Event-free survival of maralixibat-treated patients with Alagille syndrome compared to a real-world cohort from GALA.

BACKGROUND AND AIMS: Alagille syndrome (ALGS) is characterized by chronic cholestasis with associated pruritus and extrahepatic anomalies. Maralixibat, an ileal bile acid transporter inhibitor, is an approved pharmacologic therapy for cholestatic pruritus in ALGS. Since long-term placebo-controlled studies are not feasible or ethical in children with rare diseases, a novel approach was taken comparing 6-year outcomes from maralixibat trials with an aligned and harmonized natural history cohort from the G lobal AL agille A lliance (GALA) study. APPROACH AND RESULTS: Maralixibat trials comprise 84 patients with ALGS with up to 6 years of treatment. GALA contains retrospective data from 1438 participants. GALA was filtered to align with key maralixibat eligibility criteria, yielding 469 participants. Serum bile acids could not be included in the GALA filtering criteria as these are not routinely performed in clinical practice. Index time was determined through maximum likelihood estimation in an effort to align the disease severity between the two cohorts with the initiation of maralixibat. Event-free survival, defined as the time to first event of manifestations of portal hypertension (variceal bleeding, ascites requiring therapy), surgical biliary diversion, liver transplant, or death, was analyzed by Cox proportional hazards methods. Sensitivity analyses and adjustments for covariates were applied. Age, total bilirubin, gamma-glutamyl transferase, and alanine aminotransferase were balanced between groups with no statistical differences. Event-free survival in the maralixibat cohort was significantly better than the GALA cohort (HR, 0.305; 95% CI, 0.189-0.491; p <0.0001). Multiple sensitivity and subgroup analyses (including serum bile acid availability) showed similar findings. CONCLUSIONS: This study demonstrates a novel application of a robust statistical method to evaluate outcomes in long-term intervention studies where placebo comparisons are not feasible, providing wide application for rare diseases. This comparison with real-world natural history data suggests that maralixibat improves event-free survival in patients with ALGS.

Racial/Ethnic Disparities in the Management of Pediatric Acute Pancreatitis Across Children's Hospitals.

OBJECTIVES: Racial or ethnic disparities in health care delivery and resource utilization have been reported in a variety of pediatric diseases. In acute pancreatitis (AP), there is an association between Black race and increased inpatient mortality. Data on the association of race and ethnicity and resource use for managing pediatric AP are lacking. The aim of this study is to investigate this potential association in pediatric AP. METHODS: Retrospective study of children 0-18 years diagnosed with AP in the Pediatric Health Information System (PHIS) database from 2012 to 2018. Descriptive statistics were used to summarize cohort characteristics. Race/ethnicity classifications included non-Hispanic Black (NHB), non-Hispanic White (NHW, used as reference), Hispanic, and "Other." Associations between patient characteristics and race/ethnicity were determined using χ2 tests. Generalized linear mixed regression model was used to determine the association of race/ethnicity with odds of resource utilization, costs, and length of hospital stay after adjusting for covariates with a random intercept for site. RESULTS: Five thousand nine hundred sixty-three patients from 50 hospitals were included. Adjusted analysis showed that NHB children hospitalized with AP were at lower odds of receiving opioids in the first 24 hours [adjusted odds ratio (aOR) = 0.82, 95% confidence interval (CI) = 0.70-0.98] and receiving intravenous fluids during the hospitalization (aOR = 0.64, 95% CI = 0.43-0.96) when compared with NHW children. Additionally, NHB and Hispanic children had a prolonged adjusted mean length of hospital stay and higher hospital costs when compared with NHW children. Although there was no significant association between race/ethnicity and diagnosis of pancreatic necrosis or sepsis, Hispanic and "Other" children were at higher odds of receiving antibiotics during hospitalization for AP (aOR = 1.33, 95% CI = 1.13-1.57 and aOR = 1.37, 95% CI = 1.09-1.73, respectively) than NHW children. CONCLUSIONS: Disparities exist in utilization of health care interventions for pediatric AP patients by race/ethnicity. Future studies should investigate why these disparities exist and if these disparities affect outcomes.

Glucocorticoids Improve Enteral Feeding Tolerance in Pediatric Short Bowel Syndrome With Chronic Intestinal Inflammation.

