Precision nutrition in pediatric IBD: A position paper from the ESPGHAN special interest group for basic science and translational research, the IBD Porto group, and allied health professionals.

Stratified and precision nutrition refers to disease management or prevention of disease onset, based on dietary interventions tailored to a person's characteristics, biology, gut microbiome, and environmental exposures. Such treatment models may lead to more effective management of inflammatory bowel disease (IBD) and reduce risk of disease development. This societal position paper aimed to report advances made in stratified and precision nutritional therapy in IBD. Following a structured literature search, limited to human studies, we identified four relevant themes: (a) nutritional epidemiology for risk prediction of IBD development, (b) food-based dietary interventions in IBD, (c) exclusive enteral nutrition (EEN) for Crohn's disease (CD) management, and (d) pre- and probiotics for IBD management. There is scarce literature upon which we can make recommendations for precision or stratified dietary therapy for IBD, both for risk of disease development and disease management. Certain single-nucleotide polymorphisms related to polyunsaturated fatty acid (PUFA) metabolism may modify the effect dietary PUFA have in increasing the risk of IBD development. Non-colonic CD, mild-to-moderate CD, and high microbiota richness may predict success of EEN and may be used both for prediction of treatment continuation, but also for early cessation in nonresponders. There is currently insufficient evidence to make recommendations for precision or stratified dietary therapy for patients with established IBD. Despite the great interest in stratified and precision nutrition, we currently lack data to support conclusive recommendations. Replication of early findings by independent research groups and within structured clinical interventions is required.

Exercise leads to better sleep in children with inflammatory bowel disease.

BACKGROUND: The aim of our study was to investigate the effect of an exercise program on health-related quality of life (HRQoL) and sleep quality in children with inflammatory bowel disease (IBD) in remission. METHODS: A total of 42 pediatric IBD patients in remission were recruited to participate in a 6-month-long home-based exercise program. Their mean age was 15.3 years (with a range of ± 2.08 years) and there were 25 boys. With regard to disease type, 22 had Crohn's disease (CD), 18 had ulcerative colitis (UC), and two had unclassified inflammatory bowel disease (IBD-U). Prior to starting the program, and after its completion, HRQoL was assessed using the IMPACT III questionnaire, and sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI) questionnaire. Patients also wore a triaxial accelerometer for 5 consecutive days before and after the completion of the exercise program to assess physical activity (PA) objectively. RESULTS: Study participants experienced no significant increase in their IMPACT III score (from 147.6 ± 2.7 to 149.6 ± 2.7, p = 0.106) following the completion of the exercise program. The prevalence of impaired sleep quality (PSQI > 5) decreased significantly from 30.9 to 23.8% (p = 0.027). At the baseline, participants' time spent in light PA (LPA) correlated positively with their IMPACT III score (coefficient (coef.) 0.398, p = 0.013). Following the completion of the resistance training program, the changes in the IMPACT III score correlated positively with time spent in moderate-to-vigorous PA (MVPA) (coef. 0.329, p = 0.047) and negatively with changes in PSQI score (coef. -0.493, p = 0.001). CONCLUSION: The number of children with impaired sleep quality decreased significantly following the completion of a 6-month-long home-based resistance training program but improvements in HRQoL scores did not reach statistical significance.

Impact of an exercise program in children with inflammatory bowel disease in remission.

BACKGROUND: The aim of our study was to investigate the impact of a structured exercise program on bone mineral density (BMD) and body composition parameters in children and adolescents with IBD in remission. METHODS: Patients were recruited to participate in a 6-month exercise program. Total body less head (TLBH) dual energy X-ray absorptiometry (DXA) was used to measure BMD. The same method was used to assess fat mass (FM) and lean body mass (LBM) at baseline and at the completion of the program. RESULTS: Based on the baseline and endpoint TBLH DXA measurements, a total of 42 study participants (25 boys; aged 15.3 ± 2.08 years) experienced an increase in BMD (from 0.959 ± 0.023 g/cm2 to 0.988 ± 0.025 g/cm2, p < 0.001) and LBM (from 37.12 ± 1.43 kg to 38.75 ± 1.61 kg, p = 0.012). Age- and sex-based BMD Z-score increased significantly (from -0.35 ± 0.15 to -0.28 ± 0.17, p = 0.020), whilst LBM Z-score did not significantly change (from -1.78 ± 0.23 to -1.71 ± 1.49, p = 0.908). CONCLUSIONS: There was a significant improvement in BMD, age- and sex-based BMD Z-score, and LBM amongst study participants. Subgroup analysis showed that patients with CD and male study participants experienced significant improvement in all parameters, whilst patients with UC and IBD-U and female patients experienced improvement solely in BMD. IMPACT STATEMENT: Children and adolescents with IBD, regardless of disease activity, are under increased risk of secondary osteoporosis and lean body mass deficits. A 6-month home-based structured exercise program leads to a significant improvement in bone mineral density and lean body mass. Exercise therapy should be explored as a potentially adjacent to standard treatment modalities.

