Late-onset sepsis in very preterm infants in Norway in 2009-2018: a population-based study.

OBJECTIVE: To evaluate epidemiology and outcomes among very preterm infants (<32 weeks' gestation) with culture-positive and culture-negative late-onset sepsis (LOS). DESIGN: Cohort study using a nationwide, population-based registry. SETTING: 21 neonatal units in Norway. PARTICIPANTS: All very preterm infants born 1 January 2009-31 December 2018 and admitted to a neonatal unit. MAIN OUTCOME MEASURES: Incidences, pathogen distribution, LOS-attributable mortality and associated morbidity at discharge. RESULTS: Among 5296 very preterm infants, we identified 582 culture-positive LOS episodes in 493 infants (incidence 9.3%) and 282 culture-negative LOS episodes in 282 infants (incidence 5.3%). Extremely preterm infants (<28 weeks' gestation) had highest incidences of culture-positive (21.6%) and culture-negative (11.1%) LOS. The major causative pathogens were coagulase-negative staphylococci (49%), Staphylococcus aureus (15%), group B streptococci (10%) and Escherichia coli (8%). We observed increased odds of severe bronchopulmonary dysplasia (BPD) associated with both culture-positive (adjusted OR (aOR) 1.7; 95% CI 1.3 to 2.2) and culture-negative (aOR 1.6; 95% CI 1.3 to 2.6) LOS. Only culture-positive LOS was associated with increased odds of cystic periventricular leukomalacia (cPVL) (aOR 2.2; 95% CI 1.4 to 3.4) and severe retinopathy of prematurity (ROP) (aOR 1.8; 95% CI 1.2 to 2.8). Culture-positive LOS-attributable mortality was 6.3%, higher in Gram-negative (15.8%) compared with Gram-positive (4.1%) LOS, p=0.009. Among extremely preterm infants, survival rates increased from 75.2% in 2009-2013 to 81.0% in 2014-2018, p=0.005. In the same period culture-positive LOS rates increased from 17.1% to 25.6%, p<0.001. CONCLUSIONS: LOS contributes to a significant burden of disease in very preterm infants and is associated with increased odds of severe BPD, cPVL and severe ROP.

Early-onset Sepsis and Antibiotic Exposure in Term Infants: A Nationwide Population-based Study in Norway.

BACKGROUND: Sepsis is a leading cause of neonatal morbidity and mortality. Clinical suspicion may lead to overuse of antibiotics. The objective of this study was to assess the epidemiology of early-onset sepsis (EOS) and antibiotic exposure during the first week of life in Norwegian term infants. METHODS: This is a nationwide population-based study from the Norwegian Neonatal Network. During the 3-year study period (2009-2011), 20 of Norway's 21 neonatal units prospectively collected data. Among 168,877 live-born (LB) term infants born during the study period, 10,175 (6.0%) infants were hospitalized in the first week of life and included in the study. RESULTS: There were 91 cases of culture-confirmed EOS (0.54 per 1000 LB) and 1447 cases classified as culture-negative EOS (8.57 per 1000 LB). The majority of culture-confirmed EOS cases were caused by Gram-positives (83/91; 91%), most commonly group B streptococci (0.31 per 1000 LB). Intravenous antibiotics were administered to 3964 infants; 39% of all admissions and 2.3% of all LB term infants. Empiric therapy consisted of an aminoglycoside and either benzylpenicillin or ampicillin in 95% of the cases. The median (interquartile range) treatment duration was 8 (7-10) days for culture-confirmed EOS and 6 (5-7) days for culture-negative EOS. There was 1 EOS-attributable death (group B streptococcal EOS) during the study period. CONCLUSIONS: In this registry-based study, the incidence of culture-confirmed EOS was in line with previous international reports and the mortality was very low. A large proportion of infants without infection were treated with antibiotics. Measures should be taken to spare neonates unnecessary antibiotic treatment.

Effects of Natural versus Synthetic Surfactant with SP-B and SP-C Analogs in a Porcine Model of Meconium Aspiration Syndrome.

BACKGROUND: Meconium displaces surfactant from the alveolar surface and inhibits its function. The development of active synthetic surfactants is complicated, especially to synthesize the hydrophobic surfactant proteins SP-B and SP-C. A synthetic surfactant, CHF5633 containing SP-B and SP-C analogs, has been designed to act similarly to the natural surfactant poractant alfa. OBJECTIVE: To test the resistance to meconium inactivation of CHF5633 compared to poractant alfa. Secondary outcome measurements were respiratory and inflammatory parameters. METHODS: Twenty-six newborn pigs, bodyweight 1.4-2.0 kg were randomized to receive either poractant alfa or CHF5633. After anesthesia, surgery and final stabilization, meconium was instilled endotracheally followed by surfactant. Bronchial lavage fluid was obtained before intervention and every second hour. Respiratory parameters were registered and blood samples drawn before intervention and every hour. RESULTS: Surfactant was inactivated in both groups 6 h after meconium instillation, but CHF5633 was more resistant than poractant alfa in terms of lipid peroxidation. Respiratory parameters were similar in both groups. Inflammatory and hemostatic parameters differed between groups, suggesting that the surfactants may play different roles in the meconium-induced inflammatory process. Due to the differential effects and complex pattern observed, the data do not indicate that one of the surfactants was superior with respect to inflammatory and hemostatic responses. CONCLUSION: This study indicates that CHF5633 is as efficient as poractant alfa in experimental meconium aspiration syndrome.

