# PEDIATRIC INFLAMMATORY BOWEL DISEASES: LOOKING TO THE FUTURE

EDITED BY : Eytan Wine, Jeff Critch, Séamus Hussey, Víctor Manuel Navas-López and Wael El Matary PUBLISHED IN : Frontiers in Pediatrics

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ISSN 1664-8714 ISBN 978-2-88963-629-7 DOI 10.3389/978-2-88963-629-7

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# PEDIATRIC INFLAMMATORY BOWEL DISEASES: LOOKING TO THE FUTURE

Topic Editors:

Eytan Wine, University of Alberta, Canada Jeff Critch, Memorial University of Newfoundland, Canada Séamus Hussey, National Children's Research Centre, Ireland Víctor Manuel Navas-López, Hospital Materno-Infantil, Spain Wael El Matary, University of Manitoba, Canada

Citation: Wine, E., Critch, J., Hussey, S., Navas-López, V. M., El Matary, W., eds. (2020). Pediatric Inflammatory Bowel Diseases: Looking to the Future. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-629-7

# Table of Contents

Abisoye O. Akintimehin, Ríoghnach Sinead O'Neill, Conor Ring, Tara Raftery and Séamus Hussey on behalf of the DOCHAS Study

*55 Anorexia Nervosa Complicating Pediatric Crohn Disease—Case Report and Literature Review*

Aedin Collins, Elizabeth Nolan, Michelle Hurley, Antoinette D'Alton and Séamus Hussey

Rafael Martín-Masot, Pilar Ortiz Pérez, Juliana Serrano Nieto, María Martínez León, Antonia Pascual Martínez, Javier Blasco-Alonso and Victor Manuel Navas-López

*72 Stress Triggers Flare of Inflammatory Bowel Disease in Children and Adults*

Yue Sun, Lu Li, Runxiang Xie, Bangmao Wang, Kui Jiang and Hailong Cao

# Editorial: Pediatric Inflammatory Bowel Diseases: Looking to the Future

Eytan Wine1 \*, Wael El-Matary 2 , Jeff Critch3 , Víctor Manuel Navas-López 4 and Seamus Hussey 5,6

1 Division of Pediatric Gastroenterology, Departments of Pediatrics and Physiology, University of Alberta, Edmonton, AB, Canada, 2 Section of Pediatric Gastroenterology, Department of Pediatrics, Max Rady College of Medicine, University of Manitoba, Winnipeg, MB, Canada, 3 Department of Pediatrics, Memorial University, St. John's, NL, Canada, 4 Pediatric Gastroenterology and Nutrition Unit, Hospital Regional Universitario de Málaga, Málaga, Spain, 5 National Children's Research Centre, University College Dublin (UCD), Dublin, Ireland, 6 Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland

Keywords: inflammatory bowel disease, Crohn disease, ulcerative colitis, pediatrics, editorial, Research Topic

#### **Editorial on the Research Topic**

#### **Pediatric Inflammatory Bowel Diseases: Looking to the Future**

The incidence of inflammatory bowel diseases (IBD) is increasing across the globe (1), especially in children (2). This highlights not only the clinical importance of characterizing pediatric IBD (P-IBD) but also provides an opportunity to improve understanding of pathogenesis and patient outcomes. In this editorial, 12 manuscripts are reviewed, all published in the Research Topic: Pediatric Inflammatory Bowel Diseases: Looking to the Future. These papers span a diverse array of topics related to P-IBD, from rare complications, through evolving assessment tools and clinical features, to novel pathways, possibly leading to future treatments. These publications focus on features of IBD unique to children, and will serve as a resource for anyone interested in P-IBD.

Clinical expertise incorporates vigilance for recognizing unusual clinical patterns and pursuing appropriate differential diagnoses in such circumstances. Lawrence et al. reported on a 12-year-old girl with Crohn disease who presented with a short history of progressive respiratory distress and hypoxemia, requiring intensive respiratory support. Cardinal factors in this patient's case included profound lymphocytopenia, thiopurine immunosuppression, recent corticosteroid exposure, chest radiographic findings, and a sputum sample for direct microbial testing. Following a diagnosis of Pneumocystis jirovecii pneumonia (PJP), targeted therapy was successful. Current pediatric guidelines recommend considering PJP prophylaxis for patients on triple immunosuppression that includes corticosteroid therapy (3).

Collins et al. described a teenager with relapsing Crohn disease whose weight had fallen from the 25th to the 3rd percentile. Weight loss continued in hospital while on full exclusive enteral nutrition (EEN) of 2,400 kcal/day. Weight gain followed a period of 24-h nursing observation. Psychiatric evaluation led to a diagnosis of anorexia nervosa. The sentinel clinical feature was weight loss despite apparently high calorie intake. Anorexia nervosa, rather than Crohn disease, is more often considered as the potential cause of weight loss in young adults. The coexistence of both diagnoses may be associated with poor outcomes (4).

Acute neurological complications in children with IBD are rare, but should prompt consideration of intracranial vascular thrombosis, especially in the setting of active disease and thrombosis risk factors. Martín-Masot et al. describe a 5-year-old patient with refractory ulcerative colitis (UC), who developed a focal headache, upper limb paresis, and seizures within 4 days of

Edited and reviewed by: Andrew S. Day, University of Otago, New Zealand

> \*Correspondence: Eytan Wine<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Pediatric Gastroenterology, Hepatology and Nutrition, a section of the journal Frontiers in Pediatrics

Received: 26 January 2020 Accepted: 06 February 2020 Published: 26 February 2020

#### Citation:

Wine E, El-Matary W, Critch J, Navas-López VM and Hussey S (2020) Editorial: Pediatric Inflammatory Bowel Diseases: Looking to the Future. Front. Pediatr. 8:56. doi: 10.3389/fped.2020.00056

**4**

colectomy and J-pouch creation. Potential risk factors in this case included active preoperative disease resulting in reduced mobility, recent major abdomino-pelvic surgery, and central venous catheter placement. The patient had not been on thromboprophylaxis therapy during the perioperative period. A recent Pediatric clinical guideline recommends thromboprophylaxis in high risk patients, following individual risk assessment (3).

Disease aspects unique to P-IBD are discussed in three additional papers included in this Research Topic. Acute severe UC (ASUC) is a medical emergency. Pediatric-onset UC is usually more extensive and follows a more progressive course than in adults (5). Since disease severity has consistently been associated with disease extent, children are especially susceptible to severe refractory flares. With few exceptions, children with ASUC should be admitted to the hospital for intensive medical treatment. Akintimehin et al. published the results of the first population-based study of ASUC in Irish children. In this retrospective study of 55 children >41% required colectomy. Colectomy rates were higher in the pre-infliximab period (88 vs. 36%). The authors did not identify predictive factors of response to infliximab.

Endoscopy is the cornerstone in assessing mucosal healing in IBD. However, this is a relatively invasive and costly procedure. This has led to the search for non-invasive and less expensive markers of intestinal inflammation. In a single center, retrospective cohort study, El-Matary et al. examined the impact of measuring fecal calprotectin (FCAL), a stool marker for intestinal inflammation, on decision-making and clinical care of children with IBD. The majority (86%) of positive FCAL measurements (≥250µg/g of stools) resulted in treatment adjustments with subsequent clinical improvement. Conversely, 83% of those with normal FCAL remained in clinical remission over the following months without any changes in medical management. This study was limited by its retrospective design, small sample size, and limited data correlating mucosal healing with FCAL. In their commentary article, Day and Adamji highlighted the benefit of non-invasive stool markers in monitoring IBD. They proposed that serial FCAL measurements may be a more effective strategy to inform individual patient care than a one-off level.

Extraintestinal manifestations (EIM) occur in ∼1/3 of P-IBD cases. Pancreatic disease is a less frequently observed EIM, potentially manifesting in multiple ways, as highlighted by Martin-de-Carpi et al. Pancreatic involvement can present as acute pancreatitis, chronic pancreatitis, autoimmune pancreatitis, asymptomatic exocrine pancreatic insufficiency, increased pancreatic enzyme levels, structural abnormalities, and granulomatous inflammation, as well as therapy-associated pancreatitis. Clinicians caring for children with IBD should be familiar with these conditions, diagnostic considerations, and treatment options.

In a separate article, Day and Brown present their experience with using EEN, as an adjunctive therapy, in two cases with Crohn disease complicated by phlegmon to avoid resection, at least in the short term. EEN is recognized as an effective induction agent for active luminal inflammatory disease (6), but in this situation its use in complicated Crohn disease may have prevented the need for bowel resection.

This last section focuses on emerging concepts and potential pathways for future therapies in P-IBD. Multiple factors have been related to the pathogenesis of IBD, including genetic mutations, intestinal immune dysfunction, impaired intestinal microbiota, dietary factors, antibiotics, infections, type of delivery, appendectomy, urban environment, smoking, sleep disorders, and stress. Sun et al. reviewed the role of stress in IBD, explaining in detail the mechanisms by which stress affects IBD in relation to homeostasis and gut motility, intestinal microbiota, and immune and neuroendocrine dysfunction. Stress exerts a deleterious effect at different stages of IBD (onset, disease course, and prognosis). This review highlights that mechanisms related to stress should be considered as a strategy both in prevention and in the treatment of IBD.

Surgical resection is often indicated for complicated fibrostenotic and/or internally penetrating Crohn disease. Recent efforts have focused on more completely controlling inflammation to prevent disease progression and hopefully decrease the need for surgery. As Stenke et al. discuss, while no antifibrotic therapies are currently available for Crohn disease, recent research opens the possibility that targeting the profibrotic transforming growth factor-B (TGF-B) may lead to effective antifibrotic therapies in the future. This may lead to potential therapeutic options to mitigate the effects of fibrosis and reduce the need for surgical intervention.

Another key pathway, well-recognized in the pathogenesis of IBD, involves nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a nuclear transcription factor known to activate pro-inflammatory pathways. Novel approaches to regulate NF-κB and its downstream consequences are reviewed by Zaidi and Wine; these relate to recent discovery of pediatricspecific changes in A20, which is a key regulator of NF-κB, as shown in samples from children with IBD (7). The authors propose that instead of suppressing inflammation, efforts should be made to regulate key pro-inflammatory pathways, thus preventing uncontrolled inflammation to begin with.

Finally, given the recent attention to food and food additives in the pathogenesis of IBD (8), the unique role of carrageenan and carboxymethylcellulose is reviewed by Martino et al. Both chemicals are commonly-used emulsifiers and food stabilizers. Much of the evidence on the effects of these additives on gut health, and how this might relate to IBD, is based on animal studies, but the relevance of these mechanisms to human IBD is reflected in the utility of new nutritional approaches, such as the Crohn-disease exclusion diet (CDED) (9).

Together, this collection of reports presents recent advances in our understanding of P-IBD and opportunities for improving care through further research and innovation.

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

# REFERENCES

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Wine, El-Matary, Critch, Navas-López and Hussey. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Wael El-Matary1,2,3\*† , Esmail Abej4 , Vini Deora1,3, Harminder Singh2,4 and Charles N. Bernstein2,4*

*1Department of Pediatrics and Child Health, Section of Pediatric Gastroenterology, University of Manitoba, Winnipeg, MB, Canada, 2 The University of Manitoba IBD Clinical and Research Centre, Winnipeg, MB, Canada, 3 The Children's Hospital Research Institute of Manitoba, Winnipeg, MB, Canada, 4Department of Internal Medicine, Section of Gastroenterology, University of Manitoba, Winnipeg, MB, Canada*

#### *Edited by:*

*Christophe Faure, Centre Hospitalier Universitaire Sainte-Justine, Canada*

#### *Reviewed by:*

*Jan S. Suchodolski, Texas A&M University, USA Andrew S. Day, University of Otago, New Zealand Prevost Jantchou, Sainte Justine University, Canada*

> *\*Correspondence: Wael El-Matary*

<EMAIL_ADDRESS>
*† Dr. Wael El-Matary is affiliated with the University of Alexandria, Egypt.*

#### *Specialty section:*

*This article was submitted to Pediatric Gastroenterology, Hepatology and Nutrition, a section of the journal Frontiers in Pediatrics*

*Received: 29 October 2016 Accepted: 10 January 2017 Published: 25 January 2017*

#### *Citation:*

*El-Matary W, Abej E, Deora V, Singh H and Bernstein CN (2017) Impact of Fecal Calprotectin Measurement on Decision-making in Children with Inflammatory Bowel Disease. Front. Pediatr. 5:7. doi: 10.3389/fped.2017.00007*

in the management of inflammatory bowel disease (IBD) is increasing. The aim of this study was to examine the impact of FCal measurements on decision-making and clinical care of children with IBD.