Moderate-to-Vigorous Physical Activity Is Associated With Higher Bone Mineral Density in Children With Inflammatory Bowel Disease.

OBJECTIVES: Decreased mineral bone density (BMD) and reduced lean body mass (LBM) are common amongst children with inflammatory bowel disease (IBD). The aim of our cross-sectional, observational study was to evaluate the relationship between BMD, body composition and physical activity (PA) in children with IBD in remission. METHODS: Total body less head (TLBH) dual energy X-ray absorptiometry (DXA) was used to measure BMD, fat mass (FM) and lean body mass (LBM). Triaxial accelerometer for five consecutive days was used to objectivize PA. RESULTS: Forty pediatric IBD patients in clinical remission (24 boys; age 15.3 ±â€Š0.4 years; Crohn disease [CD], n = 20, ulcerative colitis [UC], n = 18, inflammatory bowel disease-unclassified [IBD-U], n = 2) were recruited. Mean BMD was 0.940 g/cm2 and mean BMD z score was -0.42 ±â€Š0.14. Patients with CD had significantly lower BMD than UC counterparts (P < 0.001). Average time spent in PA was 247.24 ±â€Š16.71 min/day with 45.73 ±â€Š8.22 min/day spent in moderate-to-vigorous PA (MVPA). We observed a significant positive correlation between the time spent in MVPA and BMD z score (P = 0.003) and LBM z score (P = 0.026). Multivariate analysis confirmed that the positive correlation of MVPA and BMD z score. There was no significant correlation between daily protein intake and BMD. Cumulative glucocorticoid dose negatively correlated with LBM z score (P = 0.003), but not with BMD z score (P = 0.069). CONCLUSIONS: This study points to a strong positive relationship between MVPA, LBM and BMD. Longitudinal studies are required in order to elucidate the modifiable processes that determine body health and favorable body composition.

Helicobacter pylori-negative Chronic Gastritis in Children: A Systematic Review.

OBJECTIVES: To systematically review the current evidence on Helicobacter pylori-negative chronic gastritis including natural history, available therapies and outcomes. METHODS: Articles providing data on the prevalence, treatment or outcomes of Helicobacter pylori-negative gastritis were identified through a systematic search in the MEDLINE and EMBASE databases. All original research articles from human studies until October 31, 2021, were included. RESULTS: A total of 54 studies were included consisted of eosinophilic gastritis (n = 9), autoimmune gastritis (n = 11), collagenous gastritis (n = 16), focally enhanced gastritis (n = 6), lymphocytic gastritis (n = 5) and other causes including idiopathic gastritis and chronic renal failure related (n = 7). Most of the included studies were either cross-sectional or longitudinal cohorts except for collagenous gastritis, which mainly included case reports and case series. The prevalence of paediatric eosinophilic gastritis ranges between 5 and 7/100,000 and patients have generally favourable outcome with 50% to 70% clinical and histological response to either corticosteroids or elimination diets. Autoimmune gastritis and collagenous gastritis are extremely rare entities, commonly present with refractory iron deficiency anaemia, while lymphocytic gastritis is relatively common (10%-45%) in children with coeliac disease. Data on treatments and outcomes of autoimmune, collagenous, and focally enhanced gastritis are lacking with limited data implying poor response to therapy in the former 2 diagnoses. CONCLUSIONS: Helicobacter pylori-negative gastritis is uncommonly reported, mainly in small cohorts, mixed adult-paediatric cohorts or as sporadic case reports. As common symptoms are not specific, thus not always result in an endoscopic evaluation, the true prevalence of these distinct disorders may be underestimated, and thus under reported.