Meconium-induced release of cytokines is mediated by the TRL4/MD-2 complex in a CD14-dependent manner.

OBJECTIVE: Meconium, the first intestinal discharge of the newborn, contains material accumulated during fetal life. Meconium activates complement and CD14 and may induce a systemic inflammatory response. Toll-like receptors are classical pattern-recognition receptors recognizing both exogenous and host-derived ligands. The cyanobacterial product CyP is a potent LPS antagonist binding to the TLR4/MD-2 complex. The aim of the present study was to investigate the role of the CD14/TLR4/MD-2 complex in meconium-induced inflammation. METHODS: Whole blood from six donors was preincubated with anti-CD14 or CyP. Meconium was added and the samples were incubated for 4h. Twenty-seven inflammatory mediators were measured in a Bioplex Array Reader. Human embryonic kidney cells transfected with plasmids containing NF-kappaB dependent luciferase reporter, human MD-2, TLR4, TLR2 and/or CD14, were incubated with meconium or LPS for 18 h. Luciferase activity in cytoplasmic extracts was measured using a Luciferase Assay System kit. RESULTS: Meconium induced formation of a broad panel of inflammatory mediators. CyP and anti-CD14 significantly (p<0.001) inhibited meconium-induced formation of (a) proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6, IFN-gamma) by 60-80% and 72-94%, respectively, (b) anti-inflammatory cytokines (IL-10, IL-1Ra) by 58-59% and 50-65%, respectively, (c) chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, eotaxin, IP-10) by 43-77% and 57-87%, respectively, and (d) growth factors (G-CSF, GM-CSF, basic FGF, PDGFbb) by 53-71% and 40-78%, respectively, with no statistical significant difference between Cyp and anti-CD14. The inflammatory response could only partly be explained by LPS, suggesting that endogenous components of meconium contribute to the inflammatory response. Meconium activated NF-kappaB dose-dependently in cells expressing TLR4/MD-2 together with CD14, while no effect was seen in cells expressing TLR4/MD-2 alone or in TLR2/CD14 transfected cells. CONCLUSIONS: The results indicate that the CD14-dependent meconium-induced inflammatory reaction is mediated through the TLR4/MD2 complex. These data may have implications for future therapeutic strategies for meconium aspiration syndrome.

Pathway-specific complement activity in pigs evaluated with a human functional complement assay.

BACKGROUND: The complement system is an important part of innate immunity. Complement deficiencies or inappropriate activation of complement may cause severe diseases. The complement functional test, Wielisa, assesses all three complement activation pathways in humans. It is important to have assays available to determine the functional complement activity in research animals. Since the pig is a relevant animal in experimental research, the aim of the present study was to evaluate the applicability of this human complement assay in pigs. METHODS: Normal pig serum was serially diluted and assayed in the Wielisa test which is based on the activation of complement detected with an antibody against activated C9. The specificity of the three pathways was assessed using purified human MBL and mouse monoclonal antibodies against human C1q and pig factor D. Sera from 103 pigs and 38 newborn pigs were analyzed. Finally, functional activity of all pathways was assessed in vitro and in vivo in the absence and presence of complement inhibitors. RESULTS: The detection antibody showed cross-reactivity against pig. Normal pig serum showed activity in all pathways however about 10-fold more serum was required to obtain values comparable to human serum. Anti-human C1q and anti-pig factor D antibodies abolished classical and alternative pathway activity, respectively. Sera with low lectin pathway activity reconstituted with purified human MBL, fully recovered this activity. No deficiencies were found in classical or alternative pathway, whereas the lectin pathway showed reduced activity in a substantial number of pigs, similar to the situation in humans. Finally, the assay was successfully used to evaluate and monitor inhibition of pig complement in vitro and in vivo. CONCLUSIONS: The human complement Wielisa test can be used for functional evaluation of all complement pathways in pig serum.

Mechanisms of complement activation and effects of C1-inhibitor on the meconium-induced inflammatory reaction in human cord blood.

