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Objectives: Acute bronchiolitis is a major public health issue with high number of infants hospitalised worldwide each year. In France, hospitalisations mostly occur between October and March and peak in December. A reduction of emergency visits for bronchiolitis has been observed at onset of the COVID-19 outbreak. We aimed to assess the pandemic effects on the hospitalisations for bronchiolitis during the 2020-2021 winter (COVID-19 period) compared with three previous winters (pre-COVID-19)., Design: Retrospective, observational and cross-sectional study., Setting: Tertiary university paediatric hospital in Paris (France)., Participants: All infants aged under 12 months who were hospitalised for acute bronchiolitis during the autumn/winter seasons (1 October to 31 March) from 2017 to 2021 were included. Clinical and laboratory data were collected using standardised forms., Results: During the COVID-19 period was observed, a 54.3% reduction in hospitalisations for bronchiolitis associated with a delayed peak (February instead of November-December). Clinical characteristics and hospitalisation courses were substantially similar. The differences during the COVID-19 period were: smaller proportion of infants with comorbidities (8% vs 14% p=0.02), lower need for oxygen (45% vs 55%, p=0.01), higher proportions of metapneumovirus, parainfluenzae 3, bocavirus, coronavirus NL63 and OC43 (all p≤0.01) and no influenza. The three infants positive for SARS-CoV-2 were also positive for respiratory syncytial virus, suggesting that SARS-CoV-2 alone does not cause bronchiolitis, despite previous assumptions., Conclusion: The dramatic reduction in infants' hospitalisations for acute bronchiolitis is an opportunity to change our future habits such as advising the population to wear masks and apply additional hygiene measures in case of respiratory tract infections. This may change the worldwide bronchiolitis burden and improve children respiratory outcomes., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Aim: We aimed to investigate the performance of procalcitonin (PCT) assay between 12 and 36 h after onset of fever (PCT H12-H36) to predict invasive bacterial infection (IBI) (ie, meningitis and/or bacteremia) in febrile neonates., Methods: We retrospectively included all febrile neonates hospitalized in the general pediatric department in a teaching hospital from January 2013 to December 2019. PCT assay ≤ 0.6 ng/ml was defined as negative. The primary outcome was to study the performance of PCT H12-H36 to predict IBI., Results: Out of 385 included neonates, IBI was ascertainable for 357 neonates (92.7%). We found 16 IBI: 3 meningitis and 13 bacteremia. Sensitivity and specificity of PCT H12-H36 in the identification of IBI were, respectively, 100% [95% CI 82.9-100%] and 71.8% [95% CI 66.8-76.6%], with positive and negative predictive values of 14.3% [95% CI 8.4-22.2%] and 100% [95% CI 98.8-100%] respectively. Of the 259 neonates who had a PCT assay within the first 12 h of fever (< H12) and a PCT assay after H12-H36, 8 had IBI. Two of these 8 neonates had a negative < H12 PCT but a positive H12-H36 PCT., Conclusions: PCT H12-H36 did not miss any IBI whereas < H12 PCT could missed IBI diagnoses. PCT H12-H36 might be included in clinical decision rule to help physicians to stop early antibiotics in febrile neonates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Romain, Guedj, Chosidow, Mediamolle, Schnuriger, Vimont, Ferrandiz, Robin, Odièvre, Grimprel and Lorrot.)

Non-pharmaceutical interventions (NPIs) against coronavirus disease 2019 were implemented in March 2020. These measures were followed by a major impact on viral and non-viral diseases. We aimed to assess the impact of NPI implementation in France on hospitalized community-acquired pneumonia (hCAP) frequency and the clinical and biological characteristics of the remaining cases in children. We performed a quasi-experimental interrupted time-series analysis. Between June 2014 and December 2020, eight pediatric emergency departments throughout France reported prospectively all cases of hCAP in children from age 1 month to 15 years. We estimated the impact on the monthly number of hCAP using segmented linear regression with autoregressive error model. We included 2,972 hCAP cases; 115 occurred during the NPI implementation period. We observed a sharp decrease in the monthly number of hCAP after NPI implementation [-63.0% (95 confidence interval, -86.8 to -39.2%); p < 0.001]. Children with hCAP were significantly older during than before the NPI period (median age, 3.9 vs. 2.3 years; p < 0.0001), and we observed a higher proportion of low inflammatory marker status (43.5 vs. 33.1%; p = 0.02). Furthermore, we observed a trend with a decrease in the proportion of cases with pleural effusion (5.3% during the NPI period vs. 10.9% before the NPI; p = 0.06). NPI implementation during the COVID-19 (coronavirus disease 2019) pandemic led not only to a strong decrease in the number of hCAP cases but also a modification in the clinical profile of children affected, which may reflect a change in pathogens involved., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rybak, Ouldali, Angoulvant, Minodier, Biscardi, Madhi, Hau, Santos, Bouvy, Dubos, Martinot, Dommergues, Gras-Le Guen, Launay, Levieux, Zenkhri, Craiu, Lorrot, Gillet, Mezgueldi, Faye, Béchet, Varon, Cohen and Levy.)