Background: The use of fecal calprotectin (FCal) as a marker of intestinal inflammation,

Materials and methods: In a retrospective cohort study, FCal, clinical activity indices, and blood markers were measured in children with established diagnoses of IBD. Pearson correlation coefficient analysis was performed to examine association between FCal and other markers. Decisions based on FCal measurements were prospectively documented and participants were evaluated 3–6 months later.

results: A total of 115 fecal samples were collected from 77 children with IBD [median age 14, interquartile range (IQR) 11–15.6 years, 42 females, 37 with Crohn's disease]. FCal positively correlated with clinical activity indices (*r* = 0.481, *P* < 0.05) and erythrocyte sedimentation rate (*r* = 0.40, *P* < 0.05) and negatively correlated with hemoglobin (*r* = −0.40, *P* < 0.05). Sixty four out of 74 (86%) positive FCal measurements (≥250 μg/g of stools) resulted in treatment escalation with subsequent significant clinical improvement while in the FCal negative group, 34 out of 41 (83%) measurements resulted in no change in treatment and were associated with remission on follow-up.

conclusion: Based on high FCal, the majority of children had treatment escalation that resulted in clinical improvement. FCal measurements were useful and reliable in decision-making and clinical care of children with IBD.

Keywords: calprotectin, children, colitis, Crohn, IBD

# INTRODUCTION

Stool markers such as fecal calprotectin (FCal) have emerged as new diagnostic tools to help in the diagnosis of intestinal inflammation (1). Fecal inflammatory markers include a biologically heterogeneous group of substances that either leak from or are actively released by the inflamed mucosa. Calprotectin is a small calcium binding protein consisting of two heavy and one light

**Abbreviations:** FCal, fecal calprotectin; IBD, inflammatory bowel disease; CD, Crohn's disease; UC, ulcerative colitis.

polypeptide chains (a heterodimer of two S100 proteins) (1). It is found in neutrophilic granulocytes, accounting for 60% of their cytosolic fraction, and in monocytes and macrophages (1–3). It has a homogeneous distribution in feces. In active inflammatory bowel disease (IBD), an increased migration of inflammatory cells including neutrophils to the inflamed intestinal mucosa has been observed. Due to leukocyte shedding in the intestinal lumen, pro-inflammatory proteins such as calprotectin can be detected and measured in the stools. FCal demonstrates good sensitivity and specificity as a marker for mucosal inflammation (1–4).

Inflammatory bowel disease is a chronic disease characterized by remissions and relapses and encompasses two related but distinct disorders of as yet unknown etiology: Crohn's disease (CD) and ulcerative colitis (UC) (5). Both adult and pediatric investigators have recognized the need to optimize and standardize methodology for assessment of disease activity. In children, active disease can have devastating consequences on growth and development. Consequently, it is prudent that disease remission is induced and maintained (6). The goal for IBD therapy is increasingly becoming mucosal healing. Therefore, the need for non-invasive, reliable, and relatively non-expensive biomarkers for IBD disease activity is growing (7).

Fecal calprotectin concentrations may correlate well with both endoscopic and histological IBD activity, which have been traditionally used to evaluate mucosal healing and response to treatment (8–10). However, the impacts of FCal measurement on decision-making and clinical outcomes in children with IBD are currently under-reported. The aim of this study was to examine the impact of FCal measurements on decision-making and clinical care of children with IBD in usual clinical care.

# MATERIALS AND METHODS

# Study Population

In a single centre cohort study, consecutive children with established diagnoses of IBD assessed between November 2013 and December 2015 in the Pediatric IBD Outpatient Clinic at the Children's Hospital, Winnipeg, MB, Canada, who had developed new symptoms that might had suggested a disease relapse were asked to bring in a stool sample for FCal measurement. Inclusion criteria included all children (<18 years) with confirmed diagnosis of IBD following the North America Society of Pediatric Gastroenterology, Hepatology and Nutrition diagnostic criteria for IBD (11).

# Covariate Data

Patients' demographics and disease characteristics and duration were collected from patients' medical records. The most significant symptom or sign as an indication for requesting FCal measurement was prospectively documented for each collected sample.

## Fecal Calprotectin

Fecal samples were collected at home and processed at the laboratory of the University of Manitoba IBD Clinical and Research Centre. FCal measurement was performed using the Quantum Blue® Lateral Flow Reader within 24 h of stool collection. FCal ≥ 250 μg/g of stools was considered a positive test, indicative of active IBD. The upper limit of test was >1,800 μg/g of stools.

### Clinical Activity Indices

Clinical activity indices [pediatric UC activity index (PUCAI) for UC and pediatric Crohn's activity index (PCDAI) for CD] (12, 13) and the impact of FCal testing on the management of IBD—investigations or changes in therapy—were assessed. Physician global assessment (PGA) for disease activity, whether it is quiescent, mild, moderate, or severe, was also documented (14).

### Other Laboratory Markers

Included hemoglobin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and serum albumin performed within 2 weeks of stool collection for FCal.

When patients were asked to bring stool samples, pediatric gastroenterologists were asked to complete a questionnaire. Other investigations such as hemoglobin, serum albumin, and CRP were known to physicians who made no changes in treatment at that time point. All patients with diarrhea had their stools tested for infection screen and only those with negative infection screen had stools for FCal measurements. The clinicians were asked to decide if they would do any additional investigations (mainly colonoscopy) or any changes in therapy, based only on the FCal results. Clinicians were given specific options to choose from for treatment escalation in case FCal comes back as positive. Once the FCal results were known, any investigations and treatment changes were documented and patients were followed up for 3–6 months. Clinical activity indices were measured in the follow-up visits.

If colonoscopy was performed following FCal results, colonoscopy findings and histopathology of collected mucosal biopsy samples performed within 1 month of collecting stool samples were examined. Presence of erythema, loss of vascularity, friability, and/or ulcerations were included in the definition of active disease on endoscopy (active vs. inactive).

# Statistical Analysis

Calculations and data analysis were performed using Statistical Package for the Social Sciences (SPSS, IBM Corp., 2013, IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY, USA). Descriptive measures [medians, interquartile range (IQR), means, ranges, and SD] were calculated for continuous variables (such as age and clinical disease activity indices), while frequencies were calculated for categorical variables (such as PGA), along with 95% confidence intervals (CIs) for the means and proportions. Variables were examined for normal distribution. Student's *t*-test was used to compare means. Categorical variable comparisons were performed using Fisher's exact test. Pearson correlation coefficient analysis was performed to examine any possible association between FCal values and clinical disease activity indices, ESR, CRP, serum albumin, hemoglobin (all continuous variables), PGA (as a categorical variable; quiescent, mild, moderate, and severe), and endoscopic activity (as a dichotomous variable; active vs. inactive). A *P* value < 0.05 was considered significant.

## Ethical Considerations

The study protocol was approved by the University of Manitoba Health Research Ethics Committee. As the study was retrospective, individual consents from participants were not needed.

# RESULTS

Over the study period, a total of 115 fecal samples were collected from 77 children with IBD (median age at FCal measurements 14, IQR 11–15.6 years, 42 females, 37 with CD), who were followed up for a median duration of 1.33 (IQR 0.5–3.1) years (mean 2.62 ± 1.84 years). Participants' demographics and disease distribution are summarized in **Table 1**. Base line medications were summarized in **Table 2**.

The most significant symptom or sign such as diarrhea, weight loss, and pallor as an indication for FCal measurement is listed in **Table 3**.

Seventy four (64%) stool samples were positive (≥250 μg/g) and 12 participants had colonoscopy within 4 weeks of FCal measurement as compared with two patients in the group with normal FCal measurements (*P* < 0.001).

# Association between FCal and Study Co-Variables

For the whole cohort, 74 (66%) out of 115 fecal samples had high FCal > 250 μg/g of stools. In 33 (44.6%) out 74 positive fecal

#### Table 1 | Demographic characteristics and disease phenotype in

*a Upper GI involvement in combination with other phenotypes.* samples, clinical activity indices were ≥10. On the other hand, 41 (34%) out 115 fecal samples had normal FCal level and only 2 (5%) out of 41 samples had clinical activity indices ≥10. Both patients were scoped and colonoscopy was normal.

Fecal calprotectin measurements had fair correlation with clinical disease activity indices (*r* = 0.483, *P* < 0.05), PGA (*r* = 0.40, *P* < 0.05), ESR (*r* = 0.40, *P* < 0.05), low hemoglobin (*r* = −0.40, *P* < *0.05*), with less correlation with low serum albumin (*r* = −0.3*, P* < 0.05) but no correlation with CRP (*r* = 0.1, *P* = 0.3).

When those with CD were compared to the UC/IBD-U group, the correlation of FCal with clinical disease activity indices was stronger for UC compared to CD. High FCal levels correlated with PUCAI (*r* = 0.80, *P* < 0.01) compared to PCDAI (*r* = 0.2, *P* = 0.09).

Correlation was limited (*r*= 0.256, *P*= 0.006) with endoscopic activity as only 14 children had colonoscopy within 4 weeks of FCal measurement.

## Impact on Management

Only 14 (12%) out of 115 samples resulted in colonoscopy as guided by physicians' discretion (likely when physicians felt that FCal value was equivocal between 100 and 250); 2 in FCal negative and 12 in the FCal positive group, i.e., for the rest of the cohort [101 (88%) samples], decisions on treatment (escalation or no treatment) were based solely on FCal measurements. Sixty four out of 74 (86%) samples with positive FCal measurements were associated with treatment escalation that resulted in improvements in clinical activity indices (**Table 4**), while in the FCal negative group, 34 out of 41 (83%) measurements were associated with no change in treatment.

**Figure 1** summarizes the impact of FCal measurements on the study cohort over time. In the first follow-up visit 3–6 months following changes in treatment in the FCal positive group, of those measurements that resulted in treatment escalation, 80%

*a Several patients were on more than one medication.*

#### Table 3 | Indications (most significant symptom/sample) for fecal calprotectin measurements in 77 children with inflammatory bowel disease.

ultimately achieved clinical remission while in the FCal negative group, 32 out 34 (94%) of those who did not have any treatment changes (including 2 patients with clinical activity indices >10), were in clinical remission at the follow-up visit.

# DISCUSSION

Fecal calprotectin is a surrogate marker for mucosal inflammation and has been proven as a very beneficial, non-invasive, and relatively an inexpensive test in the work-up of IBD with a considerable high sensitivity (15, 16). Although high FCal can be found in other gastrointestinal disorders, in addition to IBD, such as gastrointestinal infections and polyposis, proper use of this test may reduce the need for frequent colonoscopies (15).

Our study showed a fair positive correlation of high FCal with increased clinical disease activity, a finding that is concordant with other adult and pediatric studies (9, 17–19). It is noteworthy that 55% of persons with increased FCal were in clinical remission by a symptom based activity index. While this may lead some to consider that this suggests that either the FCal is nonspecific, or that a substantial proportion of children with IBD in remission have active inflammatory disease. The majority of our subjects with elevated FCal had treatment escalation (even if disease activity index suggested remission). In pediatric IBD practice, our study is unique in that we relied on FCal to direct clinical management and then followed up to determine if our FCal-directed management led to satisfactory clinical outcomes several months later.

Bunn et al. examined FCal in spot fecal samples from 37 children with IBD and 31 control children. FCal was higher in 16 children with UC and in 21 children with CD (18). Similar to our study, FCal concentration correlated negatively with serum albumin concentration (18). In another study from Italy, FCal showed a high correlation with the histologic grade of mucosal inflammation, similar to that observed for endoscopy in 58 children with IBD (19).

In our study, FCal, did not correlate that well with CRP, a finding which was also highlighted in other studies (20, 21). Schoepfer and colleagues concluded that FCal correlated better with endoscopic scoring than that with CRP (20, 21). Correlations with CRP, however, were *r* = 0.53 and 0.55 in 140 ileocolonic CD and 228 UC adult participants, respectively (18, 19). In our study, the assessment of association between high FCal and endoscopic disease activity was limited by the small number of patients who had colonoscopy and the non-validated categorical classification of endoscopic activity into two subcategories (active and inactive disease).

Our study showed a better correlation of FCal with PUCAI compared to PCDAI. FCal has been previously also found to be a stronger marker for UC compared to CD (22).

Monitoring disease activity in patients with IBD is crucial as mucosal healing has become now a new target for treatment. It has been established that calprotectin concentrations correlate well with endoscopic and histological IBD activity (8, 9). However, data on the impact of FCal directing management in an IBD population, especially in children, are sparce. In our study, 86% of positive samples resulted in escalation of treatment with subsequent improvement in the majority of participants and 88% of negative samples resulted in no change of treatment and sustained clinical remission in the majority of participants in subsequent follow-up.

In a recent study from The Netherlands, clinical activity indices, FCal, and CRP were measured in 62 teenagers with IBD (31 with CD and 31 with UC), who were asked to evaluate their disease control (100% was the best disease control) and their disease control, according to participants' opinion, was above 90% in two successive outpatient clinic visits. Unlike CRP, positive FCal was predictive of a clinical relapse in the subsequent visit in 60% of participants (23). In another prospective multicentre cohort study, stool samples from 101 children who were admitted with severe UC were obtained for measuring four different stool markers for inflammation including FCal. Repeat samples at discharge were obtained from 24 children. Although all markers were initially significantly elevated, FCal did not correlate that well with PUCAI which performed better than the fecal markers in predicting outcome following a course of intravenous corticosteroids. However, perhaps it was too soon to expect a significant drop in FCal following the short course of intravenous corticosteroids (24).

