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OBJECTIVES: To study the application value of metagenomic next-generation sequencing (mNGS) in children with severe infectious diseases. METHODS: An analysis was performed on the clinical data and laboratory test results of 29 children with severe infection who were admitted to the Second Affiliated Hospital of Wenzhou Medical University from June 2018 to December 2020. Conventional pathogen culture was performed for the 29 specimens (27 peripheral blood specimens and 2 pleural effusion specimens) from the 29 children, and mNGS pathogen detection was performed at the same time. RESULTS: Among the 29 children, 2 tested positive by conventional pathogen culture with 2 strains of pathogen, and the detection rate was 7% (2/29); however, 20 children tested positive by mNGS with 38 strains of pathogen, and the detection rate was 69% (20/29). The pathogen detection rate of mNGS was significantly higher than that of conventional pathogen culture (P<0.05), and mNGS could detect the viruses, fungi, and other special pathogens that conventional pathogen culture failed to detect, such as Orientia tsutsugamushi. The univariate analysis showed that gender, routine blood test results, C-reactive protein, procalcitonin, D-dimer, radiological findings, and whether antibiotics were used before admission did not affect the results of mNGS (P>0.05). CONCLUSIONS: Compared with conventional pathogen culture, mNGS is more sensitive for pathogen detection, with fewer interference factors. Therefore, it is a better pathogenic diagnosis method for severe infectious diseases in children.

Elevated IL-33 promotes expression of MMP2 and MMP9 via activating STAT3 in alveolar macrophages during LPS-induced acute lung injury.

BACKGROUND: Pulmonary inflammation and endothelial barrier permeability increase in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) induced by pro-inflammatory cytokines and matrix metalloproteinases (MMPs). However, the relationship between pro-inflammatory cytokines and MMPs in ALI/ARDS remains poorly understood. METHODS: A lipopolysaccharide (LPS)-induced ALI rat model was established through intratracheal instillation. The wet/dry ratios of lung tissues were measured, and bronchoalveolar lavage fluid (BALF) was collected to test protein concentrations, total cell/macrophage numbers, and pro-inflammatory cytokine levels. LPS-treated alveolar macrophages were utilized in in vitro experiments. The expression and secretion of MMPs were respectively detected using quantitative PCR, Western blotting and ELISA assays. RESULTS: The levels of IL-33 and MMP2/9 in BALF increased in all the ALI rats with severe lung injury. LPS-induced IL-33 autocrine upregulated the expression of MMP2 and MMP9 through activating STAT3. Neutralizing IL-33 in culture medium with specific antibodies suppressed the expression and secretion of MMP2 and MMP9 in LPS-treated alveolar macrophages. Consistently, eliminating IL-33 decreased the levels of MMP2 and MMP9 in BALF and alleviated lung injury in ALI rats. CONCLUSION: The IL-33/STAT3/MMP2/9 regulatory pathway is activated in alveolar macrophages during acute lung injury, which may exacerbate the pulmonary inflammation.

[Benign infantile convulsions associated with mild gastroenteritis: a clinical analysis and follow-up study].

OBJECTIVE: To study the clinical features and prognosis of benign infantile convulsions associated with mild gastroenteritis (BICE). METHODS: A retrospective analysis was performed for the clinical data of 436 children with BICE, and among these children, 206 were followed up for 1.5 to 7 years. Some parents were invited to complete the Weiss Functional Defect Scale to evaluate the long-term social function. RESULTS: The peak age of onset of BICE was 13-24 months, and BICE had a higher prevalence rate in September to February of the following year. Convulsions mainly manifested as generalized tonic-clonic seizures, which often occurred within 24 hours after disease onset and lasted for less than 5 minutes each time. Sometimes they occurred in clusters. During the follow-up of 206 children, only one had epileptiform discharge, and the other children had normal electroencephalographic results. The parents of all the 206 children thought their children had normal intelligence and had no marked changes in character. Based on the Weiss Functional Defect Scale completed by the parents of some BICE children, there was no significant difference in the long-term social function between BICE children and healthy children matched by age and sex. CONCLUSIONS: BICE mainly occurs in children aged 1-2 years, with the manifestation of transient generalized seizures in most children and cluster seizures in some children. BICE seldom progresses to epilepsy and has good prognosis.