Background: Multisystem inflammatory syndrome in children (MIS-C) is the most severe form associated with SARS-CoV-2 infection in children. To reduce the spread of SARS-CoV-2 at the population level, educational setting closure have been implemented in many countries. However, the direct benefit of school closure on the MIS-C burden remains to be explored. We aimed to assess the role of educational settings in SARS-CoV-2 transmission among children with MIS-C. Methods: We conducted a French national prospective surveillance of MIS-C, coordinated by Public Health France, from April 2020 to March 2021. During this period, we included all children with MIS-C fulfilling the WHO definition who were reported to Public Health France. For each child, we traced the source of SARS-CoV-2 transmission. The main outcome was the proportion of children with MIS-C, with educational setting-related SARS-CoV-2 infection, during the period of school opening. Results: We included 142 children fulfilling WHO criteria for MIS-C: 104 (70%) cases occurred during school opening periods. In total, 62/104 children (60%, 95%CI [50; 69]) had been contaminated by a household contact and 5/104 in educational settings (5%, 95%CI [2; 11]). Among children with MIS-C occurring during school closure periods, the proportion of household transmission remained similar (66%, 25/38). Conclusion: Children with MIS-C were mainly infected by SARS-CoV-2 within their family environment, and the educational setting played a marginal role in this transmission. This suggests that mitigating school attendance may not reduce substantially the burden of MIS-C., Competing Interests: EJ reported receiving grants from CSL Behring. CL reported receiving grants from GlaxoSmithKline, Merck Sharp & Dohme, and Sanofi and personal fees from Pfizer and Merck. RC reported receiving personal fees from GlaxoSmithKline, Pfizer, Sanofi, and Merck Sharp & Dohme. NO report travel grants from GSK, Pfizer and Sanofi, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Guenver, Oualha, Levy, Antona, Madhi, Toubiana, Lachaume, Javouhey, Lorrot, Yang, Levy, Caseris, Galeotti, Ovaert, Wiedemann, Girardin, Rybak, Cohen, Belot, Angoulvant and Ouldali.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children is generally milder than in adults, but a proportion of cases result in hyperinflammatory conditions often including myocarditis., Methods: To better understand these cases, we applied a multiparametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. Plasma cytokine and chemokine levels and blood cellular composition were measured, alongside gene expression at the bulk and single-cell levels., Findings: The most severe forms of multisystem inflammatory syndrome in children (MIS-C) related to SARS-CoV-2 that resulted in myocarditis were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomics analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis characterized by sustained nuclear factor κB (NF-κB) activity and tumor necrosis factor alpha (TNF-α) signaling and associated with decreased gene expression of NF-κB inhibitors. We also found a weak response to type I and type II interferons, hyperinflammation, and response to oxidative stress related to increased HIF-1α and Vascular endothelial growth factor (VEGF) signaling., Conclusions: These results provide potential for a better understanding of disease pathophysiology., Funding: Agence National de la Recherche (Institut Hospitalo-Universitaire Imagine, grant ANR-10-IAHU-01; Recherche Hospitalo-Universitaire, grant ANR-18-RHUS-0010; Laboratoire d'Excellence ''Milieu Intérieur," grant ANR-10-LABX-69-01; ANR-flash Covid19 "AIROCovid" and "CoVarImm"), Institut National de la Santé et de la Recherche Médicale (INSERM), and the "URGENCE COVID-19" fundraising campaign of Institut Pasteur., Competing Interests: D.D., F.R.-L., J.T., and M.M.M. are listed as inventors on a patent application related to this technology (European Patent Application no. EP21305197, entitled “Methods of predicting multisystem inflammatory syndrome [MIS-C] with severe myocarditis in subjects suffering from a SARS-CoV-2 infection”)., (© 2021 Elsevier Inc.)

