of estradiol due to placental production. Experimental data suggest that estradiol is an important modulator of the immune response. However, the role of estradiol in the pathogenesis of early-onset neonatal sepsis (EOS) is unknown. The purpose of this pilot study was to determine estradiol levels in umbilical venous blood of newborns with EOS or chorioamnionitis exposure. Methods: Estradiol concentrations were measured by enzyme immunoassay in 37 newborns with EOS, 37 newborns with chorioamnionitis and 37 controls matched for gestational age and gender. Results: Estradiol levels correlated with gestational age, birth weight, gender and mode of delivery (p < 0.05). Multivariate analysis revealed higher estradiol levels in the EOS than in the chorioamnionitis group (odds ratio 8.43, 95% CI 1.63-43.45, p = 0.01) with the highest levels in patients with proven bacteraemia (p = 0.02). No difference was found between the EOS and the control group. Exploratory analysis showed an association between lower estradiol levels and a longer duration of mechanical ventilation (n = 28, p = 0.02). Conclusions: Umbilical venous estradiol levels were similar in EOS compared to controls. Further investigation is needed to evaluate whether high estradiol levels in infants with chorioamnionitis increases the risk of developing EOS.

Reduction in the use of diagnostic tests in infants with risk factors for early-onset neonatal sepsis does not delay antibiotic treatment.

Despite a low positive predictive value, diagnostic tests such as complete blood count (CBC) and C-reactive protein (CRP) are commonly used to evaluate whether infants with risk factors for early-onset neonatal sepsis (EOS) should be treated with antibiotics. We investigated the impact of implementing a protocol aiming at reducing the number of diagnostic tests in infants with risk factors for EOS in order to compare the diagnostic performance of repeated clinical examination with CBC and CRP measurement. The primary outcome was the time between birth and the first dose of antibiotics in infants treated for suspected EOS. Among the 11,503 infants born at ≥35 weeks during the study period, 222 were treated with antibiotics for suspected EOS. The proportion of infants receiving antibiotics for suspected EOS was 2.1% and 1.7% before and after the change of protocol (p = 0.09). Reduction of diagnostic tests was associated with earlier antibiotic treatment in infants treated for suspected EOS (hazard ratio 1.58; 95% confidence interval [CI] 1.20-2.07; p <0.001), and in infants with neonatal infection (hazard ratio 2.20; 95% CI 1.19-4.06; p = 0.01). There was no difference in the duration of hospital stay nor in the proportion of infants requiring respiratory or cardiovascular support before and after the change of protocol. Reduction of diagnostic tests such as CBC and CRP does not delay initiation of antibiotic treatment in infants with suspected EOS. The importance of clinical examination in infants with risk factors for EOS should be emphasised.

Reduction in the use of diagnostic tests in infants with risk factors for early-onset neonatal sepsis does not delay antibiotic treatment

BACKGROUND: Despite a low positive predictive value, diagnostic tests such as complete blood count (CBC) and C-reactive protein (CRP) are commonly used to evaluate whether infants with risk factors for early-onset neonatal sepsis (EOS) should be treated with antibiotics. STUDY DESIGN: We investigated the impact of imple- menting a protocol aiming at reducing the number of dia- gnostic tests in infants with risk factors for EOS in order to compare the diagnostic performance of repeated clinical examination with CBC and CRP measurement. The primary outcome was the time between birth and the first dose of antibiotics in infants treated for suspected EOS. RESULTS: Among the 11,503 infants born at 35 weeks during the study period, 222 were treated with antibiotics for suspected EOS. The proportion of infants receiving an- tibiotics for suspected EOS was 2.1% and 1.7% before and after the change of protocol (p = 0.09). Reduction of dia- gnostic tests was associated with earlier antibiotic treat- ment in infants treated for suspected EOS (hazard ratio 1.58; 95% confidence interval [CI] 1.20-2.07; p <0.001), and in infants with neonatal infection (hazard ratio 2.20; 95% CI 1.19-4.06; p = 0.01). There was no difference in the duration of hospital stay nor in the proportion of infants requiring respiratory or cardiovascular support before and after the change of protocol. CONCLUSION: Reduction of diagnostic tests such as CBC and CRP does not delay initiation of antibiotic treat- ment in infants with suspected EOS. The importance of clinical examination in infants with risk factors for EOS should be emphasised.