Application of Different Sampling Methods Combined with Metagenomic Next-Generation Sequencing to Detect Pathogens in Children with Severe Pneumonia on Mechanical Ventilation.

Objective: The aim of this study was to explore the value of applying different sampling methods combined with metagenomic next-generation sequencing (mNGS) to detect pathogens in children with severe pneumonia on mechanical ventilation. Methods: Forty children with severe pneumonia on mechanical ventilation were selected, and routine endotracheal suctioning and bronchoalveolar lavage fluid (BALF) sampling methods were performed. The diagnostic efficacy of different sampling methods combined with mNGS versus traditional etiological pathogen detection strategies was compared. Results: The positive rate of mNGS pathogen detection after routine endotracheal suctioning and BALF sampling was higher than that of traditional etiological detection strategies (P < 0.05). There was no significant difference in the positive rates of pathogen detection by routine endotracheal suctioning + mNGS and BALF + mNGS (P > 0.05). Conclusion: Compared with traditional etiological detection strategies, mNGS is more efficient for diagnosing pathogens. In clinical practice, an appropriate sampling method should be selected for mNGS-based detection according to the condition of the patient. These findings could be of great importance in the diagnosis and treatment of severe pneumonia.

Clinical characteristics and prognostic analysis of acute necrotizing encephalopathy of childhood: a retrospective study at a single center in China over 3 years.

Objective: Acute Necrotizing Encephalopathy of Childhood (ANEC) is a rare, fulminant neurological disease in children with unknown mechanisms and etiology. This study summarized the clinical characteristics, treatment, and prognosis of ANEC through a retrospective analysis, providing insights into the ANEC early diagnosis and prognosis assessment. Methods: Clinical data of children diagnosed with ANEC at the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University from July 1, 2020, to June 30, 2023, were retrospectively analyzed. Results: There were 25 cases, 14 males and 11 females, with a median age of 3 years. Hospital admissions were mainly in the winter (14/25, 56%) and spring (9/25, 36%). All patients presented with varying degrees of fever and altered consciousness, with 92% (23/25) experiencing high body temperatures (>39.1°C) and 88% (22/25) having a Glasgow coma scale (GCS) score of ≤8. Seizures were observed in 88% (22/25) of patients. Laboratory findings indicated 100% B lymphocyte activation (14/14), and 78% (14/18) of patients showed cytokine storm (interleukin (IL)-6, IL-8, IL-10, interferon (IFN)-α). Neuroimaging showed symmetrical thalamus involvement, commonly involving basal ganglia and brainstem regions. Viral infection (23/24, 96%) was the predominant etiological finding, with 42% (10/24) of cases due to SARS-CoV-2 infection and 42% (10/24) to influenza A virus infection. Multi-organ dysfunction occurred in 68% (17/25) of patients, and 52% (13/25) died. Correlation analysis revealed the death group exhibited higher proportion of male, lower GCS scores, higher IL-6 level and a greater likelihood of associated brainstem impairment (p < 0.05). Conclusion: ANEC is more prevalent in the winter and spring, and its etiology may be associated with B lymphocyte activation and cytokine storm following viral infections. Clinical manifestations lack specific features, with fever, consciousness disturbances, and seizures being the main presentations, particularly in cases of high fever and hyperpyrexia. ANEC progresses rapidly and has a high mortality rate. The child's gender, GCS score, IL-6 levels, and the presence of brainstem involvement can serve as important risk factors for assessing the risk of mortality.

Application Value of Metagenomic Next-Generation Sequencing for Bloodstream Infections in Pediatric Patients Under Intensive Care.

Purpose: This study aimed to analyze the application value of metagenomic next-generation sequencing (mNGS) as a basis for the proper adjustment of the clinical treatment of bloodstream infections (BSIs) in pediatric patients under intensive care. Methods: We retrospectively enrolled 46 pediatric patients with clinically diagnosed BSIs who were hospitalized in the pediatric intensive care unit of the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University from June 2018 to July 2021. Blood samples were collected for cultivation and for mNGS detection of pathogens. Results: Among the 46 children, the average turnaround time for blood culture tests was 3.2 days, and the results revealed pathogens in three children (6.5%). The average turnaround time for mNGS was 2.2 days, and pathogens were found in 30 children (65.2%). The difference in positivity rates between blood culture and mNGS was significant (p<0.05). Blood culture tests found three pathogens, while mNGS identified 28 pathogens, indicating that mNGS detected significantly more types of pathogens than the traditional diagnostic method for pathogenic microorganisms. In some children, more than one pathogen was detected. Conclusion: mNGS can help identify pathogenic microorganisms associated with BSI in some pediatric patients under intensive care.