Functional Gastrointestinal Disorders in Mediterranean Countries According to Rome IV Criteria.

OBJECTIVES: The aim of this study was to assess the prevalence of functional gastrointestinal disorders (FGIDs) in children of Mediterranean area using Rome IV criteria and to compare the prevalence of FGIDs using Rome IV and Rome III criteria. METHODS: This was a cross-sectional study enrolling children and adolescents living in Croatia, Greece, Israel, Italy, Macedonia, and Serbia. Subjects were examined in relation to the presence of FGIDs, using the Rome IV criteria. Data were compared with the results of a previous study using Rome III data. RESULTS: We analyzed 1972 children ages 4 to 9 years (group A), and 2450 adolescents 10 to 18 years old (group B). The overall prevalence of FGIDs was 16% in group A and 26% in group B, with statistical differences among countries in both groups (P < 0.001). In group A, the prevalence of FGIDs and of functional constipation (FC) was significantly lower than in the previous study (P < 0.001), whereas in group B no significant difference was found. In both groups of age, the prevalence of abdominal migraine and irritable bowel syndrome decreased significantly (P < 0.001 and P < 0.001, respectively) using Rome IV versus Rome III criteria, conversely functional dyspepsia increased (P < 0.001). CONCLUSIONS: FGIDs are common in children and adolescents, their frequency increases with age, and there is a significant variation in the prevalence of some FGIDs among different European countries. The application of the Rome IV criteria resulted in a significantly lower prevalence of FGIDs in children compared with Rome III criteria.

Development and Validation of a Food Frequency Questionnaire for Population of Adolescents in Croatia.

RESEARCH BACKGROUND: Food frequency questionnaire (FFQ) is an important method for the estimation of dietary intake in epidemiologic studies. The aim of the study is to develop a FFQ and evaluate its relative validity for adolescents 12 to 18 years old. EXPERIMENTAL APPROACH: FFQ was developed from a previously validated youth/adolescent diet questionnaire (YAQ) by modifying it in order to include Croatian national foods. The final version of the FFQ (FFQ-m) comprised 87 food items. The reference method was a set of two 3-day food records (3DFR) administered twice during the 3 non-consecutive days, one month apart. The FFQ-m was administered approximately on the last day of the second applied dietary food record. Adolescents were recruited from randomly selected elementary and high schools in urban and rural areas of Croatia. FFQ-m was validated on a sample of 84 adolescents (70.2% female). Nutritional intake from FFQ-m and 3DFR were analysed for each participant. Spearman correlation coefficients (r) and Bland-Altman method were used to assess the validity of the FFQ-m compared to 3DFR. Anthropometric parameters were assessed in 78 adolescents. RESULTS AND CONCLUSIONS: The mean nutrient intake estimated by the FFQ-m was higher than that of the 3DFR. The average correlation coefficient for energy and nutrients in our validation study was 0.40. On average, 76.5% of adolescents were classified in the same or adjacent quartile of the nutrient intake. Bland-Altman analysis showed good agreement with all macronutrients and some micronutrients (sodium, phosphorus, potassium, calcium, magnesium and iron). A simple self-administered questionnaire completed by adolescents is a valid tool for measuring energy and nutrient intake among adolescent population. NOVELTY AND SCIENTIFIC CONTRIBUTION: This is the first FFQ developed and validated for population of adolescents in Croatia. It will contribute to further research of nutritional intake in the population of adolescents, especially those from the region.

IBD phenotype at diagnosis, and early disease-course in pediatric patients in Croatia: data from the Croatian national registry.

