Intestinal Interleukin-17 Receptor Signaling Mediates Reciprocal Control of the Gut Microbiota and Autoimmune Inflammation.

Interleukin-17 (IL-17) and IL-17 receptor (IL-17R) signaling are essential for regulating mucosal host defense against many invading pathogens. Commensal bacteria, especially segmented filamentous bacteria (SFB), are a crucial factor that drives T helper 17 (Th17) cell development in the gastrointestinal tract. In this study, we demonstrate that Th17 cells controlled SFB burden. Disruption of IL-17R signaling in the enteric epithelium resulted in SFB dysbiosis due to reduced expression of α-defensins, Pigr, and Nox1. When subjected to experimental autoimmune encephalomyelitis, IL-17R-signaling-deficient mice demonstrated earlier disease onset and worsened severity that was associated with increased intestinal Csf2 expression and elevated systemic GM-CSF cytokine concentrations. Conditional deletion of IL-17R in the enteric epithelium demonstrated that there was a reciprocal relationship between the gut microbiota and enteric IL-17R signaling that controlled dysbiosis, constrained Th17 cell development, and regulated the susceptibility to autoimmune inflammation.

Offline Two-Dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome Following Fecal Microbiota Transplantation.

Biological interpretation of untargeted LC-MS-based metabolomics data depends on accurate compound identification, but current techniques fall short of identifying most features that can be detected. The human fecal metabolome is complex, variable, incompletely annotated, and serves as an ideal matrix to evaluate novel compound identification methods. We devised an experimental strategy for compound annotation using multidimensional chromatography and semiautomated feature alignment and applied these methods to study the fecal metabolome in the context of fecal microbiota transplantation (FMT) for recurrent C. difficile infection. Pooled fecal samples were fractionated using semipreparative liquid chromatography and analyzed by an orthogonal LC-MS/MS method. The resulting spectra were searched against commercial, public, and local spectral libraries, and annotations were vetted using retention time alignment and prediction. Multidimensional chromatography yielded more than a 2-fold improvement in identified compounds compared to conventional LC-MS/MS and successfully identified several rare and previously unreported compounds, including novel fatty-acid conjugated bile acid species. Using an automated software-based feature alignment strategy, most metabolites identified by the new approach could be matched to features that were detected but not identified in single-dimensional LC-MS/MS data. Overall, our approach represents a powerful strategy to enhance compound identification and biological insight from untargeted metabolomics data.

Comparative Effectiveness of Anti-TNF in Combination With Low-Dose Methotrexate vs Anti-TNF Monotherapy in Pediatric Crohn's Disease: A Pragmatic Randomized Trial.

BACKGROUND & AIMS: Tumor necrosis factor inhibitors, including infliximab and adalimumab, are a mainstay of pediatric Crohn's disease therapy; however, nonresponse and loss of response are common. As combination therapy with methotrexate may improve response, we performed a multicenter, randomized, double-blind, placebo-controlled pragmatic trial to compare tumor necrosis factor inhibitors with oral methotrexate to tumor necrosis factor inhibitor monotherapy. METHODS: Patients with pediatric Crohn's disease initiating infliximab or adalimumab were randomized in 1:1 allocation to methotrexate or placebo and followed for 12-36 months. The primary outcome was a composite indicator of treatment failure. Secondary outcomes included anti-drug antibodies and patient-reported outcomes of pain interference and fatigue. Adverse events (AEs) and serious AEs (SAEs) were collected. RESULTS: Of 297 participants (mean age, 13.9 years, 35% were female), 156 were assigned to methotrexate (110 infliximab initiators and 46 adalimumab initiators) and 141 to placebo (102 infliximab initiators and 39 adalimumab initiators). In the overall population, time to treatment failure did not differ by study arm (hazard ratio, 0.69; 95% CI, 0.45-1.05). Among infliximab initiators, there were no differences between combination and monotherapy (hazard ratio, 0.93; 95% CI, 0.55-1.56). Among adalimumab initiators, combination therapy was associated with longer time to treatment failure (hazard ratio, 0.40; 95% CI, 0.19-0.81). A trend toward lower anti-drug antibody development in the combination therapy arm was not significant (infliximab: odds ratio, 0.72; 95% CI, 0.49-1.07; adalimumab: odds ratio, 0.71; 95% CI, 0.24-2.07). No differences in patient-reported outcomes were observed. Combination therapy resulted in more AEs but fewer SAEs. CONCLUSIONS: Among adalimumab but not infliximab initiators, patients with pediatric Crohn's disease treated with methotrexate combination therapy experienced a 2-fold reduction in treatment failure with a tolerable safety profile. CLINICALTRIALS: gov, Number: NCT02772965.

