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Children with severe acute malnutrition: new diagnostic and treatment strategies

Author: Rosalie BartelsCover artwork: Anette Tjaerby (http://anettetjaerby.net/)Cover layout: Alexandre Van DammeLayout and printed by: Optima Grafische Communicatie, Rotterdam,

the Netherlands (www.ogc.nl)ISBN: 978-94-6361-058-2

Financial support for printing this thesis was kindly provided by the Academic Medical Center, BGP products B.V., Doctors for Malawi, Pfizer B.V. and Yakult Nederland B.V.

All rights reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without the written permission of the author.

Copyright © 2018, Rosalie Bartels, Amsterdam, The Netherlands

Children with severe acute malnutrition: new diagnostic and treatment strategies

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus

prof. dr. ir. K.I.J. Maex ten overstaan van een door het College voor Promoties ingestelde commissie,

in het openbaar te verdedigen in de Agnietenkapel op donderdag 29 maart 2018, te 10.00 uur

door

Rosalie Henriëtte Bartels geboren te Amsterdam

Promotiecommissie:

Promotor(es):Prof. dr. M. Boele van Hensbroek AMC-Universiteit van Amsterdam

Copromotor(es):Dr. W.P. Voskuijl AMC- Universiteit van AmsterdamDr. R.H.J. Bandsma University of Toronto

Overige leden:Prof. dr. H.S.A. Heymans AMC- Universiteit van AmsterdamProf. dr. J.B. van Goudoever AMC- Universiteit van AmsterdamProf. dr. M.J. Manary Washington University Prof. dr. T. Ahmed University of QueenslandDr. B.G.P. Koot AMC-Universiteit van AmsterdamDr. P.F. van Rheenen Rijksuniversiteit Groningen

Faculteit der Geneeskunde

Despite

the hunger

we cannot

possess

more

than

this:

Peace

in a garden

of

our own.

from ‘Absolute trust in the goodness of the earth’ Alice Walker

COnTEnTS

Chapter 1 General Introduction and Outline of the Thesis 9

Chapter 2 The relation between malnutrition and the exocrine pancreas: a systematic review

23

Chapter 3 Both exocrine pancreatic insufficiency and signs of pancreatic inflammation are highly prevalent in children with complicated severe acute malnutrition: an observational study

85

Chapter 4 Pancreatic enzyme replacement therapy in children with severe acute malnutrition: A randomized controlled trial

101

Chapter 5 Hypoallergenic and anti-inflammatory feeds in children with complicated severe acute malnutrition: an open randomized controlled 3-arm intervention trial in Malawi

123

Chapter 6 The clinical use of longitudinal bio-electrical impedance analysis in children with severe acute malnutrition

155

Chapter 7 Summary, General Discussion and Conclusions 179

Appendices Nederlandse Samenvatting 195Abbreviations 211Contributing Authors 213Acknowledgements 216About the Author 219

Chapter 1General Introduction and Outline of the Thesis

Rosalie H. Bartels

11

1

General Introduction and Outline of the Thesis

CHIlDHOOD MORTAlITy In THe WORlD

Every day, 15000 children under the age of 5 years (under-5) died in 2016.(1) Eighty per-cent of these deaths occur among children living in sub-Saharan Africa or Southern Asia (Figure 1), (1) Furthermore, more than half of these deaths could be prevented when access to simple, affordable interventions were available.(2) In 2015, the 17 Sustainable Development Goals (SDGs), otherwise known as the Global Goals, were formulated with the aim to: ”end poverty, protect the planet and ensure that all people enjoy peace and prosperity”.(3) The third goal (“good health and well-being”) aims to: “end preventable deaths of newborns and children under-5, with all countries aiming to reduce neonatal mortality to at least as low as 12 per 1,000 live births and under-5 mortality to at least as low as 25 per 1,000 live births” by 2030.

Childhood Undernutrition in the WorldNearly half (45%) of worldwide deaths in children under-5 is attributable directly or in-directly to poor nutrition.(3,4) It was estimated in 2016 that on a Global scale 52 million children under-5 were wasted (a child who’s weight is too low for his or her height) of which , 17 million were severely wasted (Figure 2).(5) As a consequence, two of the targets of the second SDG goal (‘zero hunger’) are to: “end hunger and ensure access by all people, in particular the poor and people in vulnerable situations, including infants, to safe, nutritious and sufficient food all year round” and to: “end all forms of malnutrition, including achieving, by 2025, the internationally agreed targets on stunting and wasting in children under 5 years of age, and address the nutritional needs of adolescent girls, pregnant and lactating women and older persons” by 2030.(3) It is important that these

Figure 1. Under-five mortality rate (deaths per 1,000 live births) by country, 2016. Source: UNICEF(1)

12

Chapter 1

goals have been formulated, but it is also important to realize that up to date healthy and sufficient nutrition has been a neglected area of global health and development, accounting for less than only 1 percent of global foreign aid. This is largely due to the underlying and often hidden role malnutrition plays in childhood illnesses and deaths.(6) As a consequence, in order to reduce under-5 mortality, it is of great importance to better understand malnutrition and its causes in order to develop better preventive, diagnostic and treatment strategies.

DeFInInG SeVeRe ACUTe MAlnUTRITIOn

Different concepts and gradings of undernutrition are in use, but the World Health Or-ganization (WHO) has defined severe acute malnutrition (SAM), which is used by most researchers and clinicians, as any of the following (Figure 3):(7) - Non-edematous SAM/marasmus: a weight for height (W/H) below -3 standard devia-

tion (SD), OR a mid-upper arm circumference (MUAC) of less than 115 mm- Edematous SAM/kwashiorkor: the presence of bilateral nutritional edema - Marasmic kwashiorkor: a combination of the two above

TReATMenT AnD PROGnOSIS OF CHIlDRen WITH SeVeRe ACUTe MAlnUTRITIOn

Children with SAM are normally treated as outpatients, and receive WHO recommended rehabilitation feeds outside a hospital setting.(8) However, when they have clinical complications such as signs of severe or systemic illness and/or poor appetite, they are considered children with complicated SAM and require inpatient treatment.(8) Despite adherence to WHO and National treatment protocols the case fatality rate in children with SAM, and especially those with complicated SAM, is still unacceptably high (up to

Figure 2. Number of children under-5 who are wasted by region. Source: UNICEF-WHO-The World Bank(5)

13

1


35%).(3,4,9–12) In addition, mortality remains high after hospital discharge, which may also indicate deficits in the effectiveness of current long term management.(13,14) The above figures indicate the urgency to better understand the malnutrition ‘syndrome’ in order to improve the current SAM management and being able to identify the SAM children who are at risk of clinical deterioration and death at an early stage.

SeVeRe ACUTe MAlnUTRITIOn AnD THe exOCRIne PAnCReAS

The pathophysiology of children with SAM is complex, multifactorial and it results in many different physiological abnormalities (Figure 4).(11) One of the problems children with SAM often suffer from is severe diarrhea, which greatly increases mortality.(11,15–17) This diarrhea may be caused by: infections, intestinal epithelial dysfunction relating to malabsorption, impaired digestion or a combination of the above.(18,19) The exocrine pancreas plays a significant role in nutrient digestion by secreting enzymes (e.g. amylase, lipase, trypsinogen, etc.) that digest all macronutrients: fat, protein and carbohydrates.(20) Exocrine pancreatic insufficiency (EPI) is defined as a lack of digestive enzyme production, which can lead to impaired weight gain and growth due to protein and lipid malabsorption.(21) Its main clinical symptom is steatorrhea (the presence of excess fat in feces), caused by the inability to digest fat.(20,22) EPI is a known common complication of conditions such as Cystic Fibrosis (CF), Shwachman-Diamond syndrome, and HIV.(23–25) In children with CF, pancreatic function is an important predictor of

Figure 3. Phenotypes of SAM. Left: non-edematous SAM/marasmus. Right: edematous SAM/kwashiorkor. Source: WHO(58)

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Chapter 1

long-term survival.(26) In high income countries it is standard clinical practice to start pancreatic enzyme replacement therapy (PERT) in patients suffering from EPI with the aim of restoring nutritional status by improved digestion.(21,27) It is not well known if EPI may also be of benefit for children suffering from SAM in low-income countries.

SeVeRe ACUTe MAlnUTRITIOn AnD GUT InFlAMMATIOn

Children with SAM have intestinal pathology that is thought to result from a combina-tion of increased exposure to microbial pathogens and poor nutrition. (11,18,28–30) A significant feature of this so called ‘enteropathy’ is gut inflammation that persists despite management. (31,32) The inflammation has similarities to that which occurs in non-IgE mediated food allergy (hereafter “food allergy”; e.g. due to cow’s milk protein) and Crohn’s disease, which raises the intriguing possibility that treatments which reduce gut

Figure 4. Organ system involvement in severe malnutrition. Severe malnutrition can affect several organ systems. The functional impairments in these systems have been characterized, but the underlying mechanisms have not been fully elucidated. Source: Bhutta et al.(11)

15

1


inflammation in food allergy and Crohn’s disease may also benefit children with SAM.(33–35) In food allergy, the intestinal inflammation responds well when the causal anti-gen, if known, is excluded from the diet (e.g. cow’s milk protein) or, when the concerning antigen is not known, a hypoallergenic, elemental feed composed of single amino acids is proven to be effective both clinically and in reducing the intestinal inflammation.(35) In pediatric Crohn’s disease, first-line therapy consists of exclusive enteral nutrition, where either an elemental formula or polymeric formula is given for 6-8 weeks, while all other foods are excluded.(35–38) In limited previous research, hypoallergenic and elemental feeds were well tolerated in children with malnutrition, but evidence of benefit was limited.(39,40) If the gut inflammation in children with SAM would respond to existing treatments already being used in high income countries, this could mean a big step forward in the management of this problem that currently has not been resolved and contributes greatly to the high mortality rates of children with SAM.

SeVeRe ACUTe MAlnUTRITIOn AnD BIO-eleCTRICAl IMPeDAnCe AnAlySIS

Children with SAM are diagnosed, as described above, by measuring W/H and MUAC, and by physical examination to identify bilateral nutritional edema. These ‘anthropometric’ measurements do not provide any information on body composition (the proportion of fat mass and fat-free mass in the body). Altered body composition (in malnutrition: loss of fat-free mass) is linked to poor clinical outcome, and can be estimated by bio-electrical impedance analysis (BIA).(41) Over the past two decades, bioelectrical impedance analy-sis (BIA) has proven to be a non-invasive and inexpensive method for estimating body composition, and is widely used in various clinical situations both in adults as well as children.(41–45) Body composition is not quantified directly by BIA but is calculated from body reactance and resistance measured by changes that occur in a small alternat-ing electrical current, as it passes through the body.(46,47) Reactance arises from cell membranes, and resistance from extra- and intracellular fluid, and their combination is called ‘impedance’.(43) It provides a reliable estimate of total body water and fat free mass in healthy individuals, but requires population and disease-specific equations.(48) Although prediction equations have been recently developed for children, they have not been validated for the African pediatric population, let alone for malnourished children.(49–51) With differing phenotypes and hydration status in SAM (i.e. non-edematous SAM versus edematous SAM), knowing how BIA changes with nutritional rehabilitation in children with or without edematous severe acute malnutrition (SAM) during nutritional reha-bilitation might help the clinician. In addition to this it would help to know if BIA adds a prognostic value to clinical outcome when combined with ‘classic’ anthropometry.

16

Chapter 1

SeVeRe ACUTe MAlnUTRITIOn In MAlAWI

Malawi is a landlocked, small country in southeast Africa with an esti mated populati on of 18 million people (Figure 5).(52) It is amongst the world’s least developed countries, with a gross domesti c product per capita of $301. The economy is mostly based on agriculture, and foreign aid. There is a high prevalence of HIV (1 million people), 24000 adults and children die of AIDS annually and life expectancy is low (males: 57 years, females: 60 years).(53,54) Under-5 mortality rate in Malawi has dropped over the past 20 years, but remains among the highest in Africa with 55.1 per 1000 live births.(55) In Malawi malnutriti on is also a major contributor to under-5 mortality. Around 46 percent of children under fi ve are stunted; 21 percent are underweight; and four percent are wasted.(56) The Malawian gov-ernment has put tackling the malnutriti on problem high on their agenda. As a consequence the ‘Malawi guidelines’ on treatment of malnutriti on have been recently revised.(57) In these guidelines, community based management is encouraged, but complicated cases and children with complicated SAM should be treated in an inpati ent setti ng on, so called, Nutriti onal Rehabilitati on Units (NRU), as is similar to the management of children with complicated SAM in other low income countries. The largest NRU of Malawi is ‘Moyo’ NRU in the pediatric department of Queen Elizabeth Central Hospital in Blantyre.Moyo NRU, with a yearly admission rate of around 750 SAM children, is where the observati onal and interventi on studies in this thesis (Chapters 3-6) have been conducted between 2013-2017.

Figure 5. Malawi. Map of Malawi. Source: onestopmap.com; Openclipart.org

17

1


OUTlIne OF THe THeSIS

This thesis outlines the improvement of diagnosis and management of children with complicated SAM through improved insight into the malnutrition ‘syndrome’ and through exploring new strategies. Chapters 2-4: assessing the prevalence and treatment of EPI in children with SAM.In Chapter 2, a systematic review, we systematically synthesize current evidence con-cerning the relation between EPI and malnutrition in children. In Chapter 3 we describe the results of an observational study to assess pancreatic func-tion in children with SAM. We aim to assess whether pancreatic function: 1) is impaired in children with severe acute malnutrition (SAM), 2) is different between edematous versus non-edematous malnutrition, and 3) improves by nutritional rehabilitation. In Chapter 4 we perform a randomized controlled trial to assess the benefits of pancre-atic enzyme replacement therapy in children with complicated SAM. We look at weight gain, pancreatic function and clinical outcome after 28 days of pancreatic replacement therapy. Chapter 5 and 6: Gut inflammation and BIA:In Chapter 5 we evaluate whether therapeutic feeds that are effective in treating intesti-nal inflammation in food allergy and Crohn’s disease may also benefit children with SAM. With an open randomized controlled 3-arm intervention trial we evaluate the efficacy, tolerability and safety of a hypoallergenic and an anti-inflammatory therapeutic formula in children with complicated SAM. In Chapter 6 our focus is on the diagnostic and prognostic value of BIA in children with SAM. We aim to assess if bio-electrical impedance parameters: 1) change with nutritional rehabilitation in children with or without edematous SAM; 2) add a prognostic value to clinical outcome when combined to classic anthropometry.

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Chapter 1

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27. Taylor JR, Gardner TB, Waljee AK, Dimagno MJ, Schoenfeld PS. Systematic review: efficacy and safety of pancreatic enzyme supplements for exocrine pancreatic insufficiency. Aliment Pharmacol Ther. 2010;31(1):57–72.

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29. Prendergast A, Kelly P. Enteropathies in the developing world: neglected effects on global health. Am J Trop Med Hyg [Internet]. 2012;86(5):756–63.

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32. Jones KDJ, Hünten-Kirsch B, Laving AMR, Munyi CW, Ngari M, Mikusa J, et al. Mesalazine in the initial management of severely acutely malnourished children with environmental enteric dysfunction: a pilot randomized controlled trial. BMC Med. 2014;12:133.

33. Crittenden RG, Bennett LE. Cow’s milk allergy: a complex disorder. J Am Coll Nutr. 2005;24(6 Suppl):582S–91S.

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37. Dziechciarz P, Horvath A, Shamir R, Szajewska H. Meta-analysis: Enteral nutrition in active Crohn’s disease in children. Aliment Pharmacol Ther. 2007;26(6):795–806.

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41. Girma T, Hother Nielsen A-L, Kæstel P, Abdissa A, Michaelsen KF, Friis H, et al. Biochemical and anthro-pometric correlates of bio-electrical impedance parameters in severely malnourished children: A cross-sectional study. Clin Nutr [Internet]. 2017; Available from: http://linkinghub.elsevier.com/retrieve/pii/S0261561417300675. Accessed December 3, 2017.

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49. Nagano M, Suita S, Yamanouchi T. The validity of bioelectrical impedance phase angle for nutritional assessment in children. J Pediatr Surg. 2000;35(7):1035–9.

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57. Ministry of Health (MOH). Guidelines for Community-Based Management of Acute Malnutrition. 2nd Edition. Lilongwe, Malawi; 2016.

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Chapter 2The relation between malnutrition and the exocrine pancreas: a systematic review

Rosalie H. Bartels*, Deborah A. van den Brink*, Robert H. Bandsma, Michael Boele van Hensbroek, Merit M. Tabbers, Wieger P. Voskuijl

*These authors contributed equally

Journal of Pediatric Gastroenterology and Nutrition 2018: 66: 193–203

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Chapter 2

ABSTRACT

Objective: The relation between malnutrition and exocrine pancreatic insufficiency (EPI) has been described previously, but it is unclear if malnutrition leads to EPI or vice versa. We systematically synthesized current evidence evaluating the association between malnutrition and EPI in children.

Methods: Pubmed, Embase, and Cochrane databases were searched from inception until February 2017. We included cohort or case- controlled studies in children report-ing on prevalence or incidence of EPI and malnutrition. Data generation was performed independently by 2 authors. Quality was assessed by using quality assessment tools from the National Heart, Lung, and Blood Institute.

Results: Nineteen studies were divided into 2 groups: 10 studies showing EPI leading to malnutrition, and 9 studies showing malnutrition leading to EPI. Due to heterogeneity in design, definitions and outcome measures, pooling of results was impossible. Quality was good in 4/19 studies. Pancreatic insufficiency was linked to decreased nutritional status in 8/10 articles although this link was not specified properly in most articles. In malnourished children, improvement was seen in pancreatic function in 7/9 articles after nutritional rehabilitation. The link between the two was not further specified. Heteroge-neity exists with respect to definitions, outcome measures and study design.

Conclusions: There is sufficient evidence for an association between EPI and malnutri-tion. We could not confirm whether there is a correlation or causality between EPI or malnutrition. It was therefore not possible to draw firm conclusions from this systematic review on underlying pathophysiological mechanisms between EPI and malnutrition. More observational clinical trials are crucially needed.

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2

The relation between malnutrition and the exocrine pancreas

InTRODUCTIOn

Undernutrition is a global problem, it contributes to approximately 45% of all deaths in children less than 5 years of age while severe malnutrition is a co-morbidity in 7.8% of all under 5 deaths in children.(1–3)Malnutrition has been defined in many different ways over the past few decades and encompasses both undernutrition and overweight.(1) In this review we will discuss undernutrition only. The current definition of undernutrition is a weight for height (W/H) ≤-2 standard deviations (SD) and severe malnutrition is defined as a W/H <-3 SD and/or a mid-upper arm circumference (MUAC) of <11.5cm according to the World Health Orga-nization (WHO).(4) Severe acute malnutrition (SAM) includes two different phenotypical forms: non-edematous SAM (severe wasting or ‘marasmus’) or edematous SAM, which is nutritionally induced bilateral edema (kwashiorkor). Since mortality in SAM remains very high better understanding of the pathophysiology of SAM is needed in order to improve management. Severe diarrhea is common in children with SAM, contributes to mortality (5,6) and is not only caused by infections and intestinal epithelial dysfunction relating to malabsorption, but also by impaired pancreatic digestion.(7,8) The exocrine pancreas plays a key role in nutrient digestion by secreting digestive enzymes (i.e. amylase, lipase, and trypsin) digesting all macronutrients: fat, protein and carbohydrates.(9) Exocrine pancreatic insufficiency (EPI) is the inability to digest nutrients due to severe reduction of digestive enzymes. Its main clinical symptom is steatorrhea caused by the inability to digest fat.(9,10) Pancreatic function can be assessed by direct and indirect methods.(9) Direct methods are more invasive and include stimulation of the pancreas by secretin, followed by pancreatic duct intubation, collection and measurement of the secreted enzymes. In-direct tests measure pancreatic enzymes in serum (e.g. Serum immunoreactive trypsino-gen (IRT), lipase, and amylase), in stool (Fecal elastase-1 (FE-1) and fecal chymotrypsin (CMT)) or by using breath analysis.(9) It is current clinical practice to measure pancreatic function by FE-1 in stool.(9) EPI exists in conditions such as cystic fibrosis (CF), Shwachman-Diamond syndrome (SDS), and chronic pancreatitis (CP)(10–13) and can lead to nutrient malabsorption, undernu-trition, poor growth and mortality.(14) However, in contrast, several older studies mostly performed between 1940 and 1980, have reported that malnutrition in its turn can lead to EPI.(15–26) We have recently confirmed these findings and showed a very high preva-lence of EPI in Malawian children with SAM (93%).(27) However, it is still not clear how exactly, the relationship between malnutrition and the exocrine pancreas is. More insight into the pathophysiology underlying SAM might aid in lowering the huge malnutrition related mortality. Therefore, unraveling the association between the exocrine pancreas

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and malnutrition is of great importance. We systematically assessed the evidence con-cerning the relation between EPI and malnutrition in children. We developed the following PICOS: participants: children with malnutrition or EPI; inter-ventions: treatment for malnutrition and or EPI; comparisons: none or healthy controls; outcomes: pancreatic function and nutritional status; study design: systematic review.

METHODS

Search strategyThe databases Embase, PubMed, and Cochrane Database of Systematic Reviews were searched from inception to February 2017 (full search strategy and keywords shown in Appendix 1). To identify additional studies, reference lists of relevant studies identified in the literature search were searched by hand. During this process, the exact reporting guidelines as described in the PRISMA statement (www.prisma-statement.org) were followed.

Study selectionTwo investigators (RB and DB) independently reviewed titles and abstracts of all cita-tions in the literature results. Possible relevant studies were retrieved for full-text review. Cohort, randomized controlled trials, or case-controlled studies in children (aged 0-18 years) were included if studies were reporting on prevalence or incidence of EPI or mal-nutrition. A clear definition and assessment of EPI and malnutrition had to be provided by the authors. Study aim was to determine any relation between EPI and malnutrition. No language restriction was used. Case reports and animal studies were excluded. Dis-agreements between reviewers were adjudicated by discussion and consensus with two other authors (MT and WV).

Data extraction and analysisFor each included trial in the final analysis, data were extracted by using structured data extraction forms, which contained items such as author, participants, definitions of EPI and malnutrition, method of EPI assessment, method of malnutrition assessment, outcomes, and author’s conclusions. Methodological quality of the included articles was assessed using the National Heart, Lung, and Blood Institute (NHLBI) quality assessment tool (i.e. risk of bias).(28) Three NHLBI tools were used: one for observational cohort and cross-sectional studies, a sec-ond tool for case control studies, and thirdly a separate tool for controlled intervention studies. Using these tools, two authors (RB, DB) independently evaluated the selected articles using “yes”, “no”, “cannot determine”, “not reported”, or “not applicable”. These

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were discussed and used to frame an overall rating for the quality of each study as “good”, “fair”, or “poor”. Ratings were based on number of quality assessment questions that were confirmed with a ‘yes’: poor ≤6, fair >6 and <10, and good ≥10 questions answered with ‘yes’. For the case-control studies that had 12 questions instead of 14 we adjusted the rating: poor ≤5, fair >5 and <9, and good ≥9. A third and fourth author were consulted on any discrepancies between the two independent evaluations (MT, WV)

ReSUlTS

Study search and quality assessmentAfter deducting duplicates, the search strategy and manual search generated 1273 stud-ies that were screened for eligibility (Appendix 1). 1219 were excluded as they were not relevant to our search question (Figure 1. Flowchart). After evaluating the full text, another 35 studies were excluded for not meeting our inclusion criteria (no clear defini-tion of malnutrition (n=12), no clear definition of EPI (n=7), or both (n=3), different study design(n=4), full text non-retrievable (n=1), adult population (n=6), or not reporting on relation EPI and malnutrition (n=2)).The remaining 19 studies were divided into 2 groups: (1) studies reporting patients diagnosed with EPI who are later found to be malnourished (n=10);(29–38) (2) studies reporting patients diagnosed with malnutrition who are later found to have EPI (n=9).(16,18,21,23,24,27,39–41) Due to heterogeneity in design, definitions and outcome measures, pooling of results was impossible. Therefore, studies are discussed separately.Quality assessments are shown in supplemental tables 1 – 3. Four studies had an overall quality considered to be good (23,29,30,36), 12 were rated to be fair (16,18,24,27,31–33,35,38–41), and the remaining 3 studies were rated to be poor (21,34,37). Only four studies did account for key potential confounding variables(24,31,35,41) and just 1 study had a sample size justification.(27) Blinding of treatment of participants and researchers was only performed in one study.(36) Cohen’s κ was calculated to determine the inter-observer variation between the reviewers that assessed the articles using the quality assessment tool (RB and DB). There was moderate agreement between the two review-ers, κ=.602 (95% CI, .522 to .682), p < .0005.(42) After discussion agreement was reached in 100% of cases.