BACKGROUND: Pediatric inflammatory bowel disease (IBD) presents with extensive phenotype. The aim of this study was to determine the phenotype of pediatric IBD patients in Croatia at diagnosis and follow-up. METHODS: Children were prospectively recruited into Croatian IBD national registry. Data on diagnostic evaluation, therapy and 1-year follow-up were collected. RESULTS: A total of 51 newly diagnosed patients were recruited (19 Crohn's disease (CD), 28 ulcerative colitis (UC) and 4 IBD-unclassified (IBD-U)). Most common location in CD was ileocolonic disease (52.6%), and pancolitis in UC (53.6%). The recommended complete diagnostic algorithm was performed only in 29.4% of patients. First-line therapy used in CD was exclusive enteral nutrition for remission induction (84.2%) and azathioprine for maintenance (73.7%). In patients with UC, aminosalicylates were the most common drug used (89.3%). By the end of the first year 41.2% of CD and 53.9% of UC patients had one or more relapses and required treatment escalation. CONCLUSION: Our data confirm extensive intestinal involvement in pediatric IBD and relatively high relapse rate during the first year of follow-up. More effort should be invested on the national level to implement more stringent adherence to the current European guidelines. IMPACT: The key message of our article is that pediatric IBD in Croatia shows extensive intestinal involvement with high relapse rates in first year of follow-up. It is the first cohort study reporting on the phenotype of pediatric IBD in Croatia, but also investigates adherence to diagnostic and therapeutic European guidelines which is not commonly reported. The study is national based, thus having the greatest impact on Croatian health care,stressing out that more effort should be invested on the national level to implement more stringent adherence to the current European guidelines.

Altered Gut Microbiota Is Present in Newly Diagnosed Pediatric Patients With Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Clinical and experimental data suggest that gut microbiota plays an important role in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to determine intestinal microbiota in newly diagnosed patients with IBD and to compare it with patients' healthy siblings who share same genetic and environmental background and to healthy unrelated controls. METHODS: Molecular approach targeting 16S ribosomal RNA was employed for analyzing the gut microbiota of participants' stool samples. Terminal restriction fragment length polymorphphism analysis was performed. RESULTS: Newly diagnosed pediatric patients with IBD (n = 19, 68.4% Crohn disease [CD], mean age 14.8 ±â€Š0.65 years), their unaffected healthy siblings (n = 20, mean age 12.8 ±â€Š0.85 years), and unrelated healthy controls (n = 19, mean age 10.7 ±â€Š0.8 years) were included. Microbial diversity differed significantly between IBD patients, healthy siblings, and healthy controls (P = 0.018 for MspI digestion, P = 0.013 for HhaI digestion). No significant difference in microbial diversity was found between healthy siblings and healthy controls. In patients reduced presence of genus Eubacterium, Lactobacillus, Enterobacter and Clostridium, and increased presence of genus Streptococcus, Prevotella and Escherichia, compared with healthy siblings and healthy controls, was found. CONCLUSION: Newly diagnosed pediatric patients with IBD show significantly less diverse microbiota and microbial composition compared with healthy siblings and healthy controls.

Correction to: Nutritional status and food intake in pediatric patients with inflammatory bowel disease at diagnosis significantly differs from healthy controls.

The authors of the published original version of the above article wanted to correct the below text in the Abstract section.

Nutritional status and food intake in pediatric patients with inflammatory bowel disease at diagnosis significantly differs from healthy controls.

Nutritional status and dietary intake in pediatric-onset inflammatory bowel disease are complex and need to be further explored. Therefore, we have assessed anthropometric measures, body composition, and dietary intake of newly diagnosed pediatric patients, and compared them with healthy controls. This was a prospective cross-sectional study including newly diagnosed patients with inflammatory bowel disease (n = 89) and healthy controls (n = 159). Mean energy intake was significantly lower in healthy controls compared to patients with ulcerative colitis, but not in patients with Crohn's disease. Intake of all macronutrients, dietary fiber, and calcium was significantly lower in patients with ulcerative colitis, whereas the only intake of animal protein, fruit, and calcium differed significantly in patients with Crohn's disease. There were no significant differences in the body fat percentage between patients with ulcerative colitis or Crohn's disease vs. controls; however, lean mass-for-age z-scores were significantly lower in patients with both diseases in comparison to controls.Conclusion: Food intake of newly diagnosed pediatric patients with inflammatory bowel disease significantly differed from healthy controls. Altered anthropometry and body composition are present already at the time of diagnosis. What is Known: • Children with inflammatory bowel disease suffer from malnutrition, especially children with Crohn's disease in whom linear growth failure often precedes gastrointestinal symptoms. What is New: • This study showed significantly lower intake of energy, macronutrients, and various micronutrients in patients with ulcerative colitis compared to healthy controls, while patients with Crohn's disease have a lower intake of fruits, calcium, and animal protein at diagnosis. • Altered body composition is present in both groups of patients at the time of diagnosis.