OBJECTIVES: A group of short bowel syndrome (SBS) patients developed chronic intestinal inflammation while struggling weaning off parenteral nutrition (PN). They did not respond to standard management of SBS and food allergy. We treated them with glucocorticoids and described the outcome. METHODS: Our study is a retrospective descriptive study. We reviewed records from the intestinal rehabilitation program from 2006 to 2017. We identified 15 patients whose lab values, pathology results, and clinic notes were reviewed. RESULTS: We had more patients (n = 10) with diagnosis of gastroschisis, and more female patients (n = 9). Seven patients weaned off PN with median treatment duration of 5 months, 5 of which remained on budesonide for significant period of time (median: 7.5 months). One of these 7 patients relapsed, as the patient resumed glucocorticoids because of recurrence of chronic intestinal inflammation. Six of 15 children had significant eosinophils in their initial biopsy, 5 of these children weaned off PN whereas 1 child's gastrointestinal (GI) bleeding stopped. Four patients were not able to decrease PN calorie. Two of these patients' GI bleeding stopped, the other 2 had normalized histology. CONCLUSIONS: For SBS children with histologically confirmed chronic intestinal inflammation, glucocorticoids may help promote enteral feeding tolerance. Glucocorticoids regimen should be chosen individually. Patients are more likely to respond if initial histology has significant eosinophilic infiltration. Patients may need to remain on glucocorticoids for over 6 months.

North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Position Paper on Entrustable Professional Activities: Development of Pediatric Gastroenterology, Hepatology, and Nutrition Entrustable Professional Activities.

Quality training in pediatric gastroenterology, hepatology, and nutrition is essential for the future of our specialty from advancing the science through research to providing clinical care for children with gastrointestinal, hepatic and nutritional disorders. As educational theory has developed, both the American Board of Pediatrics (ABP) and the Accreditation Council for Graduate Medical Education (ACGME) have commissioned projects to better define training including core competencies, and milestones with the goal of competency-based assessment. Seeking to provide a clinical context for these competencies and milestones, the ABP commissioned a project for each pediatric subspecialty to develop entrustable professional activities (EPA) while at the same time developing EPAs that are common to all pediatric subspecialties. North American Society for Pediatric Gastroenterology, Hepatology, Nutrition (NASPGHAN) commissioned an EPA Task Force to develop the pediatric gastroenterology, hepatology and nutrition EPAs. This document serves as an introduction to EPAs, including their historical background, underlying educational theory, and the process used to develop the pediatric gastroenterology, hepatology and nutrition EPAs in the United States of America.

Pediatric Gastroenterology, Hepatology, and Nutrition Entrustable Professional Activities: Development of an Assessment Tool and Curricular Resources.

BACKGROUND: Entrustable professional activities (EPAs) are critical activities performed by medical professionals, which can be observed and assessed. Adding on to common EPAs for all pediatric subspecialty trainees, specialty-specific EPAs for pediatric gastroenterology, hepatology, and nutritional fellowship were developed by the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPGHAN) EPA Task Force. METHODS: Having developed specialty-specific EPAs, building EPA assessments is the next logical step, as EPAs are included under a larger umbrella of competency-based assessment. Thus, the NASPGHAN EPA Task Force and Training Committee collaborated on an assessment tool and associated curricular resources to aid in tracking trainees' progression to entrustment within individual EPAs and readiness for independent practice. RESULTS: This manuscript reports the development of an EPA assessment tool, including guiding principles and the theory behind the assessment tool, with a focus on simple, meaningful assessments that can provide crucial performance feedback to trainees. In addition, curricular resources were developed, based on the assessment tool, to support training. Ultimately, it is the hope of the NASPGHAN EPA Task Force and Training Committee that this tool can aid training programs in providing formative feedback for trainees, and can be used by training programs and clinical competency committees for summative evaluation.

Nutrition Support of Children With Chronic Liver Diseases: A Joint Position Paper of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition.