Study and patient characteristicsIn total, 2271 children were included in 19 studies (Table 1) with sample sizes ranging from 13 to 659 children (32,41) Of the included 19 studies, 12 only included children less than 5 years old.(16,18,23,24,27,30,32,36–40) These studies were conducted in 13 different countries including resource-high/developed countries: USA, Italy, Australia,

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France, Poland, UK, and Canada; as well as resource-poor/developing countries: Egypt, Ivory Coast, Malawi, Senegal, Uganda, and South Africa. Ten studies included patients that were diagnosed with a condition known to be associated with EPI such as CF, SDS, Celiac disease, human immunodeficiency virus (HIV), and CP.(29–38) Of all studies, Kolodziejczyk et al. was the only study which included CP patients, while Carrroccio et al. solely included HIV infected children.(31,35) Three studies included participants diag-nosed with SDS.(32–34) Celiac disease was studied by two separate studies conducted by Carroccio et al.(36,37) Lastly 3 studies focused on CF patients.(29,30,38) The remaining nine studies investigated malnourished children who had either moderate or severe malnutrition.(16,18,21,23,24,27,39–41)

Records identified through Pubmed and Embase searching

(n =1629)

Scre

enin

g In

clud

ed

Elig

ibili

ty

Iden

tific

atio

n

Additional records identified through other sources (hand

searched) (n =18)

Records after duplicates removed (n =1273)

Records screened (n =1273)

Records excluded (n =1219)

Full-text articles assessed for eligibility

(n = 54)

Full-text articles excluded, reasons: • no clear definition of

malnutrition (n=12), • no clear definition of EPI

(n=7), • no clear definition of

malnutrition and no clear definition of EPI (n=3),

• no cohort, controlled trial, or case-control study design (n=4),

• full text non-retrievable (n=1) • adult population (n=6), • not reporting on relation EPI

and malnutrition (n=2)

Studies included in qualitative synthesis

(n =19)

Figure 1. Flowchart of study screening and selection process. See Appendix 1, for detailed search and selection

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Pancreatic function assessment: The criterion standard test of pancreatic function is the pancreatic stimulation test,“Dreiling tube test”, (9,43) This direct pancreatic test was used in 7 studies.(16,21,32,34,36,37,39) Cutoff values were provided by the author in 1 of those studies,(32) 3 studies used control values of non-malnourished children,(16,21,39) 2 studies used control values of non-celiac children,(36,37) and one study did not provide any cutoff values at all.(34) The current most widely used pancreatic function test is an indirect test measuring faecal levels of zymogen FE-1.(9,44) This was measured in 4 studies which reported clear cutoff values.(27,29,31,33) Fat malabsorption was reported in 8 studies.(16,21,32,34,36,37,39) Of these, Kolodziejczyk et al. was the only study using a control group, while Bines et al. did not report on cutoff values at all.(35,38) Immuno-reactive trypsinogen was tested in 5 stud-ies,(24,27,32,40,41) of which two were using cutoff values,(27,32) and 3 were using a control group.(24,40,41) Serum amylase was measured in 5 studies(23,27,31,32,35) with 3 of them also measuring lipase.(23,32,35) All reported clear cutoff values except for one, El-Hodhod et al, who used values of a control group.(23) Fecal CMT was assessed in two studies with clear cutoff values.(31,32) Additional, less commonly used, tests for pancreatic function included ultrasound evaluations,(23,33,35) autopsy,(18) and endo-scopic retrograde cholangio-pancreatography (ERCP).(35)

Malnutrition assessmentAssessment of malnutrition was more consistent across the selected articles, with stud-ies using anthropometry, clinical indicators, and albumin as markers of malnutrition. Weight-for-age Z-scores (WAZ), height-for-age Z-scores (HAZ), and/or weight-for-height Z-scores (WHZ), currently recommended by WHO for defining malnutition,(45) were used in 14 studies.(16,23,24,27,29–33,36–38,40,41) Growth percentiles were used by Hill et al. and El-Hodhod et al. and a BMI ratio (Cole’s ratio: BMI actual/BMI for the 50th centile x100%) was used to classify malnutrition by Kolodziejczyk et al.(23,34,35) Four studies used clinical indicators such as pitting edema and skin lesions for malnutrition.(16,18,21,39) Lastly albumin was used as a marker of malnutrition by 4 studies, of which three used controls but El-Hodhod did not have cutoff values.(16,23,30,37)

Group 1: Articles reporting patients diagnosed with ePI who are later found to be malnourished (supplemental Table 4)(29–38)Eight out of 10 studies describe the association between EPI and malnutrition.(29,30,32–36,38) Quality assessment was rated ‘poor’ for two studies,(34,37) ‘fair’ for five stud-ies,(31–33,35,38) and ‘good’ for three studies.(29,30,36) Cohen et al. reported that CF children with no pancreatic activity (n=75/84) (FE-1<15 ug/g) had a significantly lower WAZ and more fat malabsorption compared to CF children with residual activity (n=9/84)

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(FE-1≥15 ug/g).(29) Significantly greater fat malabsorption in pancreatic insufficient CF children (n=16/29) versus pancreatic sufficient CF patients (n=13/29) was also reported by Bronstein et al. which was significantly correlated with a decrease in WAZ.(30) Bines et al. reported that pancreatic insufficiency (found in n=35/46 CF children) was strongly associated with poor weight and length gains.(38)All three studies of children with Shwachman-Diamond syndrome, reported high num-bers of EPI, (Pichler et al. 95.2%, Cipolli et al. 100%, Hill et al. 100%).(32–34) Of these, Cipolli et al. had the highest proportion of malnourished children (n=11/13, 84%). This study followed up 6 children at a mean age of 10 years, and found a significant increase in both weight and height z-scores although unclear if on pancreatic enzyme replacement therapy (PERT) or not (from -3.8 to -1.4 and from -3.6 to -1.8 respectively, both p<0.001).(32) Hill et al. reported that 64% (n=7/11) had a weight below the 3rd percentile but did not report on anthropometry during follow up.(34) Pichler et al. reported only 33% (n=7/21) to be malnourished and on follow up of unclear duration only 38% (n=5/13) experienced catch up growth.(33) Pichler et al. described that poor nutritional status in SDS is multifactorial and can be caused by several other factors than EPI, like feeding difficulties (in 43% (n=9/21) of their population) and enteropathy (50% n=7/14). None of the three SDS studies demonstrated a direct correlation between EPI and malnutrition.Carroccio et al. found EPI in 30% of HIV infected children and a significant correlation between EPI and fat malabsorption.(31) However, only 14% (n=2/14) of patients with EPI had SAM and no direct correlation between was mentioned. In children with CP, 25% (n=52/208) was malnourished and this was only significantly correlated with a higher age of onset of CP, but not with fat absorption.(35) In two different studies also carried out by Carroccio et al. pancreatic function in children with celiac disease was studied. (36,37) In one study, they found EPI in 29% (n=15/52) of the celiac children and 37% of the patients (n=19/52) had SAM but no correlation between the two was reported.(37) In the other study they investigated the effect of pancreatic enzyme therapy in children with celiac disease, and showed that 38% (n=15/40) suffered from EPI and 15% (n=6/40) from severe EPI.(36) Celiac patients who were given pancreatic enzymes had a significant increase in weight after 30 days of therapy, compared to those that did not receive therapy, but this difference disappeared after supplementation of 60 days.

Group 2: Articles reporting patients diagnosed with malnutrition who are later found to have ePI (Supplemental Table 5)(16,18,21,23,24,27,39–41)All nine studies described some association, but not always causality, between malnu-trition and EPI.(16,18,21,23,24,27,39–41) Quality assessment was rated ‘poor’ for one study,(21) ‘fair’ for seven studies,(16,18,24,27,39–41) and ‘good’ for one study.(23) Seven studies reported that EPI in children with malnutrition is correctable after nutri-

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tional rehabilitation.(16,18,21,23,24,27,39) El-Hodhod et al. showed that malnourished children (n=33) had significantly lower serum amylase, serum lipase, and pancreatic head size compared to a group of normally nourished controls (n=12), and a significant improvement was seen in all measures of pancreatic function and weight after nutri-tional rehabilitation.(23) Barbezat et al. examined pancreatic enzyme markers in gastric juice and found these to be significantly lower in children with kwashiorkor (n=14) and marasmus (n=7) than in healthy controls (n=7), and these enzymes to significantly im-prove after nutritional rehabilitation.(16) Durie et al. reported a significant correlation between severity of malnutrition (n=50) and IRT, with IRT reverting to normal in patients with improvement in nutritional status.(24) Although no statistical values were provided, Thompson et al. also showed that children with kwashiorkor had lower levels of amylase and lipase compared to controls and that these improved after nutritional rehabilitation.(18) In a study conducted in Ivory Coast and France, Sauniere et al. concluded that in chil-dren with kwashiorkor (n=25) pancreas function (based on a total of 5 different enzymes) was significantly decreased compared to healthy African (n=11), and European children (n=62) and that this disappeared after refeeding.(21) A second study by Sauniere et al. discussed pancreatic function in malnourished children in Dakar (n=13) and Abidjan (n=15) in West Africa, which was decreased compared to healthy children in France.(39) After nutritional rehabilitation pancreatic secretion levels significantly increased but remained subnormal in the children from Abidjan and no improvement was found in children from Dakar. This was similar to our own previous study in which we found EPI in 92% (n=71/77) and severe EPI in 77% (n=59/77) of children with SAM and also found an significant improvement but no normalization of pancreatic function after nutritional rehabilitation.(27) Additionally, we found the degree of EPI to be significantly worse in children with kwashiorkor compared to children with marasmus (median FE-1 of 22u/g versus 80ug/g) and elevated IRT levels in 28% (n=11/39) of the patients.Two studies reported on EPI in malnourished Australian Aboriginal children.(40,41) Similar to Durie et al., Cleghorn et al. also reported on pancreatic damage in children with malnutrition, demonstrated by a significant correlation between IRT and degree of malnutrition (n=78/198 moderately and n=63/198 severely malnourished).(40) Briars et al. also found increased IRT levels related to decreased weight z-scores but no relation to other nutritional indices like arm circumference and skinfold thickness. A potential con-founder could have been gastroenteritis in these patients potentially causing elevated IRT.(41)

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Table 1. Study Demographics (n=19)

Author Country Study Population Inclusion Criteria exclusion Criteria Methods (measurement) Main Findings Remarks

Bartels et al. 2016

Malawi -89 children with severe acute malnutrition (SAM) admitted to nutritional rehabilitation unit (NRU) (median age 21 months).

-Children 6-60 months old-Diagnosis of SAM, by World Health Organization (WHO) definitions: weight for height Z-score (WHZ) < -3 SD, mid upper arm circumference (MUAC) <115 mm, and/or presence of bilateral edema

-Previously admitted to NRU within a year-Severe hemodynamic instability, hematocrit level of ≤15%, or severe neurological symptoms

-Observational study part of nutrient prospective intervention trial 1. Clinical parameters and anthropometry daily 2. Stool samples for FE-1 analysis <200 μg/g = exocrine pancreatic insufficiency (EPI)3. S. trypsinogen and pancreatic amylase determined in subsets, n=39 and n=80 respectively stratified for HIV status

-71/77 (92%) EPI-More edematous patients had EPI, 47/48 (98%), vs. non-edematous, 24/29 (83%), (p=0.03)- Lower FE-1-levels in edematous group (p=0.009)-Severe EPI (FE-1 <100 μg/g) higher in edematous group (p=0.006)-Mortality higher non-edematous group (p=0.03) -Trypsinogen elevated, especially in edematous group (p=0.03) suggesting pancreatic inflammation, - No correlation trypsinogen and FE-1 levels (p=0.4)

-Differences in mortality between HIV reactive and non-reactive patients ns.

Pichler et al. 2015

UK -21 children with Shwachman diamond syndrome (SDS) (median age 7.8 years).

-Genetically confirmed SDS-Attended the tertiary/quaternary SDS referral center

-Not mentioned -Retrospective observational study. -Visits every 3 months1. Weight for age z-score (WAZ) and height for age Z-score (HAZ)2. FE-1, pancreatic insufficiency (PI) defined as FE-1<200 μg/g3. Ultrasound (US) for fatty replacement pancreas

Baseline results:-20/21 (95%) PI -7/21 (33%) WAZ <-2, 9/21 (43%) HAZ <-2 -Abnormal US in 7/21 (33%)

-Exact results FE-1 not shown-Longitudinal data only available for 13/21 children

Kolodziejczyk et al. 2014

Poland -208 children with chronic pancreatitis (CP) (mean age 10.8 years).

-Age <18 years-CP features verified by one imaging technique (ERCP, MRCP, CT, or US scan) and/or by EPI tests (72 h fecal fat quantification, elastase-1 stool test, breath test)-Observation period of ≥ 1 year first episode of pancreatitis

-Age > 18 years-Lack of imaging studies, or the absence of CP features -CF-Inability of long-term observation

-Patients divided into 5 groups: 1) hereditary pancreatitis (n=26), 2) CFTR and/or SPINK1 mutations without a known cause (n=46), 3) anatomic duct anomalies (n=20), 4) patients with two or more coexisting etiologic factors of CP (n=24), 5) patients with idiopathic CP (n=92);-Mean follow up 5 years1. BMI ratio to evaluate anthropometric index, classify nutritional status2. 72-hour fecal fat quantification used to diagnose EPI with fat maldigestion. 3. Cambridge classification grades CP by ECRP findings from normal (grade 1) to marked (grade 4)

-52/208 (25.0%) malnutrition (14/52 (26.9%) mild; 36/52, (69.2%) moderate; 2/52 (3.8%) severe -Fecal fat and Cambridge grades NS between the 5 groups-Mean age at disease onset higher in group 1 vs. group 2 (p<0.05)

-72 hour fecal fat quantification only measured in 152/208 (73.0%) -Vague description of histological classification

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Table 1. Study Demographics (n=19)


Bartels et al. 2016

Malawi -89 children with severe acute malnutrition (SAM) admitted to nutritional rehabilitation unit (NRU) (median age 21 months).

-Children 6-60 months old-Diagnosis of SAM, by World Health Organization (WHO) definitions: weight for height Z-score (WHZ) < -3 SD, mid upper arm circumference (MUAC) <115 mm, and/or presence of bilateral edema

-Previously admitted to NRU within a year-Severe hemodynamic instability, hematocrit level of ≤15%, or severe neurological symptoms

-Observational study part of nutrient prospective intervention trial 1. Clinical parameters and anthropometry daily 2. Stool samples for FE-1 analysis <200 μg/g = exocrine pancreatic insufficiency (EPI)3. S. trypsinogen and pancreatic amylase determined in subsets, n=39 and n=80 respectively stratified for HIV status

-71/77 (92%) EPI-More edematous patients had EPI, 47/48 (98%), vs. non-edematous, 24/29 (83%), (p=0.03)- Lower FE-1-levels in edematous group (p=0.009)-Severe EPI (FE-1 <100 μg/g) higher in edematous group (p=0.006)-Mortality higher non-edematous group (p=0.03) -Trypsinogen elevated, especially in edematous group (p=0.03) suggesting pancreatic inflammation, - No correlation trypsinogen and FE-1 levels (p=0.4)

-Differences in mortality between HIV reactive and non-reactive patients ns.

Pichler et al. 2015

UK -21 children with Shwachman diamond syndrome (SDS) (median age 7.8 years).

-Genetically confirmed SDS-Attended the tertiary/quaternary SDS referral center

-Not mentioned -Retrospective observational study. -Visits every 3 months1. Weight for age z-score (WAZ) and height for age Z-score (HAZ)2. FE-1, pancreatic insufficiency (PI) defined as FE-1<200 μg/g3. Ultrasound (US) for fatty replacement pancreas

Baseline results:-20/21 (95%) PI -7/21 (33%) WAZ <-2, 9/21 (43%) HAZ <-2 -Abnormal US in 7/21 (33%)

-Exact results FE-1 not shown-Longitudinal data only available for 13/21 children


Poland -208 children with chronic pancreatitis (CP) (mean age 10.8 years).

-Age <18 years-CP features verified by one imaging technique (ERCP, MRCP, CT, or US scan) and/or by EPI tests (72 h fecal fat quantification, elastase-1 stool test, breath test)-Observation period of ≥ 1 year first episode of pancreatitis

-Age > 18 years-Lack of imaging studies, or the absence of CP features -CF-Inability of long-term observation

-Patients divided into 5 groups: 1) hereditary pancreatitis (n=26), 2) CFTR and/or SPINK1 mutations without a known cause (n=46), 3) anatomic duct anomalies (n=20), 4) patients with two or more coexisting etiologic factors of CP (n=24), 5) patients with idiopathic CP (n=92);-Mean follow up 5 years1. BMI ratio to evaluate anthropometric index, classify nutritional status2. 72-hour fecal fat quantification used to diagnose EPI with fat maldigestion. 3. Cambridge classification grades CP by ECRP findings from normal (grade 1) to marked (grade 4)

-52/208 (25.0%) malnutrition (14/52 (26.9%) mild; 36/52, (69.2%) moderate; 2/52 (3.8%) severe -Fecal fat and Cambridge grades NS between the 5 groups-Mean age at disease onset higher in group 1 vs. group 2 (p<0.05)

-72 hour fecal fat quantification only measured in 152/208 (73.0%) -Vague description of histological classification

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Table 1. Study Demographics (n=19) (continued)


El Hodhod et al. 2005

Egypt -33 children protein energy malnutrition (PEM) (mean age 11.87±7.8 months)-12 controls (mean age 14.83±7.7 months)

- Children with PEM (according to Wellcome criteria)48

-Not mentioned -Phase 1: pre-interventional assessment -Phase 2: nutritional intervention program with breast-feeding. -Phase 3: post-intervention assessment (3-6 months after starting date)Assessments in phase 1 and 3: 1.Dietetic history, history of GI symptoms, anthropometry, clinical signs of malnutrition2. S. lipase, S. Amylase 3. US of pancreas

Phase 1: -Pancreatic head size significantly lower marasmus, kwashiorkor (KWO), marasmic kwashiorkor (MKWO), vs. controls (p<0.001, p<0.01, p<0.05)-S. amylase significantly lower all groups of PEM (p<0.001)-S. lipase significantly lower marasmus, KWO, MKWO (p<0.01, p<0.001, p<0.001) Phase 3 post-intervention: -S. amylase significantly increased all malnourished groups (p<0.001) -S. lipase significantly increased in marasmus, KWO, MKWO (p<0.001, p<0.01, p<0.001)-Pancreatic head size significantly improved in marasmus, KWO, MKWO (p<0.001, p<0.05, p<0.05)-Weight and length significantly improved all groups (p<0.001)

Cohen et al. 2005

USA -91 CF children (6-8.9 years)

-Both mild to moderate CF lung disease and PI; -CF diagnosed: sweat sodium and chloride concentrations >60 mEq/L-PI diagnosed: 72 hour fecal fat analysis <93% absorption or stool trypsin concentration <80 μg/g

-Forced expiratory volume in 1 second (FEV1) <40%-Liver disease-Diabetes type 1-Burkholderia cepacia in sputum

-12 and 24 month hospital visit-6 and 18 month home visit -Pulmonary function, anthropometric assessment, blood, urine and fecal samples1. Dietary assessment; 7-day weighed food records2. 72 hour stool samples collected annually3. Height and weight using standard techniques 4. Random stool samples; fecal elastase (FE-1) analysis

-Group with residual pancreatic activity (R-FE) higher percent coefficient of fat absorption (%CoA) than no pancreatic activity (NO-FE) group (p<0.01) [94%±3% vs. 81%±14%] at baseline. -R-FE group also had a better growth at baseline (p=0.03)

-FE-1 levels only obtained for 85 children-1 child did not complete 24 month study; excluded from analysis

Bines et al. 2002

Australia -46 CF infants (mean age 7.7 weeks)-24 controls (mean age 9 weeks)

-Positive newborn CF screening: homozygosity for ΔF508 deletion or sweat chloride concentration ≥60 mmol/L-Infants with meconium ileus studied after clinical condition stabilized

-Not mentioned 1. Prospective 3-day dietary record2. Stool microscopy and/or 3-day fecal fat balance to determine PI3. Weight and length measured; measurements converted to percentile values and Z-scores (ANTHRO Pediatric Anthropometry Software program)

-Mean weight and length significantly lower than controls or reference values (p<0.05)-PI significantly associated with lower weight and length than controls (p<0.05)

-No cutoff values provided for 3 day fecal fat balance and stool microscopy

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El Hodhod et al. 2005

Egypt -33 children protein energy malnutrition (PEM) (mean age 11.87±7.8 months)-12 controls (mean age 14.83±7.7 months)

- Children with PEM (according to Wellcome criteria)48

-Not mentioned -Phase 1: pre-interventional assessment -Phase 2: nutritional intervention program with breast-feeding. -Phase 3: post-intervention assessment (3-6 months after starting date)Assessments in phase 1 and 3: 1.Dietetic history, history of GI symptoms, anthropometry, clinical signs of malnutrition2. S. lipase, S. Amylase 3. US of pancreas

Phase 1: -Pancreatic head size significantly lower marasmus, kwashiorkor (KWO), marasmic kwashiorkor (MKWO), vs. controls (p<0.001, p<0.01, p<0.05)-S. amylase significantly lower all groups of PEM (p<0.001)-S. lipase significantly lower marasmus, KWO, MKWO (p<0.01, p<0.001, p<0.001) Phase 3 post-intervention: -S. amylase significantly increased all malnourished groups (p<0.001) -S. lipase significantly increased in marasmus, KWO, MKWO (p<0.001, p<0.01, p<0.001)-Pancreatic head size significantly improved in marasmus, KWO, MKWO (p<0.001, p<0.05, p<0.05)-Weight and length significantly improved all groups (p<0.001)

Cohen et al. 2005

USA -91 CF children (6-8.9 years)

-Both mild to moderate CF lung disease and PI; -CF diagnosed: sweat sodium and chloride concentrations >60 mEq/L-PI diagnosed: 72 hour fecal fat analysis <93% absorption or stool trypsin concentration <80 μg/g

-Forced expiratory volume in 1 second (FEV1) <40%-Liver disease-Diabetes type 1-Burkholderia cepacia in sputum

-12 and 24 month hospital visit-6 and 18 month home visit -Pulmonary function, anthropometric assessment, blood, urine and fecal samples1. Dietary assessment; 7-day weighed food records2. 72 hour stool samples collected annually3. Height and weight using standard techniques 4. Random stool samples; fecal elastase (FE-1) analysis

-Group with residual pancreatic activity (R-FE) higher percent coefficient of fat absorption (%CoA) than no pancreatic activity (NO-FE) group (p<0.01) [94%±3% vs. 81%±14%] at baseline. -R-FE group also had a better growth at baseline (p=0.03)

-FE-1 levels only obtained for 85 children-1 child did not complete 24 month study; excluded from analysis

Bines et al. 2002

Australia -46 CF infants (mean age 7.7 weeks)-24 controls (mean age 9 weeks)

-Positive newborn CF screening: homozygosity for ΔF508 deletion or sweat chloride concentration ≥60 mmol/L-Infants with meconium ileus studied after clinical condition stabilized

-Not mentioned 1. Prospective 3-day dietary record2. Stool microscopy and/or 3-day fecal fat balance to determine PI3. Weight and length measured; measurements converted to percentile values and Z-scores (ANTHRO Pediatric Anthropometry Software program)

-Mean weight and length significantly lower than controls or reference values (p<0.05)-PI significantly associated with lower weight and length than controls (p<0.05)

-No cutoff values provided for 3 day fecal fat balance and stool microscopy

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Cipolli et al. 1999

Italy -13 children with SDS (mean age at diagnosis 12.4±5.4 months)

-2 negative sweat tests to exclude CF-Diagnosis SDS in infancy-Admitted between 1980 and 1995

-Not mentioned 1. Pancreatic function; secretin stimulation test (SST), S. pancreatic [alpha]-amylase, total lipase activities, S. immunoreactive trypsinogen, fecal chymotrypsin (CMT), 2. 3 day fat balance study 3. Height Z-scores (HZ) and weight Z-scores (WZ) WHO reference standards

-At diagnosis, growth retardation. 11/13 Z-score<-2 SD (both weight and height)-12/12 (100%) patients low or absent pancreatic enzymes = EPI. -Follow up: 5/5 (100%) normal lipase values, 0/5 (0%) normal amylase, 3/5 (60%) normal or borderline trypsin and CMT-Abnormal fat balance in all assessed, improvement on follow up

-5 patients pancreatic function retested-Many missed tests- HZ, WZ interpreted as HAZ, WAZ

Carroccio et al. 1998

Italy -47 Children (aged 1-16 years, median 7.3)-45 age and sex matched children (controls), surgery (cryptorchidism, inguinal or umbilical hernia)

-HIV positive -Not mentioned 1. Pancreatic functionStool: FE-1 and chymotrypsin (CMT). Serum: total amylase and pancreatic amylase activities2. Stools collected, fat excretion analyzed (24 hours)3. WAZ Italian regional standards used

-14/47 (30%) had abnormal pancreatic function tests (7 isolated FE-1 deficiency, 3 isolated CMT deficiency, and 4 had deficiencies in both.-Mean CMT lower in HIV infected children than controls (p<0.0001)-Steatorrhea significantly associated with reduced fecal pancreatic enzymes (p<0.0.1)-Significant negative correlation steatocrit and FE-1 (p<0.03)

-Ruled out HIV drugs, other viruses as a cause of pancreatic dysfunction

Briars et al. 1998

Australia -187 aboriginal patients Mount Isa base hospital (mean age 43 months)- 472 aboriginal patients Alice Springs hospital (mean age 16 months)

-Australian aboriginal patients-Mount Isa Base hospital: age 6 months-15 years-Alice Springs: aged <36 months

-Repeat admissions: only first admission was analyzed

-Retrospective analysis

1. Nutritional assessment including anthropometry2. Immunoreactive trypsinogen (IRT) 3. Nutritional status was correlated to indications for hospital admission.

