Association of an In-Hospital Desirability of Outcomes Ranking Scale With Postdischarge Health-Related Quality of Life: A Secondary Analysis of the Life After Pediatric Sepsis Evaluation.

OBJECTIVES: To develop a desirability of outcome ranking (DOOR) scale for use in children with septic shock and determine its correlation with a decrease in 3-month postadmission health-related quality of life (HRQL) or death. DESIGN: Secondary analysis of the Life After Pediatric Sepsis Evaluation prospective study. SETTING: Twelve U.S. PICUs, 2013-2017. PATIENTS: Children (1 mo-18 yr) with septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We applied a 7-point pediatric critical care (PCC) DOOR scale: 7: death; 6: extracorporeal life support; 5: supported by life-sustaining therapies (continuous renal replacement therapy, vasoactive, or invasive ventilation); 4: hospitalized with or 3: without organ dysfunction; 2: discharged with or 1: without new morbidity to patients by assigning the highest applicable score on specific days post-PICU admission. We analyzed Spearman rank-order correlations (95% CIs) between proximal outcomes (PCC-DOOR scale on days 7, 14, and 21, ventilator-free days, cumulative 28-day Pediatric Logistic Organ Dysfunction-2 (PELOD-2) scores, and PICU-free days) and 3-month decrease in HRQL or death. HRQL was measured by Pediatric Quality of Life Inventory 4.0 or Functional Status II-R for patients with developmental delay. Patients who died were assigned the worst possible HRQL score. PCC-DOOR scores were applied to 385 patients, median age 6 years (interquartile range 2, 13) and 177 (46%) with a complex chronic condition(s). Three-month outcomes were available for 245 patients (64%) and 42 patients (17%) died. PCC-DOOR scale on days 7, 14, and 21 demonstrated fair correlation with the primary outcome (-0.42 [-0.52, -0.31], -0.47 [-0.56, -0.36], and -0.52 [-0.61, -0.42]), similar to the correlations for cumulative 28-day PELOD-2 scores (-0.51 [-0.59, -0.41]), ventilator-free days (0.43 [0.32, 0.53]), and PICU-free days (0.46 [0.35, 0.55]). CONCLUSIONS: The PCC-DOOR scale is a feasible, practical outcome for pediatric sepsis trials and demonstrates fair correlation with decrease in HRQL or death at 3 months.

Outcomes of Critically Ill Children With Acute Lymphoblastic Leukemia and Cytokine Release Syndrome Due to Chimeric Antigen Receptor T Cell Therapy: US, Multicenter PICU, Cohort Database Study.

OBJECTIVES: Cytokine release syndrome (CRS) is a potentially lethal toxicity associated with chimeric antigen receptor T cell therapy for pediatric acute lymphoblastic leukemia (ALL). Outcomes after critical illness due to severe CRS are poorly described. Our aim was to characterize critical illness outcomes across a multicenter cohort of PICU patients with ALL and CRS. DESIGN: Multicenter retrospective cohort study. SETTING: Twenty-one PICUs contributing data to Virtual Pediatric Systems, LLC (January 2020-December 2021). PATIENTS: PICU patients with ALL or unclassified leukemia and CRS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We identified 55 patients; 34 (62%) were 12 years or older, 48 (87%) were admitted from a hospital inpatient ward, and 23 (42%) received advanced organ failure support or monitoring. Fifty-one survived to PICU discharge (93%) including 19 of 23 (83%) who received advanced organ failure support or monitoring defined as receipt of noninvasive or invasive ventilation, cardiopulmonary resuscitation, extracorporeal membrane oxygenation, continuous renal replacement therapy, or placement of a tracheostomy, arterial catheter, hemodialysis catheter, or intracranial catheter. Twelve patients (22%) received invasive ventilation, nine of whom survived to PICU discharge. Two of four patients who received continuous renal replacement therapy and one of three patients who required cardiopulmonary resuscitation survived to PICU discharge. Lengths of PICU stay were median 3.0 days (interquartile range, 1.4-7.8 d) among PICU survivors, 7.8 (5.4-11.1) among those receiving advanced organ failure support or monitoring, and 7.2 days (interquartile range, 2.9-14.7 d) among nonsurvivors. Of the 51 patients who survived to PICU discharge, 48 (94%) survived the hospitalization. CONCLUSIONS: PICU patients with CRS frequently received a high level of support, and the majority survived their PICU stay and hospitalization. Additional multicenter investigations of severe CRS are necessary to inform evidence-based practice.