Chronic liver disease places patients at increased risk of malnutrition that can be challenging to identify clinically and treat. Nutrition support is a key aspect of the management of these patients as it has an impact on their quality of life, morbidity, and mortality. There are significant gaps in the literature regarding the optimal nutrition support for patients with different types of liver diseases and the impact of these interventions on long-term outcomes. This Position Paper summarizes the available literature on the nutritional aspects of the care of patients with chronic liver diseases. Specifically, the challenges associated with the nutritional assessment of these subjects are discussed, and recently investigated approaches to determining the patients' nutritional status are reviewed. Furthermore, the pathophysiology of the malnutrition seen in the context of chronic liver disease is summarized and monitoring, as well as treatment, recommendations are provided. Lastly, suggestions for future research studies are described.

Invasive pneumococcal infections in pediatric liver-small bowel-pancreas transplant recipients.

Children undergoing LSBPTx are at increased risk of IPI due to splenectomy. We aimed to describe the clinical features and outcomes of IPI in pediatric LSBPTx recipients. Between 2008 and 2016, 122 LSBPTx children at our center were retrospectively reviewed. Nine patients had 12 episodes of IPI; the median age at first infection was 3.5 years (range: 1.5-7.1 years). The median time from transplant to first infection was 3 years (range: 0.8-5.8 years). Clinical presentation included as follows: pneumonia (n = 1), bacteremia/sepsis (n = 7), pneumonia with sepsis (n = 1), meningitis with sepsis (n = 2), pneumonia and meningitis with sepsis (n = 1). The overall risk for IPI was 7.4% or 0.9% per year. The mortality rate was 22%. Seven (78%) children had received at least one dose of PCV13, four (44%) patients had received 23-valent pneumococcal polysaccharide vaccine prior to IPI. All patients were on oral penicillin prophylaxis. In conclusion, despite partial or complete pneumococcal immunization and reported antimicrobial prophylaxis, IPI in LSBPTx children can have a fatal outcome. Routine monitoring of pneumococcal serotype antibodies to determine the timing for revaccination might be warranted to ensure protective immunity in these transplant recipients.

Increased hepatic stiffness as consequence of high hepatic afterload in the Fontan circulation: a vascular Doppler and elastography study.

UNLABELLED: Hepatic dysfunction is a recognized complication after Fontan palliation of congenital heart disease. We sought to quantitatively measure hepatic stiffness and vascular Doppler indices using ultrasound (US) and shear wave elastography (SWE) in a Fontan cohort. Subjects were prospectively recruited for echocardiography and real-time hepatic duplex US with SWE for hepatic stiffness (kPa). Doppler peak velocities, velocity time integral, resistive, pulsatility, acceleration indices (RI, PI, AI), and flow volume were measured in celiac artery, superior mesenteric artery, and main portal vein (MPV). A subset underwent cardiac catheterizations with liver biopsy. Correlations were explored between SWE, duplex, hemodynamic, and histopathologic data. In all, 106 subjects were studied including 41 patients with Fontan physiology (age 13.8 ± 6 years, weight 45.4 ± 23 kg) and 65 controls (age 15.0 ± 8.4 years, weight 47.9 ± 22 kg). Patients with Fontan physiology had significantly higher hepatic stiffness (15.6 versus 5.5 kPa, P < 0.0001), higher celiac RI (0.78 versus 0.73, P = 0.04) superior mesenteric artery RI (0.89 versus 0.84, P = 0.005), and celiac PI (1.87 versus 1.6, P = 0.034); while MPV flow volume (287 versus 420 mL/min in controls, P = 0.007) and SMA AI (829 versus 1100, P = 0.002) were lower. Significant correlation was seen for stiffness with ventricular end-diastolic pressure (P = 0.001) and pulmonary artery wedge pressure (P = 0.009). Greater stiffness correlated with greater degrees of histopathologic fibrosis. No significant change was seen in stiffness or other duplex indices with age, gender, time since Fontan, or ventricular morphology. CONCLUSION: Elevated hepatic afterload in Fontan, manifested by high ventricular end-diastolic pressures and pulmonary arterial wedge pressures, is associated with remarkably increased hepatic stiffness, abnormal vascular flow patterns, and fibrotic histologic changes. The MPV is dilated and carries decreased flow volume, while the celiac and superior mesenteric arterial RI is increased. SWE is feasible in this population and shows promise as a means for predicting disease severity on liver biopsy.

Serial transverse enteroplasty allows children with short bowel to wean from parenteral nutrition.