Mount Isa study-Geometric mean IRT concentration 10.56, (95% CI, 9.56-11.67)-WZ: 107 patients normally nourished, 45 moderately malnourished (24.1%), 35 severely malnourished (18.7%)-IRT vs. WZ (ns)Alice Springs study-Geometric mean IRT concentration was 27.38 μg /L (CI 95%, 22.91-32.74)-WZ: 58 patients normally nourished, 160 moderately malnourished (33.9%), 254 severely malnourished (53.8%)-IRT vs. WZ (ns)

-Variability between hospitals, data collection and observation-Large number patients admitted with gastroenteritis which authors believe confounded results (high IRT and low WZ)-Significant maldistribution of gastroenteritis cases (p= 0.016)- WZ interpreted as WAZ

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Cipolli et al. 1999

Italy -13 children with SDS (mean age at diagnosis 12.4±5.4 months)

-2 negative sweat tests to exclude CF-Diagnosis SDS in infancy-Admitted between 1980 and 1995

-Not mentioned 1. Pancreatic function; secretin stimulation test (SST), S. pancreatic [alpha]-amylase, total lipase activities, S. immunoreactive trypsinogen, fecal chymotrypsin (CMT), 2. 3 day fat balance study 3. Height Z-scores (HZ) and weight Z-scores (WZ) WHO reference standards

-At diagnosis, growth retardation. 11/13 Z-score<-2 SD (both weight and height)-12/12 (100%) patients low or absent pancreatic enzymes = EPI. -Follow up: 5/5 (100%) normal lipase values, 0/5 (0%) normal amylase, 3/5 (60%) normal or borderline trypsin and CMT-Abnormal fat balance in all assessed, improvement on follow up

-5 patients pancreatic function retested-Many missed tests- HZ, WZ interpreted as HAZ, WAZ


Italy -47 Children (aged 1-16 years, median 7.3)-45 age and sex matched children (controls), surgery (cryptorchidism, inguinal or umbilical hernia)

-HIV positive -Not mentioned 1. Pancreatic functionStool: FE-1 and chymotrypsin (CMT). Serum: total amylase and pancreatic amylase activities2. Stools collected, fat excretion analyzed (24 hours)3. WAZ Italian regional standards used

-14/47 (30%) had abnormal pancreatic function tests (7 isolated FE-1 deficiency, 3 isolated CMT deficiency, and 4 had deficiencies in both.-Mean CMT lower in HIV infected children than controls (p<0.0001)-Steatorrhea significantly associated with reduced fecal pancreatic enzymes (p<0.0.1)-Significant negative correlation steatocrit and FE-1 (p<0.03)

-Ruled out HIV drugs, other viruses as a cause of pancreatic dysfunction

Briars et al. 1998

Australia -187 aboriginal patients Mount Isa base hospital (mean age 43 months)- 472 aboriginal patients Alice Springs hospital (mean age 16 months)

-Australian aboriginal patients-Mount Isa Base hospital: age 6 months-15 years-Alice Springs: aged <36 months

-Repeat admissions: only first admission was analyzed

-Retrospective analysis

1. Nutritional assessment including anthropometry2. Immunoreactive trypsinogen (IRT) 3. Nutritional status was correlated to indications for hospital admission.

Mount Isa study-Geometric mean IRT concentration 10.56, (95% CI, 9.56-11.67)-WZ: 107 patients normally nourished, 45 moderately malnourished (24.1%), 35 severely malnourished (18.7%)-IRT vs. WZ (ns)Alice Springs study-Geometric mean IRT concentration was 27.38 μg /L (CI 95%, 22.91-32.74)-WZ: 58 patients normally nourished, 160 moderately malnourished (33.9%), 254 severely malnourished (53.8%)-IRT vs. WZ (ns)

-Variability between hospitals, data collection and observation-Large number patients admitted with gastroenteritis which authors believe confounded results (high IRT and low WZ)-Significant maldistribution of gastroenteritis cases (p= 0.016)- WZ interpreted as WAZ

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Italy -40 children with Celiac disease. Group A (n=20) (mean age 14.2 ± 6.3 months); Group B (n=20) (mean age 14.5 ± 4.9 months)

-Celiac disease diagnosis intestinal biopsies (n=32); 8 cases positive tests anti-gluten antibodies, anti-endomysium antibodies, positive gluten test

-Not mentioned -Patients randomized to 2 groups. Group A pancreatic enzymes mix (6-10 capsules per day), group B placebo

1. Anthropometric data determined at diagnosis, 30, and 60 days. [Body weight, height, weight/height (W/H) ratio] 2. SST with intestinal biopsy. 3. Pancreatic function assessed by lipase, phospholipase, CMT4. Food recorded, weighed

-NS differences in pancreatic function, diets, between groups-Group A significant increase in relative body weight and W/H ratio after 30 days (p <0.001, and p <0.02), Group B significant increase after 60 days; in relative body weight and W/H ratio (p <0.001, and p <0.03)

-double blinding


Italy -52 children with celiac disease (6-36 months); -30 controls investigated for poor growth, normal jejunal histology (6-42 months)


-Not mentioned -Patients divided into 3 groups (W/H ratio): Group 1: W/H ratio ≤3rd percentile (n=19), mean age 16.8 months)Group 2: W/H ratio 4th - 10th percentiles (n=12), mean age 16 monthsGroup 3: W/H ratio >10th percentile (n=21) mean age 15.5 monthsGroup 4 [controls]: mean age 18.2 months (n=30)1. Exocrine pancreatic function determined by SST: lipase, CMT, phospholipase2. Body W/H ratio American national growth curves

-Lipase output was significantly lower in celiac patients compared to controls (p<0.009)-15/52 (29%) of patients presented with PI, 4/52 (8%) severe PI-W/H ratio significantly higher in controls (p<0.05)-No correlation between W/H ratio and pancreatic enzyme levels

Bronstein et al. 1992

USA -49 CF infants (admitted within 2 weeks diagnosis, 1 infant that was identified at 3.5 months to have CF)

-Positive newborn screening on basis of elevation of trypsinogen -Sweat chloride level of >60 mmol/L diagnostic of CF

-Meconium ileus -Severe respiratory distress-Family relocation to another state

1. Assessment of pancreatic insufficiency: 72-hour fat balance study, fecal nitrogen for protein malabsorption 2. Anthropometric data3. Repeat fecal collections at 6 and 12 months 4. Serum Albumin

-PI in 23/39 (59%) at diagnosis, 79% at 6 months, and 92% at 12-months.-WAZ significantly lower than normal in PI (p=0.005)-Fecal fat excretion inversely correlated with WAZ (p=0.005), weight gain (p<0.005), albumin (p<0.01)-At diagnosis: increased protein malabsorption correlated increased fat malabsorption (p<0.001)

-Some stool collections prematurely terminated due to collection difficulties or clinical complications 9/113 (7%)

Cleghorn et al. 1991

Australia -398 children admitted with acute and chronic pediatric disorders, Alice Springs Hospital (aged 6-36 months).

-Australian aboriginal children -Not mentioned 1. Grouped by Z scores, 3 nutritional groups (normal, moderate, severely malnourished) and stratified by stunted or not stunted2. Pancreatic dysfunction screened by human immunoreactive trypsinogen (IRT) assay

-57/198 (29%) normal,78/198 (39%) underweight, 63/198 (32%) severely underweight-IRT levels significantly correlated with weight Z-score (p=0.0014)-17/198 (9%) children had abnormal trypsinogen ( >89.1 μg/g)

-Analysis only on 198 children

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2





Italy -40 children with Celiac disease. Group A (n=20) (mean age 14.2 ± 6.3 months); Group B (n=20) (mean age 14.5 ± 4.9 months)


-Not mentioned -Patients randomized to 2 groups. Group A pancreatic enzymes mix (6-10 capsules per day), group B placebo

1. Anthropometric data determined at diagnosis, 30, and 60 days. [Body weight, height, weight/height (W/H) ratio] 2. SST with intestinal biopsy. 3. Pancreatic function assessed by lipase, phospholipase, CMT4. Food recorded, weighed

-NS differences in pancreatic function, diets, between groups-Group A significant increase in relative body weight and W/H ratio after 30 days (p <0.001, and p <0.02), Group B significant increase after 60 days; in relative body weight and W/H ratio (p <0.001, and p <0.03)

-double blinding


Italy -52 children with celiac disease (6-36 months); -30 controls investigated for poor growth, normal jejunal histology (6-42 months)


-Not mentioned -Patients divided into 3 groups (W/H ratio): Group 1: W/H ratio ≤3rd percentile (n=19), mean age 16.8 months)Group 2: W/H ratio 4th - 10th percentiles (n=12), mean age 16 monthsGroup 3: W/H ratio >10th percentile (n=21) mean age 15.5 monthsGroup 4 [controls]: mean age 18.2 months (n=30)1. Exocrine pancreatic function determined by SST: lipase, CMT, phospholipase2. Body W/H ratio American national growth curves

-Lipase output was significantly lower in celiac patients compared to controls (p<0.009)-15/52 (29%) of patients presented with PI, 4/52 (8%) severe PI-W/H ratio significantly higher in controls (p<0.05)-No correlation between W/H ratio and pancreatic enzyme levels


USA -49 CF infants (admitted within 2 weeks diagnosis, 1 infant that was identified at 3.5 months to have CF)

-Positive newborn screening on basis of elevation of trypsinogen -Sweat chloride level of >60 mmol/L diagnostic of CF

-Meconium ileus -Severe respiratory distress-Family relocation to another state

1. Assessment of pancreatic insufficiency: 72-hour fat balance study, fecal nitrogen for protein malabsorption 2. Anthropometric data3. Repeat fecal collections at 6 and 12 months 4. Serum Albumin

-PI in 23/39 (59%) at diagnosis, 79% at 6 months, and 92% at 12-months.-WAZ significantly lower than normal in PI (p=0.005)-Fecal fat excretion inversely correlated with WAZ (p=0.005), weight gain (p<0.005), albumin (p<0.01)-At diagnosis: increased protein malabsorption correlated increased fat malabsorption (p<0.001)

-Some stool collections prematurely terminated due to collection difficulties or clinical complications 9/113 (7%)


Australia -398 children admitted with acute and chronic pediatric disorders, Alice Springs Hospital (aged 6-36 months).

-Australian aboriginal children -Not mentioned 1. Grouped by Z scores, 3 nutritional groups (normal, moderate, severely malnourished) and stratified by stunted or not stunted2. Pancreatic dysfunction screened by human immunoreactive trypsinogen (IRT) assay

-57/198 (29%) normal,78/198 (39%) underweight, 63/198 (32%) severely underweight-IRT levels significantly correlated with weight Z-score (p=0.0014)-17/198 (9%) children had abnormal trypsinogen ( >89.1 μg/g)

-Analysis only on 198 children

40

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Sauniere et al. 1988

Senegal, Ivory Coast, France

-28 PEM children -21 African controls (relatives or stable, hospitalized children)-31 French healthy control children

-Children with PEM -Not mentioned 1. Daily weight and clinical data 2. SST: (amylase, phospholipase, lipase, trypsin, chymotrypsin analyzed)3. PEM patients randomly assigned pancreatic enzymes or placebo

-Exocrine pancreatic function in African controls significantly lower than French controls (p<0.05)-Abidjan, PI resolved after feeding -Abidjan, Placebo group- amylase, lipase, phospholipase, trypsin, CMT significantly improved (p<0.05); Pancreatic enzymes group- amylase, lipase significantly improved (p<0.05)-Dakar, pancreatic function unchanged even after 28 days, -Dakar, only amylase significantly improved pancreatic enzymes group (p<0.05)

-Feeding plan shorter in Abidjan compared to Dakar: 5 days vs. 28 in Abidjan (poor early results)-Significant difference between ages of Abidjan and Dakar control subjects, 2 groups of French controls used


France, Ivory Coast

-25 children Ivory Coast with KWO (mean age 29.15 months)- 10 children Ivory Coast with recovered KWO (mean age 21.40 months)- 3 children Ivory Coast recurrence of KWO (mean age 35 months)- 73 controls: 62 European children, (mean age= 43.89 months) & 11 children in Ivory Coast (mean age 43.86 months),hospitalized but recovered

-Children from Ivory Coast with ’active’, ‘recurrent’, and ‘recovered’ KWO based on biological and clinical indicators (loss of weight, edema, diarrhea, dehydration, low serum protein concentration, skin discoloration, and anemia)

-Not mentioned 1. SST: lipase, phospholipase, and amylase, trypsin, and CMT collected

-CMT, lipase, amylase, and phospholipase significantly lower in KWO patients vs. normal Africans and KWO patients vs. normal Africans + recovered KWO (p unknown)- Lipase and phospholipase significantly higher European controls vs. African controls (no significant difference in CMT, trypsin)

-No statistics given (authors state significance, no values)

Durie et al. 1985

Canada -50 malnourished (inadequate intake, primary intestinal/hepatic disorder, other) children (0.1-3.8 years, mean 1.25)- 38 controls: normally nourished, (mean age 1.25 years) only for IRT comparison

-Mild to severe malnutrition (varying degrees of malnutrition wasting)

-Not mentioned 1. Weekly clinical and nutritional status assessed. Target weight/length/age to sub classify patients 2. 3-5 day fecal fat balance. Fat malabsorption = fat loss >7%= fat malabsorption (>6 months), or >15% (<6 months)3. IRT: at entry, weekly intervals

-IRT in severely and moderately malnourished patients significantly higher vs. controls (p<0.001, p<0.02)-Nutritional treatment normalized IRT levels, to controls in severely malnourished (p<0.001), moderately malnourished (ns)

-Heterogenous group of patients -Lack of reference values -Fat balance in 43/50 (86%) patients-CF ruled out in 43/50 (86%) sweat tests, others no clinical evidence

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Senegal, Ivory Coast, France

-28 PEM children -21 African controls (relatives or stable, hospitalized children)-31 French healthy control children

-Children with PEM -Not mentioned 1. Daily weight and clinical data 2. SST: (amylase, phospholipase, lipase, trypsin, chymotrypsin analyzed)3. PEM patients randomly assigned pancreatic enzymes or placebo

-Exocrine pancreatic function in African controls significantly lower than French controls (p<0.05)-Abidjan, PI resolved after feeding -Abidjan, Placebo group- amylase, lipase, phospholipase, trypsin, CMT significantly improved (p<0.05); Pancreatic enzymes group- amylase, lipase significantly improved (p<0.05)-Dakar, pancreatic function unchanged even after 28 days, -Dakar, only amylase significantly improved pancreatic enzymes group (p<0.05)

-Feeding plan shorter in Abidjan compared to Dakar: 5 days vs. 28 in Abidjan (poor early results)-Significant difference between ages of Abidjan and Dakar control subjects, 2 groups of French controls used


France, Ivory Coast

-25 children Ivory Coast with KWO (mean age 29.15 months)- 10 children Ivory Coast with recovered KWO (mean age 21.40 months)- 3 children Ivory Coast recurrence of KWO (mean age 35 months)- 73 controls: 62 European children, (mean age= 43.89 months) & 11 children in Ivory Coast (mean age 43.86 months),hospitalized but recovered

-Children from Ivory Coast with ’active’, ‘recurrent’, and ‘recovered’ KWO based on biological and clinical indicators (loss of weight, edema, diarrhea, dehydration, low serum protein concentration, skin discoloration, and anemia)

-Not mentioned 1. SST: lipase, phospholipase, and amylase, trypsin, and CMT collected

-CMT, lipase, amylase, and phospholipase significantly lower in KWO patients vs. normal Africans and KWO patients vs. normal Africans + recovered KWO (p unknown)- Lipase and phospholipase significantly higher European controls vs. African controls (no significant difference in CMT, trypsin)

-No statistics given (authors state significance, no values)

Durie et al. 1985

Canada -50 malnourished (inadequate intake, primary intestinal/hepatic disorder, other) children (0.1-3.8 years, mean 1.25)- 38 controls: normally nourished, (mean age 1.25 years) only for IRT comparison

-Mild to severe malnutrition (varying degrees of malnutrition wasting)

-Not mentioned 1. Weekly clinical and nutritional status assessed. Target weight/length/age to sub classify patients 2. 3-5 day fecal fat balance. Fat malabsorption = fat loss >7%= fat malabsorption (>6 months), or >15% (<6 months)3. IRT: at entry, weekly intervals

-IRT in severely and moderately malnourished patients significantly higher vs. controls (p<0.001, p<0.02)-Nutritional treatment normalized IRT levels, to controls in severely malnourished (p<0.001), moderately malnourished (ns)

-Heterogenous group of patients -Lack of reference values -Fat balance in 43/50 (86%) patients-CF ruled out in 43/50 (86%) sweat tests, others no clinical evidence

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Hill et al. 1982

Canada -14 SDS patients (0.25-16 years old) (only 11 followed up)

-Recent diagnosis of SDS (PI by SST, and neutropenia) -only 4 diagnosed after 5 years of age.

-Not mentioned -12 year observational study1. Pancreatic function assessed by testing PST (assays of lipase, colipase, and trypsin)2. Fat balance studies3. Weight and height measured

-12 children had fat output >7%-Fat absorption improved-Lipase secretion <2% in steatorrheic patients vs. 3.7%-13.6% in non steatorrheic patients-Colipase and trypsin secretion higher in 5 patients without steatorrhea

-No clear description of the enzymes collected, cutoff values, or results for EPI classification-Very heterogenous methodology -2 patients died and 1 patient loss to follow up-Time interval between tests ranged 0.3-12.7 years

Barbezat et al. 1967

South Africa

- 14 KWO (mean age 25 months), (11/14 followed up),- 7 marasmus (mean age 20 months),- 10 chronically malnourished (mean age 102 months), -7 non malnourished, matched controls (mean age 23 months)

-Malnutrition (KWO: edema, skin lesions, growth retardation, and hypo-albuminemia or marasmus: wasting and growth retardation characterized as less than 61% of expected weight)

-Not mentioned 1. Nutritional rehabilitation2. SST: (amylase, lipase, trypsin, CMT) 3. Albumin

- Amylase lower in KWO and marasmus (p <0.01) -KWO significantly lower lipase levels (p <0.01), levels recovered after treatment-Trypsin least affected by malnutrition (only KWO affected)-CMT most affected by malnutrition. - After SST: significant positive correlation serum albumin concentration vs. enzyme output (p<0.001)

-Not all cases followed up

Thompson et al. 1952

Uganda -59 KWO patients: 40 investigated, 19 no follow up, - 24 admitted controls without signs of PEM(aged 9-58 months)

-KWO (hair and skin changes, pitting edema without cardiac or renal cause, and subnormal weight)

-Not mentioned 1. Duodenal intubation within 5 days of admission (Lipase and Amylase) 2. Feeding intervention: whole milk, skimmed milk, milk protein, or a mixture (pancreatic enzymes added)3. Reinvestigation in 40 children4. Treatment lasted 7-51 days 5. Necropsy performed on 4 children

-Extremely low levels of amylase and lipase seen in 40 KWO vs. control (p<0.001)-All groups significantly increased pancreatic enzymes after treatment (p <0.001) -Compared to controls, treatment groups significantly increased enzyme levels (p <0.01)-No significant differences in pancreas function between milk groups

-Follow up 40 cases (loss to follow up and death)-reinvestigation between 7-51 days, but not defined

%CoA, percent fecal fat absorption; CF, cystic fibrosis; CMT, chymotrypsin; CP, chronic pancreatitis; CT, computed tomography; ESPGAN, European society for paediatric gastroenterology and nutrition; ERCP, endoscopic retrograde cholangiopancreatography; EPI, exocrine pancreatic insufficiency; FE, fecal elas-tase [FE-1, fecal elastase-1]; HZ, height Z-score; HAZ, height for age Z-score; IRT, human immunoreactive trypsinogen; KWO, kwashiorkor; MKWO, marasmic kwashiorkor; MRCP, magnetic resonance cholangio-pancreatography; MUAC, mid upper arm circumference; NO-FE, no pancreatic activity; NRU, nutritional

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2




Hill et al. 1982

Canada -14 SDS patients (0.25-16 years old) (only 11 followed up)

-Recent diagnosis of SDS (PI by SST, and neutropenia) -only 4 diagnosed after 5 years of age.

-Not mentioned -12 year observational study1. Pancreatic function assessed by testing PST (assays of lipase, colipase, and trypsin)2. Fat balance studies3. Weight and height measured

-12 children had fat output >7%-Fat absorption improved-Lipase secretion <2% in steatorrheic patients vs. 3.7%-13.6% in non steatorrheic patients-Colipase and trypsin secretion higher in 5 patients without steatorrhea

-No clear description of the enzymes collected, cutoff values, or results for EPI classification-Very heterogenous methodology -2 patients died and 1 patient loss to follow up-Time interval between tests ranged 0.3-12.7 years

Barbezat et al. 1967

South Africa

- 14 KWO (mean age 25 months), (11/14 followed up),- 7 marasmus (mean age 20 months),- 10 chronically malnourished (mean age 102 months), -7 non malnourished, matched controls (mean age 23 months)

-Malnutrition (KWO: edema, skin lesions, growth retardation, and hypo-albuminemia or marasmus: wasting and growth retardation characterized as less than 61% of expected weight)

-Not mentioned 1. Nutritional rehabilitation2. SST: (amylase, lipase, trypsin, CMT) 3. Albumin

- Amylase lower in KWO and marasmus (p <0.01) -KWO significantly lower lipase levels (p <0.01), levels recovered after treatment-Trypsin least affected by malnutrition (only KWO affected)-CMT most affected by malnutrition. - After SST: significant positive correlation serum albumin concentration vs. enzyme output (p<0.001)

-Not all cases followed up


Uganda -59 KWO patients: 40 investigated, 19 no follow up, - 24 admitted controls without signs of PEM(aged 9-58 months)

-KWO (hair and skin changes, pitting edema without cardiac or renal cause, and subnormal weight)

-Not mentioned 1. Duodenal intubation within 5 days of admission (Lipase and Amylase) 2. Feeding intervention: whole milk, skimmed milk, milk protein, or a mixture (pancreatic enzymes added)3. Reinvestigation in 40 children4. Treatment lasted 7-51 days 5. Necropsy performed on 4 children

-Extremely low levels of amylase and lipase seen in 40 KWO vs. control (p<0.001)-All groups significantly increased pancreatic enzymes after treatment (p <0.001) -Compared to controls, treatment groups significantly increased enzyme levels (p <0.01)-No significant differences in pancreas function between milk groups

-Follow up 40 cases (loss to follow up and death)-reinvestigation between 7-51 days, but not defined

%CoA, percent fecal fat absorption; CF, cystic fibrosis; CMT, chymotrypsin; CP, chronic pancreatitis; CT, computed tomography; ESPGAN, European society for paediatric gastroenterology and nutrition; ERCP, endoscopic retrograde cholangiopancreatography; EPI, exocrine pancreatic insufficiency; FE, fecal elas-tase [FE-1, fecal elastase-1]; HZ, height Z-score; HAZ, height for age Z-score; IRT, human immunoreactive trypsinogen; KWO, kwashiorkor; MKWO, marasmic kwashiorkor; MRCP, magnetic resonance cholangio-pancreatography; MUAC, mid upper arm circumference; NO-FE, no pancreatic activity; NRU, nutritional

rehabilitation unit; PEM, protein energy, malnutrition; PI, pancreatic insufficiency/pancreatic insufficient; PS, pancreatic sufficiency; PST, pancreatic stimulation test; R-FE, residual pancreatic activity; SAM, severe acute malnutrition; S., serum; SD, standard deviation; SDS, Shwachman-Diamond Syndrome; SST, secre-tin stimulation test; US, ultrasound; VS, versus; W/H, weight/height; WZ, weight Z-score; WAZ, weight for age Z-score; WHO, World Health Organization; WHZ, weight for height Z-score