Eccles et al. collected data on the impact that FCal testing from 119 clinic participants including 29 with IBD. FCal testing helped in assessment of disease activity and decision-making in 25 adults with known IBD; 21 had major changes in their treatment while 4 were felt to be in remission on biological therapy (25).

*a Sixty-four changes in treatment (three samples resulted in more than one change in medication).*

In four pediatric participants with known IBD, one had a normal FCal level and was felt to be in remission but three had elevated levels that resulted in major changes in management. Similar to our study, the investigators concluded that FCal can serve as a marker for disease activity in known IBD and help in the clinical decision-making process (25).

In an open label randomized controlled study, Osterman et al. observed significantly dropping FCal levels in adults with quiescent UC after escalating their mesalamine dose (26). In another randomized study, Lasson et al. used FCal measurements to identify persons with UC who were at risk of relapse and escalated their therapy (27). Both studies, however, were experimental and not reflective of routine real-life clinical practice.

The limitations of our study include the retrospective design and small sample size especially for those who had colonoscopy. However, based on previous research, FCal is known to strongly correlate with endoscopic disease activity (9, 17) and we assumed that it did. Hence, the escalation of treatment in most participants with elevated FCal levels regardless of whether a colonoscopy was performed or not. The study main aim was how FCal measurements, as the major decisive factor, would impact on disease management. A positive FCal most often triggered a clinical response; especially a change in therapy. As we did not do colonoscopy in all subjects we cannot discern how many false negative FCal measurements were represented within our sample. Nonetheless, our results did show that the faith clinicians placed in the FCal result was rewarded with good clinical outcomes. Another limitation was a lack of measurements of FCal in the follow-up visits after implementing changes in therapy but the study outcome was intended to be clinical activity indices. Nonetheless, the study adds significantly to the current literature as little is known about the impact and outcome of FCal measurements in pediatric IBD clinical practice.

# CONCLUSION

In children with known IBD, elevated levels of FCal were more likely to be associated with elevated scores of clinical activity indices, high ESR, and low hemoglobin. However, many children with elevated FCal had normal clinical disease activity indices. Regardless of disease activity index at the time of FCal, based on abnormal measurements of FCal, the majority of children had their treatment escalated which resulted in significant clinical improvement in those with elevated disease activity index or sustained remission in those in whom the index was considered in remission. On the other hand, the majority of those with normal FCal did not have any changes in their investigations or treatment and remained in clinical remission which saved patients and health-care resources from unnecessary interventions.

# AUTHOR CONTRIBUTIONS

All authors made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically

# REFERENCES

for important intellectual content; final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

# ACKNOWLEDGMENTS

The authors are grateful to Mr. Michael Sargent for his help in fecal calprotectin measurement.

# FUNDING

This work was supported by an unrestricted grant by Abbvie Canada.

with Crohn's disease. *Inflamm Bowel Dis* (2005) 11:185–96. doi:10.1097/ 00054725-200502000-00013

27. Lasson A, Öhman L, Stotzer PO, Isaksson S, Überbacher O, Ung KA, et al. Pharmacological intervention based on fecal calprotectin levels in patients with ulcerative colitis at high risk of a relapse: a prospective, randomized, controlled study. *United European Gastroenterol J* (2015) 3:72–9. doi:10.1177/2050640614560785

**Conflict of Interest Statement:** WE-M has served as an advisory board member for Janssen Canada and AbbVie Canada and received research support from Janssen Inc. HS has consulted to Medial Cancer Screening Ltd., Israel, served on advisory board for Pendopharm, and received research funding from Merck Canada. CB is supported in part by the Bingham Chair in Gastroenterology. He has served on advisory boards for AbbVie Canada, Ferring Canada, Janssen Canada, Shire Canada, Pfizer Canada, and Takeda Canada. He has consulted to Mylan Pharmaceuticals and Bristol Myers Squibb. He has received unrestricted educational grants from AbbVie Canada, Janssen Canada, Shire Canada, and Takeda Canada. He has been on speaker's bureau for AbbVie Canada and Shire Canada. The other authors declare no conflict of interest.

*Copyright © 2017 El-Matary, Abej, Deora, Singh and Bernstein. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The Role of Carrageenan and Carboxymethylcellulose in the Development of intestinal inflammation

#### *John Vincent Martino1 , Johan Van Limbergen1,2\* and Leah E. Cahill 2,3*

*1Pediatric Gastroenterology, Hepatology and Nutrition, IWK Health Centre, Halifax, NS, Canada, 2Medicine, Dalhousie University, Halifax, NS, Canada, 3Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, USA*

Although the exact pathophysiology remains unknown, the development of inflammatory bowel disease (IBD) is influenced by the interplay between genetics, the immune system, and environmental factors such as diet. The commonly used food additives, carrageenan and carboxymethylcellulose (CMC), are used to develop intestinal inflammation in animal models. These food additives are excluded from current dietary approaches to induce disease remission in Crohn's disease such as exclusive enteral nutrition (EEN) using a polymeric formula. By reviewing the existing scientific literature, this review aims to discuss the role that carrageenan and CMC may play in the development of IBD. Animal studies consistently report that carrageenan and CMC induce histopathological features that are typical of IBD while altering the microbiome, disrupting the intestinal epithelial barrier, inhibiting proteins that provide protection against microorganisms, and stimulating the elaboration of pro-inflammatory cytokines. Similar trials directly assessing the influence of carrageenan and CMC in humans are of course unethical to conduct, but recent studies of human epithelial cells and the human microbiome support the findings from animal studies. Carrageenan and CMC may trigger or magnify an inflammatory response in the human intestine but are unlikely to be identified as the sole environmental factor involved in the development of IBD or in disease recurrence after treatment. However, the widespread use of carrageenan and CMC in foods consumed by the pediatric population in a "Western" diet is on the rise alongside a corresponding increase in IBD incidence, and questions are being raised about the safety of frequent usage of these food additives. Therefore, further research is warranted to elucidate the role of carrageenan and CMC in intestinal inflammation, which may help identify novel nutritional strategies that hinder the development of the disease or prevent disease relapse post-EEN treatment.

Keywords: carboxymethylcellulose, carrageenan, inflammatory bowel disease, microbiota, Crohn's disease, ulcerative colitis

# INTRODUCTION

Crohn's disease (CD) is a chronic relapsing and remitting inflammatory bowel disease (IBD) that causes damage to the mucosal lining of the gastrointestinal tract, resulting in abdominal pain, (bloody) diarrhea, intestinal ulceration, and often progression toward stricturing/penetrating complications requiring surgery, malnutrition, impaired growth, disability, and even mortality (1, 2). Epidemiological

#### *Edited by:*

*Séamus Hussey, Our Lady's Children's Hospital, Ireland*

#### *Reviewed by:*

*Christiane Sokollik, University Children's Hospital Bern, Switzerland Moftah Hussin Alhagamhmad, University of New South Wales, Australia*

*\*Correspondence:*

*Johan Van Limbergen<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Gastroenterology, Hepatology and Nutrition, a section of the journal Frontiers in Pediatrics*

*Received: 11 March 2017 Accepted: 13 April 2017 Published: 01 May 2017*

#### *Citation:*

*Martino JV, Van Limbergen J and Cahill LE (2017) The Role of Carrageenan and Carboxymethylcellulose in the Development of Intestinal Inflammation. Front. Pediatr. 5:96. doi: 10.3389/fped.2017.00096*

**14**

studies have shown an increasing incidence of IBD [composed of CD and ulcerative colitis (UC)] both in areas of the world where the disease has been originally more prevalent (North America, Australia, and Europe) and in countries where IBD was previously infrequent (Asia and South America) (3, 4). It is noteworthy that this increase has occurred in conjunction with the transition toward a "Western" diet characteristically high in processed foods and fats and low in fruits and vegetables (5). Although the exact etiology and pathogenesis of IBD remain obscure, it is believed to result from a combination of genetic, microbial, immunological, and environmental factors including diet (6). In the last couple of years, the field of nutrition in IBD has advanced substantially (7, 8), with several dietary aspects of interest including the food additives used to induce intestinal inflammation and ulceration in animal models of IBD (9–11).

Food additives are substances intentionally added during production, processing, packaging, transportation, or storage of commercial food products. Carrageenan is the name referring to a family of high-molecular-weight sulfated polysaccharides extracted from seaweeds and commonly used as a thickening and emulsifying food additives to improve the texture of commercial food products including infant formulas, dairy products, milk alternatives such as almond milk, processed meats, and soy-based products (9, 12). First patented in the United States in the 1930s, carrageenan's use as a food additive in the Western diet has been substantially increasing over the last 50 years (9, 13). Carboxymethylcellulose (CMC) is a derivative of cellulose, making it affordable and abundant with thickening and emulsifying qualities, and it is found vastly throughout the commercial food industry with a progressive annual increase in its usage as a food additive (14, 15).

# THE ROLE OF CARRAGEENAN IN INTESTINAL INFLAMMATION IN ANIMAL MODELS

It has been demonstrated that when guinea pigs are supplied with degraded carrageenan in their drinking water, ulcerations develop in 100% of the animals in their large intestine by the end of a 30-day period (16). The lesions induced by carrageenan in the guinea pigs' large bowel resemble features of human UC (10). Carrageenan has also produced ulcerative lesions in rabbits, mice, and rats that were associated with weight loss, anemia, diarrhea, visible or occult blood, and sometimes mucus in the feces (17). Further, macroscopic features reminiscent of human UC, such as ulcerations predominantly limited to the mucosa along with pseudopolyps and polypoidal formations, were observed, as well as features commonly seen in CD such as strictures in the small intestine leading to obstruction (17). Histological changes associated with exposure to carrageenan in animal models include acute, subacute, and chronic inflammatory changes in the mucosa; occasional crypt abscesses; cystic dilatation or distortion of mucosal glands; mucosal ulceration in various stages of progression and healing; and hyperplastic changes of the glandular epithelium (17). Pricolo et al. noted that animals fed a 2% solution of carrageenan mixed with standard rat chow developed small bowel lesions first (from 2 to 6 weeks) followed by colonic lesions that developed after 8 weeks (11), which may be of particular interest for a subgroup of human CD patients who extend their disease location (18–20).

In recent years, evidence from animal studies has been mounting to suggest that the gut microbiome plays a critical role in the development of inflammation in response to exposure to carrageenan. For example, guinea pigs fed degraded carrageenan develop cecal ulcerations within 21–30 days after carrageenan treatment (21), whereas germ-free guinea pigs fed carrageenan for 6 months or more do not develop any intestinal lesions (22). Concurrent treatment of conventional guinea pigs with carrageenan and metronidazole, an antimicrobial primarily active against anaerobic bacteria, has been shown to prevent the development of intestinal ulcerations (22, 23). Consistent with recent reports in human IBD (24, 25), microorganisms such as *Bacteroide*s *vulgatus* appear to play an important role in the development of the experimental ulcerative lesions in animals (26). For example, animals who were immunized with *B. vulgatus*, prior to carrageenan administration developed experimental disease at a faster rate and more severe lesions than animals that were administered carrageenan alone (23, 27). Mirroring the development of antibodies to components of the intestinal microbiome in human IBD (28, 29), Onderdonk et al. noted that non-immune carrageenan recipients developed antibodies to *B. vulgatus* during carrageenan treatment, implying that an adaptive immune response occurs in the animals following carrageenan-induced intestinal ulceration (26, 27). Factors present on the bacterial outer membrane may mediate these antibody responses, as one study assessed eight strains of *B. vulgatus* and observed the alpha phenon (term used to reflect strain variation of outer membrane proteins) was associated with the enhancement of colitis (30).

The specific mechanism by which carrageenan induces inflammation in experimental animal models is not clearly defined, but carrageenan has been demonstrated to decrease the amount of epithelial glycoproteins in the colon (31) and is capable of inhibiting the interaction between macrophages and lymphocytes (32). The involvement of toll-like receptor-4 (TLR4) and interleukin (IL)-6 in the innate immune response to carrageenan was demonstrated using TLR4- and myeloid differentiation primary response 88-deficient mice (33). Recently, Wei et al. showed in a trinitrobenzenesulfonic acid (TNBS) model of colitis in BALB/c mice that pretreatment with carrageenan aggravated the severity (both clinical and histological) of TNBS colitis, with a concomitant increased expression of IL-6 and tumor necrosis factor alpha (TNF)-α and a reduction in IL-10 (34). Taken together, these animal studies have led to the hypothesis that food emulsifiers such as carrageenan may act as a conditional inflammatory agent that magnifies any existing chronic inflammation of the intestinal tract provoked by pathogens (35). This hypothesis explains why carrageenan has been found to induce intestinal inflammation in most animal studies, but not all. For example, healthy neonatal pigs fed infant formula with carrageenan for 28 days had no effect on blood cytokine evaluations (IL-1β, IL-6, IL-8, and TNF-α) (36), but they would not have had any baseline inflammation and were not exposed to pathogens.