HLA DQA1*05 and risk of anti-TNF treatment failure and anti-drug antibody development in children with Crohn's Disease: HLA DQA1*05 and Pediatric Crohn's Disease.

OBJECTIVES: HLA DQA1*05 has been associated with the development of anti-drug antibodies (ADA) to tumor necrosis factor antagonists (anti-TNF) and treatment failure among adults with Crohn's disease (CD). However, findings from other studies have been inconsistent with limited pediatric data. METHODS: We analyzed banked serum from patients with CD < 21 years of age enrolled in COMBINE, a multi-center, prospective randomized trial of anti-TNF monotherapy vs. combination with methotrexate. The primary outcome was a composite of factors indicative of treatment failure. The secondary outcome was ADA development. RESULTS: A trend towards increased treatment failure among HLA DQA1*05 positive participants was not significant (HR 1.58, 95% CI 0.95-2.62; p=0.08). After stratification by HLA DQA1*05 and by methotrexate vs. placebo, patients who were HLA DQA1*05 negative and assigned to methotrexate experienced less treatment failures than HLA DQA1*05 positive patients on placebo (HR 0.31, 95% CI 0.13-0.70; p=0.005).A trend toward increased ADA development among HLA DQA1*05 positive participants was not significant (odds ratio [OR] 1.96, 95% CI 0.90-4.31, p=0.09). After further stratification, HLA DQA1*05 negative participants assigned to methotrexate were less likely to develop ADA relative to HLA DQA1*05 positive patients on placebo (OR 0.12, 95% CI 0.03-0.55; p=0.008). CONCLUSIONS: In a randomized trial of children with CD initiating anti-TNF, 40% were HLA DQ-A1*05 positive, which was associated with a trend toward increased risk of both treatment failure and ADA. These risks were mitigated, but not eliminated, by adding oral methotrexate. HLA DQ-A1*05 is an important biomarker for prognosis and risk stratification.

High Body Mass Index and Response to Anti-Tumor Necrosis Factor Therapy in Pediatric Crohn's Disease.

INTRODUCTION: Obesity is common among patients with pediatric Crohn's disease (PCD). Some adult studies suggest obese patients respond less well to anti-tumor necrosis factor (TNF) treatment. This study sought compares anti-TNF response and anti-TNF levels between pediatric patients with normal and high body mass index (BMI). METHODS: The COMBINE trial compared anti-TNF monotherapy with combination therapy with methotrexate in patients with PCD. In this secondary analysis, a comparison of time-to-treatment failure among patients with normal BMI vs BMI Z -score >1, adjusting for prescribed anti-TNF (infliximab [IFX] or adalimumab [ADA]), trial treatment assignment (combination vs monotherapy), and relevant covariates. Median anti-TNF levels across BMI category was also examined. RESULTS: Of 224 participants (162 IFX initiators and 62 ADA initiators), 111 (81%) had a normal BMI and 43 (19%) had a high BMI. High BMI was associated with treatment failure among ADA initiators (7/10 [70%] vs 12/52 [23%], hazard ratio 0.29, P = 0.007) but not IFX initiators. In addition, ADA-treated patients with a high BMI had lower ADA levels compared with those with normal BMI (median 5.8 vs 12.8 µg/mL, P = 0.02). IFX trough levels did not differ between BMI groups. DISCUSSION: Overweight and obese patients with PCD are more likely to experience ADA treatment failure than those with normal BMI. Higher BMI was associated with lower drug trough levels. Standard ADA dosing may be insufficient for overweight children with PCD. Among IFX initiators, there was no observed difference in clinical outcomes or drug levels, perhaps due to weight-based dosing and/or greater use of proactive drug monitoring.

Dual-energy CT in differentiating benign gallbladder wall thickening from wall thickening type of gallbladder cancer.