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Chapter 2

DISCUSSIOn

This is the first systematic review evaluating the association between EPI and malnutri-tion in children. Because malnutrition contributes to a high mortality in children under the age of 5 years worldwide, it is of great importance to explore potential new ways of treating malnutrition and reducing mortality. This systematic review shows that there is sufficient evidence for an association between EPI and malnutrition. Both EPI leading to malnutrition and malnutrition leading to EPI have been demonstrated in children in the existing literature. We refrained from a quantitative (pooled) analysis due to the heterogeneity of the data. Besides providing an overview of the existing literature it was not possible to draw firm conclusions on the exact link between EPI and malnutrition (correlation, causality, as-sociation).All studies describing EPI leading to malnutrition were conducted in cohorts of patients with an underlying disease: CF, CP, SDS, HIV and celiac disease.(29–38) Only two studies reported on the degree of EPI correlating with poor nutritional status.(29,30) Carroccio et al, emphasized that in celiac disease has both feature of EPI and malnutrition but no causality was reported.(37) The other studies only mentioned EPI and malnutrition but did report on a correlation. Although the exact etiology of EPI may vary, it is common practice nowadays to treat EPI with PERT. An improvement of nutritional status after PERT was described by two studies and demonstrates the influence of EPI on malnutrition.(29,36) Controversially, Pichler et al. found that in SDS patients catch-up growth was poor despite PERT.(33) All other included studies did not report on how PERT affected nutritional status. Most of the studies describing malnutrition leading to EPI were conducted in a low resource setting.(16,18,21,23,27,39) A varying degree of improvement of EPI after nu-tritional rehabilitation was reported by 7 studies, which is indicative of nutritional status influencing pancreatic function.(16,18,21,23,24,27,39) Durie et al. describe elevated IRT levels normalizing after 3 weeks to 1 year,(24) Thompson et al. report on differences in enzymes after 7 days to 51 days,(18) Sauniere et al. reports on normalization after sev-eral months(21) and Bartels et al. report on improvement of FE-1 levels only a few days after the first measurement.(27) These differences in EPI improvement can be partly explained by differences in treatment duration.This systematic review was complicated by the differences in gold standard and the defini-tions used. In addition to this, the majority of the included studies have methodologically shortcomings in terms of sample size, design, outcome measures, and statistical analysis, or used techniques that are not the (current) Gold standard. The pancreatic stimulation test (PST), a direct test, is considered the gold standard for the assessment of exocrine pancreatic function.(9) The studies included using PST are published between 1952 and

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1999.(16,18,21,32,34,36,37,39). It is not commonly used anymore because of the many disadvantages: it is invasive, impractical, burdensome to patients, and requires radiation exposure to verify positioning.(9) An indirect pancreatic tests using FE-1 currently is the most widely used test: it is noninvasive, biochemically stable, and has high sensitivity.(9) Not surprisingly, this test was used in more recent studies.(27,29,31,33) Many other different indirect tests, all less sensitive and specific for EPI than FE-1, were used as well contributing to the heterogeneity. Most studies measured serum pancreatic enzymes.(16,18,21,23,24,27,31,32,34,39–41) Abnormally low serum enzymes may indicate EPI, but these markers have low sensitivity and specificity for EPI and only support a diagnosis.(9) Fecal CMT, used in two studies,(31,32) is also less sensitive than FE-1 and requires discontinuation of PERT.(9) Fecal fat absorption, assessed in eight studies, (24,29–32,34–38) is not specific for EPI as other factors also result in a positive test (diet, malabsorption, gut transit ).(9) Other ways of measuring EPI used such as imaging and post mortem studies are non-specific to EPI and are of limited diagnostic value. Definitions of EPI and cutoff values for EPI were not consistent across studies. Several studies used healthy control groups as a comparison to estimate abnormal function instead of internationally established cutoff values. A third limitation contributing to heterogeneity was the various ways of defining malnutri-tion. Only one study(27) defined malnutrition according to the most recent WHO guide-lines (W/H ≤ -3 SD(4)), four studies defined malnutrition as a W/H ≤-2 SD(32,33,40,41) and all other studies defined it in different ways or used only clinical signs (in kwashiorkor patients). Finally, as there is a risk of publication bias as we were unable to identify un-published potential negative data on the association between EPI and malnutrition.In conclusion, this systematic review showed that there is sufficient evidence for an as-sociation between EPI and malnutrition. However, we could not confirm whether this is a correlation or a causality and therefore it was not possible to draw firm conclusions on underlying pathophysiological mechanisms between EPI and malnutrition. More observational clinical trials are crucially needed and future studies investigating EPI and malnutrition should investigate the potential role of PERT in malnourished children.

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REFEREnCES

1. Black RE, Victora CG, Walker SP et al. Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet 2013; 382: 427–451.

2. UNICEF. Levels and trends in child mortality 2015. United Nations Children’s Fund, New York: 2015. 3. UN. Sustainable Development Goals [Internet]. Available from: http://www.un.org/sustainabledevel-

opment/hunger/. Accessed December 3, 2017. 4. WHO. Guideline: Updates on the management of severe acute malnutrition in infants and children.

World Health Organization, Geneva: 2013. 5. Talbert A, Thuo N, Karisa J et al. Diarrhoea complicating severe acute malnutrition in Kenyan children:

a prospective descriptive study of risk factors and outcome. PLoS One 2012; 7: e38321. 6. Irena AH, Mwambazi M, Mulenga V. Diarrhea is a major killer of children with severe acute malnutrition

admitted to inpatient set-up in Lusaka, Zambia. Nutr J 2011; 10: 110. 7. Attia S, Versloot CJ, Voskuijl W et al. Mortality in children with complicated severe acute malnutrition

is related to intestinal and systemic inflammation: an observational cohort study. American Journal of Clinical Nutrition 2016; 104: 1441–1449.

8. Bandsma RHJ, Spoelstra MN, Mari A et al. Impaired Glucose Absorption in Children with Severe Malnu-trition. The Journal of Pediatrics 2011; 158: 282–287.e1.

9. Taylor CJ, Chen K, Horvath K et al. ESPGHAN and NASPGHAN Report on the Assessment of Exocrine Pancreatic Function and Pancreatitis in Children. J Pediatr Gastroenterol Nutr 2015; 61: 144–153.

10. Lindkvist B. Diagnosis and treatment of pancreatic exocrine insufficiency. World journal of gastroenter-ology 2013; 19: 7258–7266.

11. Nousia-Arvanitakis S. Cystic fibrosis and the pancreas: recent scientific advances. J Clin Gastroenterol 1999; 29: 138–142.

12. Shwachman H, Diamond LK, Oski FA, Khaw KT. The Syndrome of Pancreatic Insufficiency and Bone Marrow Dysfunction. J Pediatr 1964; 65: 645–663.

13. Bricaire F, Marche C, Zoubi D, Saimot AG, Regnier B. HIV and the pancreas. Lancet (London, England) 1988; 1: 65–66.

14. Littlewood JM, Wolfe SP, Conway SP. Diagnosis and treatment of intestinal malabsorption in cystic fibrosis. Pediatric pulmonology 2006; 41: 35–49.

15. Veghelyi P V. Nutritional oedema. Ann Paediatr 1950; 175: 349–377. 16. Barbezat GO, Hansen JD. The exocrine pancreas and protein-calorie malnutrition. Pediatrics 1968; 42:

77–92. 17. Tarasov NI. [External secretion of the pancreas in chronic infant nutrition disorders]. Vopr Pediatrii

1953; 21: 33–39. 18. Thompson MD, Trowell HC. Pancreatic enzyme activity in duodenal contents of children with a type of

kwashiorkor. Lancet 1952; 1: 1031–1035. 19. Blackburn WR, Vinijchaikul K. The pancreas in kwashiorkor. An electron microscopic study. Lab Invest

1969; 20: 305–318. 20. Banwell JG, Hutt MR, Leonard PJ et al. Exocrine pancreatic disease and the malabsorption syndrome in

tropical Africa. Gut 1967; 8: 388–401. 21. Sauniere JF, Sarles H, Attia Y et al. Exocrine pancreatic function of children from the Ivory Coast com-

pared to French children. Effect of kwashiorkor. Dig Dis Sci 1986; 31: 481–486. 22. Pitchumoni CS. Pancreas in primary malnutrition disorders. The American journal of clinical nutrition

1973; 26: 374–379.

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23. El-Hodhod MA, Nassar MF, Hetta OA, Gomaa SM. Pancreatic size in protein energy malnutrition: a predictor of nutritional recovery. Eur J Clin Nutr 2005; 59: 467–473.

24. Durie PR, Forstner GG, Gaskin KJ et al. Elevated serum immunoreactive pancreatic cationic trypsinogen in acute malnutrition: evidence of pancreatic damage. J Pediatr 1985; 106: 233–238.

25. Brooks SE, Golden MH. The exocrine pancreas in kwashiorkor and marasmus. Light and electron mi-croscopy. West Indian Med J 1992; 41: 56–60.

26. Bras G, Depass E, Waterlow JC. Further observations on the liver, pancreas and kidney in malnourished infants and children. I. The relation of certain histopathological changes in liver, pancreas and kidney. J Trop Pediatr (Lond) 1956; 2: 147–158.

27. Bartels RH, Meyer SL, Stehmann TA, Bourdon C, Bandsma RHJ, Voskuijl WP. Both Exocrine Pancreatic Insufficiency and Signs of Pancreatic Inflammation Are Prevalent in Children with Complicated Severe Acute Malnutrition: An Observational Study. The Journal of Pediatrics 2016; 174: 165–170.

28. Study Quality Assessment Tools [Internet]. NIH: National Heart, Lung, and Blood InstituteAvailable from: https://www.nhlbi.nih.gov/health-pro/guidelines/in-develop/cardiovascular-risk-reduction/tools

29. Cohen JR, Schall JI, Ittenbach RF, Zemel BS, Stallings VA. Fecal elastase: Pancreatic status verification and influence on nutritional status in children with cystic fibrosis. Journal of Pediatric Gastroenterology and Nutrition 2005; 40: 438–444.

30. Bronstein MN, Sokol RJ, Abman SH et al. Pancreatic insufficiency, growth, and nutrition in infants identi-fied by newborn screening as having cystic fibrosis. The Journal of Pediatrics 1992; 120: 533–540.

31. Carroccio A, Fontana M, Spagnuolo MI et al. Pancreatic dysfunction and its association with fat malab-sorption in HIV infected children. Gut 1998; 43: 558–563.

32. Cipolli M, D’Orazio C, Delmarco A, Marchesini C, Miano A, Mastella G. Shwachman’s Syndrome: Pathomorphosis and Long-term Outcome. Journal of pediatric gastroenterology and nutrition 1999; 29: 265–272.

33. Pichler J, Meyer R, Koglmeier J, Ancliff P, Shah N. Nutritional status in children with shwachman-diamond syndrome. Pancreas 2015; 44: 590–595.

34. Hill RE, Durie PR, Gaskin KJ, Davidson GP, Forstner GG. Steatorrhea and pancreatic insufficiency in Shwachman syndrome. Gastroenterology 1982; 83: 22–27.

35. Kolodziejczyk E, Wejnarska K, Dadalski M, Kierkus J, Ryzko J, Oracz G. The nutritional status and factors contributing to malnutrition in children with chronic pancreatitis. Pancreatology 2014; 14: 275–279.

36. Carroccio A, Iacono G, Montalto G et al. Pancreatic enzyme therapy in childhood celiac disease. A double-blind prospective randomized study. Digestive diseases and sciences 1995; 40: 2555–2560.

37. Carroccio A, Iacono G, Montalto G et al. Pancreatic insufficiency in celiac disease is not dependent on nutritional status. Digestive Diseases and Sciences 1994; 39: 2235–2242.

38. Bines JE, Truby HD, Armstrong DS, Phelan PD, Grimwood K. Energy metabolism in infants with cystic fibrosis. The Journal of Pediatrics 2002; 140: 527–533.

39. Sauniere JF, Sarles H. Exocrine pancreatic function and protein-calorie malnutrition in Dakar and Abi-djan (West Africa): silent pancreatic insufficiency. The American journal of clinical nutrition 1988; 48: 1233–1238.

40. Cleghorn GJ, Erlich J, Bowling FG et al. Exocrine pancreatic dysfunction in malnourished Australian aboriginal children. The Medical journal of Australia 1991; 154: 45–48.

41. Briars GL, Thornton SJ, Forrest Y, Ehrlich J, Shepherd RW, Cleghorn GJ. Malnutrition, gastroenteritis and trypsinogen concentration in hospitalised Aboriginal children. Journal of paediatrics and child health 1998; 34: 69–73.

42. Viera AJ, Garrett JM. Understanding interobserver agreement: the kappa statistic. Family medicine 2005; 37: 360–363.

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43. Dreiling DA, Hollander F. Studies in pancreatic function; preliminary series of clinical studies with the secretin test. Gastroenterology 1948; 11: 714–729.

44. Beharry S, Ellis L, Corey M, Marcon M, Durie P. How useful is fecal pancreatic elastase 1 as a marker of exocrine pancreatic disease? The Journal of Pediatrics 2002; 141: 84–90.

45. WHO, World Health Organization, United Nations Children’s Fund et al. WHO child growth standards and the identification of severe acute malnutrition in infants and children. World Health Organization, Geneva: 2009.

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Appendix 1 literature Search StrategyCochrane Search on Sept 4 2017: 39 Records 1. MeSH descriptor Nutrition Disorders explode all trees 2. Severe Acute Malnutr* or Protein Energy Malnutr* 3. MeSH descriptor Pancreatic Diseases explode all trees (Word variations have been searched 4. Exocrine Pancreatic Insufficien* 5. (#1 or #2) 6. (#3 or #4) 7. (#5 and #6)

embase Search on Feb 12 2017: 831 Records(‘pancreas exocrine insufficiency’/exp OR ‘pancreatic insufficien*’:ab,ti OR ‘pancreatic deficien*’:ab,ti) AND

(‘nutritional disorder’/exp OR ‘nutrition’/exp OR nutrition*:ab,ti) NOT (((‘animal’/exp OR ‘nonhuman’/exp) NOT ‘human’/exp) OR ‘case report’/exp) AND (‘article’/it OR ‘article in press’/it)

PubMed Search on Feb 12 2017: 798 Records(Exocrine pancreatic deficiency and Nutrition) OR (Exocrine pancreatic deficiency and developing countries):(((((“Exocrine Pancreatic Insufficiency”[Mesh] OR pancreatic insufficien*[tiab] OR pancreatic deficien*[tiab]))) AND

((“Nutrition Disorders”[Mesh] OR “Nutritional Physiological Phenomena”[Mesh] OR nutrition*[tiab])))) OR ((((“Exocrine Pancreatic Insufficiency”[Mesh] OR pancreatic insufficien*[tiab] OR pancreatic deficien*[tiab]))) AND ((((((“developing country”[tw] OR “developing countries”[tw] OR “developing nation”[tw] OR “develop-ing nations”[tw] OR “developing population”[tw] OR “developing populations”[tw] OR “developing world”[tw] OR “less developed country”[tw] OR “less developed countries”[tw] OR “less developed nation”[tw] OR “less developed nations”[tw] OR “less developed population”[tw] OR “less developed populations”[tw] OR “less developed world”[tw] OR “lesser developed country”[tw] OR “lesser developed countries”[tw] OR “lesser developed nation”[tw] OR “lesser developed nations”[tw] OR “lesser developed population”[tw] OR “lesser developed populations”[tw] OR “lesser developed world”[tw] OR “under devel-oped country”[tw] OR “under developed countries”[tw] OR “under developed nation”[tw] OR “under devel-oped nations”[tw] OR “under developed population”[tw] OR “under developed populations”[tw] OR “under developed world”[tw] OR “underdeveloped country”[tw] OR “underdeveloped countries”[tw] OR “under-developed nation”[tw] OR “underdeveloped nations”[tw] OR “underdeveloped population”[tw] OR “under-developed populations”[tw] OR “underdeveloped world”[tw] OR “middle income country”[tw] OR “middle income countries”[tw] OR “middle income nation”[tw] OR “middle income nations”[tw] OR “middle income population”[tw] OR “middle income populations”[tw] OR “low income country”[tw] OR “low income countries”[tw] OR “low income nation”[tw] OR “low income nations”[tw] OR “low income population”[tw] OR “low income populations”[tw] OR “lower income country”[tw] OR “lower income countries”[tw] OR “lower income nation”[tw] OR “lower income nations”[tw] OR “lower income population”[tw] OR “lower income populations”[tw] OR “underserved country”[tw] OR “underserved countries”[tw] OR “underserved nation”[tw] OR “underserved nations”[tw] OR “underserved population”[tw] OR “underserved populations”[tw] OR “underserved world”[tw] OR “under served country”[tw] OR “under served countries”[tw] OR “under served nation”[tw] OR “under served nations”[tw] OR “under served population”[tw] OR “under served populations”[tw] OR “under served world”[tw] OR “deprived country”[tw] OR “deprived countries”[tw] OR “deprived nation”[tw] OR “deprived nations”[tw] OR “deprived population”[tw] OR “deprived populations”[tw] OR “deprived world”[tw] OR “poor country”[tw] OR “poor countries”[tw] OR “poor nation”[tw] OR “poor nations”[tw] OR “poor population”[tw] OR “poor

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populations”[tw] OR “poor world”[tw] OR “poorer country”[tw] OR “poorer countries”[tw] OR “poorer nation”[tw] OR “poorer nations”[tw] OR “poorer population”[tw] OR “poorer populations”[tw] OR “poorer world”[tw] OR “developing economy”[tw] OR “developing economies”[tw] OR “less developed economy”[tw] OR “less developed economies”[tw] OR “lesser developed economy”[tw] OR “lesser developed economies”[tw] OR “under developed economy”[tw] OR “under developed economies”[tw] OR “underde-veloped economy”[tw] OR “underdeveloped economies”[tw] OR “middle income economy”[tw] OR “middle income economies”[tw] OR “low income economy”[tw] OR “low income economies”[tw] OR “lower income economy”[tw] OR “lower income economies”[tw] OR “low gdp”[tw] OR “low gnp”[tw] OR “low gross domestic”[tw] OR “low gross national”[tw] OR “lower gdp”[tw] OR “lower gnp”[tw] OR “lower gross domestic”[tw] OR “lower gross national”[tw] OR lmic[tw] OR lmics[tw] OR “third world”[tw] OR “lami country”[tw] OR “lami countries”[tw] OR “transitional country”[tw] OR “transitional countries”[tw]) OR (Africa[tw] OR Asia[tw] OR Caribbean[tw] OR West Indies[tw] OR South America[tw] OR Latin America[tw] OR Central America[tw] OR Afghanistan[tw] OR Albania[tw] OR Algeria[tw] OR Angola[tw] OR Antigua[tw] OR Barbuda[tw] OR Argentina[tw] OR Armenia[tw] OR Armenian[tw] OR Aruba[tw] OR Azerbaijan[tw] OR Bahrain[tw] OR Bangladesh[tw] OR Barbados[tw] OR Benin[tw] OR Byelarus[tw] OR Byelorussian[tw] OR Belarus[tw] OR Belorussian[tw] OR Belorussia[tw] OR Belize[tw] OR Bhutan[tw] OR Bolivia[tw] OR Bosnia[tw] OR Herzegovina[tw] OR Hercegovina[tw] OR Botswana[tw] OR Brasil[tw] OR Brazil[tw] OR Bulgaria[tw] OR Burkina Faso[tw] OR Burkina Fasso[tw] OR Upper Volta[tw] OR Burundi[tw] OR Urundi[tw] OR Cambodia[tw] OR Khmer Republic[tw] OR Kampuchea[tw] OR Cameroon[tw] OR Cameroons[tw] OR Cameron[tw] OR Camerons[tw] OR Cape Verde[tw] OR Central African Republic[tw] OR Chad[tw] OR Chile[tw] OR China[tw] OR Colombia[tw] OR Comoros[tw] OR Comoro Islands[tw] OR Comores[tw] OR Mayotte[tw] OR Congo[tw] OR Zaire[tw] OR Costa Rica[tw] OR Cote d’Ivoire[tw] OR Ivory Coast[tw] OR Croatia[tw] OR Cuba[tw] OR Cyprus[tw] OR Czechoslovakia[tw] OR Czech Republic[tw] OR Slovakia[tw] OR Slovak Republic[tw] OR Djibouti[tw] OR French Somaliland[tw] OR Dominica[tw] OR Dominican Republic[tw] OR East Timor[tw] OR East Timur[tw] OR Timor Leste[tw] OR Ecuador[tw] OR Egypt[tw] OR United Arab Republic[tw] OR El Salvador[tw] OR Eritrea[tw] OR Estonia[tw] OR Ethiopia[tw] OR Fiji[tw] OR Gabon[tw] OR Gabonese Republic[tw] OR Gambia[tw] OR Gaza[tw] OR Georgia Republic[tw] OR Georgian Republic[tw] OR Ghana[tw] OR Gold Coast[tw] OR Greece[tw] OR Grenada[tw] OR Guatemala[tw] OR Guinea[tw] OR Guam[tw] OR Guiana[tw] OR Guyana[tw] OR Haiti[tw] OR Honduras[tw] OR Hungary[tw] OR India[tw] OR Maldives[tw] OR Indonesia[tw] OR Iran[tw] OR Iraq[tw] OR Isle of Man[tw] OR Jamaica[tw] OR Jordan[tw] OR Kazakhstan[tw] OR Kazakh[tw] OR Kenya[tw] OR Kiribati[tw] OR Korea[tw] OR Kosovo[tw] OR Kyrgyzstan[tw] OR Kirghizia[tw] OR Kyrgyz Republic[tw] OR Kirghiz[tw] OR Kirgizstan[tw] OR “Lao PDR”[tw] OR Laos[tw] OR Latvia[tw] OR Lebanon[tw] OR Lesotho[tw] OR Basutoland[tw] OR Liberia[tw] OR Libya[tw] OR Lithuania[tw]) OR (Macedonia[tw] OR Madagascar[tw] OR Malagasy Republic[tw] OR Malaysia[tw] OR Malaya[tw] OR Malay[tw] OR Sabah[tw] OR Sarawak[tw] OR Malawi[tw] OR Nyasaland[tw] OR Mali[tw] OR Malta[tw] OR Marshall Islands[tw] OR Mauritania[tw] OR Mauritius[tw] OR Agalega Islands[tw] OR Mexico[tw] OR Micronesia[tw] OR Middle East[tw] OR Moldova[tw] OR Moldovia[tw] OR Moldovian[tw] OR Mongolia[tw] OR Montenegro[tw] OR Morocco[tw] OR Ifni[tw] OR Mozambique[tw] OR Myanmar[tw] OR Myanma[tw] OR Burma[tw] OR Namibia[tw] OR Nepal[tw] OR Netherlands Antilles[tw] OR New Caledonia[tw] OR Nicaragua[tw] OR Niger[tw] OR Nigeria[tw] OR Northern Mariana Islands[tw] OR Oman[tw] OR Muscat[tw] OR Pakistan[tw] OR Palau[tw] OR Palestine[tw] OR Panama[tw] OR Paraguay[tw] OR Peru[tw] OR Philippines[tw] OR Philipines[tw] OR Phillipines[tw] OR Phillippines[tw] OR Poland[tw] OR Portugal[tw] OR Puerto Rico[tw] OR Romania[tw] OR Rumania[tw] OR Roumania[tw] OR Russia[tw] OR Russian[tw] OR Rwanda[tw] OR Ruanda[tw] OR Saint Kitts[tw] OR St Kitts[tw] OR Nevis[tw] OR Saint Lucia[tw] OR St Lucia[tw] OR Saint Vincent[tw] OR St Vincent[tw] OR Grenadines[tw] OR Samoa[tw] OR Samoan Islands[tw] OR Navi-gator Island[tw] OR Navigator Islands[tw] OR Sao Tome[tw] OR Saudi Arabia[tw] OR Senegal[tw] OR