Lack of Benefit of Lactobacillus reuteri DSM 17938 as an Addition to the Treatment of Functional Constipation.

The aim of this study was to investigate the role of Lactobacillus reuteri DSM 17983 in the treatment of functional constipation in children. The trial was a single-center randomized, double-blind, placebo-controlled study. Patients were allocated into the 2 groups; intervention group which received L reuteri DSM 17983 and lactulose and placebo group which received placebo and lactulose. Due to small recruitment rate study was terminated prematurely; therefore, only 33 children (12 girls, median age 4.5 years, range 2-16) were randomized. There was no difference between groups in the stool frequency, stool consistency, pain, soiling rate and dose of the lactulose. This study found that L reuteri DSM 17938 adds no benefit to the treatment of constipation in children. Due to small sample size, these results, however, should be interpreted with caution.

Nutritional Management for Crohn's Disease.

Diet has been implicated in the pathogenesis of inflammatory bowel disease (IBD) and, more specifically, Crohn's disease (CD), for a long time [...].

Alterations of Nutritional Status in Children and Adolescents with Acute Lymphoblastic Leukemia.

Malnutrition is often observed in pediatric cancer patients and has been recognized as a risk factor for relapse and survival. Maintaining an appropriate nutritional status during anticancer treatment has, therefore, been more and more frequently perceived as an additional requirement for optimal therapy outcomes. The aim of our study was to establish alterations of nutritional status in 26 children and adolescents treated for acute lymphoblastic leukemia (ALL) at the Children's Hospital in Zagreb, Croatia, between 2016 and 2021, by using anthropometric measures and serum albumin levels. The majority of patients (53.8% female, median 4 years, 52.2% intermediate-risk leukemia group) had normal weight at the beginning of chemotherapy. The percentage of overweight/obese patients increased from 4.2% at diagnosis to 37.5% at the end of intensive therapy. Apart from a significant increase in body weight (BW) and body mass index (BMI) for age, a notable decline in body height/body length (BH/BL) for age in the observed period was recorded, especially in high-risk leukemia patients. The alterations in serum albumin values were not significant, nor was their correlation with BMI. Dietary consultation was offered to all patients, while children with a decline in BMI and BH/BL received additional nutritional support.

Nutritional Outcomes of Bowel Lengthening Procedure in Patients with Short Bowel Syndrome.

BACKGROUND: Although parenteral nutrition (PN) significantly improves mortality rates in pediatric short bowel syndrome (SBS), long-term PN has many possible complications and impacts quality of life. Bowel lengthening procedures (BLPs) increase the contact surface of food and the intestinal mucosa and enable the better absorption of nutrients and liquids, possibly leading to a PN decrease. METHODS: We retrospectively reviewed the data of patients with short bowel syndrome who underwent BLPs in the period from January 2016 to January 2022. Overall, eight patients, four male, five born prematurely, underwent BLPs. RESULTS: There was a significant decrease in the percentage of total caloric intake provided via PN and PN volume after the BLPs. The more evident results were seen 6 months after the procedure and at the last follow-up, which was, on average, 31 months after the procedure. Two patients were weaned off PN after their BLPs. Patients remained well nourished during the follow-up. CONCLUSIONS: The BLP led to a significant decrease in PN needs and an increase in the food intake; however, significant changes happened more than 6 months after the procedure.

Effect of Overweight and Obesity on the Response to Anti-TNF Therapy and Disease Course in Children With IBD.

BACKGROUND: This study aimed to evaluate the effect of overweight and obesity at the start of anti-TNF therapy on treatment response and relapse rate in children with inflammatory bowel disease (IBD). METHODS: This multicenter, retrospective cohort study included 22 IBD centers in 14 countries. Children diagnosed with IBD in whom antitumor necrosis factor (anti-TNF) was introduced were included; those who were overweight/obese were compared with children who were well/undernourished. RESULTS: Six hundred thirty-seven children (370 [58%] males; mean age 11.5 ±â€…3.5 years) were included; 140 (22%) were in the overweight/obese group (OG) and 497 (78%) had BMI ≤1 SD (CG). The mean follow-up time was 141 ±â€…78 weeks (median 117 weeks). There was no difference in the loss of response (LOR) to anti-TNF between groups throughout the follow-up. However, children in OG had more dose escalations than controls. Male sex and lack of concomitant immunomodulators at the start of anti-TNF were risk factors associated with the LOR. There was no difference in the relapse rate in the first year after anti-TNF introduction; however, at the end of the follow-up, the relapse rate was significantly higher in the OG compared with CG (89 [64%] vs 218 [44%], respectively, P < .001). Univariate and multivariate analysis revealed that being overweight/obese, having UC, or being of male sex were factors associated with a higher risk for relapse. CONCLUSIONS: Overweight/obese children with IBD were not at a higher risk of LOR to anti-TNF. Relapse in the first year after anti-TNF was introduced, but risk for relapse was increased at the end of follow-up.

