Evaluation of the antigen-experienced B-cell receptor repertoire in healthy children and adults

Upon antigen recognition via their B cell receptor (BR), B cells migrate to the germinal center where they undergo somatic hypermutation (SHM) to increase their affinity for the antigen, and class switch recombination (CSR) to change the effector function of the secreted antibodies. These steps are essential to create an antigen-experienced BR repertoire that efficiently protects the body against pathogens. At the same time, the BR repertoire should be selected to protect against responses to self-antigen or harmless antigens. Insights into the processes of SHM, selection, and CSR can be obtained by studying the antigen-experienced BR repertoire. Currently, a large reference data set of healthy children and adults, which ranges from neonates to the elderly, is not available. In this study, we analyzed the antigen-experienced repertoire of 38 healthy donors (HD), ranging from cord blood to 74 years old, by sequencing IGA and IGG transcripts using next generation sequencing. This resulted in a large, freely available reference data set containing 412,890 IGA and IGG transcripts. We used this data set to study mutation levels, SHM patterns, antigenic selection, and CSR from birth to elderly HD. Only small differences were observed in SHM patterns, while the mutation levels increase in early childhood and stabilize at 6 years of age at around 7%. Furthermore, comparison of the antigen-experienced repertoire with sequences from the naive immune repertoire showed that features associated with autoimmunity such as long CDR3 length and IGHV4-34 usage are reduced in the antigen-experienced repertoire. Moreover, IGA2 and IGG2 usage was increased in HD in higher age categories, while IGG1 usage was decreased. In addition, we studied clonal relationship in the different samples. Clonally related sequences were found with different subclasses. Interestingly, we found transcripts with the same CDR1-CDR3 sequence, but different subclasses. Together, these data suggest that a single antigen can provoke a B-cell response with BR of different subclasses and that, during the course of an immune response, some B cells change their isotype without acquiring additional SHM or can directly switch to different isotypes

Translating Sepsis-3 Criteria in Children: Prognostic Accuracy of Age-Adjusted Quick SOFA Score in Children Visiting the Emergency Department With Suspected Bacterial Infection

Background: Recent attempts to translate Sepsis-3 criteria to children have been restricted to PICU patients and did not target children in emergency departments (ED). We assessed the prognostic accuracy of the age-adjusted quick Sequential Organ Failure Assessment score (qSOFA) and compared the performance to SIRS and the quick Pediatric Logistic Organ Dysfunction-2 score (qPELOD-2). We studied whether the addition of lactate (qSOFA-L) would increase prognostic accuracy.Methods: Non-academic, single-center, retrospective study in children visiting the ED and admitted with suspected bacterial infection between March 2013 and January 2018. We defined suspected bacterial infection as initiation of antibiotic therapy within 24 h after ED entry. Age-adjusted qSOFA, SIRS, qPELOD-2, and qSOFA-L scores were compared by area under the receiver operating characteristics curve (AUROC) analysis. Primary outcome measure was PICU transfer and/or mortality and secondary outcome was prolonged hospital length of stay.Results: We included 864 ED visits [474 (55%) male; median age 2.5 years; IQR 9 months-6 years], of which 18 were transferred to a PICU and 6 ended in death [composite outcome PICU transfer and/or mortality; 23 admissions (2.7%)]. 179 (22.2%) admissions resulted in prolonged hospital length of stay. PICU transfer and/or death was present in 22.5% of visits with qSOFA≥2 (n = 40) compared to 2.0% of visits with qSOFA<2 (n = 444) (p < 0.01). qSOFA tends to be the best predictor of PICU transfer and/or mortality (AUROC 0.72 (95% CI, 0.57–0.86) compared to SIRS [0.64 (95% CI, 0.53–0.74), p = 0.23] and qPELOD-2 [0.60 (95% CI, 0.45–0.76), p = 0.03)]. Prolonged hospital length of stay was poorly predicted by qSOFA (AUROC 0.53, 95% CI 0.46–0.59), SIRS (0.49, 95% CI 0.44–0.54), and qPELOD-2 (0.51, 95%CI 0.45–0.57). qSOFA-L resulted in an AUROC of 0.67 (95% CI, 0.50–0.84) for PICU transfer and/or mortality and an AUROC of 0.56 (95% CI, 0.46–0.67) for prolonged hospital length of stay.Conclusion: The currently proposed bedside risk-stratification tool of Sepsis-3 criteria, qSOFA, shows moderate prognostic accuracy for PICU transfer and/or mortality in children visiting the ED with suspected bacterial infection. The addition of lactate did not improve prognostic accuracy. Future prospective studies in larger ED populations are needed to further determine the utility of the qSOFA score