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Serbia[tw] OR Montenegro[tw] OR Seychelles[tw] OR Sierra Leone[tw] OR Slovenia[tw] OR Sri Lanka[tw] OR Ceylon[tw] OR Solomon Islands[tw] OR Somalia[tw] OR Sudan[tw] OR Suriname[tw] OR Surinam[tw] OR Swaziland[tw] OR Syria[tw] OR Tajikistan[tw] OR Tadzhikistan[tw] OR Tadjikistan[tw] OR Tadzhik[tw] OR Tanzania[tw] OR Thailand[tw] OR Togo[tw] OR Togolese Republic[tw] OR Tonga[tw] OR Trinidad[tw] OR Tobago[tw] OR Tunisia[tw] OR Turkey[tw] OR Turkmenistan[tw] OR Turkmen[tw] OR Uganda[tw] OR Ukraine[tw] OR Uruguay[tw] OR USSR[tw] OR Soviet Union[tw] OR Union of Soviet Socialist Republics[tw] OR Uzbekistan[tw] OR Uzbek OR Vanuatu[tw] OR New Hebrides[tw] OR Venezuela[tw] OR Vietnam[tw] OR Viet Nam[tw] OR West Bank[tw] OR Yemen[tw] OR Yugoslavia[tw] OR Zambia[tw] OR Zimbabwe[tw] OR Rhodesia[tw]) OR (Developing Countries[Mesh:noexp] OR Africa[Mesh:noexp] OR Africa, Northern[Mesh:noexp] OR Africa South of the Sahara[Mesh:noexp] OR Africa, Central[Mesh:noexp] OR Af-rica, Eastern[Mesh:noexp] OR Africa, Southern[Mesh:noexp] OR Africa, Western[Mesh:noexp] OR Asia[Mesh:noexp] OR Asia, Central[Mesh:noexp] OR Asia, Southeastern[Mesh:noexp] OR Asia, Western[Mesh:noexp] OR Caribbean Region[Mesh:noexp] OR West Indies[Mesh:noexp] OR South America[Mesh:noexp] OR Latin America[Mesh:noexp] OR Central America[Mesh:noexp] OR Afghanistan[Mesh:noexp] OR Albania[Mesh:noexp] OR Algeria[Mesh:noexp] OR American Samoa[Mesh:noexp] OR Angola[Mesh:noexp] OR “Antigua and Barbuda”[Mesh:noexp] OR Argentina[Mesh:noexp] OR Armenia[Mesh:noexp] OR Azerbaijan[Mesh:noexp] OR Bahrain[Mesh:noexp] OR Bangladesh[Mesh:noexp] OR Barbados[Mesh:noexp] OR Benin[Mesh:noexp] OR Byelarus[Mesh:noexp] OR Belize[Mesh:noexp] OR Bhutan[Mesh:noexp] OR Bolivia[Mesh:noexp] OR Bosnia-Herzegovina[Mesh:noexp] OR Botswana[Mesh:noexp] OR Brazil[Mesh:noexp] OR Bulgaria[Mesh:noexp] OR Burkina Faso[Mesh:noexp] OR Burundi[Mesh:noexp] OR Cambodia[Mesh:noexp] OR Cameroon[Mesh:noexp] OR Cape Verde[Mesh:noexp] OR Central African Republic[Mesh:noexp] OR Chad[Mesh:noexp] OR Chile[Mesh:noexp] OR China[Mesh:noexp] OR Colombia[Mesh:noexp] OR Comoros[Mesh:noexp] OR Congo[Mesh:noexp] OR Costa Rica[Mesh:noexp] OR Cote d’Ivoire[Mesh:noexp] OR Croatia[Mesh:noexp] OR Cuba[Mesh:noexp] OR Cyprus[Mesh:noexp] OR Czechoslovakia[Mesh:noexp] OR Czech Republic[Mesh:noexp] OR Slovakia[Mesh:noexp] OR Djibouti[Mesh:noexp] OR “Democratic Republic of the Congo”[Mesh:noexp] OR Dominica[Mesh:noexp] OR Dominican Republic[Mesh:noexp] OR East Timor[Mesh:noexp] OR Ecuador[Mesh:noexp] OR Egypt[Mesh:noexp] OR El Salvador[Mesh:noexp] OR Eritrea[Mesh:noexp] OR Estonia[Mesh:noexp] OR Ethiopia[Mesh:noexp] OR Fiji[Mesh:noexp] OR Gabon[Mesh:noexp] OR Gambia[Mesh:noexp] OR “Georgia (Republic)”[Mesh:noexp] OR Ghana[Mesh:noexp] OR Greece[Mesh:noexp] OR Grenada[Mesh:noexp] OR Guatemala[Mesh:noexp] OR Guinea[Mesh:noexp] OR Guinea-Bissau[Mesh:noexp] OR Guam[Mesh:noexp] OR Guyana[Mesh:noexp] OR Haiti[Mesh:noexp] OR Honduras[Mesh:noexp] OR Hungary[Mesh:noexp] OR India[Mesh:noexp] OR Indonesia[Mesh:noexp] OR Iran[Mesh:noexp] OR Iraq[Mesh:noexp] OR Jamaica[Mesh:noexp] OR Jordan[Mesh:noexp] OR Kazakhstan[Mesh:noexp] OR Kenya[Mesh:noexp] OR Korea[Mesh:noexp] OR Kosovo[Mesh:noexp] OR Kyrgyzstan[Mesh:noexp] OR Laos[Mesh:noexp] OR Latvia[Mesh:noexp] OR Lebanon[Mesh:noexp] OR Lesotho[Mesh:noexp] OR Liberia[Mesh:noexp] OR Libya[Mesh:noexp] OR Lithuania[Mesh:noexp] OR Macedonia[Mesh:noexp] OR Madagascar[Mesh:noexp] OR Malaysia[Mesh:noexp] OR Malawi[Mesh:noexp] OR Mali[Mesh:noexp] OR Malta[Mesh:noexp] OR Mauritania[Mesh:noexp] OR Mauritius[Mesh:noexp] OR Mexico[Mesh:noexp] OR Micronesia[Mesh:noexp] OR Middle East[Mesh:noexp] OR Moldova[Mesh:noexp] OR Mongolia[Mesh:noexp] OR Montenegro[Mesh:noexp] OR Morocco[Mesh:noexp] OR Mozambique[Mesh:noexp] OR Myanmar[Mesh:noexp] OR Namibia[Mesh:noexp] OR Nepal[Mesh:noexp] OR Netherlands Antilles[Mesh:noexp] OR New Caledonia[Mesh:noexp] OR Nicaragua[Mesh:noexp] OR Niger[Mesh:noexp] OR Nigeria[Mesh:noexp] OR Oman[Mesh:noexp] OR Pakistan[Mesh:noexp] OR Palau[Mesh:noexp] OR Panama[Mesh:noexp] OR Papua New Guinea[Mesh:noexp] OR Paraguay[Mesh:noexp] OR Peru[Mesh:noexp]

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OR Philippines[Mesh:noexp] OR Poland[Mesh:noexp] OR Portugal[Mesh:noexp] OR Puerto Rico[Mesh:noexp] OR Romania[Mesh:noexp] OR Russia[Mesh:noexp] OR “Russia (Pre-1917)”[Mesh:noexp] OR Rwanda[Mesh:noexp] OR “Saint Kitts and Nevis”[Mesh:noexp] OR Saint Lucia[Mesh:noexp] OR “Saint Vin-cent and the Grenadines”[Mesh:noexp] OR Samoa[Mesh:noexp] OR Saudi Arabia[Mesh:noexp] OR Senegal[Mesh:noexp] OR Serbia[Mesh:noexp] OR Montenegro[Mesh:noexp] OR Seychelles[Mesh:noexp] OR Sierra Leone[Mesh:noexp] OR Slovenia[Mesh:noexp] OR Sri Lanka[Mesh:noexp] OR Somalia[Mesh:noexp] OR South Africa[Mesh:noexp] OR Sudan[Mesh:noexp] OR Suriname[Mesh:noexp] OR Swaziland[Mesh:noexp] OR Syria[Mesh:noexp] OR Tajikistan[Mesh:noexp] OR Tanzania[Mesh:noexp] OR Thailand[Mesh:noexp] OR Togo[Mesh:noexp] OR Tonga[Mesh:noexp] OR “Trinidad and Tobago”[Mesh:noexp] OR Tunisia[Mesh:noexp] OR Turkey[Mesh:noexp] OR Turkmenistan[Mesh:noexp] OR Uganda[Mesh:noexp] OR Ukraine[Mesh:noexp] OR Uruguay[Mesh:noexp] OR USSR[Mesh:noexp] OR Uzbekistan[Mesh:noexp] OR Vanuatu[Mesh:noexp] OR Venezuela[Mesh:noexp] OR Vietnam[Mesh:noexp] OR Yemen[Mesh:noexp] OR Yugoslavia[Mesh:noexp] OR Zambia[Mesh:noexp] OR Zimbabwe[Mesh:noexp])))))))

list of excluded articles (n=35), categorized by reason for exclusionNo Clear Criteria Malnutrition (n=12) 1. Airinei G, Gaudichon C, Bos C, et al. Postprandial protein metabolism but not a fecal test reveals protein

malabsorption in patients with pancreatic exocrine insufficiency. Clin Nutr. 2011;30:831–837. 2. Bras G, Depass E, Waterlow JC. Further observations on the liver, pancreas and kidney in malnourished

infants and children. I. The relation of certain histopathological changes in liver, pancreas and kidney. J Trop Pediatr. 1956;2(3):147-158.

3. Danus O, Urbina AM, Valenzuela I, et al. The effect of refeeding on pancreatic exocrine function in marasmic infants. J Pediatr. 1970;77(2):334-337.

4. Dorlöchter L, Aksnes L, Fluge G. Faecal elastase-1 and fat-soluble vitamin profiles in patients with cystic fibrosis in Western Norway. Eur J Nutr. 2002;41:148–152.

5. Elburg van RM, Uil JJ, van Aalderen WM, et al. Intestinal permeability in exocrine pancreatic insuf-ficiency due to cystic fibrosis or chronic pancreatitis. Pediatr Res. 1996;39:985–991.

6. Filigno SS, Robson SM, Szczesniak RD, et al. Macronutrient intake in preschoolers with cystic fibrosis and the relationship between macronutrients and growth. J Cyst Fibros. 2017;1–6.

7. Groleau V, Schall JI, Dougherty KA, et al. Effect of a dietary intervention on growth and energy expendi-ture in children with cystic fibrosis. J Cyst Fibros. 2014;13:572–578. 2014;164:1110–1115.e1.

8. Harms HK, Kennel O, Bertele RM, et al. Vitamin B12 absorption and exocrine pancreatic insufficiency in childhood. Eur J Pediatr. 1981 Mar;136:75–79.

9. Haupt ME, Kwasny MJ, Schechter MS, et al. Pancreatic enzyme replacement therapy dosing and nutri-tional outcomes in children with cystic fibrosis. J Pediatr.

10. Kawchak DA, Zhao H, Scanlin TF, et al. Longitudinal, prospective analysis of dietary intake in children with cystic fibrosis. J Pediatr. 1996;129:119–129.

11. Ooi CY, Castellani C, Keenan K, Avolio J, Volpi S, Boland M, et al. Inconclusive diagnosis of cystic fibrosis after newborn screening. Pediatrics. 2015;135(6):e1377-1385.

12. Rana M, Wong-See D, Katz T, et al. Fat-soluble vitamin deficiency in children and adolescents with cystic fibrosis. J Clin Pathol. 2014;67:605–608.

No Clear Criteria EPI (n=7) 1. Carroccio A, Guarino A, Zuin G, et al. Efficacy of oral pancreatic enzyme therapy for the treatment of fat

malabsorption in HIV-infected patients. Aliment Pharmacol Ther. 2001;15:1619–1625. 2. Cipolli M, Castellani C, Wilcken B, Massie J, et al. Pancreatic phenotype in infants with cystic fibrosis

identified by mutation screening. Arch Dis Child. 2007;92:842–846.

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3. Konstan MW, Butler SM, Wohl MEB, Stoddard M, Matousek R, Wagener JS, et al. Growth and nutritional indexes in early life predict pulmonary function in cystic fibrosis. J Pediatr. 2003;142(6):624–630.

4. Scrimshaw NS, Behar M, Arroyave G, et al. Kwashiorkor in children and its response to protein therapy. J Am Med Assoc. 1957;164:555–561.

5. Sentongo T a, Rutstein RM, Stettler N, et al. Association between steatorrhea, growth, and immunologic status in children with perinatally acquired HIV infection. Arch Pediatr Adolesc Med. 2001;155:149–153.

6. Shoff SM, Ahn H-Y, Davis L, et al. Wisconsin CF Neonatal Screening Group. Temporal associations among energy intake, plasma linoleic acid, and growth improvement in response to treatment initiation after diagnosis of cystic fibrosis. Pediatrics. 2006;117:391–400.

7. Sugito K, Furuya T, Kaneda H, et al. Long-term follow-up of nutritional status, pancreatic function, and morphological changes of the pancreatic remnant after pancreatic tumor resection in children. Pancreas. 2012;41:554–559.

No Clear Criteria Malnutrition or EPI (n=3) 1. Morin CL, van Caillie M, Roy CC, Lasalle R. Mucosal enterokinase activity in pancreatic insufficiency and

celiac disease. Pediatrics.1977;60:114–116. 2. Farrell PM, Lai HJ, Li Z, et al. Evidence on improved outcomes with early diagnosis of cystic fibrosis

through neonatal screening: enough is enough! J Pediatr. 2005;147:S30-36. 3. Brooks SE, Golden MH. The exocrine pancreas in kwashiorkor and marasmus. Light and electron mi-

croscopy. West Indian Med J.1992;41:56–60. Full text non-retrievable (n=1) 1. Tarasov NI. External secretion of the pancreas in chronic infant nutrition disorders. Vopr Pediatr.

1953;21:33–39. No Cohort, Controlled Trial, or Case-control study design (n=4) 1. Grendell JH. Nutrition and absorption in diseases of the pancreas. Clin Gastroenterol. 1983;12:551–562. 2. Jonas A, Avigad S, Diver-Haber A, et al. Transient exocrine pancreatic insufficiency in infancy. J Pediatr.

1983;102:580–582. 3. Littlewood JM. Cystic fibrosis: gastrointestinal complications. Br Med Bull. 1992;48:847–859. 4. Veghelyi PV. Nutritional oedema. Ann Paediatr. 1950;175:349-377Adult Population (n=6) 1. Chaves CRM de M, Cunha ALP da, Costa AC, et al. Nutritional status and body fat distribution in children

and adolescentes with Cystic Fibrosis. Cien Saude Colet. 2015;20:3319–3328. 2. Davies J. The essential pathology of kwashiorkor. Lancet. 1948;1:317–320. 3. Fraga C, Leite CA. Dietetic studies on malnutrition and steatorrhea caused by pancreatic insufficiency.

Prensa Med Argent. 1971;58:2101–6. 4. Kapembwa MS, Fleming SC, Griffin GE, et al. Fat absorption and exocrine pancreatic function in human

immunodeficiency virus infection. Q J Med. 1990 Jan;74:49–56. 5. Leeds JS, Hopper AD, Hurlstone DP, et al. Is exocrine pancreatic insufficiency in adult coeliac disease a

cause of persisting symptoms? Aliment Pharmacol Ther. 2007;25:265–271. 6. Zuidema PJ. Cirrhosis and disseminated calcification of the pancreas in patients with malnutrition. Trop

Geogr Med. 1959;11:70–74. Not reporting on relation EPI and malnutrition (n=2) 1. Zemel BS, Kawchak DA, Cnaan A, et al. Prospective evaluation of resting energy expenditure, nutritional

status, pulmonary function, and genotype in children with cystic fibrosis. Pediatr Res. 1996;40:578–586. 2. Zemel BS, Jawad AF, FitzSimmons S, et al. Longitudinal relationship among growth, nutritional status,

and pulmonary function in children with cystic fibrosis: Analysis of the Cystic Fibrosis Foundation National CF Patient Registry. J Pediatr. 2000;137:374–380.

54

Chapter 2

Supplemental Table 1. NHLBI Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies

Thom

pson

et a

l. 19

52

Barb

ezat

et a

l. 19

68

Hill

et a

l. 19

82

Durie

et a

l. 19

85

Saun

iere

et a

l. 19

86

Cleg

horn

et a

l. 19

91

Bron

stei

n et

al.

1992

Carr

occi

o et

al.

1994

Bria

rs e

t al.

1998

Cipo

lli e

t al.

1999

Bine

s et a

l. 20

02

Cohe

n et

al.

2005

el-H

odho

d et

al.

2005

Kolo

dzie

-jczy

k et

al.

2014

Pich

ler e

t al.

2015

Bart

els e

t al.

2016

1. Was the research question or objective in this paper clearly stated? Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

2. Was the study population clearly specified and defined? Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

3. Was the participation rate of eligible persons at least 50%? CD CD CD CD CD N Y Y Y Y Y NR CD CD Y NR

4. Were all the subjects selected or recruited from the same or similar populations (including the same time period)? Were inclusion and exclusion criteria for being in the study prespecified and applied uniformly to all participants?

Y N CD N N Y Y N Y Y Y Y Y Y Y Y

5. Was a sample size justification, power description, or variance and effect estimates provided? N N N N N N N N N N N N N N N Y

6. For the analyses in this paper, were the exposure(s) of interest measured prior to the outcome(s) being measured?

Y Y Y Y Y Y Y CD Y Y Y Y Y Y Y Y

7. Was the timeframe sufficient so that one could reasonably expect to see an association between exposure and outcome if it existed?

Y Y Y Y Y CD Y Y Y Y Y Y Y Y Y Y

8. For exposures that can vary in amount or level, did the study examine different levels of the exposure as related to the outcome (e.g., categories of exposure, or exposure measured as continuous variable)?

NA Y NA Y NA Y NA NA Y Y N Y Y Y NA NA

9. Were the exposure measures (independent variables) clearly defined, valid, reliable, and implemented consistently across all study participants?

Y Y Y Y Y Y Y Y NA N N Y Y N Y Y

10. Was the exposure(s) assessed more than once over time? N N NA Y N N Y N N N NA Y Y N NA NA

11. Were the outcome measures (dependent variables) clearly defined, valid, reliable, and implemented consistently across all study participants?

Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y

12. Were the outcome assessors blinded to the exposure status of participants? NA NA NA NA NA CD CD NA NA NA N NA N NA N NA

13. Was loss to follow-up after baseline 20% or less? Y N N N Y Y Y NA NA* N NA Y Y Y Y N

14. Were key potential confounding variables measured and adjusted statistically for their impact on the relationship between exposure(s) and outcome(s)?

N N N Y N N N N Y N N N N Y N N

Rating Fair Fair Poor Fair Poor Fair Good Poor Fair Fair Fair Good Good Fair Fair Fair

CD, cannot determine; N, no; NA, not applicable; NR, not reported; Y, yes.

55

2


Supplemental Table 1. NHLBI Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies Th

omps

on e

t al.

1952

Barb

ezat

et a

l. 19

68

Hill

et a

l. 19

82

Durie

et a

l. 19

85

Saun

iere

et a

l. 19

86

Cleg

horn

et a

l. 19

91

Bron

stei

n et

al.

1992

Carr

occi

o et

al.

1994

Bria

rs e

t al.

1998

Cipo

lli e

t al.

1999

Bine

s et a

l. 20

02

Cohe

n et

al.

2005

el-H

odho

d et

al.

2005

Kolo

dzie

-jczy

k et

al.

2014

Pich

ler e

t al.

2015

Bart

els e

t al.

2016

1. Was the research question or objective in this paper clearly stated? Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

2. Was the study population clearly specified and defined? Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

3. Was the participation rate of eligible persons at least 50%? CD CD CD CD CD N Y Y Y Y Y NR CD CD Y NR

4. Were all the subjects selected or recruited from the same or similar populations (including the same time period)? Were inclusion and exclusion criteria for being in the study prespecified and applied uniformly to all participants?

Y N CD N N Y Y N Y Y Y Y Y Y Y Y

5. Was a sample size justification, power description, or variance and effect estimates provided? N N N N N N N N N N N N N N N Y

6. For the analyses in this paper, were the exposure(s) of interest measured prior to the outcome(s) being measured?

Y Y Y Y Y Y Y CD Y Y Y Y Y Y Y Y

7. Was the timeframe sufficient so that one could reasonably expect to see an association between exposure and outcome if it existed?

Y Y Y Y Y CD Y Y Y Y Y Y Y Y Y Y

8. For exposures that can vary in amount or level, did the study examine different levels of the exposure as related to the outcome (e.g., categories of exposure, or exposure measured as continuous variable)?

NA Y NA Y NA Y NA NA Y Y N Y Y Y NA NA

9. Were the exposure measures (independent variables) clearly defined, valid, reliable, and implemented consistently across all study participants?

Y Y Y Y Y Y Y Y NA N N Y Y N Y Y

10. Was the exposure(s) assessed more than once over time? N N NA Y N N Y N N N NA Y Y N NA NA

11. Were the outcome measures (dependent variables) clearly defined, valid, reliable, and implemented consistently across all study participants?

Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y

12. Were the outcome assessors blinded to the exposure status of participants? NA NA NA NA NA CD CD NA NA NA N NA N NA N NA

13. Was loss to follow-up after baseline 20% or less? Y N N N Y Y Y NA NA* N NA Y Y Y Y N

14. Were key potential confounding variables measured and adjusted statistically for their impact on the relationship between exposure(s) and outcome(s)?

N N N Y N N N N Y N N N N Y N N

Rating Fair Fair Poor Fair Poor Fair Good Poor Fair Fair Fair Good Good Fair Fair Fair


56

Chapter 2

Supplemental Table 2. NHLBI Quality Assessment Tool for Controlled Intervention Studies



1. Was the study described as randomized, a randomized trial, a randomized clinical trial, or an RCT?

Y Y

2. Was the method of randomization adequate (i.e., use of randomly generated assignment)?

CD Y

3. Was the treatment allocation concealed (so that assignments could not be predicted)?

CD Y

4. Were study participants and providers blinded to treatment group assignment?

CD Y

5. Were the people assessing the outcomes blinded to the participants’ group assignments?

CD Y

6. Were the groups similar at baseline on important characteristics that could affect outcomes (e.g., demographics, risk factors, co-morbid conditions)?

Y Y

7. Was the overall drop-out rate from the study at endpoint 20% or lower of the number allocated to treatment?

Y Y

8. Was the differential drop-out rate (between treatment groups) at endpoint 15 percentage points or lower?

CD Y

9. Was there high adherence to the intervention protocols for each treatment group?

CD CD

10. Were other interventions avoided or similar in the groups (e.g., similar background treatments)?

Y Y

11. Were outcomes assessed using valid and reliable measures, implemented consistently across all study participants?

Y Y

12. Did the authors report that the sample size was sufficiently large to be able to detect a difference in the main outcome between groups with at least 80% power?

N N

13. Were outcomes reported or subgroups analyzed prespecified (i.e., identified before analyses were conducted)?

Y Y

14. Were all randomized participants analyzed in the group to which they were originally assigned, i.e., did they use an intention-to-treat analysis?

NA Y

Rating Fair Good


57

2


Supplemental Table 3. NHLBI Quality Assessment of Case-Control Studies


1. Was the research question or objective in this paper clearly stated and appropriate?

Y

2. Was the study population clearly specified and defined? Y

3. Did the authors include a sample size justification? N

4. Were controls selected or recruited from the same or similar population that gave rise to the cases (including the same timeframe)?

Y

5. Were the definitions, inclusion and exclusion criteria, algorithms or processes used to identify or select cases and controls valid, reliable, and implemented consistently across all study participants?

Y

6. Were the cases clearly defined and differentiated from controls? Y

7. If less than 100 percent of eligible cases and/or controls were selected for the study, were the cases and/or controls randomly selected from those eligible?

NR

8. Was there use of concurrent controls? NR

9. Were the investigators able to confirm that the exposure/risk occurred prior to the development of the condition or event that defined a participant as a case?

CD

10. Were the measures of exposure/risk clearly defined, valid, reliable, and implemented consistently (including the same time period) across all study participants?

Y

11. Were the assessors of exposure/risk blinded to the case or control status of participants?

N

12. Were key potential confounding variables measured and adjusted statistically in the analyses? If matching was used, did the investigators account for matching during study analysis?

Y

Rating Fair


58

Chapter 2

Supplemental Table 4. Articles reporting patients diagnosed with EPI who are later found to be malnourished

Study Exocrine Pancreatic Insufficiency (EPI) Malnutrition Author’s conclusions

Comments

EPI Definition: Pancreatic Markers

Pancreatic Enzymes (serum)

Fecal Enzymes Fecal Fat Other tests Definition Anthropometrics Clinical Indicators Percentage affected

Pichler et al. 2015

1. Pancreatic insufficient defined as FE-1<200 μg/g. Severe deficiency in FE-1<50 μg/g2. Ultrasound of pancreas to screen fatty replacements

20/21 (95%) patients pancreatic insufficient

7/21 (33%) abnormal US findings

WHO definitions of malnutrition: weight for height Z score (WHZ) of <-2 Other anthropometric parameters: WAZ, height for age Z-score (HAZ)

At baseline 7/21(33%) WAZ <-2, 9/21 (43%) HAZ <-2 Follow up: 5/13 (38%) catch up growth; 2/13 (15%) WAZ <-2, and 7/13 (54%) HAZ <-2

Not reported More 50% vitamin A, selenium deficiency despite PERT + supplements. Routine measurements needed.

No elaboration US findings


1. 72-h fecal fat quantification; reference value <4.0 g/day2. Endoscopic retrograde cholangiopancreatography (ERCP) diagnosing and staging chronic pancreatitis (CP). Cambridge classification grades ERCP findings normal (Grade 1) to marked (Grade 4)

Mean fecal fat output among undernourished (n=38; 6.69 g) higher than in children with BMI ratio >85% (n=114; 2.27 g/day) (ns)

ERCP: Age of CP onset significantly higher group 1 (10.8, n=38) than group 2 (8.5, n=170) (p<0.05)Mean Cambridge grade in group 1 vs group 2 ns.