BackgroundChildren have a low rate of COVID-19 and secondary severe multisystem inflammatory syndrome (MIS) but present a high prevalence of symptomatic seasonal coronavirus infections.AimWe tested if prior infections by seasonal coronaviruses (HCoV) NL63, HKU1, 229E or OC43 as assessed by serology, provide cross-protective immunity against SARS-CoV-2 infection.MethodsWe set a cross-sectional observational multicentric study in pauci- or asymptomatic children hospitalised in Paris during the first wave for reasons other than COVID (hospitalised children (HOS), n = 739) plus children presenting with MIS (n = 36). SARS-CoV-2 antibodies directed against the nucleoprotein (N) and S1 and S2 domains of the spike (S) proteins were monitored by an in-house luciferase immunoprecipitation system assay. We randomly selected 69 SARS-CoV-2-seropositive patients (including 15 with MIS) and 115 matched SARS-CoV-2-seronegative patients (controls (CTL)). We measured antibodies against SARS-CoV-2 and HCoV as evidence for prior corresponding infections and assessed if SARS-CoV-2 prevalence of infection and levels of antibody responses were shaped by prior seasonal coronavirus infections.ResultsPrevalence of HCoV infections were similar in HOS, MIS and CTL groups. Antibody levels against HCoV were not significantly different in the three groups and were not related to the level of SARS-CoV-2 antibodies in the HOS and MIS groups. SARS-CoV-2 antibody profiles were different between HOS and MIS children.ConclusionPrior infection by seasonal coronaviruses, as assessed by serology, does not interfere with SARS-CoV-2 infection and related MIS in children.

Acute arthritis is a common cause of consultation in pediatric emergency wards. Arthritis can be caused by juvenile idiopathic arthritis (JIA), septic (SA) or remain undetermined (UA). In young children, SA is mainly caused by Kingella kingae (KK), a hard to grow bacteria leading generally to a mild clinical and biological form of SA. An early accurate diagnosis between KK-SA and early-onset JIA is essential to provide appropriate treatment and follow-up. The aim of this work was to compare clinical and biological characteristics, length of hospital stays, duration of intravenous (IV) antibiotics exposure and use of invasive surgical management of patients under 6 years of age hospitalized for acute monoarthritis with a final diagnosis of JIA, SA or UA. We retrospectively analyzed data from < 6-year-old children, hospitalized at a French tertiary center for acute mono-arthritis, who underwent a joint aspiration. Non-parametric tests were performed to compare children with JIA, SA or UA. Bonferroni correction for multiple comparisons was applied with threshold for significance at 0.025. Among the 196 included patients, 110 (56.1%) had SA, 20 (10.2%) had JIA and 66 (33.7%) had UA. Patients with JIA were older when compared to SA (2.7 years [1.8-3.6] versus 1.4 [1.1-2.1], p < 0.001). Presence of fever was not different between JIA and SA or UA. White blood cells in serum were lower in JIA (11.2 × 10 9 /L [10-13.6]) when compared to SA (13.2 × 10 9 /L [11-16.6]), p = 0.01. In synovial fluid leucocytes were higher in SA 105.5 × 10 3  cells/mm 3 [46-211] compared to JIA and UA (42 × 10 3  cells/mm 3 [6.4-59.2] and 7.29 × 10 3  cells/mm 3 [2.1-72] respectively), p < 0.001. Intravenous antibiotics were administered to 95% of children with JIA, 100% of patients with SA, and 95.4% of UA. Arthrotomy-lavage was performed in 66.7% of patients with JIA, 79.6% of patients with SA, and 71.1% of patients with UA. In children less than 6 years of age with acute mono-arthritis, the clinical and biological parameters currently used do not reliably differentiate between JIA, AS and UA. JIA subgroups that present a diagnostic problem at the onset of monoarthritis before the age of 6 years, are oligoarticular JIA and systemic JIA with hip arthritis. The development of new biomarkers will be required to distinguish JIA and AS caused by Kingella kingae in these patients.

This case series examines cardiac MRI findings in four children and adolescents admitted to intensive care in April 2020 for multisystem inflammatory syndrome and Kawasaki disease-like features related to coronavirus disease 2019 (COVID-19). Acute myocarditis occurred less than 1 week after onset of fever and gastrointestinal symptoms. Physical examination showed rash and cheilitis or conjunctivitis. All patients recovered after intravenous immunoglobulin therapy. Severe acute respiratory syndrome coronavirus 2 reverse transcription polymerase chain reaction was negative in nasopharyngeal, stool, and respiratory samples and was positive on serology. Cardiac MRI showed diffuse myocardial edema on T2 short tau inversion-recovery sequences and native T1 mapping, with no evidence of late gadolinium enhancement suggestive of replacement fibrosis or focal necrosis. These findings favor postinfectious myocarditis in children and adolescents with COVID-19., (© RSNA, 2020.)

