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Abstract Background Despite improved diagnostics, pulmonary pathogens in immunocompromised children frequently evade detection, leading to significant mortality. Therefore, we aimed to develop a highly sensitive metagenomic next-generation sequencing (mNGS) assay capable of evaluating the pulmonary microbiome and identifying diverse pathogens in the lungs of immunocompromised children. Methods We collected 41 lower respiratory specimens from 34 immunocompromised children undergoing evaluation for pulmonary disease at 3 children’s hospitals from 2014–2016. Samples underwent mechanical homogenization, parallel RNA/DNA extraction, and metagenomic sequencing. Sequencing reads were aligned to the National Center for Biotechnology Information nucleotide reference database to determine taxonomic identities. Statistical outliers were determined based on abundance within each sample and relative to other samples in the cohort. Results We identified a rich cross-domain pulmonary microbiome that contained bacteria, fungi, RNA viruses, and DNA viruses in each patient. Potentially pathogenic bacteria were ubiquitous among samples but could be distinguished as possible causes of disease by parsing for outlier organisms. Samples with bacterial outliers had significantly depressed alpha-diversity (median, 0.61; interquartile range [IQR], 0.33–0.72 vs median, 0.96; IQR, 0.94–0.96; P < .001). Potential pathogens were detected in half of samples previously negative by clinical diagnostics, demonstrating increased sensitivity for missed pulmonary pathogens (P < .001). Conclusions An optimized mNGS assay for pulmonary microbes demonstrates significant inoculation of the lower airways of immunocompromised children with diverse bacteria, fungi, and viruses. Potential pathogens can be identified based on absolute and relative abundance. Ongoing investigation is needed to determine the pathogenic significance of outlier microbes in the lungs of immunocompromised children with pulmonary disease. Pulmonary infections in immunocompromised children frequently evade detection by current clinical diagnostics. We optimized metagenomic sequencing of pulmonary pathogens in immunocompromised children and show that metagenomic RNA sequencing identified pulmonary pathogens in approximately half of patients with negative clinical diagnostics.

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and the elderly. Despite its substantial global health burden, RSV-targeted treatments remain unavailable for the majority of individuals. While vaccine development is underway, a detailed understanding of the host response to RSV infection and identification of required human host factors for RSV may provide insight into combatting this pathogen. Here, we utilized single-cell RNA sequencing and functional genomics to understand the host response in both RSV-infected and bystander cells, identify what host factors mediate infection, and contextualize these findings relative to dozens of previously reported screens across 17 additional viruses.

MMPs (Matrix metalloproteinases) and their endogenous tissue inhibitors may contribute to lung injury through extracellular matrix degradation and modulation of inflammation and fibrosis. To test for an association between MMP pathway proteins and inflammation, endothelial dysfunction, and clinical outcomes. We measured MMPs in plasma collected on acute respiratory distress syndrome (ARDS) Day 1 from 235 children at five hospitals between 2008 and 2017. We used latent class analysis to identify patients with distinct MMP profiles and then associated those profiles with markers of inflammation (IL-1RA, -6, -8, -10, and -18; macrophage inflammatory protein-1α and -1β; tumor necrosis factor-α and -R2), endothelial injury (angiopoietin-2, von Willebrand factor, soluble thrombomodulin), impaired oxygenation (Pa /Fi [P/F] ratio, oxygenation index), morbidity, and mortality. In geographically distinct derivation and validation cohorts, approximately one-third of patients demonstrated an MMP profile characterized by elevated MMP-1, -2, -3, -7, and -8 and tissue inhibitor of metalloproteinase-1 and -2; and depressed active and total MMP-9. This MMP profile was associated with multiple markers of inflammation, endothelial injury, and impaired oxygenation on Day 1 of ARDS, and conferred fourfold increased odds of mortality or severe morbidity independent of the P/F ratio and other confounders (95% confidence interval, 2.1-7.6; P < 0.001). Logistic regression using both the P/F ratio and MMP profiles was superior to the P/F ratio alone in prognosticating mortality or severe morbidity (area under the receiver operating characteristic curve, 0.75; 95% confidence interval, 0.68-0.82 vs. area under the receiver operating characteristic curve, 0.66; 95% confidence interval, 0.58-0.73; P = 0.009). Pediatric patients with ARDS have specific plasma MMP profiles associated with inflammation, endothelial injury, morbidity, and mortality. MMPs may play a role in the pathobiology of children with ARDS.