BMI ratio = (BMI actual/BMI for the 50th centile) X 100 [%]Nutritional status BMI%:<75 = severe malnutrition75-85 = malnutrition86-90 = mild malnutrition91-110 = normal111-120 = overweight >120 = obesePatients divided into 2 groups: 1-clinically significant malnutrition with BMI ratio <85%, and 2 with BMI ratio >85%

52/208 (25.0%) of CP had malnutrition:14/52 (26.9%) mild malnutrition; 36/52, (69.2%) moderate malnutrition; 2/52 (3.8%) severe malnutritionClinically significant malnutrition (group 1) 38/208 (18.3%)Mean age at disease onset significantly higher group 1 vs. group 2. (p<0.05)

38/208 (18.3%)

Considerable % CP children suffer clinically significant malnutrition. Later age at CP onset predisposes to malnutrition development

Fat analysis only in 152/208 (73.0%)

Cohen et al. 2005

1. Pancreatic insufficient (PI) 72 hr. fat analyses <93% absorption or stool trypsin concentration <80 ug/g2. Fecal elastase (FE) <15 ug/g stool = no pancreatic activity (NO-FE)3. Percent fecal fat absorption (%CoA) 7-day weighted food record, 72 hour stool [compared between NO-FE and residual FE group (R-FE)]

FE<15 ug/g stool: 75/84 (89%) children = NO-FEFE≥15 ug/g stool: 9/84 (11%) = R-FE

%CoA: R-FE 94% vs 81% NO-FE = PI, (p<0.01)

Weight for Age Z-score (WAZ), Adjusted height for age Z-score (AHAZ) compared between NO-FE and R-FE (CDC growth charts as reference)

Baseline: AHAZ significantly lower NO-FE group (p=0.03)Growth over 24 months: WAZ significantly lower NO-FE group p=0.04

Not reportedSome CF children misclassified pancreatic status. R-FE children better growth, absorption

59

2


Supplemental Table 4. Articles reporting patients diagnosed with EPI who are later found to be malnourished


Comments




Pichler et al. 2015

1. Pancreatic insufficient defined as FE-1<200 μg/g. Severe deficiency in FE-1<50 μg/g2. Ultrasound of pancreas to screen fatty replacements

20/21 (95%) patients pancreatic insufficient

7/21 (33%) abnormal US findings

WHO definitions of malnutrition: weight for height Z score (WHZ) of <-2 Other anthropometric parameters: WAZ, height for age Z-score (HAZ)

At baseline 7/21(33%) WAZ <-2, 9/21 (43%) HAZ <-2 Follow up: 5/13 (38%) catch up growth; 2/13 (15%) WAZ <-2, and 7/13 (54%) HAZ <-2

Not reported More 50% vitamin A, selenium deficiency despite PERT + supplements. Routine measurements needed.

No elaboration US findings


1. 72-h fecal fat quantification; reference value <4.0 g/day2. Endoscopic retrograde cholangiopancreatography (ERCP) diagnosing and staging chronic pancreatitis (CP). Cambridge classification grades ERCP findings normal (Grade 1) to marked (Grade 4)

Mean fecal fat output among undernourished (n=38; 6.69 g) higher than in children with BMI ratio >85% (n=114; 2.27 g/day) (ns)

ERCP: Age of CP onset significantly higher group 1 (10.8, n=38) than group 2 (8.5, n=170) (p<0.05)Mean Cambridge grade in group 1 vs group 2 ns.

BMI ratio = (BMI actual/BMI for the 50th centile) X 100 [%]Nutritional status BMI%:<75 = severe malnutrition75-85 = malnutrition86-90 = mild malnutrition91-110 = normal111-120 = overweight >120 = obesePatients divided into 2 groups: 1-clinically significant malnutrition with BMI ratio <85%, and 2 with BMI ratio >85%

52/208 (25.0%) of CP had malnutrition:14/52 (26.9%) mild malnutrition; 36/52, (69.2%) moderate malnutrition; 2/52 (3.8%) severe malnutritionClinically significant malnutrition (group 1) 38/208 (18.3%)Mean age at disease onset significantly higher group 1 vs. group 2. (p<0.05)

38/208 (18.3%)

Considerable % CP children suffer clinically significant malnutrition. Later age at CP onset predisposes to malnutrition development

Fat analysis only in 152/208 (73.0%)

Cohen et al. 2005

1. Pancreatic insufficient (PI) 72 hr. fat analyses <93% absorption or stool trypsin concentration <80 ug/g2. Fecal elastase (FE) <15 ug/g stool = no pancreatic activity (NO-FE)3. Percent fecal fat absorption (%CoA) 7-day weighted food record, 72 hour stool [compared between NO-FE and residual FE group (R-FE)]

FE<15 ug/g stool: 75/84 (89%) children = NO-FEFE≥15 ug/g stool: 9/84 (11%) = R-FE

%CoA: R-FE 94% vs 81% NO-FE = PI, (p<0.01)

Weight for Age Z-score (WAZ), Adjusted height for age Z-score (AHAZ) compared between NO-FE and R-FE (CDC growth charts as reference)

Baseline: AHAZ significantly lower NO-FE group (p=0.03)Growth over 24 months: WAZ significantly lower NO-FE group p=0.04

Not reportedSome CF children misclassified pancreatic status. R-FE children better growth, absorption

60

Chapter 2

Supplemental Table 4. Articles reporting patients diagnosed with EPI who are later found to be malnourished (continued)


Comments




Bines et al. 2002

1. PI determined by stool microscopy and/or 3-day fecal fat balance

35/46 (76%) PI 1. Anthropometrics: WZ, HZ, WHZ compared to control infants and reference data

WZ and HZ significantly lower than controls (p<0.05)WHZ significantly lower than controls (p<0.05)PI significantly associated with lower WZ, HZ, WHZ (p<0.05)

Not reported Growth impairment during first weeks of life in CF infants associated with PI

EPI cutoff values not provided

Reference data for anthropometrics not given

Cipolli et al. 1999

1. Secretin stimulation test (SST) lab values:S. pancreatic alpha-amylase [1800 IU], Total lipase [216.7 IU], trypsin [34.6 IU], chymotrypsin (CMT) [32 IU] (EPI = absent or lower)2. 3 day fat balance (EPI = fat absorption <90%)3. Fecal CMT: reference value >5 units per gram feces (EPI = <3 U/g)

12/12 (100%) abnormally low or absent levels pancreatic enzymesFollow up: 5/5 (100%) normal lipase values, 0/5 (0%) normal amylase, 3/5 (60%) normal trypsin and CMT

6/7 (86%) fecal CMT values >3 U/g

- 8/8 (100%) abnormal fat balance-Follow up: 5/6 (83%) normal fat balance

Height Z-score (HZ) and Weight Z-scores (WZ) WHO reference values: malnutrition if HZ and WZ<-2

Diagnosis11/13 (84%) HZ<-2 and WZ<-2 Follow up (n=6)2/6 (33%) HZ<-2 and WZ<-2

11/13 (84%) Possibility of improvement or normalization of exocrine pancreatic function with age in SD

12/13 (92%) had SST1 patient at follow up refused SST, abnormal fat absorptionHZ, WZ interpreted as HAZ, WAZ


1. Fecal enzymes: PI= FE-1 <200 μg/g stool], CMT <7.5 U/g]2. S. total amylase and pancreatic amylase higher normal values 160 U/l and 83 U/l. 3. Fecal fat analyzed. Abnormal >2 SD above control value (value not given)

S. amylase value elevated in 12 children (1.2-4 fold higher than normal limit).No correlation between fecal enzyme vs. elevated S. amylase

14/47 (30%) low fecal enzymes 7 isolated FE-1 deficiency, 3 isolated CMT deficiency, 4 deficiency in bothMean CMT activity significantly lower than controls (p<0.0001)Mean FE-1 activity significantly lower than controls (p<0.0001)

12/47 (26%) fat malabsorption

Fat malabsorption in 8/14 (57%) with low fecal pancreatic enzymes vs. 4/33 (12%) with normal enzymes (p<0.001)Significant negative correlation steatocrit vs. FE-1 (p<0.03)

1. WAZ Italian regional standards used2. Presence of diarrhea defined as 3 or more unformed stools per day

WAZ:Abnormal pancreatic function test: median -0.66 (range -3.3 to 2.1)Normal pancreatic function test: median -0.47 (range -3.8 to 2.5)Individual results only in abnormal pancreatic function tests. Only 2 patients with abnormal WZ (-3.3 and -3.25 )

Diarrhea not significantly low pancreatic enzymes vs. normal: 4/14 (29%) vs. 4/33 (12%), (p>0.05)

*Incomplete dataAbnormal pancreatic function group:2/14(14%) z-score below -3

Abnormal pancreatic function tests frequent HIV, contributes to steatorrhea

High serum amylase attributed to salivary amylase 10/12 patients)

Individual WAZ only in 14/47 patients

61

2




Comments




Bines et al. 2002

1. PI determined by stool microscopy and/or 3-day fecal fat balance

35/46 (76%) PI 1. Anthropometrics: WZ, HZ, WHZ compared to control infants and reference data

WZ and HZ significantly lower than controls (p<0.05)WHZ significantly lower than controls (p<0.05)PI significantly associated with lower WZ, HZ, WHZ (p<0.05)

Not reported Growth impairment during first weeks of life in CF infants associated with PI

EPI cutoff values not provided

Reference data for anthropometrics not given

Cipolli et al. 1999

1. Secretin stimulation test (SST) lab values:S. pancreatic alpha-amylase [1800 IU], Total lipase [216.7 IU], trypsin [34.6 IU], chymotrypsin (CMT) [32 IU] (EPI = absent or lower)2. 3 day fat balance (EPI = fat absorption <90%)3. Fecal CMT: reference value >5 units per gram feces (EPI = <3 U/g)

12/12 (100%) abnormally low or absent levels pancreatic enzymesFollow up: 5/5 (100%) normal lipase values, 0/5 (0%) normal amylase, 3/5 (60%) normal trypsin and CMT

6/7 (86%) fecal CMT values >3 U/g

- 8/8 (100%) abnormal fat balance-Follow up: 5/6 (83%) normal fat balance

Height Z-score (HZ) and Weight Z-scores (WZ) WHO reference values: malnutrition if HZ and WZ<-2

Diagnosis11/13 (84%) HZ<-2 and WZ<-2 Follow up (n=6)2/6 (33%) HZ<-2 and WZ<-2

11/13 (84%) Possibility of improvement or normalization of exocrine pancreatic function with age in SD

12/13 (92%) had SST1 patient at follow up refused SST, abnormal fat absorptionHZ, WZ interpreted as HAZ, WAZ


1. Fecal enzymes: PI= FE-1 <200 μg/g stool], CMT <7.5 U/g]2. S. total amylase and pancreatic amylase higher normal values 160 U/l and 83 U/l. 3. Fecal fat analyzed. Abnormal >2 SD above control value (value not given)

S. amylase value elevated in 12 children (1.2-4 fold higher than normal limit).No correlation between fecal enzyme vs. elevated S. amylase

14/47 (30%) low fecal enzymes 7 isolated FE-1 deficiency, 3 isolated CMT deficiency, 4 deficiency in bothMean CMT activity significantly lower than controls (p<0.0001)Mean FE-1 activity significantly lower than controls (p<0.0001)

12/47 (26%) fat malabsorption

Fat malabsorption in 8/14 (57%) with low fecal pancreatic enzymes vs. 4/33 (12%) with normal enzymes (p<0.001)Significant negative correlation steatocrit vs. FE-1 (p<0.03)

1. WAZ Italian regional standards used2. Presence of diarrhea defined as 3 or more unformed stools per day

WAZ:Abnormal pancreatic function test: median -0.66 (range -3.3 to 2.1)Normal pancreatic function test: median -0.47 (range -3.8 to 2.5)Individual results only in abnormal pancreatic function tests. Only 2 patients with abnormal WZ (-3.3 and -3.25 )

Diarrhea not significantly low pancreatic enzymes vs. normal: 4/14 (29%) vs. 4/33 (12%), (p>0.05)

*Incomplete dataAbnormal pancreatic function group:2/14(14%) z-score below -3

Abnormal pancreatic function tests frequent HIV, contributes to steatorrhea

High serum amylase attributed to salivary amylase 10/12 patients)

Individual WAZ only in 14/47 patients

62

Chapter 2



Comments





Pancreatic function assessed SST: Lipase (range 300-6000, median 800; units/ml/min/kg)CMT (range 42-850, median 80; units/ml/min/kg Phospholipase (range 4-150, median 12; units/ml/min/kg)Severe EPI = enzymatic secretion <10% of normal value

No significant differences between groupsGroup A (treatment): 8/20 (40%) subnormal enzyme output 1 or more enzymesGroup B (placebo): 7/20 (35%) subnormal enzyme output 1 or more enzymes3/20 (15%) patients in both groups had severe EPI (6/40, 15%)

Anthropometrics: [body weight, height, weight/ height (W/H ratio)] Italian regional standards used

Increase in W/H ratioGroup A significant after 30 days (p<0.02)Group B significant after 60 days (p< 0.03)Patients with EPI:Group A (treatment): significantly higher weight vs. Group B (p<0.008)

Not reported Pancreatic enzyme therapy useful in first 30 days after celiac diagnosis


Pancreatic function assessed SST: Lipase (range 300-6000, median 800; units/ml/min/kg)CMT (range 42-850, median 80; units/ml/min/kg Phospholipase (range 4-150, median 12; units/ml/min/kg)Severe deficiency = <10% enzyme output

No statistical significance:-6/52 (12%) low phospholipase-4/52 (8%) low CMT14/52 (27%) low lipase: celiac groups statistically lower than control (p<0.009)15/52 (29%) presented pancreatic deficiency based on enzyme output 4/52 (8%) had severe EPI

Body (W/H) ratio American National growth curves, no malnutrition definition

Control group had significantly higher W/H ratio than celiac patient groups (p<0.05)No W/H difference EPI and PI patients

Not reported Mild/ moderate PI frequent in celiac patients, independent of nutrient status

-Lipase reported as 600 but is in fact 6000

63

2




Comments





Pancreatic function assessed SST: Lipase (range 300-6000, median 800; units/ml/min/kg)CMT (range 42-850, median 80; units/ml/min/kg Phospholipase (range 4-150, median 12; units/ml/min/kg)Severe EPI = enzymatic secretion <10% of normal value

No significant differences between groupsGroup A (treatment): 8/20 (40%) subnormal enzyme output 1 or more enzymesGroup B (placebo): 7/20 (35%) subnormal enzyme output 1 or more enzymes3/20 (15%) patients in both groups had severe EPI (6/40, 15%)

Anthropometrics: [body weight, height, weight/ height (W/H ratio)] Italian regional standards used

Increase in W/H ratioGroup A significant after 30 days (p<0.02)Group B significant after 60 days (p< 0.03)Patients with EPI:Group A (treatment): significantly higher weight vs. Group B (p<0.008)

Not reported Pancreatic enzyme therapy useful in first 30 days after celiac diagnosis


Pancreatic function assessed SST: Lipase (range 300-6000, median 800; units/ml/min/kg)CMT (range 42-850, median 80; units/ml/min/kg Phospholipase (range 4-150, median 12; units/ml/min/kg)Severe deficiency = <10% enzyme output

No statistical significance:-6/52 (12%) low phospholipase-4/52 (8%) low CMT14/52 (27%) low lipase: celiac groups statistically lower than control (p<0.009)15/52 (29%) presented pancreatic deficiency based on enzyme output 4/52 (8%) had severe EPI

Body (W/H) ratio American National growth curves, no malnutrition definition

Control group had significantly higher W/H ratio than celiac patient groups (p<0.05)No W/H difference EPI and PI patients

Not reported Mild/ moderate PI frequent in celiac patients, independent of nutrient status

-Lipase reported as 600 but is in fact 6000

64

Chapter 2



Comments





72-h fecal fat collection, pancreatic sufficient (PS) <15% fat malabsorption at diagnostic visit (~6 weeks) and as <10% fat malabsorption at 6 and 12 month visits

Diagnosis: 23/39 (59%) PI6 and 12 month visit: PI, 79% at 6 months, 92% at 12 months (n not given)7/49 (14%) PS in first yearOnly PI had significant fat balance ratio (p<0.05)

1. Anthropometrics: Weight and length data expressed as Z scores below 50th percentile American growth standardsChange in weight calculated as [(WAZ at diagnosis- birth weight z score) / months in age at diagnosis]Weight gain (gm/day)2. Serum Albumin lower limit ≤2.8 gm/dl

Diagnosis:-Weight gain (gm/day) (n=29) lower in PI (p=0.05)-WAZ (n=29) lower in PI(p=0.05)Fecal fat excretion inversely correlated with WAZ (p=0.005) and weight gain (p <0.005)

Frequency of hypoalbuminemia not significantly different (5/15, 33%, PI and 1/11, 9%, PS)Albumin (n=26) higher in PS (p<0.01)

Not reported PI in CF infants significant impact on growth and nutrition

Only select patients compared

Hill et al. 1982

1. PI based on PST:lipase, colipase, trypsin no reference values given2. Fecal fat: upper normal limit =7%

14/14 (100%)EPI

Lipase <2% of mean normal secretion in all steatorrheic patients. Colipase and trypsin higher in 5 patients without steatorrhea compared to 3 patients with steatorrhea

12/14 (86%) children steatorrhea.Follow up 5/12 (42%) still had steatorrhea

Growth percentiles Weight percentiles on admission:<3 n=7<10 n=2>25 n=1>50 n=1no values n=3

Height percentiles on admission:<3 n=7<10 n=2>25 n=2>50 n=0no values n=3

Not reported Fat absorption improves in majority SD patients, associated marginal improvement lipase

Follow up timeframe not consistent

%CoA, percent fecal fat absorption; AHAZ, adujsted height for age Z-score; CDC, Centers for Disease Control; CMT, chymotrypsin; ERCP, endoscopic retrograde cholangiopancreatography; FE, fecal elastase [FE-1 = fecal elastase-1]; EPI, exocrine pancreatic insufficiency; HZ, height Z-score; HAZ, height for age Z score; IRT, human immunoreactive trypsinogen; KWO, kwashiorkor; MKW0, marasmus kwashiorkor; MUAC, mid upper arm circumference; NO-FE, no pancreatic activity; NS, not significant; PEM, protein energy malnutrition; PI, pancreatic insufficiency/pancreatic insufficient; PS, pancreatic sufficiency; PST, pancreatic stimulation test; R-FE, residual pancreatic activity; S., serum; SAM, severe acute malnutrition; SD, standard deviation; SST, secretin stimulation test; US, ultrasound; W/H, weight/height; WZ, weight Z-score; WAZ, weight for age Z score; WHZ, weight for height Z score

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Comments





72-h fecal fat collection, pancreatic sufficient (PS) <15% fat malabsorption at diagnostic visit (~6 weeks) and as <10% fat malabsorption at 6 and 12 month visits

Diagnosis: 23/39 (59%) PI6 and 12 month visit: PI, 79% at 6 months, 92% at 12 months (n not given)7/49 (14%) PS in first yearOnly PI had significant fat balance ratio (p<0.05)

1. Anthropometrics: Weight and length data expressed as Z scores below 50th percentile American growth standardsChange in weight calculated as [(WAZ at diagnosis- birth weight z score) / months in age at diagnosis]Weight gain (gm/day)2. Serum Albumin lower limit ≤2.8 gm/dl

Diagnosis:-Weight gain (gm/day) (n=29) lower in PI (p=0.05)-WAZ (n=29) lower in PI(p=0.05)Fecal fat excretion inversely correlated with WAZ (p=0.005) and weight gain (p <0.005)

Frequency of hypoalbuminemia not significantly different (5/15, 33%, PI and 1/11, 9%, PS)Albumin (n=26) higher in PS (p<0.01)

Not reported PI in CF infants significant impact on growth and nutrition

Only select patients compared

Hill et al. 1982

1. PI based on PST:lipase, colipase, trypsin no reference values given2. Fecal fat: upper normal limit =7%

14/14 (100%)EPI

Lipase <2% of mean normal secretion in all steatorrheic patients. Colipase and trypsin higher in 5 patients without steatorrhea compared to 3 patients with steatorrhea

12/14 (86%) children steatorrhea.Follow up 5/12 (42%) still had steatorrhea

Growth percentiles Weight percentiles on admission:<3 n=7<10 n=2>25 n=1>50 n=1no values n=3

Height percentiles on admission:<3 n=7<10 n=2>25 n=2>50 n=0no values n=3

Not reported Fat absorption improves in majority SD patients, associated marginal improvement lipase

Follow up timeframe not consistent


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Supplemental Table 5. Articles reporting patients diagnosed with malnutrition who are later found to have Exocrine Pancreatic Insufficiency

Study Malnutrition Exocrine Pancreatic Insufficiency (EPI) Exocrine Pancreatic Insufficiency (EPI) Author’s Conclusions

Comments

Definition Anthropometrics Clinical Indicators

EPI definition: Pancreatic Markers


Fecal Enzymes Fecal Fat Other tests Percentage affected

Bartels et al. 2016

Severe acute malnutrition (SAM) defined WHZ <-3 SD and/or a mid-upper arm circumference (MUAC) of < 115 mm (non-edematous) and/or presence of bilateral edema (edematous malnutrition)

WHZ ≤-3 =51/89 (58%)MUAC <11.5 cm =45/89 (50.6%)

56/89 (63%) had edematous SAM33/89 (37%) had non edematous SAM

1. FE-1 <200 μg/g stool = EPI, Levels <100 μg/g stool =severe EPI2. S. trypsinogen in a random subset: abnormal >57 ng/mL3.S. pancreatic amylase with upper limit set at 110 U/l

Amylase:17/80 (21%) increased levels (ns between groups)

High levels of trypsinogen 11/39 (28%) patients. Non-edematous = 9/20 (45%); Edematous 2/19 (11%) (p= 0.03)

EPI on admission: 71/77 (92.2%) EPI and 59/77 (77%) severe EPI Edema patients significantly lower FE-1 levels vs non-edematous group (22 μg/g of stool vs. 80 μg/g of stool, (p=0.009)47/48 (98%) of edematous group EPI vs 24/29 (83%) of non-edematous group (p=0.03), Severe EPI 42/48 (88%) vs 17/29 (59%) in non-edematous (p=0.006)3 days post stabilization: EPI38/46 (83%) edematous 20/24 (83%) non-edematous Severe EPI 28/46 (61%) edematous 14/24 (58%) non-edematous

71/77 (92%) EPI

59/71 (83%) had severe EPI

EPI prevalent SAM children, especially edematous. Pancreatitis common SAM

Trypsinogen only measured in 39

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Supplemental Table 5. Articles reporting patients diagnosed with malnutrition who are later found to have Exocrine Pancreatic Insufficiency


Comments





Bartels et al. 2016

Severe acute malnutrition (SAM) defined WHZ <-3 SD and/or a mid-upper arm circumference (MUAC) of < 115 mm (non-edematous) and/or presence of bilateral edema (edematous malnutrition)

WHZ ≤-3 =51/89 (58%)MUAC <11.5 cm =45/89 (50.6%)

56/89 (63%) had edematous SAM33/89 (37%) had non edematous SAM

1. FE-1 <200 μg/g stool = EPI, Levels <100 μg/g stool =severe EPI2. S. trypsinogen in a random subset: abnormal >57 ng/mL3.S. pancreatic amylase with upper limit set at 110 U/l

Amylase:17/80 (21%) increased levels (ns between groups)

High levels of trypsinogen 11/39 (28%) patients. Non-edematous = 9/20 (45%); Edematous 2/19 (11%) (p= 0.03)

EPI on admission: 71/77 (92.2%) EPI and 59/77 (77%) severe EPI Edema patients significantly lower FE-1 levels vs non-edematous group (22 μg/g of stool vs. 80 μg/g of stool, (p=0.009)47/48 (98%) of edematous group EPI vs 24/29 (83%) of non-edematous group (p=0.03), Severe EPI 42/48 (88%) vs 17/29 (59%) in non-edematous (p=0.006)3 days post stabilization: EPI38/46 (83%) edematous 20/24 (83%) non-edematous Severe EPI 28/46 (61%) edematous 14/24 (58%) non-edematous

71/77 (92%) EPI

59/71 (83%) had severe EPI

EPI prevalent SAM children, especially edematous. Pancreatitis common SAM

Trypsinogen only measured in 39

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Supplemental Table 5. Articles reporting patients diagnosed with malnutrition who are later found tohave Exocrine Pancreatic Insufficiency (continued)


Comments





El-Hodhod et al. 2005

Marasmus (less than 60% of the expectedweight for age)Kwashiorkor (KWO): oedema and between 60 and 80% of the expected weight for age.Marasmus-Kwashiorkor(MKWO): less than 60% of the expected weight forage but had oedema.Weight (kg)Control 9.50±1.78

Weight (kg)Baseline:Marasmus-4.99±1.15KWO-6.00±1.89MKWO-5.51±1.67Malnourished population significantly lower than controls:Marasmus (p<0.001)KWO (p<0.01)MKWO (p<0.001)Significant improvement after nutritional rehabilitation:Marasmus (p<0.001)KWO (p<0.001)MKWO (p<0.001)

Edema mentioned, data not provided

1. Controls used as reference valuesS. amylase (U/dl) 120.28±28.90 S. lipase (U/l) 66.13±17.35 2. Ultrasound (US): Pancreatic head size (cm3) [controls]5.13±2.33

Baseline S. amylase:Marasmus 68.88 ± 28.90KWO 51.54±16.90MKWO 56.04±13.02All groups lower than controls (p<0.001)Significant improvement after nutritional rehabilitation all groups (p<0.001)Baseline S. lipase:Marasmus 38.59±21.08KWO 34.63±17.58MKWO 21.67±21.48Malnourished population lower than controls:Marasmus (p<0.01)KWO (p<0.001)MKWO (p<0.001)Significant improvement after nutritional rehabilitation. Marasmus (p<0.001)KWO (p<0.01)MKWO (p<0.001)

Baseline pancreatic head size (cm3):Marasmus 1.52±0.60KWO 2.73±0.12MKWO 3.00±0.54Malnourished population significantly lower than controls:Marasmus (p<0.001)KWO (p<0.01)MKWO (p<0.05)Significantly improved after nutritional rehabilitation:Marasmus (p<0.001)KWO- t=0.44 (p<0.05)MKWO- t=1.36 (p<0.05)

Not reported Pancreatic head size and exocrine function used to evaluate PEM and used as prognostic parameter

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Comments





El-Hodhod et al. 2005

Marasmus (less than 60% of the expectedweight for age)Kwashiorkor (KWO): oedema and between 60 and 80% of the expected weight for age.Marasmus-Kwashiorkor(MKWO): less than 60% of the expected weight forage but had oedema.Weight (kg)Control 9.50±1.78

Weight (kg)Baseline:Marasmus-4.99±1.15KWO-6.00±1.89MKWO-5.51±1.67Malnourished population significantly lower than controls:Marasmus (p<0.001)KWO (p<0.01)MKWO (p<0.001)Significant improvement after nutritional rehabilitation:Marasmus (p<0.001)KWO (p<0.001)MKWO (p<0.001)

Edema mentioned, data not provided

1. Controls used as reference valuesS. amylase (U/dl) 120.28±28.90 S. lipase (U/l) 66.13±17.35 2. Ultrasound (US): Pancreatic head size (cm3) [controls]5.13±2.33

Baseline S. amylase:Marasmus 68.88 ± 28.90KWO 51.54±16.90MKWO 56.04±13.02All groups lower than controls (p<0.001)Significant improvement after nutritional rehabilitation all groups (p<0.001)Baseline S. lipase:Marasmus 38.59±21.08KWO 34.63±17.58MKWO 21.67±21.48Malnourished population lower than controls:Marasmus (p<0.01)KWO (p<0.001)MKWO (p<0.001)Significant improvement after nutritional rehabilitation. Marasmus (p<0.001)KWO (p<0.01)MKWO (p<0.001)

Baseline pancreatic head size (cm3):Marasmus 1.52±0.60KWO 2.73±0.12MKWO 3.00±0.54Malnourished population significantly lower than controls:Marasmus (p<0.001)KWO (p<0.01)MKWO (p<0.05)Significantly improved after nutritional rehabilitation:Marasmus (p<0.001)KWO- t=0.44 (p<0.05)MKWO- t=1.36 (p<0.05)

Not reported Pancreatic head size and exocrine function used to evaluate PEM and used as prognostic parameter

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Comments





Briars et al. 1998

HZ, WZ. WHO reference valuesChildren categorized WZ-2= severely malnutrition, -1 to -2= moderate malnutrition, above -1= no malnutrition.

