Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0267-5) contains supplementary material, which is available to authorized users.
There is limited experience with vedolizumab therapy in pediatric IBD. There seems to be a marked number of subjects with clinical response in the first 6 weeks that increases further by week 22 despite the severity of disease in this cohort. Adverse events may not be directly related to vedolizumab. This study is limited by small sample size, and larger prospective studies are warranted.
Background Insight into the pathogenesis of very early onset-inflammatory bowel disease (VEO-IBD) has expanded through the identification of causative monogenic defects detected in a subset of patients. However, the clinical course of this population remains uncertain. The study objective is to determine whether VEO-IBD is associated with more severe disease, defined as increased surgical intervention and growth failure, than older pediatric IBD. Secondary outcomes included therapeutic response and hospitalizations. Methods Subjects with IBD diagnosed younger than 6 years old (VEO-IBD) were compared with children diagnosed 6 to 10 (intermediate-onset) and older than 10 years of age (older-onset IBD). Metadata obtained from the medical record included age of onset, disease phenotype and location, surgeries, medical therapy, and comorbid conditions. Length of follow-up was at least 1 year from diagnosis. Results There were 229, 221, and 521 subjects with VEO, intermediate-onset, and older-onset IBD, respectively. Very early onset-inflammatory bowel disease subjects underwent more diverting ileostomies (P < 0.001) and colectomies (P < 0.001) than the older children. There was less improvement in weight- and height-for-age Z scores during the follow-up period in subjects with VEO-IBD. Additionally, subjects with VEO-IBD had higher rates of medication failure at 1 year and were more frequently readmitted to the hospital. Targeted therapy was successfully used almost exclusively in VEO-IBD. Conclusion Patients with VEO-IBD can have a more severe disease course with increased surgical interventions and poor growth as compared with older-onset IBD patients. Further, VEO-IBD patients are more likely to be refractory to conventional therapies. Strategies using targeted therapy in these children can improve outcome and, in some cases, be curative.
Background and Aims: Children with very early onset inflammatory bowel disease [VEO-IBD] represent a unique cohort, often with a severe phenotype that is refractory to conventional medications, and some cases have underlying primary immunodeficiencies. Previous work has identified distinct histopathological patterns in the gastrointestinal tract in patients with primary immunodeficiencies. The aim of this study is to characterise the diagnostic histological findings in patients with VEO-IBD as compared with older onset paediatric IBD, and determine if there are unique pathological changes that can shed light on the driving forces of the disease, particularly immunodeficiencies. Methods: Clinical retrospective chart review, including disease characteristics and endoscopic findings, was performed on all included subjects. Two paediatric pathologists reviewed biopsies from diagnostic upper endoscopies and colonoscopies of subjects with very early onset inflammatory bowel disease and older onset inflammatory bowel disease, to evaluate for the presence of 11 histological features previously associated with inflammatory bowel disease and primary immunodeficiencies. Results: The diagnostic gastrointestinal biopsies of subjects with very early onset inflammatory bowel disease differed from those in older onset paediatric IBD, demonstrated by increased frequency of apoptosis, severe chronic architectural changes, small intestine villous blunting, and eosinophils in the crypts, lamina propria, and surface epithelium. Conclusions:The diagnostic biopsies of children with very early onset inflammatory bowel disease can identify characteristic features that may be important in guiding the diagnostic work-up in this population.
Background and Aims Dysbiosis of the gut microbiota is a well-known correlate of the pathogenesis of inflammatory bowel disease [IBD]. However, few studies have examined the microbiome in very early-onset [VEO] IBD, which is defined as onset of IBD before 6 years of age. Here we focus on the viral portion of the microbiome—the virome—to assess possible viral associations with disease processes, reasoning that any viruses potentially associated with IBD might grow more robustly in younger subjects, and so be more detectable. Methods Virus-like particles [VLPs] were purified from stool samples collected from patients with VEO-IBD [n = 54] and healthy controls [n = 23], and characterized by DNA and RNA sequencing and VLP particle counts. Results The total number of VLPs was not significantly different between VEO-IBD and healthy controls. For bacterial viruses, the VEO-IBD subjects were found to have a higher ratio of Caudovirales vs to Microviridae compared to healthy controls. An increase in Caudovirales was also associated with immunosuppressive therapy. For viruses infecting human cells, Anelloviridae showed higher prevalence in VEO-IBD compared to healthy controls. Within the VEO-IBD group, higher levels of Anelloviridae DNA were also positively associated with immunosuppressive treatment. To search for new viruses, short sequences enriched in VEO-IBD samples were identified, and some could be validated in an independent cohort, although none was clearly viral; this provides sequence tags to interrogate in future studies. Conclusions These data thus document perturbations to normal viral populations associated with VEO-IBD, and provide a biomarker—Anelloviridae DNA levels—potentially useful for reporting the effectiveness of immunosuppression.
Highlights d Multi-omics reveals markers of CDI in pediatric IBD patients d Identification of metabolites reveals distinctive features for IBD and CDI d Isocaproyltaurine is made by C. difficile and associates with active IBD d Identifies biomarkers potentially useful for distinguishing disease processes
Purpose of Review: Inflammatory bowel disease (IBD) is a multifactorial disease caused by dysregulated immune responses to commensal or pathogenic intestinal microbes, resulting in chronic intestinal inflammation. However, a subset of patients with IBD diagnosed <6 years of age, known as very early-onset (VEO)-IBD, can be phenotypically and genetically distinct from older onset IBD. We aim to review the clinical presentation of children with VEO-IBD and recent discoveries that point to the underlying genomic and immunologic drivers of disease, and the significant impact on our therapeutic decisions. Recent Findings: VEO-IBD is increasing in incidence and is associated with more severe disease, aggressive progression, and poor response to most conventional therapies. This article will review some of the genetic findings in this population and the subsequent impact on therapy, with targeted approaches. Summary: Children with VEO-IBD may present with a different phenotype and more severe disease than older children and adults. An integrated approach combining genetics, immunology, and traditional IBD evaluations can lead to the identification of causal defects that directly impact management. These strategies can also be employed in older onset refractory IBD.
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