Estradiol and progesterone strongly inhibit the innate immune response of mononuclear cells in newborns.

Newborns are particularly susceptible to bacterial infections due to qualitative and quantitative deficiencies of the neonatal innate immune system. However, the mechanisms underlying these deficiencies are poorly understood. Given that fetuses are exposed to high concentrations of estradiol and progesterone during gestation and at time of delivery, we analyzed the effects of these hormones on the response of neonatal innate immune cells to endotoxin, bacterial lipopeptide, and Escherichia coli and group B Streptococcus, the two most common causes of early-onset neonatal sepsis. Here we show that at concentrations present in umbilical cord blood, estradiol and progesterone are as powerful as hydrocortisone for inhibition of cytokine production by cord blood mononuclear cells (CBMCs) and newborn monocytes. Interestingly, CBMCs and newborn monocytes are more sensitive to the effects of estradiol and progesterone than adult peripheral blood mononuclear cells and monocytes. This increased sensitivity is associated with higher expression levels of estrogen and membrane progesterone receptors but is independent of a downregulation of Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response gene 88 in newborn cells. Estradiol and progesterone mediate their anti-inflammatory activity through inhibition of the NF-κB pathway but not the mitogen-activated protein kinase pathway in CBMCs. Altogether, these results suggest that elevated umbilical cord blood concentrations of estradiol and progesterone acting on mononuclear cells expressing high levels of steroid receptors contribute to impair innate immune responses in newborns. Therefore, intrauterine exposure to estradiol and progesterone may participate in increasing susceptibility to infection during the neonatal period.

Impaired chemical coupling of cerebral blood flow is compatible with intact neurological outcome in neonates with perinatal risk factors.

Early detection of pathophysiological factors associated with permanent brain damage is a major issue in neonatal medicine. The aim of our study was to evaluate the significance of the CO2 reactivity of cerebral blood flow (CBF) in neonates with perinatal risk factors. Fourteen ventilated neonates with perinatal risk factors (pathological cardiotocogramm, low cord pH, postpartal encephalopathy) were enrolled into this prospective study. The study was performed 18-123 h after birth. CBF was measured using the noninvasive intravenous 133Xe method. Two measurements were taken with a minimal PaCO2-difference of 5 mm Hg. From the two CBF values the CO2 reactivity was calculated. Outcome was evaluated 1 year after birth. The CBF values at a lower PaCO2 ranged from 6.6 to 115. 2 ml/100 g brain issue/min (median = 18.2) and at a higher PaCO2 level from 7.1 to 125.7 ml/100 g brain tissue/min (median = 18.75). The calculated CO2 reactivity ranged from -9.6 to 6.6% (median 1.1%) change in CBF/mm Hg change in PaCO2. CO2 reactivity correlated with lowest pH (r2 = 0.35, p = 0.02). Two infants died, one of neonatal sepsis, the other of heart failure. Neurological outcome at the age of 1 year was normal in 11 patients, 1 had severe cerebral palsy. From the 12 surviving patients the patient with severe neurological deficit showed the highest CBF values (125.7 ml/100 g/min). Impaired chemical coupling of cerebral blood flow is compatible with intact neurological outcome in neonates with perinatal risk factors. CO2 reactivity in these newborns correlates with the lowest pH and may reflect the severity of perinatal asphyxia.

High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates.