Introduction: While drug prescription should be based on established recommendations stemming from clinical trials but in pediatrics, many drugs are used without marketing authorization. Consequently recommendations are often based on clinical experience and the risk of inappropriate prescription (IP) is high. A tool for detecting IP in pediatrics-called POPI (Pediatrics: Omission of Prescriptions and Inappropriate prescriptions)-has been developed in France. However the relevance of its use at an international level is not known. Our aim has been to adapt POPI for a worldwide use., Material and Method: A two-round Delphi online questionnaire was completed and validated by international experts to identify consensual items. They were asked to rate the validity of each items taking into account the recommendations and practices in their countries. Only propositions obtaining a median score in the upper tertile with an agreement of more than 75% of the panel-for the first round-and 85%-for the second round-were retained., Results: Our panel included 11 pharmacists (55%) and 9 physicians (45%). The panelists came from 12 different countries: England, Belgium, Brazil, Canada, China, Ivory Coast, Ireland, Malaysia, Portugal, Switzerland, Turkey and Vietnam. At the end of the first round, of the 105 items of the original POPI tool, 80 items were retained including 16 items reworded and 25 items were deleted. In the second round, 14 experts participated in the study. This final international POPI tool is composed of 73 IP and omissions of prescriptions in the fields of neuropsychiatry, dermatology, infectiology, pneumology, gastroenterology, pain and fever., Discussion and Conclusion: This study highlights international consensus on prescription practice in pediatrics. The use of this tool in everyday practice could reduce the risk of inappropriate prescription. The impact of the diffusion of POPI tool will be assessed in a prospective multicentric study., Competing Interests: The authors have declared that no competing interests exist.

Nitrous oxide (N 2 O) is widely used as an anesthetic or an analgesic. N 2 O prolonged and recurrent administration is known to affect vitamin B12 metabolism with subsequent clinical consequences. We report herein the case of a 13-year-old girl with sickle cell disease exhibiting severe neurological and biochemical signs of functional vitamin B12 deficiency due to prolonged and repeated exposure to N 2 O. This was an incentive to prospectively investigate functional vitamin B12 deficiency in patients affected by sickle cell disease regularly exposed to N 2 O. We measured plasma concentrations of vitamin B12, total homocysteine, methionine and methylmalonic acid in 39 patients with sickle cell disease between 2015 and 2016. No patients developed neurological symptoms related to N 2 O administration but 19 patients (49%) had biochemical abnormalities suggesting mildly disturbed vitamin B12 metabolism e.g. decreased B12 vitamin, hypomethioninemia, or slightly increased methylmalonic acid or homocysteine. The clinical case highlight the potential severe deleterious effects of N 2 O over exposure on B12 vitamin metabolism in particular in patients affected with sickle cell disease. Conversely, when used without excess even repeatedly, there seem to be no overt clinically relevant abnormalities in vitamin B12 metabolism as observed on the cohort of 39 sickle cell disease affected patients., Competing Interests: The authors declare no competing interests., (© 2020 Published by Elsevier Inc.)

Background: The aim of this study was to identify early clinical and laboratory features that distinguish acute lymphoblastic leukemia (ALL) from juvenile idiopathic arthritis (JIA) in children presenting with persistent bone or joint pain for at least 1 month., Methods: We performed a multicenter case-control study and reviewed medical records of children who initially presented with bone or joint pain lasting for at least 1 month, all of whom were given a secondary diagnosis of JIA or ALL, in four French University Hospitals. Each patient with ALL was paired by age with two children with JIA. Logistic regression was used to compare clinical and laboratory data from the two groups., Results: Forty-nine children with ALL and 98 with JIA were included. The single most important feature distinguishing ALL from JIA was the presence of hepatomegaly, splenomegaly or lymphadenopathy; at least one of these manifestations was present in 37 cases with ALL, but only in 2 controls with JIA, for an odds ratio (OR) of 154 [95%CI: 30-793] (regression coefficient: 5.0). If the presence of these findings is missed or disregarded, multivariate analyses showed that non-articular bone pain and/or general symptoms (asthenia, anorexia or weight loss) (regression coefficient: 4.8, OR 124 [95%CI: 11.4-236]), neutrophils < 2 × 10 9 /L (regression coefficient: 3.9, OR 50 [95%CI: 4.3-58]), and platelets < 300 × 10 9 /L (regression coefficient: 2.6, OR 14 [95%CI: 2.3-83.9]) were associated with the presence of ALL (area under the ROC curve: 0.96 [95%CI: 0.93-0.99])., Conclusions: Based on our findings we propose the following preliminary decision tree to be tested in prospective studies: in children presenting with at least 1 month of osteoarticular pain and no obvious ALL in peripheral smear, perform a bone marrow examination if hepatomegaly, splenomegaly or lymphadenopathy is present. If these manifestations are absent, perform a bone marrow examination if there is fever or elevated inflammatory markers associated with non-articular bone pain, general symptoms (asthenia, anorexia or weight loss), neutrophils < 2 × 10 9 /L or platelets < 300 × 10 9 /L.