# THE ROLE OF CMC IN INTESTINAL INFLAMMATION IN ANIMAL MODELS

In mice given relatively low concentrations of CMC, low-grade inflammation and obesity/metabolic syndrome was induced in wild-type hosts and promoted robust colitis in IL-10−/− and TLR5−/− mice. In the control mice not fed CMC, the closest bacteria were shown to reside about 25 µm from epithelial cells with no bacteria observed within 10 µm, whereas CMC-treated mice exhibited some bacteria in apparent contact with the epithelium, and the average distance was reduced by more than 50% (37). In this study, CMC dramatically altered microbiota composition in both fecal and intestinal-adherent bacteria, and the authors concluded that chronic exposure to CMC resulted in erosion of the protective function of the mucus, increased bacterial adherence, and a more pro-inflammatory microbiota (37). More recently, Chassaing et al. tried to disentangle the effect of inflammation itself on microbiota composition from the effect of CMC on host parameters (that in turn promotes inflammation and subsequently alters the microbiota). They used the Mucosal Simulator of the Gastrointestinal Microbial Ecosystem (M-SHIME) model to examine the effects of emulsifiers on the human microbiota *in vitro* (38). CMC directly acted upon human microbiota to increase its pro-inflammatory potential; the CMC-induced increase in flagellin occurred after 1 day and was driven by altered microbiota gene expression rather than microbiota composition changes (38). Transfer of both emulsifier-treated M-SHIME microbiotas to germ-free recipient mice recapitulated many of the host and microbial alterations observed in mice directly treated with emulsifiers, notably the development of intestinal inflammation (38). These results suggest that the microbiota may be a key direct target of CMC to drive chronic intestinal inflammation.

Swidsinski et al. have demonstrated that exposure to 2% CMC in IBD-susceptible IL-10 gene-deficient mice results in "CD-like effects," reporting that changes in the CMC-treated mice were identical to ileal biopsy findings of CD patients (14). This was supported by the observations that mice treated with 2% CMC demonstrated increased concentrations of bacteria in the ileum, larger spaces between villi, increased amounts of bacteria adherent to the villi, increased white blood cells in the lumen, and, in some of the mice, bacterial infiltration of the epithelium (14).

These findings of the effects of commonly used emulsifiers on the microbiota, mucus layer, and epithelial barrier integrity should be considered in conjunction with the recent work by Desai et al. (39) regarding the role of dietary fiber on mucus barrier maintenance. These authors demonstrated that in gnotobiotic mice colonized with a synthetic human gut microbiota and denied dietary fiber, the microbiota will consume host-secreted mucus glycoproteins as a nutrient source in place of fiber, leading to erosion of the colonic mucus barrier (39). In turn, this erosion of the colonic mucous barrier promoted greater epithelial access and lethal colitis by the murine pathogen *Citrobacter rodentium* (39). Carrageenan and CMC are often added to commercial food products as a fiber (40), instead of other dietary fiber (such as water-insoluble cellulose and resistant starch or water-soluble fiber such as pectin and raffinose) sources. In addition, many CD patients already limit dietary fiber to avoid its bulk-forming and laxative effects. Therefore, a "perfect storm" setting in which CD patients increase their CMC/carrageenan intake but limit their intake of other dietary fibers is created, which could propagate or exacerbate the existing dysbiosis toward more mucin-degradation and which could enhance the susceptibility for mucosa-associated pathogens.

# FOOD ADDITIVES AND INTESTINAL INFLAMMATION IN HUMANS

The reports above of commonly used food additives causing intestinal inflammation in animal models have triggered investigations of how they may lead to inflammation in the human gastrointestinal tract, although perfectly corresponding human studies are of course unethical to conduct. It is likely that mechanisms by which carrageenan induces inflammation in the human intestine are similar to animals and multifactorial: the stimulation of pro-inflammatory cytokines, the disruption of the epithelial barrier, and the interference with innate mucosal immune responses to microorganisms (**Figure 1**). Community-level metabolic networks within the microbiome produce bioactive metabolites that have an established role in intestinal immune homeostasis and a healthy gut mucosal barrier (41, 42). This barrier protecting the host from luminal antigens is composed of enterocytes (epithelial cells) and the seal between them provided by the tight junctions (43). With a healthy gut mucosal barrier, an individual maintains a homeostasis where the passage of nutrients, ions, and water across the intestinal epithelium into the mesenteric blood stream is tightly regulated, while translocation of dietary antigens and components of the microbiome is prevented (43), and the active crosstalk between a diverse microbiome and the intestinal immune system leads to immune tolerance. Impaired barrier function can lead to abnormalities in the expression of tight junction protein that may trigger immune activation and the development of inflammatory disease in susceptible individuals (43, 44).

Borthakur et al. provided the first report of the inflammatory response of human intestinal epithelial cells to carrageenan exposure by demonstrating that carrageenan stimulates an inflamatory cascade in colonic epithelial cells in a pathway involving B-cell lymphoma/leukemia 10 gene (Bcl10) activation and the increased production of IL-8 (9), a key pro-inflammatory cytokine. These results were supported when Choi et al., using the human colonic adenocarcinoma cell line HCT-8, demonstrated that carrageenan led to the activation of nuclear factor kappaB, which subsequently increased the gene induction of IL-8 (12). In addition, they reported that carrageenan triggered a disruption of the epithelial barrier, decreasing the density of tight junctional protein zonula occludens (ZO)-1, causing disarray in the distribution of ZO-1 throughout the epithelium, and decreasing the gene expression of this tight junction protein (12). More recently, this group showed that carrageenan exposure triggered the expression of the proapoptotic macrophage inhibitory cytokine 1 (MIC1), which is in turn counteracted by MIC1-induced expression of activating transcription factor 3 (45).

Carrageenan has been reported to interact with the glycoprotein deleted in malignant brain tumors 1 (DMBT1). DMBT1 functions as a pattern recognition molecule with a peptide domain capable of binding and aggregating a wide spectrum of bacteria (46) and has been demonstrated to prevent the invasion of bacteria into intestinal epithelial cells *in vitro* (47). Carrageenan inhibits the bacterial aggregating function of DMBT1 *via* binding to the specific peptide that recognizes bacteria, and it has been suggested that carrageenan disrupts the mucosal protection provided by DMBT1 (48). This raises the possibility that carrageenan is capable of disrupting an innate mucosal immune function provided by DMBT1, which can then trigger the initiation or perpetuation of an inflammatory response to intestinal bacteria or bacterial antigens (48).

In human cells, carrageenan has also been demonstrated to trigger innate immune responses through pathways that involve Bc110, TLR4, NF-κB, and AP-1 (9, 35, 49), leading to the upregulation of TNF-α secretion (50). For example, Jiang et al. (51) demonstrated that carrageenan-induced TNF-α secretion is the main contributor to cellular damage in Caco-2 monolayers exposed to carrageenan. However, researchers in the field note that in these studies, the degree of inflammation caused by carrageenan alone is low, while the inflammation generated through these pathways when a pathogen is additionally present is high, supporting the hypothesis that an interaction effect is present in which carrageenan serves as a pro-inflammatory agent to amplify existing intestinal inflammation (35).

In view of the recent benchmark reports by Chassaing et al. (38) and Desai et al. (39), more studies of the intestinal response to the widely used emulsifiers, carrageenan and CMC, together with other features of a Western diet such as reduced dietary fiber intake are eagerly awaited (52). It is noteworthy that the Food and Drug Administration regulations do not contain a definition of dietary fiber but rather have relied on analytical methods for measuring levels of dietary fiber present in food. Therefore, an isolated or synthetic non-digestible carbohydrate such as carrageenan and CMC can be added to foods and quantified as a dietary fiber even if it does not provide the beneficial physiological effect to human health that dietary fiber should provide. For example, CMC is listed among the 26 most common fibers being added to food and declared on the Nutrition Facts label as dietary fiber (40).

# WIDESPREAD USE OF FOOD ADDITIVES

Patented in the United States in the 1930s, carrageenan was granted GRAS (generally regarded as safe) status in 1959 and currently remains included as a food additive that holds GRAS status in the Code of Federal Regulations in the United States (13). Carrageenan acts to thicken, stabilize, and emulsify a wide variety of foods typically consumed in the Western diet including dairy products such as chocolate milk, ice cream, cottage cheese, sour cream, and yogurt; processed meats; soymilk; almond milk; mayonnaise; and infant formula (53). Estimates regarding the average daily intake of carrageenan vary from 20 to 200 mg/day (13, 54) and are currently difficult to put into context without a standard for comparison within human studies or with animal studies.

Carboxymethylcellulose is used broadly throughout the food industry in products typically consumed by children including candy, chewing gum, "snack foods," ketchup, and various baked goods, and currently, there are no quantitative restrictions on its use nor does its addition to food require to be declared (14, 15). CMC is listed in the Food and Drug Administration's database of GRAS substances (55), and CMC is also included in the Safety and Toxicity of Excipients for Paediatrics (STEP) database (56, 57), a resource developed by the European Paediatric Formulation Initiative and the United States Paediatric Formulation Initiative in collaboration for storage and rapid/effortless access to the safety and toxicological data of commonly used excipients.

# ROLE OF DIET IN IBD: EPIDEMIOLOGY AND TREATMENT

The increasing incidence rate of IBD worldwide, with the highest incidence and prevalence being in Westernized countries (3), has been associated with increased consumption of a Western diet (5). A case–control study conducted in Japan assessed environmental risk factors for UC in 101 patients and observed that consumption of foods typical of a Western diet including bread, butter, and sausage was significantly related to an increase in UC risk (58). Moreover, D'Souza et al. demonstrated that the consumption of fried and fast foods, meats, snacks, and desserts, which they labeled as a traditional Western dietary pattern, increased the likelihood of acquiring CD in adolescent females (59). Conversely, they found that both males and females exposed to a prudent dietary pattern, consisting of mostly vegetables, fish, grains, and nuts, had a decreased likelihood of developing CD (59).

Exclusive enteral nutrition (EEN) is recommended as a firstline therapy in pediatric CD and involves the use of a liquid formula diet as the only source of nutrition over several weeks (8, 60).The mechanism of action of EEN has not been clearly established; however, it may provide efficacy *via* eliminating certain components of the Western diet and through modulation of the microbiome (61–67). More recently, novel dietary therapy regimens have been described, which involve avoidance of processed foods such as the CD exclusion diet (CDED) (68) and the Specific Carbohydrate Diet (69). Sigall-Boneh et al. demonstrated that the CDED, which avoids exposure to many components of a Western diet such as dairy products and processed foods containing food additives, led to remission in 70% of patients (8). Through large network studies such as the Canadian Institutes for Health Research supported Inflammation, Microbiome, Alimentation, GastroIntestinal and Neuropsychiatric effects (IMAGINe), considerable efforts are being made to investigate the potential contributions of dietary factors to IBD pathophysiology as a dysregulated interplay of genetic, environmental, microbial, and immunological factors (6, 59), and so more details on the role of diet, including food additives such as carrageenan and CMC, in IBD may be discovered in the near future and inform dietary recommendations and medical treatment of IBD.

# CONCLUSION

Carrageenan and CMC administered in animal models consistently result in intestinal ulcerations with histopathological features similar to human IBD. Although the set of precise mechanisms through which these emulsifiers induce lesions and inflammation remains unknown, disruption of the epithelial barrier and dysregulation of the immune response to the gut microbiome have been repeatedly implicated. These findings raise concern because carrageenan and CMC are used extensively in processed food products that are consumed by the pediatric population, and the incidence rate of childhood IBD is increasing concurrently with a rise in the adoption of a Western diet. The only successful dietary interventions to have induced CD remission exclude processed foods containing carrageenan and CMC, further supporting the possibility that carrageenan and CMC are potential triggering or magnifying substances of inflammation in IBD. Further research is warranted to clarify the role of carrageenan and CMC in the microbiome alteration of intestinal inflammation together with an improved appreciation of the complex interplay with the consumption of dietary fibers, and such studies could lead to novel nutritional strategies that help prevent the development of IBD or help induce and sustain remission.

# AUTHOR CONTRIBUTIONS

All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.

# ACKNOWLEDGMENTS

The authors would like to thank Leah Boulos from the Maritime SPOR SUPPORT Unit for her instruction to JM on literature searching and journal article extraction for this review.

# FUNDING

JVL was supported by a NASPGHAN/CCFA Young Investigator Development award (2013–2015), a Nova Scotia Health Research Foundation (NSHRF) establishment award (2015–2017), a Future Leaders in Inflammatory Bowel Disease (FLIBD) Program grant (2015–2016), a Dalhousie Medical Research Foundation equipment grant (2015–2016), a donation from the MacLeod family, an IWK Health Centre Research Associateship and Cat. B grant and a Canadian Institutes of Health Research (CIHR)- CAG-CCC New Investigator Award (2015–2020: 201412XGP-340307-205026) and a Canadian Foundation of Innovation John R. Evans Leadership fund (#35235). JVL is supported by a CIHR-SPOR-Chronic Diseases grant (Inflammation, Microbiome, and Alimentation: Gastro-Intestinal and Neuropsychiatric Effects: the IMAGINE-SPOR chronic disease network).

# REFERENCES

**Conflict of Interest Statement:** JVL has participated in an advisory board for Nestlé and has received speaking fees and research support from Nestlé. The other authors declare no conflict of interest.