OBJECTIVE: To evaluate the performance of dual-energy computed tomography (DECT) in differentiating non-acute benign from malignant gallbladder wall thickening (GBWT). METHODS: This prospective study comprised consecutive adults with GBWT who underwent late arterial phase (LAP) and portal venous phase (PVP) DECT between January 2022 and May 2023. The final diagnosis was based on histopathology or 3-6 months follow-up imaging. DECT images in LAP and PVP were assessed independently by two radiologists. The demographic, qualitative, and quantitative parameters were compared between two groups Multivariate logistic regression was performed to determine the association between the aforementioned factors and malignant GBWT. RESULTS: Seventy-five patients (mean age 56 ± 12.8 years, 46 females) were included. Forty-two patients had benign, and 33 had malignant GBWT. In the overall group, female gender (p = 0.018), lymphadenopathy (p = 0.011), and omental nodules (p = 0.044) were significantly associated with malignant GBWT. None of the DECT features differed significantly between benign and malignant GBWT in overall group. In the xanthogranulomatous cholecystitis (XGC, n = 9) vs. gallbladder cancer (GBC) (n = 33) subgroup, mean attenuation value at 140 keV LAP VMI was significantly associated with malignant GBWT [p = 0.023, area under curve 0.759 (95%CI 0.599-0.919)]. CONCLUSION: DECT-generated quantitative parameters do not add value in differentiating non-acute benign from malignant GBWT. However, DECT may have a role in differentiating XGC from GBC in a selected subgroup of patients. Further, larger studies may be necessary to confirm these findings. CLINICAL RELEVANCE STATEMENT: In patients with non-acute gallbladder wall thickening in whom there is suspicion of xanthogranulomatous cholecystitis (XGC), DECT findings may allow differentiation of XGC from wall thickening type of gallbladder cancer. KEY POINTS: Differentiation of benign and malignant gallbladder wall thickening (GBWT) at CT is challenging. Quantitative dual energy CT (DECT) features do not provide additional value in differentiating benign and malignant GBWT. DECT may be helpful in a subgroup of patients to differentiate xanthogranulomatous cholecystitis from gallbladder cancer.

The Successful Use of Upadacitinib as Monotherapy for Hidradenitis Suppurativa and Ulcerative Colitis in the Setting of Refractory Disease.

Hidradenitis suppurativa (HS) and ulcerative colitis (UC) are associated chronic inflammatory conditions with complex disease courses and potential for overlapping therapeutic management. We describe a case of severe pediatric HS and UC that were poorly controlled despite several standard-of-care therapies, including infliximab and ustekinumab. Transitioning the patient to upadacitinib monotherapy resulted in clinical improvement of both her UC and HS within 3 months, and she was then able to be weaned off her other systemic therapies. While upadacitinib is not currently FDA-approved for HS or pediatric UC, this case report shows promise for upadacitinib monotherapy for both of these complex inflammatory disorders.

Using Multidimensional Separations to Distinguish Isomeric Amino Acid-Bile Acid Conjugates and Assess Their Presence and Perturbations in Model Systems.

Bile acids play key roles in nutrient uptake, inflammation, signaling, and microbiome composition. While previous bile acid analyses have primarily focused on profiling 5 canonical primary and secondary bile acids and their glycine and taurine amino acid-bile acid (AA-BA) conjugates, recent studies suggest that many other microbial conjugated bile acids (or MCBAs) exist. MCBAs are produced by the gut microbiota and serve as biomarkers, providing information about early disease onset and gut health. Here we analyzed 8 core bile acids synthetically conjugated with 22 proteinogenic and nonproteogenic amino acids totaling 176 MCBAs. Since many of the conjugates were isomeric and only 42 different m/z values resulted from the 176 MCBAs, a platform coupling liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) was used for their separation. Their molecular characteristics were then used to create an in-house extended bile acid library for a combined total of 182 unique compounds. Additionally, ∼250 rare bile acid extracts were also assessed to provide additional resources for bile acid profiling and identification. This library was then applied to healthy mice dosed with antibiotics and humans having fecal microbiota transplantation (FMT) to assess the MCBA presence and changes in the gut before and after each perturbation.

Fecal Microbiota Transplantation Across the Lifespan: Balancing Efficacy, Safety, and Innovation.

