Proteomic profiling reveals diagnostic signatures and pathogenic insights in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a severe disease that emerged during the COVID-19 pandemic. Although recognized as an immune-mediated condition, the pathogenesis remains unresolved. Furthermore, the absence of a diagnostic test can lead to delayed immunotherapy. Using state-of-the-art mass-spectrometry proteomics, assisted by artificial intelligence (AI), we aimed to identify a diagnostic signature for MIS-C and to gain insights into disease mechanisms. We identified a highly specific 4-protein diagnostic signature in children with MIS-C. Furthermore, we identified seven clusters that differed between MIS-C and controls, indicating an interplay between apolipoproteins, immune response proteins, coagulation factors, platelet function, and the complement cascade. These intricate protein patterns indicated MIS-C as an immunometabolic condition with global hypercoagulability. Our findings emphasize the potential of AI-assisted proteomics as a powerful and unbiased tool for assessing disease pathogenesis and suggesting avenues for future interventions and impact on pediatric disease trajectories through early diagnosis.

Accuracy of FAST in detecting intraabdominal bleeding in major trauma with pelvic and/or acetabular fractures: a retrospective cohort study.

PURPOSE: The Focused Assessment with Sonography for Trauma (FAST) is a tool to rapidly detect intraabdominal and intrapericardial fluid with point-of-care ultrasound. Previous studies have questioned the role of FAST in patients with pelvic fractures. The aim of the present study was to assess the accuracy of FAST to detect clinically significant intraabdominal hemorrhage in patients with pelvic fractures. METHODS: We included all consecutive patients with pelvic and/or acetabular fractures treated our Level 1 trauma center from 2009-2020. We registered patient and fracture characteristics, FAST investigations and CT descriptions, explorative laparotomy findings, and transfusion needs. We compared FAST to CT and laparotomy findings, and calculated true positive and negative findings, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS: We included 389 patients. FAST had a sensitivity of 75%, a specificity of 98%, a PPV of 84%, and a NPV of 96% for clinically significant intraabdominal bleeding. Patients with retroperitoneal hematomas were at increased risk for laparotomy both because of True-negative FAST and False-positive FAST. CONCLUSION: FAST is accurate to identify clinically significant intraabdominal blood in patients with severe pelvic fractures and should be a standard asset in these patients. Retroperitoneal hematomas challenge the FAST interpretation and thus the decision making when applying FAST in patients with pelvic fractures.

Distinct clinical parameters were associated with shorter spontaneous resolution in children with non-tuberculous mycobacterial lymphadenitis.

AIM: Non-tuberculous mycobacteria (NTM) lymphadenitis typically resolves spontaneously, yet factors influencing the duration remain explored. We aimed to identify clinical parameters associated with shorter spontaneous resolution. METHODS: This cohort study included children with NTM lymphadenitis from 1 January 2015 to 1 March 2021 at Copenhagen University Hospital. Time-to-event analysis assessed clinical parameters associated with the duration of NTM lymphadenitis. RESULTS: Sixty children (57% boys) with a median age of 24 months (range 11-84) were included; 13 (22%) received primary surgery, 13 (22%) underwent surgery after a wait-and-see period and 34 (57%) received no intervention. In children without intervention, the median duration was 10 months (range 2-25). Faster resolution was associated with parental-reported lymph node enlargement within 2 weeks (HR 2.3, 95% CI 1.0-5.0; p = 0.044), abscess on ultrasound examination (HR 3.3, 95% CI 1.5-7.3; p = 0.003) and skin discoloration and/or perforation within 3 months of onset (HR 4.3, 95% CI 1.3-14.4; p = 0.017 and HR 3.7, 95% CI 1.5-9.1; p = 0.005). CONCLUSION: Knowledge of predictors for shorter spontaneous resolution of NTM lymphadenitis, such as rapid initial lymph node enlargement, abscess on ultrasound examination, and skin discoloration and/or perforation within 3 months of disease onset, may guide clinical management decisions concerning surgery versus a conservative approach.

