Background. The profile of cellular immunological responses of children across the spectrum of COVID-19, ranging from acute SARS-CoV-2 infection to full recovery or Long COVID, has not yet been fully investigated. Methods. We examined and compared cytokines in sera and cell subsets in peripheral blood mononuclear cells (B and regulatory T lymphocytes) collected from four distinct groups of children, distributed as follows: younger than 18 years of age with either acute SARS-CoV-2 infection (n = 49); fully recovered from COVID-19 (n = 32); with persistent symptoms (Long COVID, n = 51); and healthy controls (n = 9). Results. In the later stages after SARS-CoV-2 infection, the cohorts of children, both with recovered and persistent symptoms, showed skewed T and B subsets, with remarkable differences when compared with children at the onset of the infection and with controls. The frequencies of IgD⁺CD27⁻ naïve B cells, IgD⁺IgM⁺ and CD27⁻IgM⁺CD38^dim B cells were higher in children with recent infection than in those with an older history of disease (p < 0.0001 for all); similarly, the total and natural Tregs compartments were more represented in children at onset when compared with Long COVID (p < 0.0001 and p = 0.0005, respectively). Despite the heterogeneity, partially due to age, sex and infection incidence, the susceptibility of certain children to develop persistent symptoms after infection appeared to be associated with the imbalance of the adaptive immune response. Following up and comparing recovered versus Long COVID patients, we analyzed the role of circulating naïve and switched B and regulatory T lymphocytes in counteracting the evolution of the symptomatology emerged, finding an interesting correlation between the amount and ability to reconstitute the natural Tregs component with the persistence of symptoms (linear regression, p = 0.0026). Conclusions. In this study, we suggest that children affected by Long COVID may have a compromised ability to switch from the innate to the adaptive immune response, as supported by our data showing a contraction of naïve and switched B cell compartment and an unstable balance of regulatory T lymphocytes occurring in these children. However, further prospective immunological studies are needed to better clarify which factors (epigenetic, diet, environment, etc.) are involved in the impairment of the immunological mechanisms in the Long COVID patients.

We provide preliminary evidence that, also in children, Long coronavirus disease (COVID) may be characterized by a proinflammatory signature. Ten Long COVID patients, 7 convalescent subjects after COVID infection and 4 healthy controls were enrolled. When adjusted for sex, children with long COVID had statistically significant differences in the levels of Flt3L, CD5, uPA, CCL23, CD40 and TGFα. When adjusted for age, CCL23 levels remained statistically significant.

Background: Bloodstream infections affect up to 20% of pediatric cancer patients receiving intensive care, contributing significantly to morbidity and mortality, with infection-related mortality rates reported to be as high as 16%. Methods: The identification of microorganisms directly from whole blood is difficult due to several factors, such as interference from host genomic material, low bacterial load, the endogenous components of whole blood and exogenous substances, which can interfere with the identification process. Nevertheless, rapid microbial diagnosis remains of paramount importance in these patients. Results and Conclusion: Here, we present the first case of bacterial pathogen identification directly from whole blood using MALDI-TOF mass spectrometry in an onco-hematological pediatric patient affected by sepsis and admitted to Bambino Gesù Children’s Hospital (IRCCS) in Rome, Italy.

Background/Objectives: Paediatric osteoarticular infections (POAIs) present unique diagnostic and therapeutic challenges. Microbiological culture (MC) is typically time-consuming and lacks sensitivity, especially when patients have received antibiotics. The BIOFIRE^® Joint Infection Panel (BJIP) is a syndromic molecular assay for the direct identification of most pathogens causing POAIs. Methods: We evaluated BJIP in 17 synovial fluids, and then, we retrospectively assessed its utility in 93 off-label specimens (i.e., 25 purulent fluids/biopsies and 68 whole blood samples). All specimens were collected from October 2022 to March 2024 from paediatric patients admitted at the Bambino Gesù Children’s Hospital in Rome. Results: A bacterial pathogen was isolated in only one of 17 synovial fluid cultures, while BJIP identified eight additional microorganisms in MC-negative cases. The most frequently detected pathogen was S. aureus (44.5%, 4/9). BJIP performance in synovial fluids showed an overall positive percentage agreement (PPA) and negative percentage agreement (NPA) of 100% and 88.1%, respectively, compared to MC. All positive results (n/N = 9/17) were considered medically significant, with an increase in NPA to 100%. In purulent fluids/biopsies, BJIP and MC were concordant in 72% of cases (n/N = 18/25), with a per-sample PPA and NPA of 90% and 60%, respectively. For whole blood samples, almost all samples were negative by both methods (i.e., reference blood culture and BJIP), and the molecular test did not enable any further microbiological diagnosis. Conclusions: The BIOFIRE^® Joint Infection Panel rapidly and accurately enabled or excluded a diagnosis of a POAI (~1 vs. 24–96 h for MC), optimising antimicrobial therapy.