An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities.

Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.

Parental Mental Health Care After Their Child's Pediatric Intensive Care Hospitalization.

OBJECTIVES: Post-traumatic stress disorder, depression, and anxiety have all been found in parents of PICU survivors. How these research findings translate to actual use of mental health services by parents remains unknown. DESIGN: Retrospective observational cohort study. SETTING: Insurance claims data from 2006 to 2013 obtained from the IBM MarketScan Commercial Database. PATIENTS: Parents of PICU survivors. INTERVENTIONS: We examined rates of: 1) mental health diagnoses, 2) outpatient mental health visits, and 3) prescriptions for antidepressants and anxiolytics among parents, 6 months before and 6 months after their child's PICU admission, using each parent as their own control. MEASUREMENTS AND MAIN RESULTS: Of the 95,070 parents identified, 9.5% received a new mental health diagnosis in the 6 months after their child's PICU hospitalization, which represented a 110% increase from pre-PICU rates. A smaller proportion of parents were given new prescriptions for antidepressants (3.4%) and anxiolytics (3.9%) in the 6 months after their child's PICU hospitalization. Mothers were twice as likely to receive a new mental health diagnosis and be taking a new medication than fathers in the post-PICU period. The parental diagnosis of acute stress disorder or post-traumatic stress disorder increased by 87% from the pre-PICU to the post-PICU period. CONCLUSIONS: After their child's PICU hospitalization, the proportion of parents with a new mental health diagnosis nearly doubled. Mothers were at nearly twice the risk of receiving a new mental health diagnosis and receiving a new mental health medication compared with fathers. The proportion of parents receiving mental healthcare is much lower than the proportion reporting mental health symptoms in long-term outcomes studies. Whether this indicates a gap in healthcare delivery for parents with mental health symptoms remains unknown.

A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici.

Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used for the control of a broad range of fungal diseases. This has been the most rapidly expanding fungicide group in terms of new molecules discovered and introduced for agricultural use over the past fifteen years. A particular pattern of differential sensitivity (resistance) to the stretched heterocycle amide SDHIs (SHA-SDHIs), a subclass of chemically-related SDHIs, was observed in naïve Zymoseptoria tritici populations not previously exposed to these chemicals. Subclass-specific resistance was confirmed at the enzyme level but did not correlate with the genotypes of the succinate dehydrogenase (SDH) encoding genes. Mapping and characterization of the molecular mechanisms responsible for standing SHA-SDHI resistance in natural field isolates identified a gene paralog of SDHC, termed ZtSDHC3, which encodes for an alternative C subunit of succinate dehydrogenase, named alt-SDHC. Using reverse genetics, we showed that alt-SDHC associates with the three other SDH subunits, leading to a fully functional enzyme and that a unique Qp-site residue within the alt-SDHC protein confers SHA-SDHI resistance. Enzymatic assays, computational modelling and docking simulations for the two SQR enzymes (altC-SQR, WT_SQR) enabled us to describe enzyme-inhibitor interactions at an atomistic level and to propose rational explanations for differential potency and resistance across SHA-SDHIs. European Z. tritici populations displayed a presence (20-30%) / absence polymorphism of ZtSDHC3, as well as differences in ZtSDHC3 expression levels and splicing efficiency. These polymorphisms have a strong impact on SHA-SDHI resistance phenotypes. Characterization of the ZtSDHC3 promoter in European Z. tritici populations suggests that transposon insertions are associated with the strongest resistance phenotypes. These results establish that a dispensable paralogous gene determines SHA-SDHIs fungicide resistance in natural populations of Z. tritici. This study paves the way to an increased awareness of the role of fungicidal target paralogs in resistance to fungicides and demonstrates the paramount importance of population genomics in fungicide discovery.