Overweight and obese children with inflammatory bowel disease required more frequent dose escalations, but overall loss of response to anti-TNF therapy was not increased. Furthermore, in the long term, they tend to have a higher risk for relapse.

Importance of dietary fiber in children.

Dietary fibers (DFs) are essential components of human nutrition and are principally defined as non-digestible carbohydrates (oligosaccharides and polysaccharides) usually classified by their physicochemical and physiological characteristics (water solubility, viscosity, fermentability, and bulking effect). Unfortunately, there is limited information on dietary fiber recommendations for children, and the evidence on their effect on health and symptom control is mainly available for the adult population. Therefore, this review aims to give a comprehensive overview of the characteristics and dietary sources of dietary fiber and their potential health benefits in healthy children but also their potential use in the treatment of sick children.

The Frequency and Impact of Self-Imposed Elimination Diets on the Nutritional Status and Clinical Course of Disease in Children with Inflammatory Bowel Disease.

BACKGROUND AND AIMS: From the patients' perspective, diet has a relevant role in triggering symptoms of inflammatory bowel disease (IBD). There is a lack of prospective studies regarding the diet of children with IBD. The aim of this study was to assess the frequency and impact of self-imposed elimination diets on the nutritional status and clinical course of disease in the pediatric population. METHODS: This was a prospective case-control study that included newly diagnosed patients with IBD and healthy controls (age/sex-matched peers and siblings) over a one-year period. The participants were examined in three categories: (1) anthropometric data and nutritional status; (2) dietary intake, as obtained by a Food Frequency Questionnaire (FFQ); and (3) dietary beliefs and elimination diets, as obtained by a structured questionnaire. RESULTS: Overall, one-hundred and thirty-seven participants were included (twenty-eight with Crohn's disease, sixteen with ulcerative colitis, three with IBD-unclassified, and seventy healthy controls). Only 15% of patients followed the self-imposed elimination diet upon the diagnosis, which increased to 47.6% by the end of the follow-up. The elimination diet did not influence the nutritional status and quality of the diet. Self-imposed elimination diets were not a risk factor for disease relapse. Most of the patients received nutritional counseling during the follow-up. CONCLUSIONS: The number of patients following self-imposed elimination diets had increased during the disease course but with no influence on nutritional status or relapse risk.

Gut Microbiota Composition Changes following Discontinuation of Exclusive Enteral Nutrition in Children with Crohn's Disease.

This study aims to determine changes in the intestinal microbiota of children with Crohn's disease (CD) before and during exclusive enteral nutrition (EEN) and after its discontinuation. A total of 14 newly diagnosed children with CD (median age 16.0 years; 43% female) were included in this study. Patients were initially treated with EEN and were followed for one year after EEN discontinuation. Stool samples were taken at the time of diagnosis (before EEN introduction), the second day of EEN, the last day of EEN, and every two months for one year after the discontinuation of EEN. A molecular approach targeting 16S ribosomal RNA was used for analysing the gut microbiota. No change was found in the Shannon diversity index before, during, and after EEN cessation (HhaI-digestion p = 0.82; MspI-digestion p = 0.87). According to the PCO, on the basis of the dissimilarity matrices of OTUs, a clear separation of patients at different time points, forming two clusters (before and during EEN as opposed to after EEN), was evident. No clear separation was noted between patients who achieved sustained remission as opposed to those who did not achieve sustained remission during EEN and at the follow-up. In conclusion, a distinct change in the microbiota composition already occurred after two months of EEN discontinuation and remained mostly unchanged over a year of follow-up.