Background: Infant meningitis, particularly caused by Escherichia coli, remains a life-threatening condition, especially in premature and low-weight infants. Infections of the central nervous system can be fatal, necessitating prompt diagnosis and appropriate treatment. Acute infections caused by various pathogens, including E. coli, often present with similar clinical symptoms. The rapid identification of pathogens and their antimicrobial resistance mechanisms is critical for timely and effective treatment. We report the case of an 8-month-old patient who presented with fever, diarrhea, and convulsive seizures and was subsequently diagnosed with meningitis. Despite initial empirical treatment with ceftriaxone, the patient’s condition worsened. Methods: At Bambino Gesù Children’s Hospital, molecular diagnostic tools, including the FilmArray Meningitis/Encephalitis and Blood Culture Identification panels, were employed. Results: Although the Meningitis panel did not detect any pathogens due to the lack of the specific bacterial target, the off-label use of the Blood Culture Identification panel identified a non-K1 Escherichia coli strain carrying the CTX-M resistance gene, an extended-spectrum beta-lactamase (ESBL). Despite the rapid diagnostic approach and adjustment of antibiotic therapy, the patient succumbed to the infection due to the strain’s high virulence and multidrug resistance. Whole-genome sequencing further characterized the strain, revealing that it belonged to the ST131 group, a highly resistant and virulent strain associated with sepsis. Conclusions: This case highlights the importance of integrating advanced molecular diagnostics, such as whole-genome sequencing, with traditional methods to improve pathogen detection, especially in cases of emerging resistant strains that are not covered by standard diagnostic panels. It also emphasizes the need for the continuous adaptation of diagnostic tools to include non-K1 E. coli strains for more comprehensive and timely meningitis diagnosis.

Commercially available Interferon-γ release assays (IGRAs), including the last-generation QuantiFERON TB-Plus (QFT-Plus), are effective in aiding the diagnosis of tuberculosis (TB) infection but cannot distinguish latent TB subjects from active TB patients. The aim of this study was to prospectively evaluate the performance of an HBHA-based IGRA, combined with commercially available IGRAs, to assess their usefulness as a prognostic biomarkers and aid in the monitoring of TB treatment in children. Following clinical, microbiological, and radiological assessment, children younger than 18 years of age classified as either LTBI or active TB were tested at baseline and during treatment by the QuantiFERON TB-Plus (QFT) assay and an aliquot of whole-blood was stimulated with HBHA. Among the 655 children evaluated, 559 (85.3%) were classified as “Non TB”, 44 patients (6.7%) with active TB, and 52 (7.9%) with LTBI. The median HBHA-IGRA IFN-gamma responses were able to discriminate active TB from LTBI (0.13 IU/ml vs 1.995, (p < 0,0001), those with asymptomatic TB from those with symptomatic TB (1.01 IU/ml vs 0.115 IU/ml, p 0.017), or more severe TB (p 0.022), and significantly raised during successful TB treatment (p < 0.0001). Conversely, CD4 + and CD8 + responses were similar in all groups of patients, although active TB patients had higher CD4 + responses and LTBI higher CD8 + responses.  Conclusion: HBHA-based IGRA, combined with CD4 + and CD8 + responses assessed by commercially available IGRAs, is a useful support in the characterization of the TB spectrum in children and monitoring of TB-therapy. What is Known:• Current immune diagnostics are not able to discriminate active and latent Ttuberculosis, including the recently approved QFT-PLUS..• New immunological assays with prognostic value are highly needed. What is New:• HBHA-based IGRA, combined with CD4+ and CD8+ responses assessed by commercially available IGRAs, is a useful support for the differentiation of active and latent TB in children..• HBHA-based IGRA, combined with CD4+ and CD8+ responses assessed by commercially available IGRAs, is a useful support in the monitoring of TBtherapy in children..