The vulnerability to infection of newborns is associated with a limited ability to mount efficient immune responses. High concentrations of adenosine and prostaglandins in the fetal and neonatal circulation hamper the antimicrobial responses of newborn immune cells. However, the existence of mechanisms counterbalancing neonatal immunosuppression has not been investigated. Remarkably, circulating levels of macrophage migration inhibitory factor (MIF), a proinflammatory immunoregulatory cytokine expressed constitutively, were 10-fold higher in newborns than in children and adults. Newborn monocytes expressed high levels of MIF and released MIF upon stimulation with Escherichia coli and group B Streptococcus, the leading pathogens of early-onset neonatal sepsis. Inhibition of MIF activity or MIF expression reduced microbial product-induced phosphorylation of p38 and ERK1/2 mitogen-activated protein kinases and secretion of cytokines. Recombinant MIF used at newborn, but not adult, concentrations counterregulated adenosine and prostaglandin E2-mediated inhibition of ERK1/2 activation and TNF production in newborn monocytes exposed to E. coli. In agreement with the concept that once infection is established high levels of MIF are detrimental to the host, treatment with a small molecule inhibitor of MIF reduced systemic inflammatory response, bacterial proliferation, and mortality of septic newborn mice. Altogether, these data provide a mechanistic explanation for how newborns may cope with an immunosuppressive environment to maintain a certain threshold of innate defenses. However, the same defense mechanisms may be at the expense of the host in conditions of severe infection, suggesting that MIF could represent a potential attractive target for immune-modulating adjunctive therapies for neonatal sepsis.

Continuous positive airway pressure causes lung injury in a model of sepsis.

Continuous positive airway pressure, aimed at preventing pulmonary atelectasis, has been used for decades to reduce lung injury in critically ill patients. In neonatal practice, it is increasingly used worldwide as a primary form of respiratory support due to its low cost and because it reduces the need for endotracheal intubation and conventional mechanical ventilation. We studied the anesthetized in vivo rat and determined the optimal circuit design for delivery of continuous positive airway pressure. We investigated the effects of continuous positive airway pressure following lipopolysaccharide administration in the anesthetized rat. Whereas neither continuous positive airway pressure nor lipopolysaccharide alone caused lung injury, continuous positive airway pressure applied following intravenous lipopolysaccharide resulted in increased microvascular permeability, elevated cytokine protein and mRNA production, and impaired static compliance. A dose-response relationship was demonstrated whereby higher levels of continuous positive airway pressure (up to 6 cmH(2)O) caused greater lung injury. Lung injury was attenuated by pretreatment with dexamethasone. These data demonstrate that despite optimal circuit design, continuous positive airway pressure causes significant lung injury (proportional to the airway pressure) in the setting of circulating lipopolysaccharide. Although we would currently avoid direct extrapolation of these findings to clinical practice, we believe that in the context of increasing clinical use, these data are grounds for concern and warrant further investigation.

Interleukin-33 safeguards neutrophils in sepsis.

Cytokines have a fundamental role in orchestrating innate immune responses to bacterial infections. Interleukin-33 (IL-33) is now shown to protect from sepsis by promoting neutrophil influx into the focus of infection

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008.