Invasive group A streptococcal infections in children and adolescents in Denmark during 2022-23 compared with 2016-17 to 2021-22: a nationwide, multicentre, population-based cohort study.

BACKGROUND: A historic increase in paediatric invasive group A streptococcal (iGAS) infections was reported globally in 2022. iGAS infections can lead to severe manifestations (eg, pleural empyema, necrotising fasciitis, toxic shock syndrome, osteomyelitis, septic arthritis, and meningitis). We aimed to compare the incidence and severity of iGAS infections overall, for distinct clinical phenotypes, and for GAS emm variants in Denmark in 2022-23 with reference to the previous six seasons (ie, 2016-17, 2017-18, 2018-19, 2019-20, 2020-21, and 2021-22). METHODS: In this nationwide, multicentre, population-based cohort study, we included all children and adolescents in Denmark aged 0-17 years with a positive culture of GAS or GAS confirmed through PCR-based methods from otherwise sterile sites in 2022-23 and the previous six seasons from 2016-17 to 2021-22. For all seven seasons, data were obtained from week 21 to week 20 of the next year. Patients at all 18 paediatric hospital departments in Denmark were identified through the Danish Microbiology Database, in which iGAS isolates from sterile sites are prospectively registered, including emm typing. We obtained electronic medical health records for each patient admitted with a diagnosis of iGAS. We calculated the incidence of iGAS per 1 000 000 inhabitants aged 0-17 years in each season from week 21 to week 20 of the next year and the risk ratios (RRs) for incidence of iGAS, distinct disease manifestations, and emm variants in 2022-23 versus the three pre-COVID-19 seasons in 2016-17, 2017-18, and 2018-19 using Fisher's exact test and Pearson's χ2 test. FINDINGS: Among the Danish population of 1 152 000 children and adolescents aged 0-17 years, 174 with iGAS disease were included. 76 children and adolescents with iGAS during 2022-23 were identified; 31 (41%) of 76 were female and 45 (59%) were male. 98 children and adolescents with iGAS during 2016-17 to 2021-22 were identified; 41 (42%) of 98 were female and 57 (58%) were male. There was an increase in incidence of iGAS from mean 22·6 (95% CI 14·7-33·1) per 1 000 000 children and adolescents during 2016-17 to 2018-19 to 66·0 (52·0-82·6) per 1 000 000 during 2023-23 (RR 2·9, 95% CI 1·9-4·6; p<0·0001). During the COVID-19 pandemic in 2019-20, 2020-21, and 2021-22, the mean incidence of iGAS was 6·1 (95% CI 2·4-12·5) per 1 000 000 children and adolescents. In 2022-23, there was a 9·5-fold increase in emm-12 (95% CI 2·2-40·8; p=0·0002) and a 2·7-fold increase in emm-1 (1·3-5·5; p=0·0037). The most common clinical manifestations of iGAS in 2022-23 were soft-tissue infections, which increased by 4·5-fold (1·9-10·9; p=0·0003), and complicated pneumonia with parapneumonic effusion, which increased by 4·0-fold (1·4-11·4; p=0·0059), both compared with the three pre-COVID-19 seasons. Overall, there was no increased severity of iGAS in 2022-23 compared with the previous six seasons as measured by median duration of hospital stay (8 days, IQR 4-14 vs 9 days, 5-15; p=0·39), paediatric intensive care unit (PICU) admission (17 [22%] of 76 vs 17 [17%] of 98; p=0·53), duration of stay in PICU (4 days, IQR 2-10 vs 4 days, 2-11; p=0·84), or mortality (three [4%] of 76 vs three [3%] of 98; p=1·00). In 2022-23, there was a 3·6-fold (95% CI 1·8-7·3; p=0·0001) increase in children with a preceding upper respiratory tract infection and a 4·6-fold (1·5-14·1; p=0·0034) increase in children with a preceding varicella-zoster infection, both compared with the three pre-COVID-19 seasons. INTERPRETATION: In Denmark, the incidence of paediatric iGAS increased in 2022-23 compared with the three pre-COVID-19 seasons of 2016-17, 2017-18, and 2018-19. However, the course of iGAS disease in children and adolescents in 2022-23 was not more severe than in previous seasons. The high morbidity across all seasons highlights iGAS as a major invasive bacterial infection in children and adolescents. FUNDING: Innovation Fund Denmark.