Patients with immune dysregulation may present with varying combinations of autoimmunity, autoinflammation, immunodeficiency, atopy, lymphoproliferation, and/or malignancy, often with multisystem involvement. Recognizing specific patterns of immune dysregulation, coordinating and interpreting complex diagnostic testing, and choosing initial (often empiric) treatment can be challenging. Centers are increasingly assembling multidisciplinary teams (MDTs) to standardize evaluation and optimize treatment of patients with complex immune dysregulation (immune dysregulation MDTs [immMDTs]). However, published information on the composition and function of immMDTs is sparse, and there is little guidance for those seeking to establish or optimize an immMDT. To inform this review, we assembled a panel of 24 pediatric providers from multiple specialties who actively participate in immMDTs to provide expert opinion. We also conducted a search of the available information on pediatric immMDTs from PubMed. Based on these insights, we summarize the structure and function of active immMDTs across the United States and focus on best practices and context-dependent solutions that may enable institutions with varying goals, patient populations, and resources to establish an immMDT.

Mucosal immunity plays an important role in the control of viral respiratory infections like SARS-CoV-2. While systemic immune responses against the SARS-2-CoV-2 have been studied in children, there is no information on mucosal antibody response, especially in the lower respiratory tract of children coronavirus disease 2019 (COVID-19) and post-infectious multisystem inflammatory syndrome in children (MIS-C) against emerging SARS-CoV-2 variants. Therefore, we evaluated neutralizing antibody responses in paired plasma and endotracheal aspirates of pediatric severe, acute COVID-19 or MIS-C patients against SARS-CoV-2 WA1/2020, as well as against variants of concern (VOCs). Neutralizing antibody responses against the SARS-CoV-2 WA1/2020 strain in pediatric plasma were 2-fold or 35-fold higher compared with the matched endotracheal aspirate in COVID-19 or MIS-C patients, respectively. In contrast to plasma, neutralizing antibody responses against the VOCs and variants of interest (VOIs) in endotracheal aspirates were lower, with only one endotracheal aspirate demonstrating neutralizing titers against the Iota, Kappa, Beta, Gamma, and Omicron variants. In conclusion, our findings suggest that children and adolescents with severe COVID-19 or MIS-C have weak mucosal neutralizing antibodies in the trachea against circulating SARS-CoV-2 Omicron and other VOCs, which may have implications for recovery and for re-infection with emerging SARS-CoV-2 variants.

[...]of the clear need for enhanced LRTI diagnostics in HCT recipients, we sequentially enrolled 22 adult HCT recipients hospitalized for acute respiratory illnesses who underwent bronchoscopy and BAL between January 25, 2012, and May 20, 2013, under University of Michigan protocol HUM00043287. Standard-of-care BAL microbiologic testing was uniformly performed on all patients and included semiquantitative cultures for bacteria, mycobacteria, fungi, and cytomegalovirus; Aspergillus galactomannan assay; silver stain for Pneumocystis jirovecii; multiplex polymerase chain reaction influenza A/B, respiratory syncytial virus and human metapneumovirus; and human herpesvirus-6 polymerase chain reaction, as detailed in the Methods in the online supplement (4). With respect to potential bacterial pathogens, mNGS identified Streptococcus mitis in patient 13, an oropharyngeal microbe known to cause bacteremia and acute respiratory distress in HCT recipients (6), and Corynebacterium proprinquum, one of the few virulent Corynebacterium species associated with LRTI (7). mNGS identified a diversity of DNA viruses including human herpesvirus-6, cytomegalovirus, herpes simplex virus, Epstein-Barr virus, human papilloma virus, and torque teno viruses; however, only five of these also had well-defined evidence of active replication marked by detectable RNA transcripts (Table 1). mNGS identified microbes of uncertain pathogenicity in nine patients (Table 1) who had coexisting clinical diagnoses of graft-versus-host disease (patients 11, 22, 23, 24, 25, 31, 34, 35, and 37) or bacteremia/sepsis (patient 3), which could have contributed to respiratory symptoms resulting from noninfectious pulmonary inflammation. Because asymptomatic carriage of respiratory pathogens is well described (8), establishing biomarkers of genuine infection is critical for determining the significance of a given microbiologic finding. [...]our limited sequencing depth did not yield the human transcriptome coverage that would be desired for optimal differential gene expression analyses, although we were able to rigorously evaluate a composite metric of immunity genes.

In this study evaluating BNT162b2, vaccine effectiveness against hospitalization for Covid-19 in the delta-predominant period among adolescents 12 to 18 years of age was more than 90%; during the omicron period, vaccine effectiveness was 40% against hospitalization and 79% against critical illness. Vaccine effectiveness against hospitalization was 68% among children 5 to 11 years of age.

In this study, maternal vaccination with an mRNA vaccine during pregnancy was less common among infants hospitalized for Covid-19 than among controls. The effectiveness of maternal vaccination against Covid-19 hospitalization of infants was 52% overall and was greater when delta, rather than omicron, was predominant.

Investigators used a case–control, test-negative design to assess the effectiveness of the BNT162b2 vaccine in adolescents for the prevention of Covid-19–related hospitalization, ICU admission, or receipt of life support. Among 445 case patients and 777 controls, of the 180 patients admitted to an ICU, only 2 had been fully vaccinated; all 7 deaths occurred in unvaccinated patients.