Evaluation of a clinical decision rule to guide antibiotic prescription in children with suspected lower respiratory tract infection in The Netherlands

BACKGROUND: Optimising the use of antibiotics is a key component of antibiotic stewardship. Respiratory tract infections (RTIs) are the most common reason for antibiotic prescription in children, even though most of these infections in children under 5 years are viral. This study aims to safely reduce antibiotic prescriptions in children under 5 years with suspected lower RTI at the emergency department (ED), by implementing a clinical decision rule. METHODS AND FINDINGS: In a stepped-wedge cluster randomised trial, we included children aged 1-60 months presenting with fever and cough or dyspnoea to 8 EDs in The Netherlands. The EDs were of varying sizes, from diverse geographic and demographic regions, and of different hospital types (tertiary versus general). In the pre-intervention phase, children received usual care, according to the Dutch and NICE guidelines for febrile children. During the intervention phase, a validated clinical prediction model (Feverkidstool) including clinical characteristics and C-reactive protein (CRP) was implemented as a decision rule guiding antibiotic prescription. The intervention was that antibiotics were withheld in children with a low or intermediate predicted risk of bacterial pneumonia (≤10%, based on Feverkidstool). Co-primary outcomes were antibiotic prescription rate and strategy failure. Strategy failure was defined as secondary antibiotic prescriptions or hospitalisations, persistence of fever or oxygen dependency up to day 7, or complications. Hospitals were randomly allocated to 1 sequence of treatment each, using computer randomisation. The trial could not be blinded. We used multilevel logistic regression to estimate the effect of the intervention, clustered by hospital and adjusted for time period, age, sex, season, ill appearance, and fever duration; predicted risk was included in exploratory analysis. We included 999 children (61% male, median age 17 months [IQR 9 to 30]) between 1 January 2016 and 30 September 2018: 597 during the pre-intervention phase and 402 during the intervention phase. Most children (77%) were referred by a general practitioner, and half of children were hospitalised. Intention-to-treat analyses showed that overall antibiotic prescription was not reduced (30% to 25%, adjusted odds ratio [aOR] 1.07 [95% CI 0.57 to 2.01, p = 0.75]); strategy failure reduced from 23% to 16% (aOR 0.53 [95% CI 0.32 to 0.88, p = 0.01]). Exploratory analyses showed that the intervention influenced risk groups differently (p < 0.01), resulting in a reduction in antibiotic prescriptions in low/intermediate-risk children (17% to 6%; aOR 0.31 [95% CI 0.12 to 0.81, p = 0.02]) and a non-significant increase in the high-risk group (47% to 59%; aOR 2.28 [95% CI 0.84 to 6.17, p = 0.09]). Two complications occurred during the trial: 1 admission to the intensive care unit during follow-up and 1 pleural empyema at day 10 (both unrelated to the study intervention). Main limitations of the study were missing CRP values in the pre-intervention phase and a prolonged baseline period due to logistical issues, potentially affecting the power o

Educational paper: Primary immunodeficiencies in children: a diagnostic challenge

Primary immunodeficiencies (PIDs) are characterized by an increased susceptibility to infections due to defects in one ore more components of the immune system. Although most PIDs are relatively rare, they are more frequent than generally acknowledged. Early diagnosis and treatment of PIDs save lives, prevent morbidity, and improve quality of life. This early diagnosis is the task of the pediatrician who encounters the child for the first time: he/she should suspect potential PID in time and perform the appropriate diagnostic tests. In this educational paper, the first in a series of five, we will describe the most common clinical presentations of PIDs and offer guidelines for the diagnostic process, as well as a brief overview of therapeutic possibilities and prognosis

Technical validity and usability of a novel smartphone-connected spirometry device for pediatric patients with asthma and cystic fibrosis

Background: Diagnosis and follow-up of respiratory diseases traditionally rely on pulmonary function tests (PFTs), which are currently performed in hospitals and require trained personnel. Smartphone-

Repertoire sequencing of B cells elucidates the role of UNG and mismatch repair proteins in somatic hypermutation in humans