The pharmacokinetic profile of most drugs is dependent on the patient's covariates and may be influenced by the disease. Cefotaxime is frequently prescribed in pediatric patients with sickle cell disease (SCD), characterized by vaso-occlusive complications, chronic hemolytic anemia, and a defective immunological function predisposing the individual to severe infection. Data on the impact of the disease on the disposition of cefotaxime are missing. In the present study, our aims were to determine cefotaxime pharmacokinetics when prescribed to children with SCD for suspected or proven bacterial infection, identify significant covariates, and perform Monte Carlo simulations to optimize the drug dosage. Cefotaxime serum concentrations were measured in 78 pediatric SCD patients receiving cefotaxime intravenously at a daily dose of 200 mg/kg of body weight in three or four divided doses over 30 min. A total of 107 concentrations were available for pharmacokinetic analysis. A population pharmacokinetic model was developed with NONMEM software and used for Monte Carlo simulations. Cefotaxime concentrations ranged from 0.05 to 103.7 mg/liter. Cefotaxime pharmacokinetics were best described by a one-compartment model: the median estimated weight-normalized volume of distribution and clearance were 0.42 liter/kg (range, 0.2 to 1.1 liter/kg) and 0.38 liter/h/kg (range, 0.1 to 1.2 liter/h/kg). Cefotaxime clearance increased by 22% in patients with acute chest syndrome. Dosing optimization, performed using EUCAST MIC susceptibility breakpoints, showed that a dose of 100 mg/kg/6 h should be used, depending on the patient's characteristics and clinical presentation, in order to reach a value of the percentage of time that the drug concentration exceeded the MIC under steady-state pharmacokinetic conditions of 80% in 80% of the patients when targeting sensitive Gram-positive cocci and Gram-negative bacilli with MICs of 1 mg/liter or below., (Copyright © 2018 American Society for Microbiology.)

Objectives: To assess the management of febrile urinary-tract infection (FUTIs) due to extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) in children, the Pediatric Infectious Diseases Group of the French Pediatric Society set up an active surveillance network in pediatric centers across France in 2014., Materials and Methods: We prospectively analysed data from 2014 to 2016 for all children < 18 years old who received antibiotic treatment for FUTI due to ESBL-E in 24 pediatric centers. Baseline demographic, clinical features, microbiological data and antimicrobials prescribed were collected., Results: 301 children were enrolled in this study. The median age was 1 year (IQR 0.02-17.9) and 44.5% were male. These infections occurred in children with history of UTIs (27.3%) and urinary malformations (32.6%). Recent antibiotic use was the main associated factor for FUTIs due to ESBL-E, followed by a previous hospitalization and travel history. Before drug susceptibility testing (DST), third-generation cephalosporins (3GC) PO/IV were the most-prescribed antibiotics (75.5%). Only 13% and 24% of children received amikacine alone for empirical or definitive therapy, respectively, whereas 88.7% of children had isolates susceptible to amikacin. In all, 23.2% of children received carbapenems in empirical and/or definitive therapy. Cotrimoxazole (24.5%), ciprofloxacin (15.6%) and non-orthodox clavulanate-cefixime combination (31.3%) were the most frequently prescribed oral options after obtaining the DST. The time to apyrexia and length of hospital stay did not differ with or without effective empirical therapy., Conclusions: We believe that amikacin should increasingly take on a key role in the choice of definitive therapy of FUTI due to ESBL-E in children by avoiding the use of carbapenems.