OBJECTIVE: To analyze the effects of serial transverse enteroplasty (STEP) on parenteral and enteral calories in children with short bowel syndrome, and examine short- and long-term complications. STUDY DESIGN: A retrospective analysis of prospectively-collected data from a large single center cohort of patients undergoing STEP procedure was analyzed. Baseline demographic and clinical information, operative data, and short- and long-term complications were recorded. Detailed growth and nutritional data were obtained for 6 months prior and 12 months following STEP procedure. RESULTS: Sixty-eight procedures were performed in 51 patients over a 68-month period. Median bowel length at first STEP was 51 cm with a median length gain of 54%. Repeat STEP patients had longer initial length (77 cm) and reduced length gain (20%). Operative times and blood loss were low, with few complications. Parenteral calorie requirement was stable or rising for 6 months prior to STEP, but decreased to median <20 kCal/kg/d at 1 year postop. Longer length gains were associated with higher risk of stricture formation. Seven children were transplanted, and 60% of nontransplanted children were enterally independent, with the remainder making ongoing progress; 48/51 children are alive at a median of 39 months follow-up. CONCLUSIONS: STEP is shown to be safe, well tolerated, and to have definitive benefit in reducing parenteral calorie requirements over the first year following the procedure. It has an important role in achieving enteral independence in children with short bowel syndrome.

Liver transplant for congenital factor VII deficiency.

Congenital factor VII (FVII) deficiency is a rare, autosomal recessive bleeding disorder with a spectrum of phenotypes ranging from asymptomatic to life-threatening intra-cranial hemorrhage (ICH). Orthotopic liver transplantation has been described for definitive treatment in a few patients with severe manifestations. We report a patient with congenital FVII deficiency and recurrent ICH, despite twice-weekly prophylaxis with recombinant activated FVII. At 17 months of age, he underwent an orthotopic liver transplant. He is now 1-year post-transplant, on maintenance immunosuppression with no hemorrhage or other complications.

Intestinal transplantation in children with multiple intestinal atresias and immunodeficiency.

GVHD has been reported in 8-10% of children after small bowel transplant (SBTx). Immunodeficient children may be predisposed to aggressive, steroid-resistant GVHD. There exists a unique association of immunodeficiency in children with MIA (MIAI). We report on our SBTx experience in patients with the diagnosis of MIAI, their high incidence of GVHD, and the possible role of stem cell transplantation in these patients. We performed a review of records from children that underwent SBTx or that we evaluated for SBTx at our institution. We focused on the diagnoses of atresia, multiple intestinal atresia, immunodeficiency, and GVHD in our patient population. Children with MIAI are likely to experience severe GVHD following SBTx. MIAI correlated with a 100% incidence of GVHD in these patients. Of the five patients with MIAI that underwent SBTx, three succumbed to severe GVHD within 1-6 months after SBTx. One patient received stem cell transplant prior to SBTx and did not develop severe GVHD, but died from influenza nine months after SBTx. Our unique patient survives long-term, with engraftment of donor γ δ T cells. He has mild, persistent chronic GVHD. Atresia is a common referral diagnosis for SBTx. Patients with multiple atresias, especially MIAI, are at significant risk for the complication of GVHD following SBTx. We recommend careful immunologic assessment and antecedent stem cell transplant in children with MIAI prior to SBTx.

Gallbladder remnant: A potential source for biliary stones postcholecystectomy; a case report in a child with sickle cell disease.

Stone formation in a gallbladder remnant is a rare postcholecystectomy complication. This report describes the case of gallstones in a gallbladder remnant of an adolescent with sickle cell disease (SCD) years after laparoscopic cholecystectomy. A 15-year-old female with SCD presented to our gastroenterology clinic with concerns of recurrent choledocholithiasis despite cholecystectomy 2 years before presentation. About 4 months before presentation to our clinic, she was evaluated at the referring physician's emergency department for recurrent severe abdominal pain of 1 month duration. After admission to the hospital, common bile duct stones were seen on magnetic resonance cholangiopancreatography (MCRP) imaging and subsequently removed via endoscopic retrograde cholangiopancreatography (ERCP). On review of her MRCP and ERCP at our hospital, a remnant of gallbladder containing multiple stones was identified. She subsequently underwent a laparoscopic resection of the gallbladder remnant. Clinicians should consider biliary duct imaging in children with biliary colic following cholecystectomy, especially those with history of chronic hemolysis.

