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.

Metagenomic, metabolomic, and lipidomic shifts associated with fecal microbiota transplantation for recurrent Clostridioides difficile infection.

Recurrent C. difficile infection (rCDI) is an urgent public health threat for which the last resort and lifesaving treatment is a fecal microbiota transplant (FMT). However, the exact mechanisms which mediate a successful FMT are not well understood. Here we use longitudinal stool samples collected from patients undergoing FMT to evaluate changes in the microbiome, metabolome, and lipidome after successful FMTs. We show changes in the abundance of many lipids, specifically acylcarnitines and bile acids, in response to FMT. These changes correlate with Enterobacteriaceae, which encode carnitine metabolism genes, and Lachnospiraceae, which encode bile salt hydrolases and baiA genes. LC-IMS-MS revealed a shift from microbial conjugation of primary bile acids pre-FMT to secondary bile acids post-FMT. Here we define the structural and functional changes in successful FMTs. This information will help guide targeted Live Biotherapeutic Product development for the treatment of rCDI and other intestinal diseases.

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.

Gut Microbial Changes Following Fecal Microbiota Transplantation for D-Lactic Acidosis in Two Children.

D-lactic acidosis (D-LA) is an uncommon complication of short bowel syndrome characterized by elevated plasma D-lactate and encephalopathy. Treatments include rehydration, dietary carbohydrate restriction, and antibiotics to alter the gut microbiota. Fecal microbiota transplantation (FMT) has recently been used in children to successfully treat D-LA. We compared the clinical course and then utilized metagenomic shotgun sequencing to describe changes in the composition and function of the intestinal microbiome following FMT in 2 patients with recurrent D-LA. FMT altered the composition of the fecal microbiota in these 2 patients with recurrent D-LA, though not necessarily in a consistent manner. Importantly, microbial metabolic pathways were also impacted by FMT, which may be critical for achieving desired clinical outcomes. While sample size limits the generalizability of our results, these findings set the stage for further understanding of the role of microbes in the pathogenesis of recurrent D-LA.

Giardia hinders growth by disrupting nutrient metabolism independent of inflammatory enteropathy.

Giardia lamblia (Giardia) is among the most common intestinal pathogens in children in low- and middle-income countries (LMICs). Although Giardia associates with early-life linear growth restriction, mechanistic explanations for Giardia-associated growth impairments remain elusive. Unlike other intestinal pathogens associated with constrained linear growth that cause intestinal or systemic inflammation or both, Giardia seldom associates with chronic inflammation in these children. Here we leverage the MAL-ED longitudinal birth cohort and a model of Giardia mono-association in gnotobiotic and immunodeficient mice to propose an alternative pathogenesis of this parasite. In children, Giardia results in linear growth deficits and gut permeability that are dose-dependent and independent of intestinal markers of inflammation. The estimates of these findings vary between children in different MAL-ED sites. In a representative site, where Giardia associates with growth restriction, infected children demonstrate broad amino acid deficiencies, and overproduction of specific phenolic acids, byproducts of intestinal bacterial amino acid metabolism. Gnotobiotic mice require specific nutritional and environmental conditions to recapitulate these findings, and immunodeficient mice confirm a pathway independent of chronic T/B cell inflammation. Taken together, we propose a new paradigm that Giardia-mediated growth faltering is contingent upon a convergence of this intestinal protozoa with nutritional and intestinal bacterial factors.

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.

Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut.

Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile.

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.

Agr2-associated ER stress promotes adherent-invasive E. coli dysbiosis and triggers CD103+ dendritic cell IL-23-dependent ileocolitis.

Endoplasmic reticulum (ER) stress is associated with Crohn's disease (CD), but its impact on host-microbe interaction in disease pathogenesis is not well defined. Functional deficiency in the protein disulfide isomerase anterior gradient 2 (AGR2) has been linked with CD and leads to epithelial cell ER stress and ileocolitis in mice and humans. Here, we show that ileal expression of AGR2 correlates with mucosal Enterobactericeae abundance in human inflammatory bowel disease (IBD) and that Agr2 deletion leads to ER-stress-dependent expansion of mucosal-associated adherent-invasive Escherichia coli (AIEC), which drives Th17 cell ileocolitis in mice. Mechanistically, our data reveal that AIEC-induced epithelial cell ER stress triggers CD103+ dendritic cell production of interleukin-23 (IL-23) and that IL-23R is required for ileocolitis in Agr2-/- mice. Overall, these data reveal a specific and reciprocal interaction of the expansion of the CD pathobiont AIEC with ER-stress-associated ileocolitis and highlight a distinct cellular mechanism for IL-23-dependent ileocolitis.

Pediatric Inflammatory Bowel Disease for General Surgeons.

Key differences exist in pediatric and adult inflammatory bowel disease (IBD), and a multidisciplinary approach focused on meeting these needs should be implemented. In an emergency situation, surgical management of pediatric IBD should focus on patient stabilization with an eye toward future intestinal function.

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.

Increased colonic expression of ACE2 associates with poor prognosis in Crohn's disease.