Aim: Kingella kingae osteoarticular (KKO) infections are frequently associated with upper respiratory tract infections. However, no comparative studies detecting respiratory viruses had ever been performed between KKO and non-KKO (NKO)., Patients & Methods: Eighteen viruses were searched by FilmArray(®) Respiratory Panel (BioFire Diagnostics, UT, USA) in the oropharynx of 6-to-48-month-children admitted for KKO and NKO in 2013., Results: At least one virus was detected in the oropharynx of 19/21 (90.5%) KKO and 3/8 (37.5%) NKO cases (p = 0.008). In KKO group, human rhinovirus was predominant (12/21; 57.1%), especially during winter (7/11; 63.6%) despite its low concomitant circulation (<10%). Human rhinovirus was found in 2/8 (25%) in NKO group., Conclusion: Higher prevalence of respiratory virus in oropharynx was observed in KKO than NKO, strengthening their putative role in KKO pathophysiology.

Background: Based on European recommendations of ESPGHAN/ESPID from 2008, first line therapy for dehydration caused by acute gastroenteritis (AGE) is oral rehydration solution (ORS). In case of oral route failure, nasogastric tube enteral rehydration is as efficient as intra-venous rehydration and seems to lead to fewer adverse events. The primary objective was to describe rehydration strategies used in cases of AGE in pediatric emergency departments (PEDs) in Belgium, France, The Netherlands, and Switzerland., Methods: An electronic survey describing a scenario in which a toddler had moderate dehydration caused by AGE was sent to physicians working in pediatric emergency departments. Analytical data were analyzed with descriptive statistics and Kruskal -Wallis Rank test., Results: We analyzed 68 responses, distributed as follows: Belgium N = 10, France N = 37, The Netherlands N = 7, and Switzerland N = 14. Oral rehydration with ORS was the first line of treatment for 90% of the respondents. In case of first line treatment failure, intravenous rehydration was preferred by 95% of respondents from France, whereas nasogastric route was more likely to be used by those from Belgium (80%), The Netherlands (100%) and Switzerland (86%). Serum electrolyte measurements were more frequently prescribed in France (92%) and Belgium (80%) than in The Netherlands (43%) and Switzerland (29%). Racecadotril was more frequently used in France, and ondansetron was more frequently used in Switzerland. No respondent suggested routine use of antibiotics., Conclusion: We found variations in practices in terms of invasiveness and testing. Our study supports the need for further evaluation and implementation strategies of ESPGHAN/ESPID guidelines. We plan to extend the study throughout Europe with support of the Young ESPID Group.

Economic evaluation of vaccination programs can be challenging and does not always fully capture the benefits provided. Reasons for this include the difficulties incurred in accurately capturing the health and economic impact of infectious diseases and how different diseases may interact with each other. Rotavirus infection, for example, peaks at a similar time than other infectious diseases, such as RSV and influenza, which can cause hospital overcrowding and disruption, and may pose a risk to more vulnerable children due to limited availability of isolation facilities. Another challenge, specific to evaluating childhood vaccination, is that QoL cannot be accurately measured in children due to a lack of validated instruments. Childhood diseases also incur a care giver burden, due to the need for parents to take time off work, and this is important to consider. Finally, for diseases such as RVGE, cost-effectiveness analyses in which longer time horizons are considered may not reflect the short-term benefits of vaccination. Further quantification of the economic impact of childhood diseases is thus required to fully highlight the true benefits of childhood vaccination that may be realized. Herein we explore the limitations of existing economic evaluations for childhood vaccination, and how economic analyses could be better adapted in future.

We describe two atypical cases of Kingella kingae infection in children diagnosed by PCR, one case involving a soft tissue abscess and one case a femoral Brodie abscess. Both patients had concomitant human rhinovirus infection. K. kingae strains, isolated from an oropharyngeal swab, were characterized by multilocus sequence typing and rtxA sequencing.

Inoculation of blood culture vials with joint fluid samples has revealed the important pathogenic role of Kingella kingae in pediatric arthritis. However, recent studies based on broad-range 16S ribosomal DNA PCR and real-time PCR without a probe suggest that conventional methods remain suboptimal. We developed a new real-time PCR method with a probe that is highly specific for K. kingae and applied it to joint fluid samples collected from 89 children with suspected arthritis admitted to our institution during a 2-year period. Real-time PCR was also applied to blood samples obtained before surgery and to joint drainage fluid samples obtained during several days after surgery. Thirty-six (40%) of the 89 cases of suspected septic arthritis had positive culture. Staphylococcus aureus was the main isolate (n = 19/36, 53%), followed by K. kingae (n = 7/36, 19%). Specific real-time PCR identified K. kingae in 24 of the 53 culture-negative cases. Thus, K. kingae was present in 31 (52%) of the 60 documented cases, making it the leading pathogen. Real-time PCR on all 15 blood DNA extracts from patients with K. kingae infection was negative, demonstrating that joint fluid positivity did not result from DNA circulating in blood. Real-time PCR amplification of drainage fluid samples showed that the pathogen could be detected for up to 6 days after antibiotic initiation. K. kingae real-time PCR applied to DNA extracted from joint fluid samples, but not from blood samples, markedly improved the etiological diagnosis of septic arthritis in children. Retrospective diagnosis is feasible for up to 6 days after treatment initiation.

