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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
...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.
Angiopoietin-2 (Ang-2) is a key mediator of pulmonary vascular permeability. This study tested the association between plasma Ang-2 and mortality in pediatric acute respiratory distress syndrome ...(ARDS), with stratification for prior hematopoietic cellular transplantation (HCT), given the severe, yet poorly understood, ARDS phenotype of this subgroup. We enrolled 259 children <18 years of age with ARDS; 25 had prior HCT. Plasma Ang-2, von Willebrand Factor antigen (vWF), and vascular endothelial growth factor (VEGF) were measured on ARDS days 1 and 3 and correlated with patient outcomes. Day 1 and day 3 Ang-2 levels were associated with mortality independent of age, sex, race, and P/F ratio odds ratio (OR) 3.7, 95% CI 1.1-11.5, P = 0.027; and OR 10.2, 95% confidence interval (CI) 2.2-46.5, P = 0.003, for each log10 increase in Ang-2. vWF was associated with mortality (P = 0.027), but VEGF was not. The association between day 1 Ang-2 and mortality was independent of levels of both vWF and VEGF (OR 3.6, 95% CI 1.1-12.1, P = 0.039, for each log10 increase in Ang-2). 45% of the cohort had a rising Ang-2 between ARDS day 1 and 3 (adjusted mortality OR 3.3, 95% CI 1.2-9.2, P = 0.026). HCT patients with a rising Ang-2 had 70% mortality compared with 13% mortality for those without (OR 16.3, 95% CI 1.3-197.8, P = 0.028). Elevated plasma levels of Ang-2 were associated with mortality independent of vWF and VEGF. A rising Ang-2 between days 1 and 3 was strongly associated with mortality, particularly in pediatric HCT patients, suggesting vulnerability to ongoing endothelial damage.
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.
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.
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.
Purpose
Up to 38 % of children with cancer require pediatric intensive care unit (PICU) admission within 3 years of diagnosis, with reported PICU mortality of 13–27 % far exceeding that of the ...general PICU population. PICU outcomes data for individual cancer types are lacking and may help identify patients at risk for poor clinical outcomes.
Methods
We performed a retrospective multicenter analysis of 10,365 PICU admissions of cancer patients no greater than 21 years old among 112 PICUs between 1 January 2009 and 30 June 2012. We evaluated the effect of cancer type, age, gender, genetic syndrome, stem cell transplantation, PRISM3 score, infections, and critical care interventions on PICU mortality.
Results
After excluding scheduled perioperative admissions, cancer patients represented 4.2 % of all PICU admissions (10,365/246,346), had overall mortality of 6.8 % (708/10,365) vs. 2.4 % (5,485/230,548) in the general PICU population (RR = 2.9, 95 % CI 2.7–3.1,
p
< 0.001), and accounted for 11.4 % of all PICU deaths (708/6,215). Hematologic cancer patients had greater median PRISM3 score (8 vs 2,
p
< 0.001), rates of sepsis (27 vs 9 %, RR = 2.9, 95 % CI 2.6–3.1,
p
< 0.001), and mortality (9.6 vs 4.5 %, RR = 2.1, 95 % CI 1.8–2.5,
p
< 0.001) compared to solid cancer patients. Among hematologic cancer patients, stem cell transplantation, diagnosis of acute myeloid leukemia, PRISM3 score, and infection were all independently associated with PICU mortality.
Conclusions
Children with cancer account for 4.2 % of PICU admissions and 11.4 % of PICU deaths. Hematologic cancer patients have significantly higher admission illness severity, rates of infections, and PICU mortality than solid cancer patients. These data may be useful in risk stratification for closer monitoring and patient counseling.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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