Fecal microbiota transplantation (FMT) is a rapidly growing therapy aimed at reconstituting the dysbiotic microbiota of a patient with the beneficial stool microbiota of a healthy individual. The efficacy rates of FMT are very robust for recurrent Clostridioides difficile infection in both children and adults. Although complications of FMT have been reported, it is generally believed to be a safe procedure. Novel indications for FMT are being studied, with the hope that ultimately it may be useful for a variety of disorders. As this field continues to grow, however, it is necessary to consider efficacy, safety, and innovation across the lifespan. There are unique concerns regarding FMT as it pertains to children, adults, and the elderly. In this review, we seek to update clinicians, researchers, and regulators on how these factors must be balanced across the lifespan as we move forward with this innovative therapy.

Multiparametric MR imaging with diffusion-weighted, intravoxel incoherent motion, diffusion tensor, and dynamic contrast-enhanced perfusion sequences to assess gallbladder wall thickening: a prospective study based on surgical histopathology.

OBJECTIVE: To investigate the diagnostic performance of a multiparametric magnetic resonance imaging (MRI) protocol comprising quantitative MRI (diffusion-weighted imaging (DWI), intravoxel incoherent motion (IVIM), diffusion tensor imaging (DTI), and dynamic contrast-enhanced (DCE) perfusion MRI) and conventional MRI in the characterization of gallbladder wall thickening (GWT). METHODS: This prospective study comprised consecutive adults with GWT who underwent multiparametric MRI between July 2020 and April 2022. Two radiologists evaluated the MRI independently. The final diagnosis was based on surgical histopathology. The association of MRI parameters with malignant GWT was evaluated. The area under the curve (AUC) for the quantitative MRI parameters and diagnostic performance of conventional, and multiparametric MRI were compared. The interobserver agreement between two radiologists was calculated. RESULTS: Thirty-five patients (mean age, 56 years, 23 females) with GWT (25 benign and ten malignant) were evaluated. The quantitative MRI parameters significantly associated with malignant GWT were apparent diffusion coefficient on DWI (p = 0.007) and mean diffusivity (MD) on DTI (p = 0.013), perfusion fraction (f) on IVIM (p = 0.033), time to peak enhancement (TTP, p = 0.008), and wash in rate (p = 0.049) on DCE-MRI. TTP had the highest AUC of 0.790, followed by MD (0.782) and f (0.742) (p = 0.213) for predicting malignant GWT. Multiparametric MRI had significantly higher sensitivity (90% vs. 80%, p = 0.045) than conventional MRI for diagnosing malignant GWT. The two radiologists' reading had substantial to near-perfect agreement (kappa = 0.639-1) and moderate to strong correlation (interclass correlation coefficient = 0.5-0.88). CONCLUSION: Multiparametric protocol incorporating advanced sequences improved the diagnostic performance of MRI for differentiating benign and malignant GWT. KEY POINTS: • Multiparametric MRI had 90% sensitivity and 88% specificity for diagnosing malignant GWT, compared to 80% sensitivity and 88% specificity for conventional CE-MRI. • Among the quantitative MRI parameters, TTP (perfusion-MRI) had the highest AUC of 0.790, followed by MD (0.782) and IVIM-f (0.742). • For most quantitative MRI parameters, there was moderate to strong agreement (ICC = 0.5-0.88).

Irgm1-deficiency leads to myeloid dysfunction in colon lamina propria and susceptibility to the intestinal pathogen Citrobacter rodentium.

IRGM and its mouse orthologue Irgm1 are dynamin-like proteins that regulate vesicular remodeling, intracellular microbial killing, and pathogen immunity. IRGM dysfunction is linked to inflammatory bowel disease (IBD), and while it is thought that defective intracellular killing of microbes underscores IBD susceptibility, studies have yet to address how IRGM/Irgm1 regulates immunity to microbes relevant to intestinal inflammation. Here we find that loss of Irgm1 confers marked susceptibility to Citrobacter rodentium, a noninvasive intestinal pathogen that models inflammatory responses to intestinal bacteria. Irgm1-deficient mice fail to control C. rodentium outgrowth in the intestine, leading to systemic pathogen spread and host mortality. Surprisingly, susceptibility due to loss of Irgm1 function was not linked to defective intracellular killing of C. rodentium or exaggerated inflammation, but was instead linked to failure to remodel specific colon lamina propria (C-LP) myeloid cells that expand in response to C. rodentium infection and are essential for C. rodentium immunity. Defective immune remodeling was most striking in C-LP monocytes, which were successfully recruited to the infected C-LP, but subsequently underwent apoptosis. Apoptotic susceptibility was induced by C. rodentium infection and was specific to this setting of pathogen infection, and was not apparent in other settings of intestinal inflammation. These studies reveal a novel role for Irgm1 in host defense and suggest that deficiencies in survival and remodeling of C-LP myeloid cells that control inflammatory intestinal bacteria may underpin IBD pathogenesis linked to IRGM dysfunction.