Association between BMI Change, Transaminases, and Other Metabolic Parameters in Children with Nonalcoholic Fatty Liver Disease.

Background: Weight loss and lifestyle interventions are the mainstay of treatment in pediatric NAFLD. There are gaps in the literature on the objective improvement in BMI to meaningfully impact NAFLD in children. Aim: To determine the decrease in BMI associated with a significant decline in ALT and other metabolic parameters. Methods: Retrospective chart review of pediatric patients with the diagnosis of NAFLD. Data were collected at the baseline and 6 and 12 months. A linear regression model was used to assess the percent change in BMI predictive of change in ALT and other metabolic parameters. Results: 281 charts were included. 71% of patients who had up to a 2.5% loss in BMI at 6 months had a decrease in ALT of up to 10 U/L compared to 43% patients who did not have a decrease in BMI up to 2.5% loss at the same time period (P=0.01). The linear regression model showed that 6-month and 12-month percent changes in BMI are predictive of 6-month and 12-month ALT changes (P=0.01 and 0.02), respectively. ALT normalization was achieved on 12% of patients with a ≥2.5% decrease in BMI at 6 months compared to 1% of patients that had no decrease of ≥2.5% decrease in BMI at 6 months (P=0.01). The mean BMI Z-score decline was 0.18 (P=0.001) in the group with a ≥2.5% decrease in BMI at 6 months. Conclusions: BMI loss of up to 2.5% and the mean BMI Z-score 0.18 are associated with a significant decrease in ALT of up to 10 U/L. BMI percent change at 6 months and 12 months is predictive of changes in ALT. These results should help guide providers in clinical practice set objective goals for the management of children with NAFLD resulting from obesity.

Successful rehabilitation in pediatric ultrashort small bowel syndrome.

OBJECTIVE: To examine treatment outcomes in pediatric patients with ultrashort small bowel (USSB) syndrome in an intestinal rehabilitation program (IRP). STUDY DESIGN: We reviewed IRP records for 2001-2011 and identified 28 children with USSB (≤ 20 cm of small bowel). We performed univariate analysis using the Fisher exact test and Wilcoxon rank-sum test to compare characteristics of children who achieved parenteral nutrition (PN) independence with intact native bowel and those who did not. Growth, nutritional status, and hepatic laboratory test results were compared from the time of enrollment to the most recent values using the Wilcoxon signed-rank test. RESULTS: Of the 28 patients identified, 27 (96%) survived. Almost one-half (48%) of these survivors achieved PN independence with their native bowel. The successfully rehabilitated patients were more likely to have an intact colon and ileocecal valve (P = .01). Significant improvements in PN kcal/kg, total bilirubin, and height and weight z-scores were seen in all patients, but serum hepatic transaminase levels did not improve in the nonrehabilitated patients. CONCLUSION: Enrollment in an IRP provides an excellent probability of survival for children with USSB. The presence of an intact ileocecal valve and colon are positively associated with rehabilitation in this population, but are not requisite. Approximately one-half of patients with USSB can achieve rehabilitation, with a median time to PN independence of less than 2 years. The USSB population can attain reduced PN dependence, improvement of PN-associated liver disease, and enhanced growth with the aid of an IRP.

Hepatic fibrosis persists and progresses despite biochemical improvement in children treated with intravenous fish oil emulsion.

OBJECTIVES: Intestinal failure-associated liver disease (IFALD) is a multifactorial process, which can culminate in cirrhosis and need for transplantation. Fish oil-based lipid emulsions (FOE) reportedly reverse hyperbilirubinemia, but there are little data on their effect on the histopathology of IFALD. METHODS: We blindly examined sequential liver biopsy data on 6 children receiving FOE, with scoring of cholestasis, inflammation, fibrosis, and ductal proliferation based on standardized systems. This information was correlated with biochemical and clinical data to determine any possible relations between biologic and histologic improvement. RESULTS: The median gestational age was 35 weeks, median birth weight 2064 g, and common most reason for intestinal loss was gastroschisis (5/6 children). Median intestinal length was 26 cm beyond the ligament of Treitz and most children had roughly 2 of 3 of their colonic length. It was observed that although hyperbilirubinemia reversed and hepatic synthetic function was preserved across timepoints, fibrosis was persistent in 2 cases, progressive in 3 cases, and regressed in only 1. It remained severe (grade 2 or higher) in 5 of 6 children at last biopsy. Histologic findings of cholestasis improved in all patients and inflammation improved in 5 of 6 children. There were mixed effects on ductal proliferation and steatosis. CONCLUSIONS: In children treated with FOE, reversal of hyperbilirubinemia is not reflected by a similar histologic regression of fibrosis at the timepoints studied. Children with IFALD should have active ongoing treatment and be considered for early referral to an Intestinal Failure Program even with a normalized bilirubin.