165/659 (25.0%) normally nourished205/659 (31.1%) moderately malnourished289/659 (43.9%) severely malnourished

IRT compared among study populationNo reference value

IRT value (95% CI) Mount Isa study:Mean: 10.56 μg/L (9.56-11.67)normally nourished: 9.59 μg/L (8.49-10.82)moderately malnourished: 10.92 μg/L (8.64-13.8)severely malnourished: 13.62 μg/L (10.89-17.02)IRT correlation with HZ, WZ (ns)Alice Springs study:Mean: 27.38 μg/L (22.91-32.74)normally nourished: 30.21 μg/L (24.86-36.7)moderately malnourished: 32.6 μg/L (28.36-37.52)severely malnourished: 29.22 μg/L (25.16-33.93)IRT correlation with HZ, WZ (ns)

Not reported High IRT in low WZ confounding effect of gastroenteritis, may result subclinical pancreatic disease in gastroenteritis

WZ interpreted as WAZ

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Comments





Briars et al. 1998

HZ, WZ. WHO reference valuesChildren categorized WZ-2= severely malnutrition, -1 to -2= moderate malnutrition, above -1= no malnutrition.

165/659 (25.0%) normally nourished205/659 (31.1%) moderately malnourished289/659 (43.9%) severely malnourished

IRT compared among study populationNo reference value

IRT value (95% CI) Mount Isa study:Mean: 10.56 μg/L (9.56-11.67)normally nourished: 9.59 μg/L (8.49-10.82)moderately malnourished: 10.92 μg/L (8.64-13.8)severely malnourished: 13.62 μg/L (10.89-17.02)IRT correlation with HZ, WZ (ns)Alice Springs study:Mean: 27.38 μg/L (22.91-32.74)normally nourished: 30.21 μg/L (24.86-36.7)moderately malnourished: 32.6 μg/L (28.36-37.52)severely malnourished: 29.22 μg/L (25.16-33.93)IRT correlation with HZ, WZ (ns)

Not reported High IRT in low WZ confounding effect of gastroenteritis, may result subclinical pancreatic disease in gastroenteritis

WZ interpreted as WAZ

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Comments






WAZ:Normal: weight >-1, moderate: -1 to -2, severely underweight <-2.

Mean WAZ -1.6 (range -3.5 to 1.5) 57/198 (29%) normal, 78 /198 (39%) underweight, 63/198 (32%) severely underweight

Serum immunoreactive trypsinogen, (IRT) normal values obtained from population group, (upper normal = 89.1 μg/L

Serum cationic trypsinogen: Normal; 0/57 (0%) elevated levels.Moderately Underweight; 11/78 (14%) elevated levels. Severely Underweight; 6/63 (10%) elevated levelsSignificant correlation WAZ vs. IRT (p=0.0014) IRT significantly higher severely underweight vs. normal (p<0.05)

17/198 (9%)elevated IRT = EPI

Pancreatic dysfunction may be common and overlooked to ongoing malnutrition and disease in Australian Aboriginal children

Only 198/ 398 had IRT analyzed, subsequent comparisons only of 198

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Comments






WAZ:Normal: weight >-1, moderate: -1 to -2, severely underweight <-2.

Mean WAZ -1.6 (range -3.5 to 1.5) 57/198 (29%) normal, 78 /198 (39%) underweight, 63/198 (32%) severely underweight

Serum immunoreactive trypsinogen, (IRT) normal values obtained from population group, (upper normal = 89.1 μg/L

Serum cationic trypsinogen: Normal; 0/57 (0%) elevated levels.Moderately Underweight; 11/78 (14%) elevated levels. Severely Underweight; 6/63 (10%) elevated levelsSignificant correlation WAZ vs. IRT (p=0.0014) IRT significantly higher severely underweight vs. normal (p<0.05)

17/198 (9%)elevated IRT = EPI

Pancreatic dysfunction may be common and overlooked to ongoing malnutrition and disease in Australian Aboriginal children

Only 198/ 398 had IRT analyzed, subsequent comparisons only of 198

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Comments






Malnutrition defined on clinical and anthropometric symptoms of PEM: decreased weight and height, diarrhea, loss of appetite, edema

28/28 (100%) PEM.

SST: Reference values for amylase, lipase, trypsin, CMT not given. Instead values compared to controls in same setting, and controls in France.Dakar control: U/mlamylase 49.8±18lipase 24.6±4.5phospholipase 1.2±0.2trypsin 2.0±0.4CMT 12.6±3.6Abidjan control: U/mlamylase 54.2±10.3lipase 164.4±44.6phospholipase 4.8±1.0trypsin 6.2±1.3CMT 27.8±6.6

Dakar PEM vs. Dakar control nsPI not improved after 28 daysAbidjan PEM significantly lower phospholipase, lipase, trypsin, CMT activity vs. Abidjan controls (p<0.05)Abidjan: placebo-lipase, trypsin, CMT, amylase, phospholipase significantly improved (p<0.05)Amylase and lipase significantly improved treatment group vs. placebo (p<0.05)

Can’t determine

West Africa, latent PI involving water, electrolytes, and enzymes. PI neither aggravated by kwashiorkor nor corrected by feeding

Authors compared enzyme measurements with both controls in same area and French controls

Dakar controls significantly lower vs. French controls

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Comments






Malnutrition defined on clinical and anthropometric symptoms of PEM: decreased weight and height, diarrhea, loss of appetite, edema

28/28 (100%) PEM.

SST: Reference values for amylase, lipase, trypsin, CMT not given. Instead values compared to controls in same setting, and controls in France.Dakar control: U/mlamylase 49.8±18lipase 24.6±4.5phospholipase 1.2±0.2trypsin 2.0±0.4CMT 12.6±3.6Abidjan control: U/mlamylase 54.2±10.3lipase 164.4±44.6phospholipase 4.8±1.0trypsin 6.2±1.3CMT 27.8±6.6

Dakar PEM vs. Dakar control nsPI not improved after 28 daysAbidjan PEM significantly lower phospholipase, lipase, trypsin, CMT activity vs. Abidjan controls (p<0.05)Abidjan: placebo-lipase, trypsin, CMT, amylase, phospholipase significantly improved (p<0.05)Amylase and lipase significantly improved treatment group vs. placebo (p<0.05)

Can’t determine

West Africa, latent PI involving water, electrolytes, and enzymes. PI neither aggravated by kwashiorkor nor corrected by feeding

Authors compared enzyme measurements with both controls in same area and French controls

Dakar controls significantly lower vs. French controls

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Comments






KWO- clinical and biological abnormalities: weight loss (not quantified by authors), edema, diarrhea, dehydration, skin discoloration, low serum protein concentration, anemia

28/28 (100%) KWO clinical symptoms (25 first admittance, 3 readmitted KWO)

SST: Pancreatic enzymes (Units/ml in 15min) Lipase Amylase Phospholipase CMTTrypsinCases compared with African controls, Parisian controls, and African controls + recovered kwashiorkor

Absolute numbers not reportedLipase, amylase, and CMT significantly higher normal Africans vs. KWO AfricansAmylase, lipase, phospholipase, CMT significantly lower KWO Africans vs. normal and recovered KWONo significant differences normal Africans and healed KWO children

Not reported PI in KWO reversible, trypsin more resistant, no relationship KWO and tropical pancreatitis

Amylase not analyzed French controls

Durie et al. 1985

Patients sub-classified according degree malnutrition: ideal weight/length/age ‘severe’: weight < 75%‘moderate’: 75% to 85%‘mild’: 85% to 95%‘normal’ >90%

Severely malnourished: 25/50 (50%)moderately malnourished: 23/50 (46%)mildly malnourished: 2/50 (4%)

1. IRT values based on controls: 32.3±10.4 ng/ml2. 3-5 day fat balance studies. Fat malabsorption if fat losses >7% in those 6 months or older, >15% in those younger than 6 months

IRT:severe malnutrition (compared to controls): 77.4±42.0 ng/ml(p<0.001)moderately malnutrition: 54.2±16.1 ng/ml (p<0.02)mild malnutrition: control levels (ns)

Fat malabsorption 17/43 (40%)

36/50 (72%) IRT in malnourished may be pancreatic acinar cell damage or obstructed pancreatic ducts.IRT normal after improvement nutritional status

13/17 (76%) steatorrhea due to non-pancreatic cause

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Comments






KWO- clinical and biological abnormalities: weight loss (not quantified by authors), edema, diarrhea, dehydration, skin discoloration, low serum protein concentration, anemia

28/28 (100%) KWO clinical symptoms (25 first admittance, 3 readmitted KWO)

SST: Pancreatic enzymes (Units/ml in 15min) Lipase Amylase Phospholipase CMTTrypsinCases compared with African controls, Parisian controls, and African controls + recovered kwashiorkor

Absolute numbers not reportedLipase, amylase, and CMT significantly higher normal Africans vs. KWO AfricansAmylase, lipase, phospholipase, CMT significantly lower KWO Africans vs. normal and recovered KWONo significant differences normal Africans and healed KWO children

Not reported PI in KWO reversible, trypsin more resistant, no relationship KWO and tropical pancreatitis

Amylase not analyzed French controls

Durie et al. 1985

Patients sub-classified according degree malnutrition: ideal weight/length/age ‘severe’: weight < 75%‘moderate’: 75% to 85%‘mild’: 85% to 95%‘normal’ >90%

Severely malnourished: 25/50 (50%)moderately malnourished: 23/50 (46%)mildly malnourished: 2/50 (4%)

1. IRT values based on controls: 32.3±10.4 ng/ml2. 3-5 day fat balance studies. Fat malabsorption if fat losses >7% in those 6 months or older, >15% in those younger than 6 months

IRT:severe malnutrition (compared to controls): 77.4±42.0 ng/ml(p<0.001)moderately malnutrition: 54.2±16.1 ng/ml (p<0.02)mild malnutrition: control levels (ns)

Fat malabsorption 17/43 (40%)

36/50 (72%) IRT in malnourished may be pancreatic acinar cell damage or obstructed pancreatic ducts.IRT normal after improvement nutritional status

13/17 (76%) steatorrhea due to non-pancreatic cause

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Comments





Barbezat and Hansen 1968

1. MalnutritionKWO: edema, skin lesions, growth retardation, and hypoalbuminemiaMarasmus and Chronic malnourished: growth retardation, absence of edema and skin lesions, wasted little or no subcutaneous fat, slight hypoalbuminemia (chronic previous KWO patients)2. Percent expected weight (50th Boston percentile control values): 94.04% SD 5.093. Serum albumin concentration: mean control value 3.72 gm/100ml SD 0.40

Mean value of percent expected weight KWO 68.40% SD 11.07Marasmus 52.61% SD 7.32Chronically malnourished 67.99% SD 12.71

KWO: 14Marasmus: 7Chronic malnutrition: 10

Serum AlbuminKWO: 1.67 gm/100ml SD 0.47Marasmus: 2.15 gm/100ml SD 0.40Chronic malnutrition: 3.39 gm/100ml SD 0.43

SST:Analyzed enzymes amylase, lipase, trypsin, and CMT.Values compared to controls and malnourished subgroups

No absolute values:Amylase: lower in KWO and marasmus (p<0.01). KWO group significantly improved after treatment (p<0.01)Lipase: KWO significantly lower lipase levels (p< 0.01)Trypsin: KWO lower vs. controls and recovered (p<0.02)CMT: KWO, marasmus patients lower (p<0.01) (most affected enzyme)Chronically malnourished less CMT than recovered KWO(p<0.02)

Not reported Pancreatic enzyme output grossly deficient in KWO and marasmus. Complete restoration pancreatic function after dietary therapy

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Comments





Barbezat and Hansen 1968

1. MalnutritionKWO: edema, skin lesions, growth retardation, and hypoalbuminemiaMarasmus and Chronic malnourished: growth retardation, absence of edema and skin lesions, wasted little or no subcutaneous fat, slight hypoalbuminemia (chronic previous KWO patients)2. Percent expected weight (50th Boston percentile control values): 94.04% SD 5.093. Serum albumin concentration: mean control value 3.72 gm/100ml SD 0.40

Mean value of percent expected weight KWO 68.40% SD 11.07Marasmus 52.61% SD 7.32Chronically malnourished 67.99% SD 12.71

KWO: 14Marasmus: 7Chronic malnutrition: 10

Serum AlbuminKWO: 1.67 gm/100ml SD 0.47Marasmus: 2.15 gm/100ml SD 0.40Chronic malnutrition: 3.39 gm/100ml SD 0.43

SST:Analyzed enzymes amylase, lipase, trypsin, and CMT.Values compared to controls and malnourished subgroups

No absolute values:Amylase: lower in KWO and marasmus (p<0.01). KWO group significantly improved after treatment (p<0.01)Lipase: KWO significantly lower lipase levels (p< 0.01)Trypsin: KWO lower vs. controls and recovered (p<0.02)CMT: KWO, marasmus patients lower (p<0.01) (most affected enzyme)Chronically malnourished less CMT than recovered KWO(p<0.02)

Not reported Pancreatic enzyme output grossly deficient in KWO and marasmus. Complete restoration pancreatic function after dietary therapy

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Comments






Established KWO defined as pitting edema, without obvious renal or cardiac cause, changes in hair, and subnormal weight. Other noted features were hyperpigmentation of skin, raw weeping areas, diarrhea and wasting

59/59 (100%) children fulfilled the criteria for kwashiorkor

Diarrhea present in 85%

1. Pancreatic stimulation test (PST): lipase and amylase extracted. Reference values based on control values Amylase units/ml;mean: 2.92 SD: 1.62Lipase units per 0.1 ml; mean: 3.84 SD: 1.042. Necropsy performed on children who died: histopathology of pancreas noted

Group 1= followed up (n=40)group 2= not followed up (n=10)group 3= died (n=8)group 4=not treated (n=1)Amylase at admissiongroup 1: 0.40 units/ml; group 2: 0.50 units/ml; group 3: 0.53 units/ml; group 4: 0.05 units/mlDischargegroup 1: 4.33 units/ml; group 4: 0.1 units/mlLipase at admission group 1: 1.0 units/0.1ml; group 2: 1.3 units/0.1ml; group 3: 0.81 units/0.1ml; group 4: 1.0 units/0.1mlDischarge:group 1: 3.88 units/ml; group 4: 0.9 units/0.1mlOn admission amylase and lipase significantly lower in KWO (p<0.001). Fully recovered after nutritional rehabilitation (p<0.01)

Histopathology:2 children that died: loss of cytoplasm in acinar cells, collapse of acinar structure, and increase in fibrous tissue. 2 children who died from intercurrent infection histologically normal

100% (58/58)

KWO children well below normal amylase and lipase, reversible with treatment

40 /59 (68%) children who were re-evaluated after treatment

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Comments






Established KWO defined as pitting edema, without obvious renal or cardiac cause, changes in hair, and subnormal weight. Other noted features were hyperpigmentation of skin, raw weeping areas, diarrhea and wasting

59/59 (100%) children fulfilled the criteria for kwashiorkor

Diarrhea present in 85%

1. Pancreatic stimulation test (PST): lipase and amylase extracted. Reference values based on control values Amylase units/ml;mean: 2.92 SD: 1.62Lipase units per 0.1 ml; mean: 3.84 SD: 1.042. Necropsy performed on children who died: histopathology of pancreas noted

Group 1= followed up (n=40)group 2= not followed up (n=10)group 3= died (n=8)group 4=not treated (n=1)Amylase at admissiongroup 1: 0.40 units/ml; group 2: 0.50 units/ml; group 3: 0.53 units/ml; group 4: 0.05 units/mlDischargegroup 1: 4.33 units/ml; group 4: 0.1 units/mlLipase at admission group 1: 1.0 units/0.1ml; group 2: 1.3 units/0.1ml; group 3: 0.81 units/0.1ml; group 4: 1.0 units/0.1mlDischarge:group 1: 3.88 units/ml; group 4: 0.9 units/0.1mlOn admission amylase and lipase significantly lower in KWO (p<0.001). Fully recovered after nutritional rehabilitation (p<0.01)

Histopathology:2 children that died: loss of cytoplasm in acinar cells, collapse of acinar structure, and increase in fibrous tissue. 2 children who died from intercurrent infection histologically normal

100% (58/58)

KWO children well below normal amylase and lipase, reversible with treatment

40 /59 (68%) children who were re-evaluated after treatment

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Chapter 3Both exocrine pancreatic insufficiency and signs of pancreatic inflammation are highly prevalent in children with complicated severe acute malnutrition: an observational study

Rosalie H. Bartels, Sophie L. Meyer, Tijs A. Stehmann, Céline Bourdon, Robert H.J. Bandsma, Wieger P. Voskuijl

Journal of Pediatrics 2016: 174:165–70

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ABSTRACT

Objectives: To assess whether pancreatic function: 1) is impaired in children with severe acute malnutrition (SAM), 2) is different between edematous versus non-edematous malnutrition, and 3) improves by nutritional rehabilitation.

Study design: We followed 89 children with SAM admitted to Queen Elizabeth Central Hospital in Blantyre, Malawi. Stool and blood samples were taken on admission and three days after initial stabilization to determine exocrine pancreatic function via fecal elastase-1 (FE-1) and serum trypsinogen and amylase levels.

Results: 33 children (37.1%) had non-edematous SAM whereas 56 (62.9%) had edema-tous SAM. On admission, 92% of patients showed evidence of pancreatic insufficiency as measured by FE-1 < 200 μg/g of stool. Patients with edematous SAM were more likely to have low FE-1 (98 vs. 82.8%, p=0.026). FE-1 levels remained low in these individuals throughout the assessment period. Serum trypsinogen was elevated (>57 ng/ml) in 28% and amylase in 21% (>110 U/l) of children, suggesting pancreatic inflammation.

Conclusions: Exocrine pancreatic insufficiency is highly prevalent in children with SAM and especially in children with edematous SAM. In addition, biochemical signs suggestive of pancreatitis are common in children with SAM. These results have implications for standard rehabilitation treatment of children with SAM who may benefit from pancreatic enzyme replacement therapy.

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exocrine pancreatic insufficiency in children with severe acute malnutrition

InTRODUCTIOn

Despite a decrease over the last decade, mortality rates in children remain high with up to forty-seven percent of deaths in children under five years occurring in Sub-Saharan Africa (1,2). An estimated forty-five percent of deaths worldwide are attributable to under-nutrition defined as a weight for height (W/H) less than or equal to -2 standard deviations (SD) from the norm (3,4) despite protocolized WHO treatment(5–7).Diarrhea is commonly found in children with severe acute malnutrition (SAM) and is associated with increased risk of death (8–10). The broad etiologies of diarrhea in SAM include enteropathy related to malabsorption leading to osmotic diarrhea and infectious secretory diarrhea (11). Extra-intestinal factors such as changes in bile acid secretion or exocrine pancreatic function have not been well studied in the etiology of diarrhea in SAM. Previous studies have suggested that severely malnourished children may suffer from exocrine pancreatic insufficiency (EPI) (12–23). EPI is defined as a lack of digestive en-zyme production; this in turn leads to impaired weight gain and growth due to protein and lipid malabsorption (24). EPI is a common complication in diseases such as Cystic Fibrosis (CF) (25), Shwachman-Diamond syndrome (26), and HIV (27). In children with CF, pancreatic function is an important predictor of long-term survival (28). Nearly all studies on pancreatic (dys-) function in malnourished children were done in the 1940s -1980s in small groups of children (12–14). These studies showed that mal-nourished children had reduced pancreatic enzymatic output compared to reference ranges (15,16,19) with evidence that pancreatic function recovered after refeeding (13,15,16,29). Autopsy studies on children with malnutrition describe a combination of pancreatic atrophy and loss of zymogen-secreting pancreatic acinar cells in SAM (16–19,30–32). Since these studies, the assessment of pancreatic function has advanced and warrants reassessment.The current diagnosis of EPI relies on ‘direct’ or ‘indirect tests’ of exocrine pancreatic function (33). Direct tests are expensive and invasive, which limits their use in children and in low-resource settings or routine clinical practice (34). More feasible tests are in-direct test via the measurement of pancreatic enzymes in serum (trypsinogen, amylase), in stool (fecal elastase-1, fecal chymotrypsin), or the detection of C13-mixed-triglyceride in a breath test (33). Fecal elastase-1 (FE-1) is a clinically validated marker with good specificity and sensitivity to diagnose severe EPI and is currently recommended as a screening tool of EPI (33,35,36). The role of trypsinogen in detecting pancreatic insuf-ficiency is valuable in the assessment of pancreatic function in patients with CF (37,38). Both serum trypsinogen and amylase levels are released by damaged pancreatic cells and are therefore used as a marker of pancreatitis (39,40). Their use in diagnosing EPI is limited by their low sensitivity and specificity (33).

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The aim of this study was to assess pancreatic function children with SAM. We hypoth-esized that pancreatic function in children with SAM as assessed by FE-1 and serum trypsinogen and amylase levels is: 1) impaired, 2) correlates to clinical outcomes of duration of hospital stay and number of days from admission to clinical stabilization, 3) differs between edematous versus non-edematous malnutrition, and 4) improves during nutritional rehabilitation.

METHODS

Study design and population This observational study was completed within the framework of a nutrient prospective intervention trial (ISRCTN 13916953). This “TranSAM Study” was conducted at the MOYO Nutritional Rehabilitation Unit (NRU) of the Pediatric Department at Queen Elizabeth Central Hospital in Blantyre, Malawi. Sample size calculations were originally based on numbers needed to assess the primary outcome of the TranSAM study (carbohydrate malabsorption). The TranSAM study aimed to determine whether the use of transition phase diets with different carbohydrate contents affected fecal pH, length of stay and other clinical outcomes in severely malnourished children. For the TranSAM study, children were randomly assigned to treatment with either F75 + RUTF (Ready-to-use therapeutic foods), RUTF only or F100 after ‘clinical stabilization’ (absence of acute life-threatening conditions, return of appetite, improvement of gastrointestinal losses and edema, and absence of WHO ‘danger signs’). Accounting for contingencies, the study aimed to recruit a total of 108 patients to detect a 20% difference in primary outcome with a=0.05 and 80% power based on previous findings (41). The study was approved by the Malawi College of Medicine Research and Ethics Committee and carried out accord-ing to Good Clinical Practice guidelines which are based on the Declaration of Helsinki (42). Children with SAM admitted to the NRU between January 2013 and July 2013 (n=509) were screened for recruitment. Informed consent was obtained from parents or guard-ians by verbal and printed explanations in Chichewa, the main local language in Malawi, or English with witnessed consent by signature or by thumbprint for those unable to write.Inclusion criteria were: children aged 6 – 60 months admitted with a diagnosis of severe acute malnutrition as defined by WHO by a weight-for-height (W/H) of less than – 3 SD and/or a mid-upper arm circumference (MUAC) of less than 115 mm (non-edematous malnutrition) and/or presence of bilateral edema (edematous malnutrition) (43). Both HIV positive and negative children were included. We excluded children who were pre-viously admitted to the NRU within the year or presented with severe hemodynamic

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instability, a hematocrit level of ≤15%, or severe neurological symptoms. After admission children were treated according to WHO guidelines (5).