Implications of Paneth cell dysfunction on gastrointestinal health and disease.

PURPOSE OF REVIEW: Paneth cells are specialized, secretory epithelial cells located in the small intestine. Although their existence was first described in 1872, their precise role in the gut remained unclear for over a century. Over the past few decades, elegant studies have shown Paneth cells play a key role enhancing gut barrier function, as niche cells for the intestinal stem cell compartment and via secreting antimicrobial peptides to establish an antimicrobial barrier at the epithelial surface. This review describes what is known about Paneth cell biology from human and animal studies with a focus on their putative role in clinical gastrointestinal disease. RECENT FINDINGS: Recent work has demonstrated important associations of dysfunctional Paneth cells with several gastrointestinal disorders. These include Crohn's disease, enteric infections, graft-versus-host disease, necrotizing enterocolitis, and environmental enteric dysfunction. Ongoing studies are examining precisely how Paneth cell biology is altered in these various disease states. SUMMARY: By understanding the mechanisms of Paneth cell regulation - and how these processes go awry in specific gastrointestinal diseases - we set the stage for using Paneth cells as biomarkers for disease progression and developing novel therapeutics that augment Paneth cell function to treat a spectrum of gastrointestinal disorders.

Diagnosis and Management of Oral Extraintestinal Manifestations of Pediatric Inflammatory Bowel Disease.

ABSTRACT: Inflammatory bowel diseases (IBD) represent a group of chronic inflammatory disorders of the gastrointestinal tract that lead to impaired quality of life and substantial health care costs. Up to 50% of pediatric IBD cases present with manifestations in the oral cavity. These may develop in nearly every oral tissue, including the soft tissues, tongue, lips, teeth, and lymph nodes. The goal of this review is to offer a systematic approach to diagnose and manage commonly encountered oral manifestations of pediatric IBD. This knowledge is critical for enhancing the comprehensive care and quality of life of children with these debilitating diseases.

Diagnostic accuracy of Liver Imaging Reporting and Data System locoregional treatment response criteria: a systematic review and meta-analysis.

OBJECTIVE: There is increasing adoption of Liver Imaging Reporting and Data System (LI-RADS) treatment response (LR-TR) criteria. However, there is still a relative lack of evidence evaluating the performance of these criteria. We performed this study to assess the diagnostic accuracy of LI-RADS LR-TR criteria. METHODS: A thorough search of PubMed, Embase, Scopus, and Cochrane Central Register of Controlled Trials for studies reporting diagnostic accuracy of LI-RADS LR-TR criteria was conducted through 30 June 2020. The meta-analytic summary of sensitivity, specificity, and diagnostic odds ratio of LI-RADS LR-TR criteria was computed using explant histopathology as the reference standard. The quality of the studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. RESULTS: Four studies were found eligible for meta-analysis. The total number of LR-TR observations was 462 (240 patients, 82.5% males). Different locoregional therapies (LRTs), including bland embolization, chemoembolization, radiofrequency ablation, and microwave ablation, had been used. The mean time interval between LRT and liver transplantation ranged from 181 to 219 days. There was a moderate to good inter-reader agreement for LR-TR criteria. The pooled sensitivity and specificity of LR-TR criteria for viable disease were 62% (95% CI, 49-74%; I2 = 69%) and 87% (95% CI, 76-93%; I2 = 57%), respectively. The pooled diagnostic odds ratio and area under the curve were 9.83 (95% CI, 5.34-18.08; I2 = 19%) and 0.80. CONCLUSIONS: LI-RADS LR-TR criteria have acceptable diagnostic performance for the diagnosis of viable tumor after LRT. Well-designed prospective studies evaluating criteria of equivocal lesions and effect of different LRTs should be performed. KEY POINTS: • The pooled sensitivity and specificity of LI-RADS LR-TR criteria for the diagnosis of viable tumor were 62% and 87%, respectively. • The pooled diagnostic odds ratio and area under the curve were 9.83 and 0.80. • LR-TR criteria had a moderate to good inter-reader agreement.