Within-Host Recombination in the Foot-and-Mouth Disease Virus Genome.

Recombination is one of the determinants of genetic diversity in the foot-and-mouth disease virus (FMDV). FMDV sequences have a mosaic structure caused by extensive intra- and inter-serotype recombination, with the exception of the capsid-encoding region. While these genome-wide patterns of broad-scale recombination are well studied, not much is known about the patterns of recombination that may exist within infected hosts. In addition, detection of recombination among viruses evolving at the within-host level is challenging due to the similarity of the sequences and the limitations in differentiating recombination from point mutations. Here, we present the first analysis of recombination events between closely related FMDV sequences occurring within buffalo hosts. The detection of these events was made possible by the occurrence of co-infection of two viral swarms with about 1% nucleotide divergence. We found more than 15 recombination events, unequally distributed across eight samples from different animals. The distribution of these events along the FMDV genome was neither uniform nor related to the phylogenetic distribution of recombination breakpoints, suggesting a mismatch between within-host evolutionary pressures and long-term selection for infectivity and transmissibility.

Full Genome Sequencing Reveals New Southern African Territories Genotypes Bringing Us Closer to Understanding True Variability of Foot-and-Mouth Disease Virus in Africa.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hooved animals that poses a constant burden on farmers in endemic regions and threatens the livestock industries in disease-free countries. Despite the increased number of publicly available whole genome sequences, FMDV data are biased by the opportunistic nature of sampling. Since whole genomic sequences of Southern African Territories (SAT) are particularly underrepresented, this study sequenced 34 isolates from eastern and southern Africa. Phylogenetic analyses revealed two novel genotypes (that comprised 8/34 of these SAT isolates) which contained unusual 5′ untranslated and non-structural encoding regions. While recombination has occurred between these sequences, phylogeny violation analyses indicated that the high degree of sequence diversity for the novel SAT genotypes has not solely arisen from recombination events. Based on estimates of the timing of ancestral divergence, these data are interpreted as being representative of un-sampled FMDV isolates that have been subjected to geographical isolation within Africa by the effects of the Great African Rinderpest Pandemic (1887–1897), which caused a mass die-out of FMDV-susceptible hosts. These findings demonstrate that further sequencing of African FMDV isolates is likely to reveal more unusual genotypes and will allow for better understanding of natural variability and evolution of FMDV.

Deep Sequencing of Foot-and-Mouth Disease Virus Reveals RNA Sequences Involved in Genome Packaging.

Nonenveloped viruses protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. Packaging and capsid assembly in RNA viruses can involve interactions between capsid proteins and secondary structures in the viral genome, as exemplified by the RNA bacteriophage MS2 and as proposed for other RNA viruses of plants, animals, and human. In the picornavirus family of nonenveloped RNA viruses, the requirements for genome packaging remain poorly understood. Here, we show a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA, we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem-loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. This study provides evidence for the involvement of predicted RNA structures in picornavirus packaging and offers a readily transferable methodology for identifying packaging requirements in many other viruses.IMPORTANCE In order to transmit their genetic material to a new host, nonenveloped viruses must protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. For many nonenveloped RNA viruses the requirements for this critical part of the viral life cycle remains poorly understood. We have identified RNA sequences involved in genome packaging of the picornavirus foot-and-mouth disease virus. This virus causes an economically devastating disease of livestock affecting both the developed and developing world. The experimental methods developed to carry out this work are novel, simple, and transferable to the study of packaging signals in other RNA viruses. Improved understanding of RNA packaging may lead to novel vaccine approaches or targets for antiviral drugs with broad-spectrum activity.