Activation of the IL-1ß/KLF2/HSPH1 pathway promotes STAT3 phosphorylation in alveolar macrophages during LPS-induced acute lung injury.

Acute lung injury (ALI) is a lethal disease with diffuse lung inflammation, in which JAK/STAT3 signaling has been well recognized for its role in initiating and amplifying inflammatory processes. However, the mechanism for the enhancement and maintenance of signal transducer and activator of transcription 3 (STAT3) activation has not yet been clearly demonstrated in ALI. In the present work, we established a lipopolysaccharide (LPS)-induced ALI rat model through intratracheal instillation and isolated the alveolar macrophages (AMs) from the rats in the model. We demonstrated that the expression of Kruppel-like factor 2 (KLF2) significantly decreased in the AMs from LPS-induced ALI rats (LPS-AMs) as compared with the AMs from control rats (NC-AMs). Overexpressing KLF2 in LPS-AMs inhibited the phosphorylation of STAT3 and reduced the levels of STAT3 target genes, including matrix metalloproteinase (MMP)-2/9 (MMP-2/9). Further investigation indicated that KLF2 trans-inhibited heat shock protein H1 (HSPH1), which interacted with STAT3 and enhanced its phosphorylation. As a crucial inflammatory mediator in ALI, interleukin-1ß (IL-1ß) induced the down-regulation of KLF2 in LPS-AMs, as interrupting IL-1ß signaling in LPS-AMs by antibody neutralization or IL1R1 knockdown rescued the expression of KLF2. Consistently, stimulating NC-AMs with IL-1ß decreased KLF2 and increased HSPH1, while overexpression of KLF2 suppressed IL-1ß-induced HSPH1. Additionally, in vivo studies showed that treatment with an IL-1ß antibody or HSPH1 inhibitor alleviated lung injury in ALI rats, as well as decreased the levels of p-STAT3 and MMP-2/9. In conclusion, activation of the IL-1ß/KLF2/HSPH1 pathway facilitated STAT3 phosphorylation in AMs, which exacerbated pulmonary inflammation in ALI.

Performance of Three Mortality Prediction Scores and Evaluation of Important Determinants in Eight Pediatric Intensive Care Units in China.

Background: The mortality prediction scores were widely used in pediatric intensive care units. However, their performances were unclear in Chinese patients and there were also no reports based on large sample sizes in China. This study aims to evaluate the performances of three existing severity assessment scores in predicting PICU mortality and to identify important determinants. Methods: This prospective observational cohort study was carried out in eight multidisciplinary, tertiary-care PICUs of teaching hospitals in China. All eligible patients admitted to the PICUs between Aug 1, 2016, and Jul 31, 2017, were consecutively enrolled, among whom 3,957 were included for analysis. We calculated PCIS, PRISM IV, and PELOD-2 scores based on patient data collected in the first 24 h after PICU admission. The in-hospital mortality was defined as all-cause death within 3 months after admission. The discrimination of mortality was assessed using the area under the receiver-operating characteristics curve (AUC) and calibrated using the Hosmer-Lemeshow goodness-of-fit test. Results: A total of 4,770 eligible patients were recruited (median age 18.2 months, overall mortality rate 4.7%, median length of PICU stay 6 days), and 3,957 participants were included in the analysis. The AUC (95% confidence intervals, CI) were 0.74 (0.71-0.78), 0.76 (0.73-0.80), and 0.80 (0.77-0.83) for PCIS, PRISM IV, and PELOD-2, respectively. The Hosmer-Lemeshow test gave a chi-square of 3.16 for PCIS, 2.16 for PRISM IV and 4.81 for PELOD-2 (p ≥ 0.19). Cox regression identified five predictors from the items of scores better associated with higher death risk, with a C-index of 0.83 (95%CI 0.79-0.86), including higher platelet (HR = 1.85, 95% CI 1.59-2.16), invasive ventilation (HR = 1.40, 1.26-1.55), pupillary light reflex (HR = 1.31, 95% CI 1.22-1.42) scores, lower pH (HR 0.89, 0.84-0.94), and extreme PaO2 (HR 2.60, 95% CI 1.61-4.19 for the 1st quantile vs. 4th quantile) scores. Conclusions: Performances of the three scores in predicting PICU mortality are comparable, and five predictors were identified with better prediction to PICU mortality in Chinese patients.