The generation of high-affinity antibodies depends on somatic hypermutation (SHM). SHM is initiated by the activation-induced cytidine deaminase (AID), which generates uracil (U) lesions in the B-cell receptor (BCR) encoding genes. Error-prone processing of U lesions creates a typical spectrum of point mutations during SHM. The aim of this study was to determine the molecular mechanism of SHM in humans; currently available knowledge is limited by the number of mutations analyzed per patient. We collected a unique cohort of 10 well-defined patients with bi-allelic mutations in genes involved in base excision repair (BER) (UNG) or mismatch repair (MMR) (MSH2, MSH6, or PMS2) and are the first to present next-generation sequencing (NGS) data of the BCR, allowing us to study SHM extensively in humans. Analysis using ARGalaxy revealed selective skewing of SHM mutation patterns specific for each genetic defect, which are in line with the five-pathway model of SHM that was recently proposed based on mice data. However, trans-species comparison revealed differences in the role of PMS2 and MSH2 in strand targeting between mice and man. In conclusion, our results indicate a role for UNG, MSH2, MSH6, and PMS2 in the generation of SHM in humans comparable to their function in mice. However, we observed differences in strand targeting between humans and mice, emphasizing the importance of studying molecular mechanisms in a human setting. The here developed method combining NGS and ARGalaxy analysis of BCR mutation data forms the basis for efficient SHM analyses of other immune deficiencies

Detectable A Disintegrin and Metalloproteinase With Thrombospondin Motifs-1 in Serum Is Associated With Adverse Outcome in Pediatric Sepsis.

ImportanceA Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 is hypothesized to play a role in the pathogenesis of invasive infection, but studies in sepsis are lacking.ObjectivesTo study A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 protein level in pediatric sepsis and to study the association with outcome.DesignData from two prospective cohort studies.Setting and participantsCohort 1 is from a single-center study involving children admitted to PICU with meningococcal sepsis (samples obtained at three time points). Cohort 2 includes patients from a multicenter study involving children admitted to the hospital with invasive bacterial infections of differing etiologies (samples obtained within 48 hr after hospital admission).Main outcomes and measuresPrimary outcome measure was mortality. Secondary outcome measures were PICU-free days at day 28 and hospital length of stay.ResultsIn cohort 1 (n = 59), nonsurvivors more frequently had A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels above the detection limit than survivors at admission to PICU (8/11 [73%] and 6/23 [26%], respectively; p = 0.02) and at t = 24 hours (2/3 [67%] and 3/37 [8%], respectively; p = 0.04). In cohort 2 (n = 240), A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels in patients within 48 hours after hospital admission were more frequently above the detection limit than in healthy controls (110/240 [46%] and 14/64 [22%], respectively; p = 0.001). Nonsurvivors more often had detectable A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels than survivors (16/21 [76%] and 94/219 [43%], respectively; p = 0.003), which was mostly attributable to patients with Neisseria meningitidis.Conclusions and relevanceIn children with bacterial infection, detection of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 within 48 hours after hospital admission is associated with death, particularly in meningococcal sepsis. Future studies should confirm the prognostic value of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 and should study pathophysiologic mechanisms

Educational paper: Primary antibody deficiencies

Primary antibody deficiencies (PADs) are the most common primary immunodeficiencies and are characterized by a defect in the production of normal amounts of antigen-specific antibodies. PADs represent a heterogeneous spectrum of conditions, ranging from often asymptomatic selective IgA and IgG subclass deficiencies to the severe congenital agammaglobulinemias, in which the antibody production of all immunoglobulin isotypes is severely decreased. Apart from recurrent respiratory tract infections, PADs are associated with a wide range of other clinical complications. This review will describe the pathophysiology, diagnosis, and treatment of the different PADs

Educational paper: The expanding clinical and immunological spectrum of severe combined immunodeficiency

Severe combined immunodeficiency (SCID) is one of the most severe forms of primary immunodeficiency characterized by absence of functional T lymphocytes. It is a paediatric emergency, which is life-threatening when recognized too late. The clinical presentation varies from the classical form of SCID through atypical SCID to Omenn syndrome. In addition, there is a considerable immunological variation, which can hamper the diagnosis. In this educational review, we describe the immunopathological background, clinical presentations and diagnostic process of SCID, as well as the therapeutic possibilities