Comparison of virtual computed tomography enteroscopy using carbon dioxide with small-bowel enteroclysis and capsule endoscopy in patients with small-bowel tuberculosis.

OBJECTIVE: Comparison of virtual CT enteroscopy (VCTE) using carbon dioxide with small-bowel enteroclysis (SBE) and capsule endoscopy (CE) in small-bowel tuberculosis (SBTB). METHODS: This prospective study comprised consecutive patients suspected to have SBTB. VCTE and SBE were performed on the same day and evaluated by independent radiologists. CE was performed within 2 weeks. VCTE was performed following insufflation of carbon dioxide via catheters in the jejunum and anorectum. A contrast-enhanced CT was followed by a delayed non-contrast CT. Image processing was done using virtual colonoscopy software. Findings on VCTE, SBE, and CE were compared. The final diagnosis of SBTB was based on either histopathological or cytological findings, response to antitubercular treatment, or a combination of these. RESULTS: Of the 55 patients in whom VCTE was performed, complete data was available in 52 patients. A final diagnosis of SBTB was established in 37 patients. All patients had VCTE and SBE. CE was performed in 34 patients. Adequate luminal distension was achieved in all patients with SBE and 35 patients with VCTE. SBE showed more strictures in jejunum (10.8%) and ileum (75.7%) compared with VCTE (jejunum, 8.1%, and ileum, 64.9%) and CE (jejunum, 5.9%, and ileum, 61.8%). However, difference was not statistically significant. VCTE revealed a greater length of strictures in both the jejunum and ileum compared with SBE and CE. CONCLUSION: VCTE allows adequate evaluation of the bowel in most patients with SBTB. It allows detection of greater length of abnormality in jejunum and ileum compared with SBE and CE. KEY POINTS: • The use of VCTE using CO2 bowel insufflation in patients with SBTB should be considered. • VCTE allows detection of a greater length of abnormality in the jejunum and ileum.

Unusual Sites of Necrotic Collections in Acute Necrotizing Pancreatitis: Association with Parenchymal Necrosis and Clinical Outcomes.

BACKGROUND: The presence of necrotic collection in acute necrotizing pancreatitis (ANP) at intra-abdominal sites other than the retroperitoneum has not been systematically studied. AIM: To investigate unusual sites of necrotic collections at computed tomography (CT) and to evaluate association with pancreatic necrosis and clinical outcomes. METHODS: This retrospective study comprised of consecutive patients with ANP evaluated between January 2018 and March 2019. Based on CT findings, patients were divided into two groups: collections at unusual sites (small bowel mesentery, mesocolon, omentum, subcapsular collections along liver and spleen, pelvis, anterior abdominal wall, and inguinoscrotal regions) and collections at usual retroperitoneal locations (lesser sac, gastrosplenic location, anterior and posterior pararenal spaces, and paracolic gutters). The differences in CT findings and clinical outcomes (need for drainage, length of hospitalization, intensive care unit admission, surgery, and death) between the two groups were evaluated. RESULTS: A total of 75 patients with ANP were evaluated. There were 25 (33.3%) patients with collections in unusual locations. These included mesentery (n = 17), splenic subcapsular location (n = 7), omentum (n = 6), hepatic subcapsular location (n = 4), anterior abdominal wall (n = 3), pelvis (n = 2), and inguinoscrotal location (n = 1). Compared to patients with collections at usual locations (n = 50), there were no differences in the CT findings except complete parenchymal necrosis (32% vs. 0%, P = .001). There were no statistically significant differences in the clinical outcomes between the two groups. CONCLUSIONS: Mesenteric collections are frequent in ANP. The other non-retroperitoneal sites are infrequently involved. There is no association between unusual sites of collection and clinical outcomes.

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

Outcome of percutaneous drainage in patients with pancreatic necrosis having organ failure.