*Copyright © 2017 Martino, Van Limbergen and Cahill. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Commentary: Impact of Fecal Calprotectin measurement on Decision-making in Children with Inflammatory Bowel Disease

*Andrew S. Day1,2\* and Mustafa Adamji1*

*1Department of Paediatrics, Christchurch Hospital, Christchurch, New Zealand, 2 Department of Paediatrics, University of Otago, Christchurch, New Zealand*

Keywords: children, inflammatory bowel disease, fecal markers, calprotectin, Crohn disease

#### **A Commentary on**

### **Impact of Fecal Calprotectin Measurement on Decision-Making in Children with Inflammatory Bowel Disease**

*by El-Matary W, Abej E, Deora V, Singh H, Bernstein CN. Front Pediatr (2017) 5:7. doi: 10.3389/ fped.2017.00007*

#### *Edited by:*

*Séamus Hussey, Our Lady's Children's Hospital, Ireland*

#### *Reviewed by:*

*Juan Cristobal Ossa, Universidad de Chile, Chile Matthew Wyatt Carroll, University of Alberta, Canada*

#### *\*Correspondence:*

*Andrew S. Day<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Gastroenterology, Hepatology and Nutrition, a section of the journal Frontiers in Pediatrics*

*Received: 23 March 2017 Accepted: 22 May 2017 Published: 06 June 2017*

#### *Citation:*

*Day AS and Adamji M (2017) Commentary: Impact of Fecal Calprotectin Measurement on Decision-Making in Children with Inflammatory Bowel Disease. Front. Pediatr. 5:133. doi: 10.3389/fped.2017.00133*

In their recent publication, El-Matary et al. (1) describe the utility of measurement of fecal calprotectin (FC) in the ongoing management of children with known inflammatory bowel disease (IBD). In this cohort of 77 children, FC was measured upon presentation with key changes in symptoms (most commonly abdominal pain and hematochezia). The child's management was then adjusted according to the level of the FC, with a cutoff of 250 µg/g stool. Almost 90% of those with elevated FC had a change in their management, which resulted in a reduction in clinical activity indices over the subsequent 3–6 months. Repeated FC measurements were not available. Conversely, the majority of those with low FC (below the cutoff) had no change in management in the following months. Reassuringly, 94% of these children were judged to be in remission at their follow-up visit.

Non-invasive markers, especially those measured in stool, have become increasingly important and relevant in recent years in the management of IBD. Numerous markers have now been described. Although calprotectin has been the most utilized to date, other promising markers include S100A12, osteoprotegerin, lactoferrin, and M2 pyruvate kinase (2–4).

Generally, markers such as calprotectin provide greater specificity and sensitivity for the presence of gut inflammation than standard serum-based markers. In one assessment of routine serum markers at the time of diagnosis, erythrocyte sedimentation rate, C-reactive protein, albumin, and platelet counts were each normal in 19 (13%) and all abnormal in just 52 (36%) of 146 children with CD (5). Overall, the platelet count was most often abnormal in this group. Both FC and fecal S100A12 provided much greater utility than any of the same four serum markers in an earlier cohort of 31 children with IBD (6).

Although fecal markers have particular roles in the investigation of an individual with undifferentiated symptoms (to assess the need for further investigations and to reach a diagnosis), they also clearly have important roles in individuals diagnosed with IBD. Such roles include the monitoring of disease control, assessment of response to an intervention, assessment of mucosal healing (MH), and to provide an assessment of the risk of relapse in the coming months.

The current report focused upon children with a change in symptoms, such as the development of rectal bleeding. The key issues in this context are to ascertain whether the change in symptoms is related to an exacerbation of disease, or due to other factors such as an intercurrent infection or due to functional overlap. Both enteric infections and irritable bowel syndrome can result in an elevated FC. Prompt access to FC measurement clearly provides guidance as to the clinical management required. In practice, however, one would otherwise consider the pattern of symptoms, the routine blood tests, and examination findings and anthropometry as well. It would seem reasonable and appropriate to consider FC measurement in addition to these assessments, rather than instead.

There are some data suggesting that FC levels may vary according to disease location, with it being less reliable in small bowel inflammation (7). Assessment at baseline, at the time of initial diagnostic endoscopy, along with serial measurements over time may be of assistance for the individual patient. Accordingly, a change in FC from a prior measurement may be more helpful than a one-off level.

The other potential roles of fecal markers in monitoring disease progress are also important, especially in children. Several reports indicate that the serial measurement of S100A12 or FC in individuals in clinical remission may predict the risk of a future relapse (8, 9). In addition, fecal markers may enable the early prediction of recurrence after ileo-colonic resection (10). The role of FC in predicting the achievement of MH is less clear: the data are not yet conclusive as to the correlation between FC and MH (11). In addition, FC may have a role in guiding

# REFERENCES

the appropriate indication for repeat endoscopic assessment in children with established disease: optimizing the timing of endoscopy in children will clearly be of benefit to health administrations (given the cost of endoscopy) and also for children and their parents (given the inconvenience of endoscopy for children).

Clearly, the potential roles for FC and other non-invasive markers are likely to expand. Further understanding of the differential information provided by the various markers may lead to the use of more than one marker, or indeed to the use of a panel of markers.

In conclusion, the work of El-Matary and colleagues (1) provides further important information of the utility of FC in children with IBD. Additional prospective assessments are required, ideally with comparisons between available markers and with serial estimations over time.

# AUTHOR CONTRIBUTIONS

AD formulated the plan for this commentary along with initial draft manuscript. MA reviewed the draft manuscript and assisted in revisions of the drafts of the manuscript.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Day and Adamji. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Crohn's Strictures—Moving Away from the Knife

*Emily Stenke1 \*, Billy Bourke1,2 and Ulla Knaus 1 \**

*1School of Medicine, Conway Institute, University College Dublin, Dublin, Ireland, 2Department of Pediatric Gastroenterology, Our Lady's Children's Hospital, Dublin, Ireland*

Crohn's disease (CD) is a lifelong inflammatory bowel disease with a rapidly rising incidence in the pediatric population. A common complication of CD is the development of fibrotic strictures, which may be present at initial diagnosis or develop many years later. Clinical presentation depends on stricture location and degree of obstruction, and strictures frequently contain a mixture of inflammatory and fibrotic tissue. Histological examination of Crohn's strictures shows thickening of the muscular layers and the submucosa, where increased collagen deposition by activated myofibroblasts is concentrated around islands of smooth muscle cells and at the superficial margin of the muscularis propria. No antifibrotic therapies for Crohn's strictures exist. Profibrotic transforming growth factor-β (TGFβ)/bone morphogenetic protein signaling stimulates myofibroblast differentiation and extracellular matrix deposition. Understanding and targeting TGFβ1 downstream signaling is the main focus of current research, raising the possibility of specific antifibrotic therapy in CD becoming available in the future.

#### *Edited by:*

*Jeff Critch, Memorial University of Newfoundland, Canada*

#### *Reviewed by:*

*Andrew S. Day, University of Otago, New Zealand Amit Assa, Schneider Children's Hospital, United States*

*\*Correspondence:*

*Emily Stenke<EMAIL_ADDRESS>Ulla Knaus<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Gastroenterology, Hepatology and Nutrition, a section of the journal Frontiers in Pediatrics*

*Received: 16 March 2017 Accepted: 02 June 2017 Published: 16 June 2017*

#### *Citation:*

*Stenke E, Bourke B and Knaus U (2017) Crohn's Strictures—Moving Away from the Knife. Front. Pediatr. 5:141. doi: 10.3389/fped.2017.00141*

Keywords: Crohn's disease, inflammatory bowel disease, fibrosis, intestine, reactive oxygen species, NADPH oxidase

# INTRODUCTION

The incidence of Crohn's disease (CD) is rapidly rising in the pediatric population (1). Crohn's inflammation is transmural and can occur throughout the gastrointestinal tract, unlike ulcerative colitis where mucosal inflammation is confined to the colon. CD is phenotyped according to disease behavior (inflammatory/penetrating/stricturing) and location using the Paris classification for pediatric patients (2), a modification of the Montreal classification (3). Fibrotic narrowing of the intestinal tract (stricturing disease) is present in approximately 10–17% of children at diagnosis (4), affecting up to 40% by 10 years after diagnosis (5–7). Strictures occur predominantly in the ileum, reflecting the common distribution of inflammation, but can arise throughout the digestive tract (8). Presenting features vary from overt intestinal obstruction to subacute and non-specific symptoms. The conventional conceptual framework of the pathobiology of fibrosis in CD is one of chronic inflammation leading to ongoing frustrated attempts at healing with formation of disorganized tissue. If this occurs circumferentially in a relatively narrow diameter organ such as the small intestine, the result is a fibrotic stricture. Here, we will describe current knowledge and recent advances regarding the diagnosis and pathogenesis of intestinal fibrosis and will review potential future therapies.

# CLINICAL AND PATHOLOGICAL DIAGNOSIS

The clinical presentation of stricturing disease is highly variable, depending on the location and degree of obstruction. Presentations range from non-specific symptoms including abdominal discomfort, poor appetite, energy and/or growth, to overt obstruction with abdominal pain, vomiting, and reduced bowel movements. Strictures may be inflammatory, fibrotic, or more commonly a

**23**

combination of both (8). Most strictures are in the small bowel, inaccessible to standard endoscopy, and even when accessible, endoscopy can only provide information (visual, histological) on the mucosa, while the fibrotic collagen deposition occurs submucosally. Capsule endoscopy can provide visual information but no tissue samples and is contraindicated in the presence of suspected strictures due to the risk of retention. Therefore, clinicians usually rely on radiological tools for the diagnosis of stenotic disease.

A fibrotic stricture is inferred by the presence of a narrowed lumen with proximal dilation. Computed tomography (CT) and magnetic resonance enterogram (MRE) are preferred to X-rays with enteral contrast [small-bowel follow through (SBFT)] as they allow assessment of extraintestinal as well as intestinal complications of CD and have similar sensitivity and accuracy. CT and MRE permit a transmural assessment of the bowel wall, enabling the classification of strictures as inflammatory, fibrotic, or mixed. Inflammation is suggested by avid enhancement and mesenteric inflammation (hypervascularity, fat stranding), whereas fibrosis is characterized by a thickened bowel wall with a featureless appearance, minimal or no enhancement and the absence of mesenteric inflammation. MRE has comparable (9–12) or superior (13) sensitivity and specificity to CT for the detection of fibrosis in the presence of inflammation in adult and pediatric patients, while MRE has greater sensitivity for the detection of fibrosis alone (10). A recent prospective pediatric study demonstrated 73% sensitivity and 81% specificity for the detection of strictures using MRE compared to 42% sensitivity and 68% specificity for CT enterograms (12). Due to its lack of ionizing radiation and its capacity to provide accurate diagnosis of both intra- and extraintestinal complications, MRE is the preferred modality in the pediatric population, although technical limitations (institutional access, need for general anesthesia) ensure a retained role for CT and SBFT. Recent advances in ultrasound elastography as a measurement of tissue stiffness have shown promising results in the differentiation between inflammatory and fibrotic strictures (14, 15), but have not yet reached routine clinical practice. As sensitivity does not reach 100% with any modality, clinical judgment is required in conjunction with imaging findings to decide about surgical exploration/intervention.

# HISTOPATHOLOGY

Fibrosis is defined as the permanent and abnormal deposition of extracellular matrix (ECM), primarily collagen, within tissues, resulting in a distortion of structure and impeding normal tissue and organ function. It is understood to be an aberrant response to ongoing inflammation, where tissue remodeling in response to injury, through ECM deposition and subsequent breakdown, becomes self-perpetuating. In the normal small bowel, the mucosa consists of a single layer of epithelium, lamina propria, and basement membrane. Deep to the intact mucosa is the muscularis mucosa, a thin layer of smooth muscle cells, and then the submucosa. The submucosa is a loose connective tissue layer with fibroblasts as main cell type within an ECM traversed by blood vessels and nerves (16). The ECM is a complex biochemical structure where collagen types I and III predominate (17). Deep to the submucosa is the muscularis propria with its inner circular and outer longitudinal layers of smooth muscle cells. The collagen strands of the submucosa tend to be concentrated at its border with the muscularis propria.

Histological examination of CD strictures reveals abnormalities in both the submucosal space and muscular layers (**Figure 1**). The submucosa is increased in volume and density, with increased collagen deposition being concentrated around islands of smooth muscle cells and at the superficial margin of the muscularis propria. Studies have shown an increase in total protein and collagen content, especially collagen subtypes I, III, and V in these strictures. Although increased in absolute terms, the relative proportions of types I and III collagen appear to be comparable between strictures and healthy intestine, and although the proportion of type V collagen is relatively amplified, type I collagen remains dominant (16, 17). Myofibroblasts, differentiated from fibroblasts, are the primary source of ECM production and secretion. Increased numbers of local fibroblasts and myofibroblasts in fibrosis have been attributed to a variety of mechanisms including the proliferation of existing fibroblasts in the local area (18), the induction of epithelial-to-mesenchymal transition (19), the recruitment and differentiation of bone marrow-derived fibrocytes (20), as well as endothelial-to-mesenchymal transition (21), but the relative contribution of each process is unknown. Thickening of the muscularis mucosa and muscularis propria as well as smooth muscle cell proliferation within the submucosa itself have been observed. In some instances, the proliferation of smooth muscle cells within the submucosa can be so pronounced that it results in the obliteration of the submucosa (22). The combined overall effect of increased ECM deposition and muscular hypertrophy is transmural thickening and stiffening, which when occurring circumferentially causes narrowing and obstruction of the intestinal lumen.