The host receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small intestine. Our aim was to study colonic ACE2 expression in Crohn's disease (CD) and non-inflammatory bowel disease (non-IBD) controls. We hypothesized that the colonic expression levels of ACE2 impacts CD course. We examined the expression of colonic ACE2 in 67 adult CD and 14 NIBD control patients using RNA-seq and quantitative (q) RT-PCR. We validated ACE2 protein expression and localization in formalin-fixed, paraffin-embedded matched colon and ileal tissues using immunohistochemistry. The impact of increased ACE2 expression in CD for the risk of surgery was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator. To provide critical support for the generality of our findings, we analyzed previously published RNA-seq data from two large independent cohorts of CD patients. Colonic ACE2 expression was significantly higher in a subset of adult CD patients which was defined as the ACE2-high CD subset. IHC in a sampling of ACE2-high CD patients confirmed high ACE2 protein expression in the colon and ileum compared to ACE2-low CD and NIBD patients. Notably, we found that ACE2-high CD patients are significantly more likely to undergo surgery within 5 years of CD diagnosis, and a Cox regression analysis found that high ACE2 levels is an independent risk factor for surgery (OR 2.17; 95% CI, 1.10-4.26; p = 0.025). Increased intestinal expression of ACE2 is associated with deteriorated clinical outcomes in CD patients. These data point to the need for molecular stratification that can impact CD disease-related outcomes.

The "Gum-Gut" Axis in Inflammatory Bowel Diseases: A Hypothesis-Driven Review of Associations and Advances.

In modern medicine, the oral cavity has often been viewed as a passive conduit to the upper airways and gastrointestinal tract; however, its connection to the rest of the body has been increasingly explored over the last 40 years. For several diseases, the periodontium and gingiva are at the center of this oral-systemic link. Over 50 systemic conditions have been specifically associated with gingival and periodontal inflammation, including inflammatory bowel diseases (IBD), which have recently been elevated from simple "associations" to elegant, mechanistic investigations. IBD and periodontitis have been reported to impact each other's progression via a bidirectional relationship whereby chronic oral or intestinal inflammation can impact the other; however, the precise mechanisms for how this occurs remain unclear. Classically, the etiology of gingival inflammation (gingivitis) is oral microbial dysbiosis in the subgingival crevice that can lead to destructive periodontal disease (periodontitis); however, the current understanding of gingival involvement in IBD is that it may represent a separate disease entity from classical gingivitis, arising from mechanisms related to systemic inflammatory activation of niche-resident immune cells. Synthesizing available evidence, we hypothesize that once established, IBD can be driven by microbiomial and inflammatory changes originating specifically from the gingival niche through saliva, thereby worsening IBD outcomes and thus perpetuating a vicious cycle. In this review, we introduce the concept of the "gum-gut axis" as a framework for examining this reciprocal relationship between the periodontium and the gastrointestinal tract. To support and explore this gum-gut axis, we 1) provide a narrative review of historical studies reporting gingival and periodontal manifestations in IBD, 2) describe the current understanding and advances for the gum-gut axis, and 3) underscore the importance of collaborative treatment and research plans between oral and GI practitioners to benefit this patient population.

Increased Colonic Expression of ACE2 Associates with Poor Prognosis in Crohn's disease.

BACKGROUND AND AIMS: The host receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small intestine. Our aim was to study colonic ACE2 expression in Crohn's disease (CD) and non-inflammatory bowel disease (non-IBD) controls. We hypothesized that the colonic expression levels of ACE2 impacts CD course. METHODS: We examined the expression of colon ACE2 using RNA-seq and quantitative (q) RT-PCR from 69 adult CD and 14 NIBD control patients. In a subset of this cohort we validated ACE2 protein expression and localization in formalin-fixed, paraffin-embedded matched colon and ileal tissues using immunohistochemistry. The impact of increased ACE2 expression in CD for the risk of surgery was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator. To provide critical support for the generality of our findings, we analyzed previously published RNA-seq data from two large independent cohorts of CD patients. RESULTS: Colonic ACE2 expression was significantly higher in a subset of adult CD patients (ACE2-high CD). IHC in a sampling of ACE2-high CD patients confirmed high ACE2 protein expression in the colon and ileum compared to ACE2-low CD and NIBD patients. Notably, we found that ACE2-high CD patients are significantly more likely to undergo surgery within 5 years of diagnosis, with a Cox regression analysis finding that high ACE2 levels is an independent risk factor (OR 2.18; 95%CI, 1.05-4.55; p=0.037). CONCLUSION: Increased intestinal expression of ACE2 is associated with deteriorated clinical outcomes in CD patients. These data point to the need for molecular stratification that may impact CD disease-related outcomes.

Mucosal Inflammatory and Wound Healing Gene Programs Reveal Targets for Stricturing Behavior in Pediatric Crohn's Disease.

BACKGROUND AND AIMS: Ileal strictures are the major indication for resective surgery in Crohn's disease (CD). We aimed to define ileal gene programs present at diagnosis linked with future stricturing behavior during five year follow-up, and to identify potential small molecules to reverse these gene signatures. METHODS: Antimicrobial serologies and pre-treatment ileal gene expression were assessed in a representative subset of 249 CD patients within the RISK multicenter pediatric CD inception cohort study, including 113 that are unique to this report. These data were used to define genes associated with stricturing behavior and for model testing to predict stricturing behavior. A bioinformatics approach to define small molecules which may reverse the stricturing gene signature was applied. RESULTS: 19 of the 249 patients developed isolated B2 stricturing behavior during follow-up, while 218 remained B1 inflammatory. Using deeper RNA sequencing than in our prior report, we have now defined an inflammatory gene signature including an oncostatin M co-expression signature, tightly associated with extra-cellular matrix (ECM) gene expression in those who developed stricturing complications. We further computationally prioritize small molecules targeting macrophage and fibroblast activation and angiogenesis which may reverse the stricturing gene signature. A model containing ASCA and CBir1 serologies and a refined eight ECM gene set was significantly associated with stricturing development by year five after diagnosis (AUC (95th CI) = 0.82 (0.7-0.94)). CONCLUSION: An ileal gene program for macrophage and fibroblast activation is linked to stricturing complications in treatment naïve pediatric CD, and may inform novel small molecule therapeutic approaches.

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.

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.

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.