Oral-only antibiotics for bone and joint infections in children: study protocol for a nationwide randomised open-label non-inferiority trial.

INTRODUCTION: Children with bone and joint infections are traditionally treated with intravenous antibiotics for 3-10 days, followed by oral antibiotics. Oral-only treatment has not been tested in randomised trials. METHODS AND ANALYSIS: Children (3 months to 18 years) will be randomised 1:1 with the experimental group receiving high-dose oral antibiotics and the control group receiving intravenous antibiotics with a shift in both groups to standard oral antibiotics after clinical and paraclinical improvement. Children in need of acute surgery or systemic features requiring intravenous therapy, including septic shock, are excluded. The primary outcome is defined as a normal blinded standardised clinical assessment 6 months after end of treatment. Secondary outcomes are non-acute treatment failure and recurrent infection. Outcomes will be compared by a non-inferiority assumption with an inferiority margin of 5%. ETHICS AND DISSEMINATION: The trial has the potential to reduce unnecessary hospitalisation and use of intravenous antibiotics in children with bone or joint infections. Due to the close follow-up, exclusion of severely ill children and predefined criteria for discontinuation of the allocated therapy, we expect the risk of treatment failure to be minimal. TRIAL REGISTRATION NUMBER: NCT04563325.

Sulfatide inhibits fibroblast growth, activation and oxidative stress induced by ectopic insulin.

AIM: To study the effect of sulfatide on gene expression and proliferation of human primary fibroblasts induced by insulin, insulin-like growth factor-1 and human growth hormone. MATERIALS AND METHODS: Human primary fibroblasts were exposed to 1, 3 and 30 µM of sulfatide or its precursor galactosylceramide (GalCer). Proliferation was determined by 3 H-thymidine incorporation and gene expression via microarray analysis. RESULTS: Sulfatide and GalCer reduced the growth rate of fibroblasts by 32%-82% when exposed to 0.5 nM insulin. After challenge with 120 µM of H2 O2 , sulfatide reduced membrane leakage. Fibroblast gene expression was altered by sulfatide in gene pathways associated with cell cycle/growth, transforming growth factor-ß function, and encoding of proteins involved in intracellular signalling. NFKBIA, a key control element in NF-кB regulation, was decreased 2-fold by sulfatide. CONCLUSIONS: Sulfatide strongly inhibits fibroblast growth. We therefore suggest the addition of sulfatide to injectable commercial insulin formulations, which would reduce adverse fibroblast growth and improve well-being in patients with diabetes.

Ogilvie Syndrome in Patients With Traumatic Pelvic and/or Acetabular Fractures: A Retrospective Cohort Study.

OBJECTIVES: To assess the incidence, risk factors, and clinical outcomes of Ogilvie syndrome (OS) in patients with pelvic and/or acetabular fractures. DESIGN: Retrospective cohort study. SETTING: Level 1 trauma center. PATIENTS: One thousand sixty patients with pelvic and/or acetabular fractures treated at Rigshospitalet, Copenhagen, between 2009 and 2020. INTERVENTION: Interventions comprised the treatment of pelvic and/or acetabular fractures with emergency external and/or internal fixation. MAIN OUTCOME MEASUREMENTS: Outcomes included diagnosis of OS, perioperative complications, ICU stay and length, length of admission, and mortality. RESULTS: We identified 1060 patients with pelvic and/or acetabular fractures. Of these, 25 patients were diagnosed with OS perioperatively, corresponding to incidences of 1.6%, 2.7%, and 2.6% for acetabular, pelvic, and combined fractures, respectively. Risk factors included congestive heart failure, diabetes, concomitant traumatic lesions, head trauma, fractures of the cranial vault and/or basal skull, retroperitoneal hematomas and spinal cord injuries, and emergency internal fixation and extraperitoneal packing. Six (24%) patients underwent laparotomy, and all patients had ischemia or perforation of the cecum for which right hemicolectomy was performed. Ogilvie syndrome was associated with a significant increase in nosocomial infections, sepsis, pulmonary embolism, ICU stay, and prolonged hospital admission. CONCLUSION: Ogilvie syndrome in patients with pelvic and/or acetabular fractures is associated with increased risk of perioperative complications and prolonged hospital and ICU stays, resulting in an increased risk of morbidity and mortality. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Lower urinary tract injuries in patients with pelvic fractures at a level 1 trauma center - an 11-year experience.