# SIGNALING AND THE FIBROTIC RESPONSE

Various growth factors and cytokines have been implicated in the development of fibrosis including IL-13, platelet-derived growth factor (PDGF), connective tissue growth factor, basic fibroblast growth factor, insulin-like growth factor, bone morphogenetic proteins (BMPs), and transforming growth factor-β (TGFβ) (23). The TGFβ family is secreted by a wide variety of cells throughout the body and its effects are highly varied and complex. As well as being responsible for cell proliferation and differentiation, anti-inflammatory control, wound healing, tumor suppression in healthy tissues and cancer progression in neoplastic tissues, TGFβ is the primary cytokine driving the development of fibrosis in tissues throughout the body. Differences in TGFβ subtype, receptors, cofactors, and signaling pathways allow this group of cytokines to display such versatility (24). TGFβ1 is the most common subtype and its role in the development of tissue fibrosis through the recruitment of fibroblasts, transdifferentiation to myofibroblasts, and stimulation of ECM secretion has been convincingly demonstrated in many organs including in intestinal fibrosis (25–28).

TGFβ1 is a homodimeric signaling molecule produced by myofibroblasts and inflammatory cells (e.g., macrophages),

platelets, and parenchymal cells during the hemostasis and inflammation phases of tissue injury and healing. This para- and autocrine signaling is anti-inflammatory and supports wound healing but drives fibrosis. The TGFβ receptor is a transmembrane complex of two dimers: two type I receptors (ALK5, also called TGFBR1 or TβTI) and two type II receptors (TGFBR2); both are serine/threonine kinases and binding of TGFβ results in phosphorylation of ALK5 by TGFBR2 with subsequent binding and phosphorylation of downstream signaling proteins SMAD2 and SMAD3 by ALK5 (**Figure 2**). Once phosphorylated, SMAD2 and SMAD3 form a trimer with SMAD4, which translocates to the nucleus and binds to target DNA. The transcriptional targeting, nuclear translocation, and longevity of these SMAD transcription factors is modulated by the binding of a wide variety of effector molecules. SMAD2/3 binding to ALK5 is antagonized by SMAD7. SMAD7 antisense oligonucleotides (mongersen) showed promise in the treatment of inflammatory CD by restoring the TGFβ1–SMAD2/3 pathway (29); no associated increase in fibrosis has been observed but longer term follow-up is required. Several non-canonical TGFβ signaling pathways have been described (30). Phosphorylated TGFBR1 and TGFBR2 lead to activation of mitogen-activated protein kinases (MAPKs), which regulate multiple pathways including those leading to transcription. MAPKs also regulate the canonical TGFβ pathway by phosphorylating active SMAD to promote its proteasomal degradation. TGFβ activates phosphoinositide 3-kinase, leading to downstream activation of Akt, mTOR, and upregulation of protein translation. Akt interacts directly with SMAD3 to prevent its activation and indirectly inhibits SMAD-mediated transcription through phosphorylation of FoxO transcription factors, thus blocking their translocation to the nucleus. A more detailed review of the complexity of TGFβ signaling can be found in the study by Massague (24).

The pleiotropic and multifunctional effects of TGFβ1 signaling make it an unattractive therapeutic target in fibrosis. Substantial effort has been directed at characterizing downstream signaling pathways of TGFβ where fibrosis-specific pharmacologic intervention might be achievable. Reactive oxygen species (ROS) have been implicated as mediators of the profibrotic effect of TGFβ1. ROS can be produced non-enzymatically (e.g., by radiation, toxic chemicals) or enzymatically. Most cellular ROS is produced in the mitochondria during cellular respiration. Other ROS sources include the cytochrome P450 family, cyclooxygenases, peroxisomal oxidases, xanthine oxidoreductase, and the family of NADPH oxidases (NOX). The NOX family is unique as its only known biological role is ROS production, and NOX enzymes appear to be intimately involved in fibrogenesis. NADPH oxidase 4 (NOX4) is a constitutively active, transcriptionally regulated producer of H2O2. An accumulating body of evidence suggests that TGFβ1 upregulates NOX4 expression and that NOX4, through the production of H2O2, promotes myofibroblast differentiation and secretion of ECM proteins (31–33). In a rat model of renal fibrosis and in cell-based assay systems, TGFβ1 stimulation was associated with increased NOX4 expression and H2O2 production, while reduced NOX4 expression by siRNA-mediated knockdown decreased ROS production and expression of profibrotic proteins including collagen, α-SMA, and fibronectin (34). These findings have been replicated using cell-based assays, mouse models, and patient samples in the lung (35–38), skin (39), and liver (40–42). A study by Jung et al. using fibroblasts and a mouse model of renal

fibrosis suggested that NOX4 upregulation by TGFβ1 depends on activation of SMAD2 and Akt (43). Latella and coworkers reported that SMAD3 was necessary for the development of intestinal fibrosis in a chronic TNBS model (44), suggesting that NOX4 mediates the effect of TGFβ1 through canonical and non-canonical pathways. Somewhat contradictorily, NOX4 was protective in a renal fibrosis model (45), and there is evidence from mice and patient data that NOX4 may be protective against atherosclerosis (46–49). Another mouse model showed preserved myofibroblast differentiation but impaired wound healing in NOX4−/− mice (50), suggesting that the requirement of NOX4 for myofibroblast differentiation and collagen secretion may be tissue and/or context dependent (acute versus chronic insult). Despite the growing evidence for a fundamental role of NOX4 in fibrosis, only limited studies exist for the intestine. Hotta and coworkers demonstrated a reduction in TGFβ1-dependent collagen I production by murine intestinal myofibroblasts treated with a pan-NOX inhibitor or NOX4 siRNA (51). Data from RNA-Seq analysis of intestinal fibroblasts showed variable upregulation of NOX4 transcription in three patients with CD compared to three healthy controls (52). BMPs belong to the TGFβ superfamily and signal *via* phosphorylation and complex formation of SMAD 1, 5, and 8 (53). BMP7 may protect against colitis and prevent fibrosis by antagonizing TGFβ signaling (54–56). Angiopoietin-like protein 2 (ANGPTL2) modulates BMP signaling and initial studies suggest that organ damage in ANGPTL2 knockdown mice is linked to NOX4 (57, 58).

TGFβ1 signaling stimulated the production of mitochondrial ROS (mtROS), possibly by inhibition of complex III and IV and *via* the mTOR signaling pathway, with subsequent increase in profibrotic gene expression. Reduction of mtROS by the antioxidant MitoQ reduced TGFβ1 expression, SMAD2 and SMAD3 activation, and collagen deposition in a liver fibrosis model (59). mtROS and NOX4 may interact through a positive feedback loop to promote TGFβ1-driven fibrosis. NOX4-derived ROS caused mitochondrial dysfunction and increased mtROS, while mtROS amplified the TGFβ1-mediated increase in NOX4 expression (60). Additionally, increased TGFβ1 inhibited the antioxidant response, thereby exacerbating the prooxidant shift and further driving fibrosis (61).

Key enzymes in cross-linking and stabilizing the network of collagen fibrils are H2O2-generating lysyl oxidase (LOX) and the lysyl oxidase-like proteins (LOXL1–4), which are copperdependent amine oxidases that oxidatively modify the ε-amino group of lysine side chains in collagen and elastin for formation of inter- and intrachain cross-links. Clinical and animal-based studies in the liver and myocardium demonstrate that LOXs promote tissue stiffening through cross-linking of existing collagen and elastin fibrils and that inhibition of LOXL2 may inhibit and even reverse fibrosis (62–65). Studies in rat and human lung fibroblasts, human trabecular cells, and human osteoblasts suggest that TGFβ1 upregulates LOXs, which in turn modify the actions of TGFβ1 (66–69). It is important to note that TGFβ1 is secreted and stored extracellularly bound to a latent TGF-β-binding protein and a latency-associated peptide. This inactive complex is bound to the ECM *via* integrins, and active TGFβ is released by protease cleavage or conformational changes caused by increased stiffness of the ECM (61, 70). This provides a possible explanation for TGFβ1's effects in the absence of active inflammation (71, 72) as well as the finding that a stiff matrix is required for myofibroblast differentiation (62, 73).

# CURRENT TREATMENTS AND FUTURE POSSIBILITIES

Although improved management of CD inflammation by anti-TNFα therapy (infliximab, adalimumab) appears to reduce the rate of stricture development (74), there is currently no medical therapy directly targeting fibrosis in CD. Patients whose strictures fail to respond to anti-inflammatory therapies (aimed at any inflammation and edema coexisting with fibrosis) require surgical intervention. Endoscopic balloon dilatation (EBD) is an option for single, short, and uncomplicated strictures accessible by endoscopy, for instance, stricture recurrence at ileocecal anastomoses. Although technical success rates are high, with a low rate of complications, retrospective data from adult patients demonstrate that 42–70% of patients will require repeat EBD or surgical intervention by 5 years (75–77). Pediatric data are limited, but support the feasibility and safety of EBD (78, 79). Given the predominance of mixed fibrotic/inflammatory strictures over purely fibrotic strictures, intrastricture injection of corticosteroids has been proposed as an adjunct to balloon dilation. A prospective randomized control trial including nine adult patients showed no difference in stricture recurrence rates at 1 year (80), while a prospective RCT including 29 pediatric patients showed earlier stricture recurrence in patients treated with placebo (79). Due to the small trials and number of patients studied, the benefit of intralesional steroid injection has not been confirmed (81). Surgery is the mainstay of treatment for fixed Crohn's strictures. Simple, short strictures can be treated with bowel preserving strictureplasty; longer, multiple, or complicated strictures (e.g., significant inflammation, penetrating disease, and suspected cancer) are treated with resection and primary anastomosis. Resection carries its own risks, including anastomotic dehiscence and disease recurrence at the site of the anastomosis, malabsorptive issues following terminal ileal resection including vitamin B12 deficiency and bile salt malabsorption, and, in cases requiring repeated resections for recurrent strictures, short bowel syndrome leading to a dependence on parenteral nutrition.

Medical therapies to prevent and reverse fibrosis are eagerly sought and much of the focus for new therapies has been for pulmonary and hepatic disease. As our understanding of the pathophysiology of fibrosis improves, we are discovering more potential drug targets. Pirfenidone, a growth factor inhibitor, has been licensed for use in idiopathic pulmonary fibrosis (IPF), based on positive results from Phase 2 and 3 trials (82, 83). Nintedanib, a kinase inhibitor that acts on vascular endothelial growth factor receptors, PDGF receptors, and fibroblast growth factor receptors, showed efficacy and safety in Phase 2 and 3 trials and is licensed for use in IPF (83). Specific NOX4 inhibitors are still not available, albeit a NOX inhibitor (GKT137831) performed well in preclinical models (35, 42) and a Phase 1 clinical trial (84). A Phase 2 trial confirmed safety but not efficacy in the treatment of diabetic kidney disease; recruitment for a Phase 2 trial in patients with primary biliary cirrhosis is ongoing. Integrin αvβ6 is another target of interest; it mediates the conformational release of active TGFβ from its latent complex (72) and Phase 2 trials of a monoclonal antibody in the treatment of IPF are ongoing (85). Given the complex interaction between TGFβ secretion and tissue stiffness mediated by myofibroblast contraction, ECM deposition, and cross-linking with further TGFβ release, it is possible that the effective treatment of fibrosis will require combination therapy. For example, both Simtuzumab, a monoclonal antibody to LOXL2, and Relaxin (an inhibitor of myofibroblast contraction) have individually lacked efficacy for fibrotic disease in Phase 2/3 trials (86). However, a recent preclinical trial demonstrated a reduction in airway fibrosis using Relaxin and anti-LOXL2 antibody together (87).

Clinical trials assessing antifibrotic efficacy of currently available drugs in CD have not yet been initiated. Three times daily oral doses of pirfenidone commenced at time of transplantation and continued for 7 days reduced TGFβ expression and intestinal fibrosis in an intestinal transplant mouse model (88), suggesting that it may prevent fibrosis in CD. Animal models of intestinal fibrosis will provide an opportunity for preclinical testing of future drugs that target signaling pathways (23, 89), and therapies to reverse as well as prevent fibrosis will be required (35). A separate but related challenge still to be addressed will be the development of biomarkers for the accurate categorization of patients at risk of, or in the early stages of fibrosis, in order to intervene in CD at an early stage with existing or future antifibrotic drugs.

# AUTHOR CONTRIBUTIONS

ES: conception and drafting of article. ES, BB, and UK: revision and final approval of article.

# FUNDING

This work was funded by the Health Research Board, Ireland (HPF 2016-1677) (ES) and the Science Foundation Ireland (UK).