BACKGROUND: Meconium aspiration syndrome has a complex pathophysiology. Meconium activates the complement system and meconium-induced cytokine formation is differentially mediated by complement and CD14. C1-inhibitor (C1-INH) regulates complement and contact-system activation mainly by protease inhibition, but may reduce inflammation by other mechanisms as well. OBJECTIVE: The aim of the study was to investigate the initial mechanisms of meconium-induced complement activation and to study the effect of C1-INH on the meconium-induced inflammatory reaction. METHODS: Human serum from five donors was preincubated with an anti-MBL monoclonal antibody and then incubated with meconium for 30 min at 37 degrees C. Human cord whole blood, anticoagulated with lepirudin, from six donors was preincubated with C1-INH and then incubated with meconium for 30 min and 4h at 37 degrees C. Complement activation products specific for the different pathways were measured by ELISAs: classical pathway C1rs/C1-INH complexes, classical and lectin pathway C4d, alternative pathway C3bBbP, and terminal pathway sC5b-9 complex (TCC). A Bio-Plex Array Reader was used to measure 27 inflammatory mediators. RESULTS: The anti-MBL monoclonal antibody significantly reduced meconium-induced formation of C4d by 63% (p=0.0159) and TCC by 27% (p=0.0079). C1-INH dose-dependently inhibited meconium-induced formation of C1rs/C1-INH complexes, C4d, C3bBbP, and TCC compared to albumin (p<0.002 for all). C1-INH induced a dose-dependent and substantial inhibition of meconium-induced formation of the proinflammatory cytokines TNFalpha, IL-1 beta, IL-6 and IFN-gamma (p<0.01 for all), the chemokines IL-8, MCP-1, MIP-1 alpha, MIP-1 beta, and eotaxin (p<0.02 for all), the growth factors G-CSF, GM-CSF, basic FGF, and PDGFbb (p<0.05 for all), and the anti-inflammatory cytokine IL-1ra (p<0.001). CONCLUSIONS: Meconium activated the lectin complement pathway as well as the alternative pathway. C1-INH efficiently reduced a broad spectrum of inflammatory mediators even at the lowest concentration. Administration of C1-INH may thus reduce the inflammatory response in MAS.

Albumin lavage does not improve the outcome of meconium aspiration syndrome.

OBJECTIVE: Meconium aspiration syndrome is still a serious condition with high mortality and morbidity. No specific treatment is yet available, although surfactant is known to reduce the need for extracorporeal membrane oxygenation and surfactant lavage has shown promising results in animal studies. Our group has previously shown reduced oxygenation index in an experimental model of meconium aspiration syndrome in newborn pigs when mixing albumin with meconium before endotracheal instillation. Lung compliance increased when albumin was instilled after meconium as a rescue. The aim of this study was to combine the effect of albumin and lavage. METHODS: Sixteen newborn pigs (six in the meconium-albumin group, six in the meconium group, and four control animals) were anesthetized and tracheotomized. Meconium 4 mL/kg was instilled endotracheally. After five minutes, albumin 15 mL/kg was instilled in the meconium-albumin group followed by endotracheal suctioning. The observation time was six hours. Respiratory and hemodynamic parameters were measured. The terminal complement complex and proinflammatory cytokines were analyzed in plasma. RESULTS: Oxygenation index, ventilatory index, and the terminal complement complex (sC5b-9) increased significantly in both groups, but significantly more in the meconium-albumin group. Compliance decreased, but significantly more in the meconium-albumin group. The terminal sC5b-9 complex increased in both groups, but significantly more in the meconium-albumin group. Tumor necrosis factor-alpha, interleukin (IL)- 1beta, and IL-6 increased significantly in both groups. CONCLUSION: Albumin-lavage did not improve the outcome of experimental meconium aspiration syndrome.

Role of complement and CD14 in meconium-induced cytokine formation.

OBJECTIVE: Meconium aspiration syndrome has a complex, poorly defined pathophysiology. Meconium is a potent activator of complement in vitro and in vivo; the latter is associated with a systemic inflammatory response. The complement system and Toll-like receptors are 2 important upstream components of the innate immune system that act partly independently in the inflammatory network. The aim of this study was to investigate the relative role of complement and CD14 in meconium-induced cytokine production. METHODS: Human adult (n = 6) and cord whole blood (n = 6) anticoagulated with lepirudin was collected and distributed into tubes that contained inhibitory antibodies (anti-CD14, anti-C2, anti-factor D, or combinations thereof). The tubes were preincubated for 5 minutes before addition of meconium or buffer and then incubated for 4 hours at 37 degrees C. Complement activation was measured by quantification of the terminal sC5b-9 complement complex by enzyme-linked immunosorbent assay. A panel of 27 inflammatory mediators (cytokines, chemokines, and growth factors) was measured by using multiplex technology. RESULTS: Fourteen of the 27 mediators measured were induced by meconium both in cord and adult blood. In cord blood, 2 additional chemokines were induced and the inflammatory response was, in general, more potent. Blocking of complement or CD14 differentially reduced the formation of most mediators, anti-CD14 being more effective. Notably, the combined inhibition of complement and CD14 almost completely abolished meconium-induced formation of the cytokines and the chemokines and markedly reduced the formation of growth factors. The endogenous lipopolysaccharide content of meconium could not explain the CD14-mediated response. CONCLUSIONS: Meconium-induced triggering of the cytokine network is differentially mediated by complement and CD14. A combined inhibition of these effector mechanisms may be an alternative approach to reduce the inflammatory reaction in meconium aspiration syndrome.