We used real-time PCR to examine the persistence of Bordetella pertussis DNA in serial nasopharyngeal aspirates from 22 children treated for pertussis. After 5 days of treatment, PCR was positive for all 21 assessable patients. After 14 and 21 days, PCR was still positive for 83% (10/12) and 66% (4/6) of assessable patients, respectively. One patient was tested 1 month after treatment initiation, and B. pertussis DNA was still detectable. Quantitative analysis showed that the DNA concentration diminished during treatment in all except one case. The PCR cycle threshold at which B. pertussis DNA became detectable increased by a mean of 1.7 cycles per day (range, 0.86 to 3.68 cycles per day). Real-time PCR can thus be used to diagnose pertussis in young children for up to 3 weeks after treatment initiation. Its potential value for assessing the treatment outcome remains to be determined.

Rotavirus is the major cause of infantile gastroenteritis and each year causes 611,000 deaths worldwide. The virus infects the mature enterocytes of the villus tip of the small intestine and induces a watery diarrhea. Diarrhea can occur with no visible tissue damage and, conversely, the histological lesions can be asymptomatic. Rotavirus impairs activities of intestinal disaccharidases and Na+-solute symports coupled with water transport. Maldigestion of carbohydrates and their accumulation in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water reabsorption can lead to a malabsorption component of diarrhea. Since the discovery of the NSP4 enterotoxin, diverse hypotheses have been proposed in favor of an additional secretion component in the pathogenesis of diarrhea. Rotavirus induces a moderate net chloride secretion at the onset of diarrhea, but the mechanisms appear to be quite different from those used by bacterial enterotoxins that cause pure secretory diarrhea. Rotavirus failed to stimulate Cl- secretion in crypt, whereas it stimulated Cl- reabsorption in villi, questioning, therefore, the origin of net Cl- secretion. A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of rotavirus infection. Also, the overall chloride secretory response is regulated by a phospholipase C-dependent calcium signaling pathway induced by NSP4. However, the overall response is weak, suggesting that NSP4 may exert both secretory and subsequent anti-secretory actions, as did carbachol, hence limiting Cl- secretion. All these characteristics provide the means to make the necessary functional distinction between viral NSP4 and bacterial enterotoxins.

The direct effect of the rotavirus NSP4 114-135 and Norovirus NV464-483 peptides on 36Cl uptake was studied by using villus cell brush border membrane (BBM) isolated from young rabbits. Both peptides inhibited the Cl-/H+ symport activity about equally and partially. The interaction involved one peptide-binding site per carrier unit. Whereas in vitro NSP4 114-135 caused nonspecific inhibition of the Cl-/H+ symporter, the situation in vivo is different. Because rotavirus infection in young rabbits accelerated both Cl- influx and Cl- efflux rates across villi BBM without stimulating Cl- transport in crypt BBM, we conclude that the NSP4 114-135 peptide, which causes diarrhea in young rodents, did not have any direct, specific effect on either intestinal absorption or secretion of chloride. The lack of direct effect of NSP4 on chloride transport strengthens the hypothesis that NSP4 would trigger signal transduction pathways to enhance net chloride secretion at the onset of rotavirus diarrhea.

We describe two cases of aseptic meningitis occurring some time after pneumococcal meningitis. Both cases may have resulted from an inflammatory response to persistent pneumococcal cell membrane components, as the cerebrospinal fluid samples were positive by the Binax NOW Streptococcus pneumoniae antigen test. Potential mechanisms and diagnostic impact are discussed.