# REFERENCES

strictures in Crohn's disease. *Aliment Pharmacol Ther* (2012) 36(2):151–8. doi:10.1111/j.1365-2036.2012.05146.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Stenke, Bourke and Knaus. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# *Pneumocystis jirovecii* pneumonia in pediatric Inflammatory Bowel Disease: a Case Report and Literature Review

#### *Sally J. Lawrence1 \*, Manish Sadarangani2 and Kevan Jacobson1*

*1Department of Pediatric Gastroenterology, Hepatology and Nutrition, BC Children's Hospital, University of British Columbia, Vancouver, BC, Canada, 2Vaccine Evaluation Center, BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada*

Immunosuppressive therapy is a known risk factor for opportunistic infections. We report the first case of severe *Pneumocystis jirovecii* infection requiring intensive care in a pediatric patient with inflammatory bowel disease (IBD). The literature was reviewed and there were 92 reported cases of *Pneumocystis* pneumonia (PCP) in patients with IBD. Most sources were case reports and there was likely reporting bias toward patients receiving immunomodulators, anti-tumor necrosis factor (anti-TNF) therapy, and those who died. Overall, 56% of patients were males and 58% had Crohn's disease. The median age was 45 years (interquartile range 30–68, range 8–78) and 86% of patients were lymphopenic. The case-fatality rate was 23%. Corticosteroids were used as IBD treatment in 88% of patients who subsequently developed PCP, 42% received thiopurines, 44% used anti-TNF therapy, and 15% received either cyclosporine or tacrolimus. Rates of mono, dual, triple, and quadruple immunosuppression therapy were 35, 35, 29, and 2%, respectively. This report highlights the importance of considering PCP in immunosuppressed lymphopenic pediatric IBD patients who present with unusual symptoms. Moreover, it should give gastroenterologists the impetus to limit immunosuppressive therapy to its minimal effective dose and consider options such as exclusive enteral nutrition wherever possible. Although there is no place for global PCP prophylaxis in IBD given the low incidence, in an era when there is increasing use of biologic agents with combination immunosuppressive therapy, the risk-benefit profile of PCP chemoprophylaxis should be revisited in selected cohorts such as patients on triple immunosuppression with corticosteroids, thiopurines, and a biological agent or calcineurin inhibitor, especially in lymphopenic individuals.

Keywords: *Pneumocystis jirovecii*, pneumocystis pneumonia, inflammatory bowel disease, pediatric, opportunistic infection, immunosuppressive therapy, lymphopenia

# INTRODUCTION

Immunosuppressive therapy is a known risk factor for opportunistic infections (1). We report a case that highlights the importance of considering opportunistic infection in immunosuppressed pediatric patients with inflammatory bowel disease (IBD) who present with unusual symptoms. We present the first case of severe *Pneumocystis jirovecii* (*P. jirovecii*) infection requiring intensive care in a pediatric patient with IBD.

#### *Edited by:*

*Eytan Wine, University of Alberta, Canada*

### *Reviewed by:*

*Moftah Hussin Alhagamhmad, University of New South Wales, Australia Nikhil Pai, McMaster University, Canada Duška Tješic´ -Drinkovic´ , University of Zagreb, Croatia*

> *\*Correspondence: Sally J. Lawrence<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Gastroenterology, Hepatology and Nutrition, a section of the journal Frontiers in Pediatrics*

*Received: 28 April 2017 Accepted: 06 July 2017 Published: 24 July 2017*

### *Citation:*

*Lawrence SJ, Sadarangani M and Jacobson K (2017) Pneumocystis jirovecii Pneumonia in Pediatric Inflammatory Bowel Disease: A Case Report and Literature Review. Front. Pediatr. 5:161. doi: 10.3389/fped.2017.00161*

**31**

# CASE REPORT

A 12-year-old Caucasian girl was diagnosed with gastric and ileocolonic Crohn's disease (CD), Paris classification L3, L4a, B1, G1, having presented with growth failure and abdominal pain and having undergone endoscopic and magnetic resonance enterography assessment. She failed exclusive enteral nutrition (EEN) and was treated with oral prednisone tapered over 3 months with maintenance azathioprine (AZA) (2 mg/kg/day). Treatment resulted in resolution of clinical symptoms and improved biochemical markers. She had recurrence of symptoms after 9 months with abdominal pain and diarrhea. Infective work-up was negative and she was commenced on oral budesonide 9 mg and her AZA dose was increased (2.5 mg/kg/day). Thiopurine metabolites at this stage were normal [6-TGN 278 pmol/8 × 108 (normal range 230–400), 6-MMPN 734 pmol/8 × 108 (normal range <5,700)].

Three months later, she presented with a 3-day history of fever, dry cough, and progressive dyspnea. On examination, she was hypoxic with SaO2 90% despite 15 l/min high flow oxygen. She had severe lymphopenia (0.0 × 109 /l on manual count), elevated white cell count (10.8 × 109 /l), raised C-reactive protein (268 mg/l), and lactate dehydrogenase was 763 U/l. Chest X-ray showed bilateral interstitial infiltrates (**Figure 1**). She required intensive care for bi-level positive airway pressure respiratory support. AZA and budesonide were discontinued and she was commenced on piperacillin–tazobactam, clarithromycin, and oseltamivir to provide empiric coverage against bacteria (including atypical) and influenza, with minimal improvement in symptoms. An induced sputum sample was negative for bacterial and viral pathogens [culture and broad panel polymerase chain reaction (PCR)] but revealed *P. jirovecii* on silver stain (**Figure 2**). Intravenous (IV) trimethoprim–sulfamethoxazole (TMP–SMX) 20 mg/kg TMP/100 mg/kg SMX and methylprednisone 1 mg/kg twice daily were initiated, which resulted in improvement in respiratory status and weaning of respiratory support over 7 days.

Prior to discharge, she was investigated for an underlying primary immunodeficiency. Human immunodeficiency (HIV)

Figure 1 | Chest X-ray demonstrating bilateral pulmonary infiltrates caused by pneumocystis pneumonia.

testing was negative and immunoglobulin levels were normal. B and T cell panel revealed a low absolute count of CD3, CD4, and CD8 (0.47, 0.36, 0.14 × 109 /l respectively) with normal number of B cells (0.43 × 109 /l), normal CD4/CD8 ratio (2.43), and a good response to previous diphtheria and tetanus vaccines. She remained lymphopenic during admission, but this slowly improved. Notably, at IBD diagnosis, she had normal lymphocyte levels [lymphocyte count 1.7–2.6 (normal range 0.9–3.5 × 109 /l)]; however, after AZA initiation, she had intermittent lymphopenia (0.4–1.2 × 109 /l). Thiopurine methyltransferase activity testing was unfortunately unavailable at diagnosis.

She was discharged on oral TMP–SMX for 21 days, having had 3 days of IV therapy. She received 3 days of methylprednisone followed by 5 days of oral prednisone, which was tapered over 2.5 months. AZA was restarted at a reduced dose (1.5 mg/kg/day). Within 2 months, she had normal lymphocyte numbers with a normal absolute CD4 count (0.56 × 109 /l) and normal T-cell numbers. She developed an urticarial rash thought to be secondary to TMP–SMX 4 days post discharge and was changed to oral clindamycin and primaquine to complete the 21-day treatment course. She was then started on prophylactic oral dapsone, which was discontinued when she developed arthralgia. She has not subsequently taken PCP prophylaxis.

One year post-PCP admission, she was commenced on adalimumab monotherapy due to ongoing poor growth, abdominal pain, and an elevated fecal calprotectin (>1,800 μg/g). This resulted in resolution of symptoms, catch-up growth, and progression through puberty. She has had no further significant infections in the last 5 years and her lymphocyte count has remained stable.

# LITERATURE REVIEW AND DISCUSSION

## Overview of *P. jirovecii* Pneumonia

*Pneumocystis jirovecii* (formerly known as *Pneumocystis carinii*) is a ubiquitous opportunistic fungus, which causes

Figure 2 | Grocott–Gomori's methenamine silver stain of sputum specimen showing "cup shaped" *Pneumocystis jirovecii* cysts in small aggregates.

pneumonia [*Pneumocystis* pneumonia (PCP)]. It was first described in 1940s malnourished infants (2). In the 1980s, it was associated with HIV-infected patients with low CD4 counts (3). PCP continues to be an acquired immune deficiency syndrome-defining illness although the advent of antiretroviral therapy has resulted in incidence reduction (4). PCP has been recognized as a disease in children with primary cellmediated immunodeficiency, patients receiving chemotherapy for hematological malignancies, solid organ, and bone marrow transplant recipients and in patients requiring immunosuppressants (5). Notably, the incidence in the immunosuppressed non-HIV population is increasing with the escalating use of immunosuppressive agents (4, 6, 7). Inflammatory autoimmune conditions such as IBD account for up to 20% of PCP in HIV-negative patients (8).

*Pneumocystis jirovecii* exposure appears to occur early in life and is often asymptomatic (9, 10). Reinfection of the immunosuppressed host through environmental or person-to-person transmission rather than reactivation from latency appears to be the major mode of acquisition in immunosuppressed patients (10). Effective host defenses against *P. jirovecii* are mediated by the innate, T-cell, and to a lesser extent, humoral immune responses. In immunosuppressed patients, the infection results in a dysfunctional immune response, composed of mononuclear cells, CD8 lymphocytes, and activated macrophages, which causes diffuse lung damage (11). Moreover, the inhaled *Pneumocystis* trophozoites inhibit epithelial repair processes within the alveoli resulting in severe lung damage (12).

Adult data show that unlike in HIV-infected individuals where presentation can be slow and insidious, non-HIV infected immunocompromised individuals can have an aggressive course often culminating in respiratory failure over several days (4, 13–15). PCP is characterized by a non-productive cough, fever, and dyspnea. On chest X-ray, diffuse bilateral interstitial pulmonary infiltrates are most common (4). In equivocal cases, high resolution chest computed tomography is more sensitive and commonly shows diffuse ground glass opacity (16).

*Pneumocystis jirovecii* cannot be cultured, therefore, definitive diagnosis is made by visualization of the organism's cysts and trophozoites. This can be achieved using induced sputum, bronchioalveolar lavage specimens, or lung tissue stained with Grocott–Gomori's methenamine silver or visualized using immunofluorescence. Unfortunately, sensitivity is <67% in the non-HIV infected patient due to low numbers of *P. jirovecii* (17). PCR testing of *P. jirovecii* nucleic acid in respiratory samples is more sensitive for diagnosis; however, discrimination between colonization and actual disease has been challenging. Quantitative PCR is thought to help differentiate between these two entities better than qualitative PCR with recent research focusing on oral washes as a less invasive diagnostic tool (18, 19).

First line treatment of PCP is IV TMP–SMX (15–20 mg/kg/ 24 h of the trimethoprim component in three divided doses) due to excellent tissue penetration, rapid response, and low cost (4). Adverse reactions include leukopenia, thrombocytopenia, and rashes including Stevens–Johnson syndrome (5). However, reactions appear less common in children (20). Alternative therapy includes clindamycin plus primaquine, pentamidine, and dapsone (21). Atovaquone has been used for mild disease as it is less effective than TMP–SMX, but better tolerated (22). Corticosteroids have been used as an adjuvant treatment in severe disease, reducing respiratory failure and the length of intensive care unit stay (23).

PCP mortality rates are between 20 and 60% in immunocompromised non-HIV infected individuals in contrast to rates of 10–20% in HIV-infected patients (4, 7, 13, 15, 24, 25). The more severe course is thought to be secondary to a more disseminated pulmonary inflammatory response and diagnostic delay (4, 26).

## PCP and IBD

A retrospective cohort study determined that IBD patients were at an elevated risk of PCP compared to the general population with an increased relative risk (hazard ratio 2.96; 95% CI 1.75–4.29) but low absolute risk (0.03%) (26). The incidence of PCP in immunosuppressed IBD patients [thiopurines, methotrexate, calcineurin inhibitors, anti-tumor necrosis factor (anti-TNF) agents, or steroids] was 32/100,000 patient-years (PY) compared to 5.5/100,000 PY for non-immunosuppressed IBD patients (26). The risk appeared greater in CD compared with ulcerative colitis (UC) (26). In another population-based IBD cohort, double immunosuppression resulted in a higher risk of PCP than monotherapy (0.6/100 vs. 0.3/100 PY). There were few patients on triple therapy making risk analysis challenging (27).

There is only one published case of a child with IBD developing PCP. The 8-year-old CD patient, on infliximab monotherapy, developed PCP with concurrent disseminated histoplasmosis after 15 months therapy. His disease course was not severe. He did not require respiratory support and responded to TMP–SMX. He was maintained on budesonide with no PCP prophylaxis (28). PCP has been described in adult IBD patients on corticosteroids, calcineurin inhibitors, thiopurines, and anti-TNF agents (8, 26, 29–33).