BACKGROUND: Percutaneous catheter drainage (PCD) is an effective way of drainage in acute pancreatitis (AP) and its role in persistent organ failure (OF) has not been studied. This study assessed the outcome of severe AP managed with PCD. METHODS: We retrospectively analysed outcome of AP patients undergoing PCD for persistent OF with respect to success of PCD, etiology, severity scores, OF, imaging features and PCD parameters. Success of PCD was defined as resolution of with PCD and survived without surgical necrosectomy. RESULTS: Between January 2016 and May 2018, 83 patients underwent PCD for persistent OF at a mean duration of 25.59 ± 21.2 days from pain onset with successful outcome in 47 (56.6%) patients. Among PCD failures, eleven (13.25%) patients underwent surgery. Overall mortality was 31 (37.3%). On multivariate analysis, pancreatic necrosis <50% and absence of extrapancreatic infection (EPI) predicted the success of PCD. Presence of infected necrosis did not affect the outcome of PCD in organ failure. CONCLUSION: PCD improves the outcome in patients with OF even when done early irrespective of the status of infection of necrosis. Therefore, PCD may be considered early in the course of patients with OF.

Reappraisal of the classical abdominal anatomical landmarks using in vivo computerized tomography imaging.

INTRODUCTION: With expanding scope of interventions it becomes mandatory to have correct and evidence-based knowledge of surface anatomy of internal abdominal structures. Information available in text books is derived from work done on cadaveric studies. Current study was designed to provide data of key abdominal surface anatomical landmarks and their variations in living subjects using CT imaging of adult population. MATERIALS AND METHODS: Cross-sectional study was conducted using 100 abdominopelvic CT scans of patients of Indian origin. RESULTS: Vertebral levels of origin of celiac trunk varied from T11 to L1/2 intervertebral disc, superior mesenteric artery from T12 to L2, inferior mesenteric artery from L2 to L4 and aortic bifurcation from L3 to L5. Origin of both renal arteries varied between T12 and L2 and the formation of inferior vena cava varied from L3 to L5. Vertebral levels of upper pole of both kidneys ranged from T11 to upper L3. Spleen was related to 9th to 11th ribs in 36% and 10th to 12th ribs in 34% scans. Most common vertebral levels of subcostal plane, planum supracristale and planum intertuberculare noticed were lower L2, L4 and lower L5, respectively. CONCLUSIONS: Data derived from imaging investigations of living subjects and variations from the conventional descriptions observed in the current study might be helpful for clinicians.

Serum Protein Biomarkers of Fibrosis Aid in Risk Stratification of Future Stricturing Complications in Pediatric Crohn's Disease.

OBJECTIVES: Avoiding fibrostenotic complications is of paramount concern in the management of Crohn's disease (CD). We sought to investigate the association of candidate biomarkers of fibrosis collected at diagnosis with the future development of fibrostenotic CD. METHODS: Using the Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's Disease cohort, a multicenter prospective observational pediatric inception cohort, subjects with an inflammatory phenotype (B1) at diagnosis who later converted to a stricturing phenotype (B2) within 3 years were compared with those who remained B1. Serum collected at diagnosis underwent both parallel reaction monitoring-targeted proteomic analysis and conventional enzyme-linked immunosorbent assay for 10 candidate biomarkers of intestinal fibrosis. Cox proportional hazard regression was used for multivariable analysis of time-dependent outcomes. RESULTS: In 116 subjects 58 subjects with verified B1 phenotype at diagnosis who later converted to B2 disease were compared with 58 subjects who remained B1 over 3 years of follow-up. Extracellular matrix protein 1 (ECM1) levels in the upper quartile (hazard ratio [HR] 3.43, 95% confidence limit [CL] 1.33, 8.42) were associated with future fibrostenotic disease. ASCA IgA (HR 4.99, 95% CL 1.50, 16.68) and CBir levels (HR 5.19, 95% CL 1.83, 14.74) were also associated with future intestinal fibrostenosis, although ECM1 continued to demonstrate independent association with conversion to B2 even with adjustment for serologies in multivariable analysis (HR 5.33, 95% CL 1.29, 22.13). CONCLUSIONS: ECM1 and other biomarkers of fibrosis may aid in determining the risk of uncomplicated inflammatory disease converting to B2 stricturing phenotypes in children with CD. Prospective validation studies to verify test performance and optimize clinical utilization are needed before clinical implementation.