OBJECTIVE: To provide an update to the original Surviving Sepsis Campaign clinical management guidelines, "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," published in 2004. DESIGN: Modified Delphi method with a consensus conference of 55 international experts, several subsequent meetings of subgroups and key individuals, teleconferences, and electronic-based discussion among subgroups and among the entire committee. This process was conducted independently of any industry funding. METHODS: We used the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations. A strong recommendation (1) indicates that an intervention's desirable effects clearly outweigh its undesirable effects (risk, burden, cost) or clearly do not. Weak recommendations (2) indicate that the tradeoff between desirable and undesirable effects is less clear. The grade of strong or weak is considered of greater clinical importance than a difference in letter level of quality of evidence. In areas without complete agreement, a formal process of resolution was developed and applied. Recommendations are grouped into those directly targeting severe sepsis, recommendations targeting general care of the critically ill patient that are considered high priority in severe sepsis, and pediatric considerations. RESULTS: Key recommendations, listed by category, include early goal-directed resuscitation of the septic patient during the first 6 hrs after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm potential source of infection (1C); administration of broad-spectrum antibiotic therapy within 1 hr of diagnosis of septic shock (1B) and severe sepsis without septic shock (1D); reassessment of antibiotic therapy with microbiology and clinical data to narrow coverage, when appropriate (1C); a usual 7-10 days of antibiotic therapy guided by clinical response (1D); source control with attention to the balance of risks and benefits of the chosen method (1C); administration of either crystalloid or colloid fluid resuscitation (1B); fluid challenge to restore mean circulating filling pressure (1C); reduction in rate of fluid administration with rising filing pressures and no improvement in tissue perfusion (1D); vasopressor preference for norepinephrine or dopamine to maintain an initial target of mean arterial pressure > or = 65 mm Hg (1C); dobutamine inotropic therapy when cardiac output remains low despite fluid resuscitation and combined inotropic/vasopressor therapy (1C); stress-dose steroid therapy given only in septic shock after blood pressure is identified to be poorly responsive to fluid and vasopressor therapy (2C); recombinant activated protein C in patients with severe sepsis and clinical assessment of high risk for death (2B except 2C for postoperative patients). In the absence of tissue hypoperfusion, coronary artery disease, or acute hemorrhage, target a hemoglobin of 7-9 g/dL (1B); a low tidal volume (1B) and limitation of inspiratory plateau pressure strategy (1C) for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure in acute lung injury (1C); head of bed elevation in mechanically ventilated patients unless contraindicated (1B); avoiding routine use of pulmonary artery catheters in ALI/ARDS (1A); to decrease days of mechanical ventilation and ICU length of stay, a conservative fluid strategy for patients with established ALI/ARDS who are not in shock (1C); protocols for weaning and sedation/analgesia (1B); using either intermittent bolus sedation or continuous infusion sedation with daily interruptions or lightening (1B); avoidance of neuromuscular blockers, if at all possible (1B); institution of glycemic control (1B), targeting a blood glucose < 150 mg/dL after initial stabilization (2C); equivalency of continuous veno-veno hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1A); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding using H2 blockers (1A) or proton pump inhibitors (1B); and consideration of limitation of support where appropriate (1D). Recommendations specific to pediatric severe sepsis include greater use of physical examination therapeutic end points (2C); dopamine as the first drug of choice for hypotension (2C); steroids only in children with suspected or proven adrenal insufficiency (2C); and a recommendation against the use of recombinant activated protein C in children (1B). CONCLUSIONS: There was strong agreement among a large cohort of international experts regarding many level 1 recommendations for the best current care of patients with severe sepsis. Evidenced-based recommendations regarding the acute management of sepsis and septic shock are the first step toward improved outcomes for this important group of critically ill patients.

Gas6 and its receptors are implicated in sepsis as modulators of innate immunity

Gas6 downregulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. We aimed to determine whether Gas6 is involved in sepsis. We measured Gas6 plasma levels in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Gas6 level was higher in septic patients than in control groups (P 0.0001). The sensitivity and specificity of Gas6 levels to predict fatal outcome were 83% and 88%. We next investigated whether Gas6 affects cytokine production and outcome in experimental models of endotoxemia and peritonitis in wild-type (WT) and Gas6-/- mice. Circulating levels of Gas6 after LPS 25mg/kg i.p. peaked at 1 hour (P<0.001). Similarly, TNF- was higher in Gas6-/- than in WT mice 1 hour after LPS (P<0.05). Furthermore, 62 anti- and pro-inflammatory cytokines were quantified in plasma after LPS injection. Their levels were globally higher in Gas6-/- plasma after LPS, 47/62 cytokines being at least 50% higher in Gas6-/- than in WT plasma after 1 hour. Mortality induced by 25mg/kg LPS was 25% in WT versus 87% in Gas6-/- mice (P<0.05). LPS-induced mortality in Gas6 receptors Axl-/-, Tyro3-/- and Merkd was also enhanced when compared to WT mice (P<0.001). In peritonitis models (cecal ligation and puncture, CLP, and i.p. injection of E. coli), Gas6 plasma levels increased and remained elevated at least 24 hours. CLP increased mortality in Gas6-/- mice. Finally, we explored the role of Gas6 in LPS-treated macrophages. We found that Gas6 was released by LPS-stimulated WT macrophages and that Gas6-/- macrophages produced more TNF- and IL-6 than WT macrophages. Cytokine release by Gas6-/- macrophages was higher than by WT macrophages (cytokine array). Adjunction of recombinant Gas6 to the culture medium of Gas6-/- macrophages diminished the cytokine production to WT levels. In LPS-treated Gas6-/- macrophages, Akt and Erk1/2 phosphorylation was reduced whereas p38 and NF B activation was enhanced. Thus, in septic patients, elevated Gas6 levels were associated with fatal outcome. In mice, they raised in experimental endotoxemia and peritonitis models, and correlated also with sepsis severity. However, Gas6-/- mice survival in these models was reduced compared to WT. Gas6 secreted by macrophages in response to LPS activated Akt and restrained p38 and NF B activation, thereby dampening macrophage activation. Altogether these data suggest that, during endotoxemia, Gas6-/- mice phenotype resembles that of mice which have undergone PI3K inhibition, indicating that Gas6 is a major modulator of innate immunity.