We performed a literature review of PCP in IBD. A database search for studies on MEDLINE, EMBASE, and the Cochrane Controlled Trials Registry was performed. References of included articles were searched for further studies. **Table 1** summarizes the details of published IBD studies reporting patients with PCP. Most sources were case reports [evidence level 5 (34)], and there was likely reporting bias toward immunomodulators, anti-TNF therapy, and patients who died. There were 92 reported cases of PCP in patients with IBD in the English literature, of which 56% were males and 58% had CD. The median age was 45 years [interquartile range (IQR) 30–68, range 8–78]. There was little documented information about CD4 counts at PCP diagnosis; however, 86% (12/14) of patients were lymphopenic. The case-fatality rate (CFR) was 23% (7/31) based on the reported outcome data. Where medication was documented, rates of mono, dual, triple, and quadruple immunosuppression therapy were 35% (18/52), 35% (18/52), 29% (15/52), and 2% (1/52), respectively. The numbers were too small to comment on the effect of incremental risk with increasing numbers of immunosuppressants. Corticosteroids, as mono, dual or triple therapy, were used as IBD treatment, in 88% (46/52) of patients who developed PCP. 42% (22/52)

Table 1 | Summary of published literature of inflammatory bowel disease patients who developed pneumocystis pneumonia (*n* = 92).

*CS, corticosteroids; T, thiopurines (azathioprine/6-Mercaptopurine); Tac, tacrolimus; CSA, cyclosporine; anti-TNF, anti-tumor necrosis factor therapy (adalimumab or infliximab); MTX, methotrexate; MMF, mycophenolate mofetil; IMs, immunomodulators (thiopurine, tacrolimus); ND, not documented; CD, Crohn's disease; UC, ulcerative colitis; IQR, interquartile range.*

*a Data on gender and age only available for the one patient who died.*

received thiopurines, 44% (23/52) took anti-TNF therapy, and 15% (8/52) used either cyclosporine or tacrolimus.

# PCP and Corticosteroids

Corticosteroids are known to reduce CD4 lymphocytes, which predisposes to PCP development (51). Corticosteroids have emerged as a major contributor to PCP in the non-HIV immunosuppressed population and the risk is particularly increased at or above 16 mg of prednisolone (50–52). The median therapy duration prior to PCP was 8–12 weeks (50, 51). Interestingly, in some studies, the disease only became apparent when corticosteroids were tapered (24, 50, 51).

# PCP and Thiopurines

Thiopurines (AZA and 6-mercaptopurine) can inhibit cellmediated immunity, which influences PCP development. In the literature, two IBD patients on thiopurine monotherapy developed PCP, 10 were on dual therapy, as was the case with our patient, and 10 were on triple therapy. The CFR in this group was 19% (3/16), where outcome data were available.

# PCP and Calcineurin Inhibitors

Cyclosporine works by inhibiting production of IL-2 by helper T-cells and by affecting T-cell, B-cell, neutrophil, and mast cell function. In the literature, 50% (4/8) of IBD patients with PCP died (32, 37, 38). Such cases have prompted a discussion regarding the role of prophylactic antibiotics in patients on cyclosporine; however, the limited cases and reporting bias needs to be considered (37, 53, 54).

# PCP and Methotrexate

Three patients in the IBD literature developed PCP while on methotrexate in combination with corticosteroid and anti-TNF therapy. Methotrexate has been implicated in the development of PCP in rheumatoid arthritis with 28 documented cases, 25% of whom died (55, 56).

# PCP and Anti-TNF Agents

Cytokines inhibited by anti-TNF agents are involved in the host response to PCP resulting in reduced PCP clearance. Moreover, anti-TNF therapy can lower CD4 counts making patients more susceptible to PCP (57). Review of the Food and Drug Administration Adverse Event Reporting System data between 1998 and 2003 identified 84 patients with PCP associated with infliximab, with a mean age of 55 years; 19% of patients had IBD. Concomitant immunosuppressive agents included immunomodulators (66%), corticosteroids (50%), and cyclosporine (5%). The CFR was 27% (33). PCP has been reported to occur 9–14 weeks after the first infliximab induction dose (33, 57, 58). Notably, Colombel et al. did not report any cases of PCP associated with 3,160 patients (in six global clinical trials) on adalimumab (59). However, there are two case reports of PCP in IBD patients on adalimumab (8, 40). In total, 162 cases of PCP are reported in the IBD and rheumatology literature associated with anti-TNF therapy, which includes pediatric patients. Of the 138 patients with outcome information available, 20% died. Unfortunately, studies do not always specify whether single, double, or triple immunosuppression was used (8, 26–28, 33, 40–49, 56, 58, 60, 61). Data on the incidence of PCP in patients on anti-TNF agents are largely based on rheumatoid arthritis studies and most are population based. The majority of studies report incidence rates <50 cases per 100,000 PY (62). Results are hampered by heterogeneity in the method of PCP diagnosis, moreover, discrimination between *P. jirovecii* colonization and actual disease can be challenging.

There are no published data on PCP associated with vedolizumab, ustekinumab, or other novel IBD treatments (63, 64). More long-term data are required to assess safety profiles of these agents.

# Other Risk Factors for PCP in Non-HIV Infected Immunosuppressed Patients

The use of multiple immunosuppressive agents incrementally increases the risk of opportunistic infection in IBD; moreover, malnutrition and surgery can play a role, although this has not been specifically addressed in PCP (1, 27, 40, 51, 52, 62). As documented in our case, lymphopenia (especially CD4 count <300 cells/mm3 ) has been associated with increased risk of PCP in 60–95% of cases (5, 24, 50, 65). Notably, not all immunosuppressed patients will present with lymphopenia (24, 65).

Long et al. reviewed a case series of 38 IBD patients who developed PCP and showed that rates of hospitalization at some stage within 60 days of PCP were high (50%) (26). Moreover, patients had higher rates of comorbidities, principally, lung disease and diabetes mellitus, compared to the background population. Advanced age >65 years was found to be an additional risk (26).

# PCP in Other Pediatric Non-HIV Immunosuppressive Conditions

A population-based cohort study in juvenile idiopathic arthritis patients reported an incidence of 7/100,000 PY (66). Multiple immunosuppressive agents and lymphopenia are important risk factors in rheumatic diseases (67). The risk of PCP in pediatric cancer depends on the malignancy type and chemotherapy category. Lymphoid malignancies have the highest risk with rates of 22–45% (68). The overall risk of PCP post solid organ transplant has been estimated to be 5–15% in the absence of PCP prophylaxis (5). Risk factors include malnutrition, previous cytomegalovirus infection, and underlying lung disease. Medications such as steroids, antilymphocyte agents, calcineurin inhibitors, and biological agents such as alemtuzumab (anti-CD52 monoclonal antibody) have also been implicated (5).

# Prophylaxis against PCP

In a 2012 survey of PCP prophylaxis practice by gastroenterologists, 11% prescribed prophylaxis to their patients with IBD on combination therapy. Gastroenterologists were more likely to prescribe if they had previous practical experience of PCP, or practiced in an academic center (69).

The effects of PCP can be severe, however, prevention entails using drugs with adverse effects that may counterbalance the benefits, as occurred in our patient. The guidelines for PCP prophylaxis in HIV-infected patients have been universally adopted, but there is a lack of consensus on prophylaxis with TMP–SMX in IBD patients. A recent Cochrane meta-analysis of prophylactic treatment with TMP–SMX in non-HIV infected patients included 1,412 patients, of which 520 were children. The authors were unable to find published literature addressing prophylaxis in IBD. In patients with hematological cancers and transplant recipients, they reported an 85% reduction in PCP incidence with prophylaxis and PCP-related mortality reduced by 83% with few adverse events reported (20). No children in the included studies had a severe adverse event compared to 3.1% of adults suggesting a lower probability of harm in children.

There is insufficient evidence to recommend PCP prophylaxis for all IBD patients on immunosuppressive monotherapy; however, with increasing number of immunosuppressive agents, the risk of opportunistic infection is known to increase (1, 27, 62). Large studies addressing the specific incremental increased risk associated with multiple immunosuppressive agents especially triple therapy have not been done. This needs to be considered, as the use of multiple immunosuppressive agents are increasing. A recent study showed that approximately half of patients started on anti-TNF therapy were already on two immunosuppressive agents (40). Dietary therapies (EEN, partial enteral nutrition, and exclusion diets) in CD are increasingly being used and investigated and are attractive options to limit immunosuppression and improve nutrition in this population (70–72).

A study using simulation modeling to address cost and comparative effectiveness of PCP prophylaxis in CD concluded that at the present incidence, routine chemoprophylaxis was not cost effective but, based on limited data, it may be effective in triple immunosuppressive therapy (73). The 2014 European Crohn's and Colitis opportunistic infection guidelines recommend PCP prophylaxis in IBD patients on triple immunosuppression including either a calcineurin inhibitor or anti-TNF therapy. This recommendation is based on expert gastroenterology and infectious disease opinion (74). Other practical approaches proposed for patients on high-dose steroids and multiple immunosuppressants include measurement of CD4 counts in those patients with a total lymphocyte count of <600 cells/mm3 as a means of identifying patients at risk of infections, although not all patients who develop PCP have lymphopenia, therefore, precluding its use as an isolated risk identification guide (24, 43, 65, 75). Evaluating the utility of measuring CD4 counts in IBD patients on multiple immunosuppressants should be considered.

# CONCLUSION

We described a case of severe PCP in an immunosuppressed lymphopenic pediatric IBD patient. This case reiterates the importance of limiting immunosuppression to its minimal effective dose. Although the incidence of PCP in the IBD population is low, it is an aggressive condition that has a higher relative risk in IBD patients compared to the general population. Moreover, it is associated with significant morbidity and mortality. PCP should be considered in the differential diagnosis of immunosuppressed pediatric IBD patients who develop respiratory symptoms, with a low threshold for treatment. Although there is no place for global PCP prophylaxis in IBD, in an era when there is increasing use of biologic agents

# REFERENCES

with combination immunosuppressive therapy, the risk-benefit profile of PCP prophylaxis should be revisited in selected cohorts such as patients on triple immunosuppression with corticosteroids, thiopurines, and a biological agent or calcineurin inhibitor especially in lymphopenic individuals. Further studies are required to guide definitive PCP prophylaxis in high risk subgroups of IBD patients.

# INFORMED CONSENT

Written informed consent for publication of the case report and figures was obtained from the patient and parents.

# AUTHOR CONTRIBUTIONS

SL performed the literature review and wrote the manuscript. MS and KJ critically reviewed the manuscript.

# ACKNOWLEDGMENTS

The authors thank Dr. Oana Popescu for pathology slide images and Dr. Orlee Guttman for providing case information.

pneumonia in patients with AIDS. *N Engl J Med* (1993) 328:1521–7. doi:10.1056/NEJM199305273282103

of remission in children and young adults with Crohn's disease. *Inflamm Bowel Dis* (2014) 20(8):1353–60. doi:10.1097/MIB.0000000000000110

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Lawrence, Sadarangani and Jacobson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The Adjunctive Role of Nutritional Therapy in the Management of Phlegmon in Two Children with Crohn's Disease

*Andrew S. Day\* and Stephanie C. Brown*

*Department of Paediatrics, University of Otago, Christchurch, New Zealand*

Crohn's disease may be complicated by the development of penetrating (fistulizing) or structuring complications. The presentation of an intra-abdominal phlegmon or abscess with or without an associated fistula has traditionally required surgical intervention. This series of two cases illustrates a beneficial role of non-surgical management, with parenteral and then enteral nutrition playing central roles. This report further elaborates the potential adjunctive role of enteral nutrition in the management of this complication of CD.

### *Edited by:*

*Eytan Wine, University of Alberta, Canada*

#### *Reviewed by:*

*Amit Assa, Schneider Children's Hospital, United States Wael El Matary, University of Manitoba, Canada Jason Silverman, University of Alberta, Canada*

#### *\*Correspondence:*

*Andrew S. Day<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Pediatric Gastroenterology, Hepatology and Nutrition, a section of the journal Frontiers in Pediatrics*

*Received: 07 June 2017 Accepted: 30 August 2017 Published: 20 September 2017*

#### *Citation:*

*Day AS and Brown SC (2017) The Adjunctive Role of Nutritional Therapy in the Management of Phlegmon in Two Children with Crohn's Disease. Front. Pediatr. 5:199. doi: 10.3389/fped.2017.00199*

Keywords: children, Crohn disease, exclusive enteral nutrition, nutritional treatment, phelgmon

# INTRODUCTION

Crohn's disease (CD), one type of inflammatory bowel disease (IBD), may present at any age, with up to a quarter of cases diagnosed in childhood or adolescence (1). CD may be complicated by the development of fistulae or penetrating disease, which may lead to an extra-intestinal collection, or phlegmon. Generally, fistulizing disease is uncommon at diagnosis in children, with most children having uncomplicated inflammatory disease at that time (2). Typically, the management of a phlegmon might include drainage and/or early resection.

Exclusive enteral nutrition (EEN) is a well-established therapy to induce remission in pediatriconset CD (3). This intervention comprises a period of a liquid diet, using a polymeric or elemental formula, with the exclusion of normal foods during this time. EEN induces remission in more than 85% of individuals and is associated with numerous other benefits, including high rates of mucosal healing, nutritional improvements, and enhanced bone health (3).