BACKGROUND: Urological injuries can occur in patients with pelvic fractures. Treatment recommendations lack solid evidence and is often pragmatical. There is a continuous need to describe short- and long-term morbidity following lower urinary tract trauma. OBJECTIVE: To describe incidence, diagnosis, treatment, and morbidity following lower urinary tract injuries in pelvic fractures. PATIENTS AND METHODS: Retrospective study including patients with pelvic, including acetabular, fractures admitted to a Level I Trauma Centre covering 2.8 million citizens between 2009 and 2020. Outcome measurements comprised primary management, treatment trajectory, short- and long-term complications and outcomes. RESULTS: A total of 39 (5%) patients with pelvic fractures had concomitant urethral and/or bladder injuries, and one patient with an acetabular fracture had a bladder injury. The management of urethral injuries varied vastly, and complete urethral ruptures were associated with severe short- and long-term complications. Only one patient with bladder injury experienced severe long-term complications. CONCLUSIONS: Management of lower urinary tract injuries in patients with major pelvic fractures remains a major challenge. Special attention should be focused on urethral injuries where we uncovered an unsystematic treatment and follow-up even in a highly experienced centre, although this is also attributed to complicated multidisciplinary patient trajectories. There is a continuous need to reduce long-term complications following urethral trauma which should be addressed in multicenter studies.

[Crocodile bites].

Crocodile bites are one of the most powerful bites of any creature. Bite wounds are usually deep, with severe tissue damage, frequently contaminated with unusual microorganisms, and are difficult to treat. This case report describes the treatment of a middle-aged man admitted after a crocodile attack to the left distal end of humerus, elbow and proximal forearm. He was successfully treated with empirical broad-spectrum antibiotics and repeated wound debridement. The patient was discharged after nine days of intensive care at a level 1 trauma unit, good outcomes were seen eight months later.

Fatal SARS-CoV-2-Associated Panton-Valentine Leukocidin-producing Staphylococcal Bacteremia: A Nationwide Multicenter Cohort Study.

We reviewed all cases of Panton-Valentine leukocidin-producing Staphylococcus aureus (PVL-SA) bacteremia in Danish children between 2016 and 2021. We found 2 fatal cases with preceding viral prodrome due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given the usual benign course of SARS-CoV-2 infection in children, awareness of possible superinfection with PVL-SA in a child with rapid deterioration is crucial to ensure adequate treatment, including antimicrobial drugs with antitoxin effect.

Immunity to vaccine-preventable diseases among paediatric healthcare workers in Denmark, 2019.

BackgroundHealthcare workers (HCW) have been identified as index cases in disease outbreaks of vaccine-preventable diseases (VPD) in hospitals.AimWe investigated whether Danish paediatric HCW were protected against selected serious VPD.MethodsWe included 90% of staff members from two paediatric departments. All 555 HCW (496 women) supplied a blood sample for serology and filled in a questionnaire. Antibodies were measured with enzyme immunoassay against measles, mumps, rubella (MMR), varicella zoster, pertussis toxin and diphtheria toxin.ResultsProtective levels of IgG were found for measles (90.3%), mumps (86.5%), rubella (92.3%), varicella (98.6%) and diphtheria (80.5%). We found seropositivity for all three MMR components in 421 (75.9%) HCW, lowest in those younger than 36 years (63.3%). Only 28 (5%) HCW had measurable IgG to pertussis. HCW with self-reported immunity defined as previous infection or vaccination, had protective levels of IgG against measles, mumps, rubella and varicella in 87.4-98.8% of cases, not significantly higher than in those not reporting immunity. Previous history of disease had a high positive predictive value (PPV) of 96.8-98.8%. The PPV for previous vaccination ranged from 82.5% to 90.3%. In contrast, negative predictive values of self-reported history of disease and vaccination were remarkably low for all diseases.ConclusionThe immunity gaps found primarily in young HCW indicate a need for a screening and vaccination strategy for this group. Considering the poor correlation between self-reported immunity and seropositivity, efforts should be made to check HCW's immune status in order to identify those who would benefit from vaccination.