Macrophage Migration Inhibitory Factor Deficiency Is Associated With Impaired Killing of Gram-Negative Bacteria by Macrophages and Increased Susceptibility to Klebsiella pneumoniae Sepsis.

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIF's regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

Diagnostic accuracy of G-CSF, IL-8, and IL-1ra in critically ill children with suspected infection.

OBJECTIVE: To elucidate the diagnostic accuracy of granulocyte colony-stimulating factor (G-CSF), interleukin-8 (IL-8), and interleukin-1 receptor antagonist (IL-1ra) in identifying patients with sepsis among critically ill pediatric patients with suspected infection. DESIGN AND SETTING: Nested case-control study in a multidisciplinary neonatal and pediatric intensive care unit (PICU) PATIENTS: PICU patients during a 12-month period with suspected infection, and plasma available from the time of clinical suspicion (254 episodes, 190 patients). MEASUREMENTS AND RESULTS: Plasma levels of G-CSF, IL-8, and IL-1ra. Episodes classified on the basis of clinical and bacteriological findings into: culture-confirmed sepsis, probable sepsis, localized infection, viral infection, and no infection. Plasma levels were significantly higher in episodes of culture-confirmed sepsis than in episodes with ruled-out infection. The area under the receiver operating characteristic curve was higher for IL-8 and G-CSF than for IL-1ra. Combining IL-8 and G-CSF improved the diagnostic performance, particularly as to the detection of Gram-negative sepsis. Sensitivity was low (<50%) in detecting Staphylococcus epidermidis bacteremia or localized infections. CONCLUSIONS: In this heterogeneous population of critically ill children with suspected infection, a model combining plasma levels of IL-8 and G-CSF identified patients with sepsis. Negative results do not rule out S. epidermidis bacteremia or locally confined infectious processes. The model requires validation in an independent data-set.

Macrophage migration inhibitory factor and host innate immune defenses against bacterial sepsis.

Macrophages are essential effector cells of innate immunity that play a pivotal role in the recognition and elimination of invasive microorganisms. Mediators released by activated macrophages orchestrate innate and adaptive immune host responses. The cytokine macrophage migration inhibitory factor (MIF) is an integral mediator of the innate immune system. Monocytes and macrophages constitutively express large amounts of MIF, which is rapidly released after exposure to bacterial toxins and cytokines. MIF exerts potent proinflammatory activities and is an important cytokine of septic shock. Recent investigations of the mechanisms by which MIF regulates innate immune responses to endotoxin and gram-negative bacteria indicate that MIF acts by modulating the expression of Toll-like receptor 4, the signal-transducing molecule of the lipopolysaccharide receptor complex. Given its role in innate immune responses to bacterial infections, MIF is a novel target for therapeutic intervention in patients with septic shock.