Childhood obesity for pediatric gastroenterologists.

Obesity in childhood is one of the major health issues in pediatric health care today. As expected, the prevalence of obesity-related comorbidities has risen in parallel with that of obesity. Consultation regarding these concomitant diseases and subsequent management by subspecialists, including pediatric gastroenterologists, is now common and has resulted in obesity being recognized as a chronic disease requiring coordination of care. Although medications and even surgery may provide effective, though often temporary, treatments for obesity and its comorbidities, behavioral interventions addressing healthy dietary and physical activity habits remain a mainstay in the obesity treatment paradigm. Therefore, the issue of weight management must be addressed by both general practitioner and subspecialist alike. In this report, we review select aspects of pediatric obesity and obesity-related management issues because it relates in particular to the field of pediatric gastroenterology and hepatology.

Immunological complications beyond rejection after intestinal transplantation.

PURPOSE OF REVIEW: Intestinal transplantation (ITx) represents a major immunological challenge as the bidirectional exchange of donor's and recipient's immune cells delivers a graft highly chimeric and immunogenic. Therefore, there is a higher risk for immunological complications. It is important to understand the prevalence, natural history and mechanisms of other immunological complications, beyond rejection, to better understand the implications of ITx. RECENT FINDINGS: In recent studies, graft-versus-host disease (GVHD) holds a high mortality (43-70%). Apart from increasing or modifying immunosuppression, there are not other new therapies at this time. Inflammatory bowel disease (IBD) in solid organ transplant recipients has been estimated to be 10 times that of the general population. In the ITx recipient, it is not clear whether IBD is an autonomous disorder or a different phenotype of acute rejection. There are some studies suggesting that anti-tumour necrosis factor α (anti-TNFα) may have a role in preventing or treating rejection. According to recent studies, the incidence of autoimmune hemolytic anemia is 11% and a large proportion of these patients will respond to conventional therapy. SUMMARY: Rejection, GVHD, IBD-like disorder, autoimmune disorders and food allergies are among the more significant immunological complications after ITx. We need further studies to comprehend the risk factors and pathophysiology that will lead to better markers and innovative therapies that will impact survival.

Repeat serial transverse enteroplasty leads to reduction in parenteral nutrition in children with short bowel syndrome.

BACKGROUND/PURPOSE: Following a serial transverse enteroplasty (STEP) procedure some children develop redilation of the small intestine leading to impaired enteral tolerance and inability to wean parenteral nutrition (PN). The benefit of a second STEP procedure (2STEP) has been controversial. METHODS: We performed a retrospective review of our experience (2008-2018) performing 2STEP, with comparative analysis of nutritional outcomes pre- and postsurgery. RESULTS: During this period 2STEP was performed in 23 patients (13 F:10 M) at a median (25%-75%) age of 2.2 (1.2-3.6) years. Median intestinal length was 68 (40-105) cm before and 85 (40-128) cm after 2STEP. Leading up to 2STEP, PN provided almost 75% of estimated calorie needs. By 24 weeks following 2STEP drops in mean PN percent approached statistical significance (p = 0.07) and at most recent follow up the mean PN percentage was statistically better than at the time of operation or 4 weeks prior to 2STEP, and was nearly significant compared with 12 weeks (p = 0.07) and 24 weeks (p = 0.06) prior. Thirteen children were completely off parenteral support. CONCLUSION: When small intestine redilation occurs following a STEP procedure and where PN cannot otherwise be weaned we believe these data support performing a 2STEP. We cannot predict preoperatively which children will ultimately benefit. LEVEL OF EVIDENCE: 3 (retrospective comparative study).