Rotavirus is a major cause of infantile gastroenteritis worldwide. However, the mechanisms underlying fluid and electrolyte secretion associated with diarrhea remain largely unknown. We investigated the hypothesis that loss of Cl(-) into the luminal contents during rotavirus infection may be caused by a dysfunction in the chloride absorptive capacity across the intestinal brush-border membrane (BBM). The luminal Cl(-) concentrations in the entire small intestine of young rabbits infected with lapine rotavirus decreased at 1 and 2 days postinfection (dpi), indicating net Cl(-) absorption. At 7 dpi, luminal Cl(-) concentrations were slightly increased, indicating a moderate net Cl(-) secretion. By using a rapid filtration technique, (36)Cl uptake across BBM was quantified by modulating the alkali-metal ion, electrical, chloride, and/or proton gradients. Rotavirus infection caused an identical, 127% +/- 24% increase in all Cl(-) uptake activities (Cl(-)/H(+) symport, Cl(-) conductance, and Cl(-)/anion exchange) observed across the intestinal BBM. The rotavirus activating effects on the symporter started at 1 dpi and persisted up to 7 dpi. Kinetic analyses revealed that rotavirus selectively affected the capacity parameter characterizing the symporter. We report the novel observation that rotavirus infection stimulated the Cl(-) reabsorption process across the intestinal BBM. We propose that the massive Cl(-) reabsorption in villi could partly overwhelm chloride secretion in crypt cells, which possibly increases during rotavirus diarrhea, the resulting imbalance leading to a moderate net chloride secretion.

The hydrodynamic diameters of native rotavirus particles, bovine RF and simian SA11 strains, were determined by quasielastic light scattering. By using this method and agarose gel electrophoresis, the Ca(2+) dissociation constant, K(Ca), governing the transition from triple-layer particles (TLPs) to double-layer particles (DLPs), was shown to increase, at constant pH, as the temperature and/or the ionic strength of the incubation medium increased. We report the novel observation that, under physiological conditions, K(Ca) values for both RF and SA11 rotaviruses were well above the intracytoplasmic Ca(2+) concentrations of various cells, which may explain why TLP uncoating takes place within vesicles (possibly endosomes) during the entry process. A correlation between TLP uncoating and cell membrane permeabilization was found, as shown by the release of carboxyfluorescein (CF) from CF-loaded intestinal brush-border membrane vesicles. Conditions stabilizing the virion in the TLP form inhibited CF release, whereas conditions favoring the TLP-to-DLP transformation activated this process. We conclude that membrane permeabilization must be preceded by the loss of the outer-capsid proteins from trypsinized TLP and that physiological ionic strength is required for permeabilization to take place. Finally, the paper develops an alternative explanation for the mechanism of rotavirus entry, compatible with the Ca(2+)-dependent endocytic pathway. We propose that there must be an iterative process involving tight coupling in time between the lowering of endosomal Ca(2+) concentration, virion decapsidation, and membrane permeabilization, which would cause the transcriptionally active DLPs to enter the cytoplasm of cells.

Lysteriolysin O (LLO) induces a microtubule-dependent activation of mucin exocytosis in the human mucin-secreting HT29-MTX. Cholesterol inhibits the LLO-induced mucin exocytosis, whereas the oxidized form of cholesterol had no inhibitory effect. LLO-induced mucin exocytosis inhibited by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. Inhibition of cholesterol synthesis in HT29-MTX cells results in a decrease in the LLO-induced mucin exocytosis. Other lipids such as gangliosides are able to inhibit the LLO-induced mucin exocytosis, suggesting that the binding of the toxin occurs at a multiplicity of membrane-associated lipids acting as receptors. Incubation of the toxin with lipids such as cholesterol or gangliosides does not decrease binding of LLO to target membranes. The present work also provides evidence that the LLO-induced mucin exocytosis develops independently of the pore-forming activity of the toxin. Finally, we demonstrated that the toxin associates with detergent-insoluble glycolipid microdomains (DIGs) containing VIP/21 caveolin, allowing internalization of the toxin and subsequent activation of the mucin exocytosis.

To gain further insight into the mechanism by which lactobacilli develop antimicrobial activity, we have examined how Lactobacillus acidophilus LB inhibits the promoted cellular injuries and intracellular lifestyle of Salmonella enterica serovar Typhimurium SL1344 infecting the cultured, fully differentiated human intestinal cell line Caco-2/TC-7. We showed that the spent culture supernatant of strain LB (LB-SCS) decreases the number of apical serovar Typhimurium-induced F-actin rearrangements in infected cells. LB-SCS treatment efficiently decreased transcellular passage of S. enterica serovar Typhimurium. Moreover, LB-SCS treatment inhibited intracellular growth of serovar Typhimurium, since treated intracellular bacteria displayed a small, rounded morphology resembling that of resting bacteria. We also showed that LB-SCS treatment inhibits adhesion-dependent serovar Typhimurium-induced interleukin-8 production.