The Effect of Medical Student Volunteering in a Student-Run Clinic on Specialty Choice for Residency.

INTRODUCTION:  Student-run free clinics (SRFCs) are a recent popular addition to medical school education, and a subset of studies has looked at the influence of SRFC volunteering on the medical student's career development. The majority of the research done in this area has focused on understanding if these SRFCs produce physicians who are more likely to practice medicine in underserved communities, caring for the uninsured. The remainder of the research has investigated if volunteering in an SRFC influences the specialty choice of medical school students. The results of these specialty choice studies give no definitive answer as to whether medical students chose primary or specialty care residencies as a result of their SRFC experience. Keeping Neighbors in Good Health through Service (KNIGHTS) is the SRFC of the University of Central Florida College of Medicine (UCF COM). Both primary and specialty care is offered at the clinic. It is the goal of this study to determine if volunteering in the KNIGHTS SRFC influences UCF COM medical students to choose primary care, thereby helping to meet the rising need for primary care physicians in the United States. METHODS:  A survey was distributed to first, second, and third-year medical students at the UCF COM to collect data on demographics, prior volunteering experience, and specialty choice for residency. Responses were then combined with records of volunteer hours from the KNIGHTS Clinic and analyzed for correlations. We analyzed the frequency and Pearson's chi-squared values. A p value of less than 0.05 was considered statistically significant. RESULTS:  Our survey had a total response rate of 39.8%. We found that neither the act of becoming a KNIGHTS Clinic volunteer nor the hours volunteered at the KNIGHTS Clinic influenced the UCF COM student's choice to enter a primary care specialty (p = NS). Additionally, prior volunteering/clinical experience or the gender of the medical school student did not influence a student's choice to volunteer at the KNIGHTS Clinic. DISCUSSION:  Volunteering at KNIGHTS Clinic did not increase student choice to enter primary care, with students choosing other specialties at equal rates, probably due to the variety of specialties present at the KNIGHTS Clinic. This suggests that the volunteer attending physicians present at an SRFC may influence the choice of residency for students. It also suggests that SFRCs are not a viable tool to increase the number of primary care doctors in the United States.

Characterization of Heterogeneity in Childhood Immunization Coverage in Central Florida Using Immunization Registry Data.

Despite high vaccine coverage in the United States in general, and in the State of Florida specifically, some children miss scheduled vaccines due to health system failures or vaccine refusal by their parents. Recent experiences with outbreaks in the United States suggest that geographic clustering of un(der)vaccinated populations represent a threat to the elimination status of some vaccine-preventable diseases. Immunization registries continue to expand and play an important role in efforts to track vaccine coverage and use. Using nearly 700,000 de-identified immunization records from the Florida Department of Health immunization information system (Florida SHOTS™) for children born during 2003-2014, we explored heterogeneity and potential clustering of un(der)vaccinated children in six counties in central Florida-Brevard, Lake, Orange, Oseola, Polk, and Seminole-that represent a high-risk area for importation due to family tourist attractions in the area. By zip code, we mapped the population density, the percent of children with religious exemptions, the percent of children on track or overdue for each vaccine series without and with exemptions, and the numbers of children with no recorded dose of each vaccine. Overall, we found some heterogeneity in coverage among the counties and zip codes, but relatively consistent and high coverage. We found that some children with an exemption in the system received the vaccines we analyzed, but exemption represents a clear risk factor for un(der)immunization. We identified many challenges associated with using immunization registry data for spatial analysis and potential opportunities to improve registries to better support future analyses.

A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq.

BACKGROUND: Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. RESULTS: The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5' genomic termini and area immediately flanking the poly(C) region. CONCLUSIONS: We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

Management of psychotropic medication side effects in children and adolescents.

This article is a review of several of the most concerning side effects of psychotropic medications in children and adolescents. An emphasis is placed on review of the prevalence, presentation, monitoring, and evidence-based management of these side effects.