Self-reported immunity and opinions on vaccination of hospital personnel among paediatric healthcare workers in Denmark.

BACKGROUND: Denmark has no general recommendations for vaccination of healthcare workers (HCWs). We explored the self-reported immunity to varicella, measles, mumps, and rubella, reasons for receiving the influenza vaccine or not, and opinions on vaccination of HCWs against varicella, MMR, pertussis, diphtheria, and influenza among staff from departments with a high risk of exposure to infectious agents. METHODS: From May 2019 to August 2019, a structured questionnaire was distributed to clinical and non-clinical HCWs at a tertiary and a general paediatric department in Denmark. Self-reported immunity was defined as either previous infection or vaccination against the disease. RESULTS: Of 619 employed HCWs, 555 (90%) were included. A large proportion were unsure of or denied previous vaccination or infection with measles (20.1%), mumps (30.2%), rubella (21.4%), varicella (12.1%), pertussis (44.1%), and diphtheria (32.1%). Non-clinical personnel and employees born in 1974-1983 had the lowest level of self-reported immunity. Mandatory vaccination of non-immune HCWs was approved by 54-68.9% of participants, and any kind of vaccination (mandatory or as an offer at hospitals) was approved of up to 95.3% of all participants depending on the disease. During the season 2018/19, 214 (38.6%) HCWs received the influenza vaccine, including 20.3% of non-clinical staff, 34.8% of nurses and 56.5% of doctors (P < 0.001). Reasons for lack of vaccine uptake were mainly employees considering themselves rarely sick, the vaccine was not regarded as necessary, forgetfulness or lack of time. Only 37.8% was in favour of mandatory influenza vaccination. CONCLUSIONS: A large proportion of paediatric HCWs were not aware of their immune status against important vaccine-preventable diseases. >90% supported vaccination of HCWs, with two out of three supporting mandatory MMR, pertussis and diphtheria vaccination. Better information and an official immunisation policy of non-immune HCWs in Denmark is warranted.

Clinical Manifestations in Children with Staphylococcal Bacteremia Positive for Panton-Valentine Leucocidin: A Nationwide Survey in a Low Methicillin-Resistance Setting.

A total of 714 pediatric cases of Staphylococcus aureus bacteremia were identified from 2008 to 2015 in Denmark; 98% were methicillin-susceptible S. aureus (MSSA). Fifteen isolates (2,1%) were Panton-Valentine leucocidin positive (0.17/100,000 children/year) and 87% MSSA. Eight cases (53%) were severe, including all pneumonia cases. Panton-Valentine leucocidin positive Staphylococcus aureus bacteremia is rare in our setting with high MSSA-prevalence. Half of the cases were uncomplicated.

Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase.

BACKGROUND: PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. RESULTS: In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. CONCLUSIONS: In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA-amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.

Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins.

The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omics" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.

[Rickets and anaemia caused by coeliac disease in a three-year-old girl].

We present a case report of a three-year-old girl of Pakistani origin with coeliac disease. The first symptom was spontaneous fracture of the fibula. Vitamin D deficiency rickets and iron deficiency anaemia were demonstrated. An intestinal biopsy and elevated antibody levels confirmed the coeliac disease. Rickets is a known but rare presentation of coeliac disease, but this case report illustrates, how coeliac disease in children often presents with non-gastrointestinal symptoms. The importance of investigations for malabsorp-tion, if iron deficiency is not easily corrected with sufficient iron supplements, is underlined as well as the importance of screening for coeliac disease.