Blockage of thymic stromal lymphopoietin signaling improves acute lung injury in mice by regulating pulmonary dendritic cells.

OBJECTIVES: To investigate the effects of blockage of thymic stromal lymphopoietin (TSLP) signaling by TSLP receptor (TSLPR)-immunoglobulin (Ig) on acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: C57BL/6 mice received TSLPR-Ig or controlled-Ig before being induced ALI. Lung wet/dry (W/D) weight ratio was recorded. Neutrophil number and albumin concentration of bronchoalveolar lavages fluids (BALF) were determined. Besides, bone marrow dendritic cells (BMDCs) were separated and cultured with medium, TSLP, TSLP plus TSLPR-Ig or TSLP plus controlled-Ig. Protein expression levels of TSLP in lung tissues, phosphorylation extracellular regulated protein kinases (pERK) 1/2, p38, and signal transducers and activators of transcription (STAT) 3 in BMDCs were analyzed using Western blotting. Expression of CD40, CD80 and CD86 on pulmonary DCs and BMDCs was determined using flow cytometry (FCM). RESULTS: The W/D ratio, neutrophil number and albumin concentration were significantly decreased in the TSLPR-Ig group compared with the controlled-Ig and model group. Moreover, there was a noticeable decrease in CD40, CD80 or CD86 expression by TSLPR-Ig on both pulmonary DCs and BMDCs. The protein levels of TSLP, pERK1 and STAT3 were significantly decreased by TSLPR-Ig. However, no significant differences were found in p38 and pERK2. CONCLUSION: These results suggest that TSLP may be involved in ALI, and blockage of TSLP signaling using TSLPR-Ig improves ALI at least in part by regulation of DCs functions. The underling downstream signaling mediated by TSLP might be associated with activating the ERK1 and STAT3 signaling pathway.

[A multicenter prospective clinical study on continuous blood purification in treating childhood severe sepsis].

OBJECTIVE: To evaluate efficacy of continuous blood purification (CBP) in childhood severe sepsis through the analysis of organ function, inflammatory mediators and prognosis. METHOD: Forty-seven children with severe sepsis aged 29 days -16 years who were treated in PICU of Shanghai and Zhejiang five hospitals during October 1, 2011 and September 30, 2012 were enrolled; 30 cases treated with CBP were recorded as logged group , 17 cases without CBP as unlogged group. Changes in the cardiovascular, respiratory function, renal function, inflammatory markers, PRISM score III, PCIS and survival were observed and compared between the two groups at baseline (d0), first days (d1), second days (d2), third days (d3), fifth days (d5). RESULT: (1) Cardiovascular function: In d3 and d5, heart rate (HR) and mean arterial pressure (MAP) were improved as compared to unlogged group (121, 119 vs. 138, 137; 71, 80 mmHg vs. 63, 62 mmHg, P < 0.05), with no statistical significance in arterial blood lactate concentration. (2) Oxygenation index (PaO2/FiO2) and arterial oxygen saturation (SaO2) increased as compared to unlogged group, but did not reach statistical significance. (3) Blood urea nitrogen (BUN) and creatinine (Cr) were improved as compared with unlogged group from d1 (P < 0.05). (4) Inflammatory mediators did not show significant differences. (5) Twenty-eight days survival rate: logged group was 70.0%, unlogged group was 52.9%, but the difference was not statistically significant (P = 0.242). CONCLUSION: CBP can improve circulatory function, oxygenation, and renal function in children with severe sepsis. No evidence was found that CBP could decrease the level of inflammatory mediators, improve critical score and 28 days survival rate.