Clinical data and sample collection All children admitted to MOYO NRU had a thick blood film examined for parasitemia (malaria) and a hematocrit count done. HIV antibody test was offered with appropriate pre- and post-counseling. After initial anthropometry, the following clinical data were collected daily: weight, stool frequency and consistency, number of days until clinical stabilization, and duration of hospital stay. Blood was collected at admission and stool collected on admission and three days after initial clinical stabilization. Upon collection, stool samples were immediately homogenized and frozen at -80oC until further analysis.Diarrhea was defined according to WHO standards (3 or more loose or watery stools in the past 24 hours) (44). Severe diarrhea was defined as 10 or more loose or watery stools in the past 24 hours. Information about stool frequency, consistency and (severe) diarrhea was obtained from the mother or guardian through verbal recall.

laboratory Analysis FE-1 levels were determined using enzyme-linked immune assay (ELISA) at the clinical laboratory of the University Medical Center Groningen, the Netherlands. The same procedures were applied to watery and non-watery stool. In accordance with standard practice, exocrine pancreatic insufficiency was defined as FE-1 levels below 200 μg/g of stool and severe exocrine pancreatic insufficiency as FE-1 levels below 100 μg/g of stool (35,45).Serum trypsinogen concentrations were determined in a random subset of patients (N=39) by the clinical laboratory of the Hospital for Sick Children, Toronto, Canada us-ing a radioimmunoassay as described previously (46). For this study, reference values for normal trypsinogen levels were 10 - 57 ng/ml (Hospital for Sick Children, Toronto, Canada). Serum pancreatic amylase concentrations were determined in 80 patients by ELISA (Abcam, Cambridge, UK). The upper limit of normal was set at 110 U/l.

Data and Statistical AnalysesData were collected on standardized forms and analyzed with IBM® SPSS® Statistics Ver-sion 22.0.0.0 Software (47) and with R statistical software (Version 3.2.2). Descriptive statistics were used to show baseline characteristics of study participants. Median dif-ferences in levels of serum trypsinogen, serum amylase, and fecal FE-1 were tested by Wilcoxon rank sum test. Fisher’s Exact test was used to analyze differences in patient numbers between clinical groups. Mixed effects logistic regression models were used to compare FE-1 levels of patients at admission and three days post stabilization to account for within-patient measures; i.e. patient set as a random factor and accounting for miss-

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ing values. Non-parametric Spearman’s correlation was used to relate FE-1, trypsinogen, and amylase. All tests were conducted at 95% level of significance.

ReSUlTS

Baseline characteristics In total, 89 children with SAM were included in this study; clinical characteristics are detailed in Table 1. In this cohort, most children were diagnosed with edematous SAM as opposed to the non-edematous form. Thirty-three patients were HIV-reactive of whom twenty-seven were newly diagnosed and four already on antiretroviral therapy at admis-sion; two patients had missing information on HIV treatment status. Mortality amongst the study cohort was 15.7% (n=14) and was significantly higher in the non-edematous group compared to the edematous group (27.3 vs. 8.9%, p=0.03). No significant differ-ences in mortality were found in HIV non-reactive versus HIV-reactive patients (12.5% vs. 21.2%, p=0.4).

Table 1. Patient characteristics

N=89

Edematous, n (%) 56 (62.9%)

Male, n (%) 39 (44.3%)

Age (months) 21 (16-27)

HIV reactive, n (%) 33 (37.1%)

Weight on admission (kg) 7.3 (5.5-8.7)

Weight for Height (SD) <= -3, n (%) 51 (58.0%)

MUAC (cm) < 11.5, n (%) 45 (50.6%)

Duration of illness before admission (days) 14 (7-28)

Duration of hospital stay (days) 9 (8-12)

Discharged alive, n (%) 71 (79.8%)

Died, n (%) 14 (15.7%)

Absconded, n (%) 4 (4.5%)

Median (inter quartile range); missing values sex (1, 1.1%), Weight for Height (1, 1.1%), duration of ill-ness (4, 4.5%), MUAC is mid upper arm circumference.

Pancreatic insufficiency in patients with SAM On admission, overall levels of FE-1 were markedly reduced, specifically in the edema-tous group (Table 2). Evidence of pancreatic insufficiency (FE-1 < 200 μg/g of stool) was seen in 92.2% of patients, while prevalence of severe pancreatic insufficiency (FE-1 < 100 μg/g of stool) was 76.6% on admission. Edematous SAM patients had significantly

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lower FE-1 levels on admission as compared to patients without edema (median 22 μg/g of stool, IQR 15 – 57.5 vs. median 80 μg/g of stool, IQR 19-150, p=0.009; Figure). Severe pancreatic insufficiency was significantly higher in the edematous group compared to the non-edematous group (n=42, 87.5% vs. n=17, 58.6%, p=0.006). During hospital ad-mission, FE-1 levels increased only modestly across all patients from 35 μg/g of stool (IQR 15-90) on admission to 69 μg/g of stool (IQR 15-160) three days after clinical stabilization (p=0.03). FE-1 levels remained abnormally low in the majority of children (82.9%). FE-1 levels can appear misleadingly low in patients with watery diarrhea (33), therefore we conducted an analysis in which they were excluded (n=17). FE-1 was significantly lower (p=0.004) in patients with watery stools than in those with semi-formed or normal stools (median of 15 μg/g stool, IQR 15 – 20 vs. 43 μg/g stool, IQR 16 – 115, p=0.004); however, 57 children (90.5%) without watery stools had low FE-1 levels and 46 (73.0%) showed severe pancreatic insufficiency (FE-1 < 100 μg/g of stool). This study was not powered to test differences in mortality, and no differences were found between pan-creatic sufficient versus insufficient patient groups. We did not find any relation between FE-1 levels and sex, age, HIV status, length of hospital stay, or number of days until clinical stabilization.

Table 2. SAM patients with abnormal measurements of markers of pancreatic insufficiency and pan-creatic inflammation

Abnormal, n (%)

Admission n= Clinical Cut-off All patients Non-edematous Edematous p-value

Trypsinogen (ng/mL) 39 > 57 ng/ml 11/39 (28%) 9/20 (45%) 2/19 (11%) 0.03 *

Amylase U/l 80 > 110 U/l 17/80 (21%) 9/31 (29%) 8/49 (16%) 0.3

Fecal Elastase-1 (μg/g) 77 < 200 ug/g 71/77 (92%) 24/29 (83%) 47/48 (98%) 0.03 *

< 100 ug/g 59/77 (77%) 17/29 (59%) 42/48 (88%) 0.006 **

3 days Post-Stabilization n= Clinical Cut-off All patients Non-edematous Edematous p-value

Fecal Elastase-1 (μg/g) 70 < 200 ug/g 58/70 (83%) 20/24 (83%) 38/46 (83%) 1

< 100 ug/g 42/70 (60%) 14/24 (58%) 28/46 (61%) 1

Number of patients out of total patients, with or without edema, that present with abnormal pancreatic markers. Clinical cut-off for fecal elastase of <200 ug/g represents pancreatic insufficiency, <100 ug/g is deemed severe pancreatic insufficiency. p-values obtained with Fisher’s exact test; significance code: * <0.05, ** <0.01.

Pancreatic inflammation in patients with SAM We measured levels of serum trypsinogen in a random subset of children (n=39). High levels (>57 ng/ml) were found in 11 patients (28.2%); this group had median serum tryp-sinogen values of 130.7 ng/ml (IQR; 70.8 – 239.2). The number of patients showing high

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trypsinogen levels differed between edematous and non-edematous groups (10.5%, n=2 vs. 45%, n=9, p=0.03). Trypsinogen levels did not correlate with FE-1 (P=0.15, p=0.4). No significant difference in mortality was found between those with normal vs. abnormal trypsinogen levels at admission. We found no relation between serum trypsinogen levels and sex, age, HIV status, length of hospital stay, or duration in days until clinical stabiliza-tion. Serum pancreatic amylase levels were determined in 80 patients. Levels above 110 U/l were found in 21% of children (n=17) with a median level of 148.4 U/l (IQR; 119.5 – 202.3). Serum amylase levels did not significantly differ between non-edematous and edematous patients (p=0.4). A significant correlation was found between amylase and trypsinogen levels (P=0.36, p=0.03). Serum amylase levels did not differ with sex, age, HIV status, length of hospital stay, days until clinical stabilization, or mortality.

Figure 1 Fecal elastase-1 levels in severely acute malnourished children with or without edema on admission and 3 days post-discharge. Dark grey shaded boxes indicate admission, light grey: 3 days post-stabilization. Boxplot midline indicates median; whiskers, the inter-quartile range, and outlying values indicated with black dots. Difference be-tween time points (i.e. admission and 3 days post-stabilization) tested by mixed effect logistic regression with patient as a random factor. Significance code: * <0.05.

DISCUSSIOn

To our knowledge, this study is the largest to investigate pancreatic function in children with severe acute malnutrition and is the first to assess their pancreatic function using clinically validated tests. The results strongly support our hypothesis that pancreatic

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function is impaired in children with SAM. Our data suggest that both exocrine pancreatic insufficiency and pancreatic inflammation are common in children with SAM.Pancreatic function can be tested both direct and indirectly. Direct tests are expensive, invasive, not well standardized for children and unavailable in most low resource set-tings like Malawi. Therefore, we chose to use indirect clinically validated tests. These can be divided in three categories; those that: 1) assess urine or serum for markers of pancreatic enzyme processing; 2) analyze unabsorbed or undigested fat in feces or, 3) use breath tests and measures of pancreatic enzymes in stool or serum (36). FE-1 is the most sensitive indirect test and the most widely used clinically to evaluate EPI in high-income settings (33). Immunoreactive trypsinogen measured in serum is currently used for newborn screening of CF and is elevated when the pancreas is inflamed (33). However, the reliability of trypsinogen as a marker for EPI below the age of seven has been questioned (36,48). Serum amylase concentrations are used to diagnose pancreatic inflammation, i.e. pancreatitis, but only have a supporting role in the diagnosis of EPI in conjunction with additional clinical and biochemical information (33,49). Increased concentrations of amylase can also be found with intestinal obstruction or reduced renal clearance (50).As compared to clinical reference values, our data indicate that the majority of children with SAM have EPI. Significant differences in the degree of EPI were found between children with and without edematous SAM, as both moderate and severe EPI were more prevalent in the edematous group. In addition, our data suggest that pancreatic insuf-ficiency persists beyond initial clinical stabilization. The high prevalence of EPI seen in our cohort of children with SAM is consistent with previous studies that revealed pancreatic insufficiency via duodenal aspirates and post-mortem investigations (12-22); however, these studies lack clear reference values and are mostly published between 1940 – 1980 with varying sample sizes. These studies do not offer information on the differences in the prevalence of EPI between edematous and non-edematous SAM. In 2005, a small study by el-Hodhod et al. reported exocrine pancreatic dysfunction in SAM patients by relating pancreatic size determined by ultrasound to changes in serum amylase and lipase levels (23). The elevated trypsinogen and amylase concentrations found in a significant proportion of children with SAM suggest that in addition to EPI, pancreatic inflammation is also present. Pancreatic inflammation has not been reported in children with SAM, but it has been reported in case studies of patients with eating disorders leading to malnutrition (51). The presence of pancreatic inflammation may be related to the presence of a systemic pro-inflammatory state in children with SAM due to increased pro-inflammatory cytokines (52) perhaps in combination with decreased anti-oxidant concentrations (53,54). For this study, we chose an amylase concentration above 110 U/l as abnormal, which has been used before. However, this cut-off value is

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conservative, as our measures of pancreatic amylase are consistently lower than those found in healthy children (55). The differences in amylase and trypsinogen levels in edematous vs. non-edematous SAM phenotypes suggest the possibility of differences in the pathophysiology leading to pancreatic damage in these children. Brooks et al. reviewed histological sections of pancreas and liver from 65 children who died of malnutrition-related causes and found a high rate of pancreatic atrophy (20); it is possible that this pancreatic atrophy and fibrosis seen after longstanding malnutrition in children with edematous SAM can result in very low levels of trypsinogen production (18). Our study did not find relations between the degree of pancreatic insufficiency or inflam-mation and clinical outcomes of duration of hospital stay or length to reach stabilization. This is likely due to the small number with normal FE-1 levels (>200ug/ml) (n=6). In addi-tion, our study was not designed to capture clinical symptoms normally associated with pancreatitis such as vomiting, anorexia, or epigastric abdominal pain. One of the limitations of our study was the lack of a control group of healthy Malawian children. However, previous research has shown that EPI is significantly more prevalent in children with SAM versus population controls (13,15,21). A second limitation is the re-liability of FE-1 in watery stools, which can be low due to dilution rather than decreased production (33). However, when evaluating FE-1 levels in the subset of children without diarrhea, the prevalence of EPI was only marginally lower than those with diarrhea; this suggests that FE-1 reveals EPI in most children with SAM. A third limitation is the short time that passed between stool sample collection for FE-1 analysis. To study the long-term influence of nutritional rehabilitation on pancreatic function, it would have been interesting to repeat our measures after a longer period of time. A fourth limitation was that the trypsinogen testing could only be conducted in 39 patients out of the 89 as the quantity of samples obtained were insufficient to complete all assays in all patients. Finally, we did not have enough biological specimens to determine other markers of pan-creatic function or inflammation, such as serum lipase concentrations. Lipase is known to be a more sensitive marker of pancreatic inflammation than amylase in infants and toddlers (56). In conclusion, our study shows that EPI is highly prevalent in children with SAM and more common in children with edematous malnutrition. Furthermore, our study is the first to document signs of pancreatic inflammation in these children. We additionally showed that short-term nutritional rehabilitation improves but does not normalize exocrine pancreatic function. Future research is needed to investigate whether this patient popu-lation would benefit from the addition of treatments addressing EPI such as pancreatic exocrine replacement therapy (PERT) or the use of a diet adjusted to account for an altered digestion secondary to pancreatitis.

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ACKnOWleDGeMenTS

We would like to thank all study participants and their guardians for their participation and the nursing staff and clinical officers of the MOYO NRU at Queen Elizabeth Central Hospital in Blantyre, Malawi, for their hard work in conducting high-quality patient care and research.

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52. González-Torres C, González-Martínez H, Miliar A, Nájera O, Graniel J, Firo V, et al. Effect of Malnutrition on the Expression of Cytokines Involved in Th1 Cell Differentiation. Nutrients. 2013;5(2):579–93.

53. Sive AA, Subotzky EF, Malan H, Dempster WS, Heese HD. Red blood cell antioxidant enzyme concentra-tions in kwashiorkor and marasmus. Ann Trop Paediatr. 1993;13(1):33–8.

54. Tatli MM, Vural H, Koc A, Kosecik M, Atas A. Altered anti-oxidant status and increased lipid peroxidation in marasmic children. Pediatr Int Off J Japan Pediatr Soc. 2000;42(3):289–92.

55. Kelly J, Raizman JE, Bevilacqua V, Chan MK, Chen Y, Quinn F, et al. Complex reference value distribu-tions and partitioned reference intervals across the pediatric age range for 14 specialized biochemi-cal markers in the CALIPER cohort of healthy community children and adolescents. Clin Chim Acta. 2015;450:196–202.

56. Coffey MJ, Nightingale S, Ooi CY. Diagnosing acute pancreatitis in children: What is the diagnostic yield and concordance for serum pancreatic enzymes and imaging within 96 h of presentation? Pancreatol-ogy. 2014;14(4):251–6.

Chapter 4Pancreatic enzyme replacement therapy in children with severe acute malnutrition: A randomized controlled trial

Rosalie H. Bartels, Céline Bourdon, Isabel Potani, Brian Mhango, Deborah A. van den Brink, John S. Mponda, Anneke C. Muller Kobold, Robert H. Bandsma, Michael Boele van Hensbroek, Wieger P. Voskuijl

Journal of Pediatrics 2017: 190:85–92.e2

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ABSTRACT

OBJeCTIVe: To assess the benefits of pancreatic enzyme replacement therapy (PERT) in children with complicated SAM.

STUDy DeSIGn: At Queen Elizabeth Central Hospital in Malawi, we conducted a random-ized controlled trial in 90 children aged 6-60 months with complicated SAM. All children received standard care, the intervention group also received PERT for 28 days. Outcome measures were: percentage of weight change, EPI measured by levels of Fecal Elastase-1 (FE-1), duration of hospital stay, mortality and digestive function reflected by fecal fatty acid split-ratios.

ReSUlTS: Children treated with PERT for 28 days did not gain more weight than controls (13.7±9.0% in controls vs. 15.3±11.3% in PERT, p=0.56). EPI was present in 83.1% of patients on admission and FE-1 levels increased during hospitalization mostly seen in children with non-edematous SAM (p<0.01). Although this study was not powered to detect differences in mortality, mortality was significantly lower in the intervention group treated with pancreatic enzymes. Children that died had low fecal fatty acid split-ratios at admission. EPI was not improved by PERT, but children receiving PERT were more likely to be discharged with every passing day (p=0.02) compared to controls.

COnClUSIOnS: PERT does not improve weight gain in severely malnourished children but does increase the rate of hospital discharge. Mortality was lower in patients on PERT: a finding that needs to be investigated in a larger cohort with stratification for edematous and non-edematous malnutrition. Mortality in SAM is associated with markers of poor digestive function.

Trial registration ISRCTn.com: 57423639

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Pancreatic enzyme replacement therapy in children with severe acute malnutrition

InTRODUCTIOn

Although childhood-mortality globally decreased by 53% between 1990 and 2015, six-teen thousand children under the age of five still died every day in 2015 (1). Sub-Saharan Africa has the highest share of global under-five mortality (47%) (1). Under-nutrition, as defined by low weight for height (W/H ≤ -2 standard deviations (SD)), contributes to approximately 45% and severe wasting (W/H < -3 SD) to 7.8% of these deaths (2,3). Even if World Health Organization (WHO) treatment protocols are followed rigorously, case fatality rates remain high which underlines the urgent need for better treatment strate-gies (3–5).Severe diarrhea is common in children with severe acute malnutrition (SAM) and greatly increases mortality (6–8). Diarrhea in SAM is not only caused by infections (9) and intes-tinal epithelial dysfunction relating to malabsorption (10), but also by impaired digestion. The exocrine pancreas plays a central role in nutrient digestion by secreting digestive enzymes (e.g. amylase, lipase, trypsinogen, etc.). Exocrine pancreatic insufficiency (EPI) in conditions such as cystic fibrosis (CF) is linked to nutrient malabsorption, poor nu-tritional status and mortality (11). Several ‘classic’ studies, mostly performed between 1940 and 1980, have suggested that children with SAM also have EPI (12–23). We have recently confirmed these findings using contemporary techniques and showed that the prevalence of EPI was 93% in Malawian children with SAM (24). Also, those with the edematous form of SAM (i.e., presenting with nutritional bilateral pitting edema) had more severe EPI than those with non-edematous SAM (i.e., severe wasting) (24). In EPI patients with other underlying etiologies than SAM (25–27), it is standard clinical practice to start pancreatic enzyme replacement therapy (PERT) (28,29) with the aim of restoring nutritional status by improved digestion. The benefits of using PERT to treat children with SAM have not been thoroughly inves-tigated. Only one study, performed in 1988 by Sauniere et al., attempted to assess PERT as a potential treatment (30). The study did not report improvements of pancreatic func-tion but was limited by a small sample size (n=7 and n=8), inadequate dosage and short duration of PERT treatment. The primary objective of this study was to assess the effect of PERT on weight gain of children hospitalized for severe acute malnutrition. Secondary objectives were to com-pare the effect of PERT on: 1) exocrine pancreatic function as assessed by fecal elastase-1 levels (FE-1), 2) duration of hospital stay and 3) mortality and, 4) digestive function as-sessed by free fatty acids (FFA) and triglycerides (TG).

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SUBJeCTS AnD MeTHODS

Study design and populationThis prospective, randomized, single blinded pilot study (OPTIMISM trial) was conducted at the Nutrition Rehabilitation Unit (NRU) in the Pediatric Department of Queen Elizabeth Central Hospital in Blantyre, Malawi (ISRCTN57423639). The study was approved by the Malawi College of Medicine Research and Ethics Committee (COMREC nr P.11/12/1306) and conducted according to guidelines of Good Clinical Practice which are based on the principles of the Declaration of Helsinki(31). Between February and September 2014, we screened children with complicated SAM (i.e. those with signs of severe clinical illness and/or poor appetite(32)) that were admit-ted to the NRU. Parents/guardians were informed about the study both verbally and with printed information in Chichewa or English. Before performing any research procedures, written consent was obtained, and for those unable to read or write the information was read to them in Chichewa. Consent was confirmed through a signature or thumbprint by the parent or guardian, witnessed by a staff member of the study. All staff were fully trained prior to starting the study. All authors had access to the study data and reviewed and approved the final manuscript.

Inclusion and exclusion criteriaInclusion criteria were: children aged 6 – 60 months, admitted to hospital with a diagno-sis of SAM. SAM was defined according to WHO standards, by any of the following: a W/H below -3 SD (non-edematous SAM/marasmus), a mid-upper arm circumference (MUAC) of less than 115 mm (non-edematous SAM/marasmus), or the presence of bilateral edema (edematous SAM/kwashiorkor) (33).Patients were excluded if they had malaria (assessed by a positive blood smear), or signs suggestive of severe underlying systemic illness such as sepsis, severe pneumonia or severe diarrhea.

Randomization and selection processAn independent researcher prepared sealed envelopes using a computerized randomiza-tion program (34) and these were used to assign patients to treatment groups. The study was stratified for HIV status to ensure equal distribution of HIV reactive patients between groups.

Inpatient careFor all children admitted to the NRU, a thick blood film was examined for parasitemia and hematocrit counts. All patients were offered an HIV antibody test with appropri-ate pre- and post-counseling. During hospital stay, all children were treated according

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to national/WHO guidelines (32,35). Baseline characteristics were obtained and health questionnaires completed. Appetite, gastrointestinal losses, degree of edema, hydration state and vital signs were recorded daily. The presence and severity of diarrhea, defined as having 3 or more loose or watery stools per day(36), was assessed using standard-ized departmental pro-forma. An assigned nurse recorded daily weight; she was both unaware of treatment groups and not involved in the everyday care of the study patients (single blinded). Body weight was measured using a Marsden 4201 digital scale, which was calibrated daily. Supine length was taken using a measure board.

InterventionPatients assigned to the PERT group were prescribed 3000 Units of Lipase/kilogram of bodyweight to be taken 3 times a day with an upper limit dose of 10,000 Units Lipase/kg bodyweight per day (37). Each PERT capsule contains enteric-coated mini-microspheres of porcine-derived lipase (10.000 PhEur units), amylase (8.000 PhEur units) and protease (600 PhEur units). PERT was administered immediately before a feed. To enhance intake, capsules were opened and granules mixed into a spoonful of apple-sauce (pH <5.5). This acidity avoids the dissolution of the protective enteric coating of the granules. PERT intake was monitored. Serious adverse events (SAEs), defined as: skin rash, pruritus/urticaria, anaphylaxis or any episode of clinical deterioration accompanied by shock or respira-tory distress (respiratory rate > 60/min) or oxygen requirement (O2 saturation <94%) or impaired consciousness (Blantyre coma score <4) or hypoglycemia (serum glucose of <3 mmol/L), were recorded and would lead to patient withdrawal from the study; one child in the PERT group was withdrawn because of urticaria.

laboratory investigationsFecal samples were obtained on admission, day-14 and -28 and homogenized prior to storage at -80oC. To measure EPI, FE-1 levels were determined in stool using an enzyme-linked immunosorbent assay (pancreatic elastase ELISA, Bioserv Diagnostics GmbH, Rostock, Germany) at the clinical laboratory of the University Medical Center Groningen in the Netherlands. EPI was defined as FE-1 levels below 200 ug/g of stool, and severe EPI as below 100 ug/g of stool (38,39). Digestive function was measured by split-ratios of free fatty acids (FFA) and triglycerides (TG) on admission and day 28. This ratio can reflect failed fatty acid breakdown (i.e. high proportion of TG in total fecal fatty acids) and/or failed absorption (i.e. high proportion of FFA in total fecal fatty acids) (40). FFA and TG were measured with Fourier transform infrared (FTIR) spectroscopy using a simple hex-ane extraction procedure for stool (41). Briefly, an aliquot of the extracted hexane layer was directly injected into the measurement cell of the spectrophotometer, a BioRad Excalibur Series, Model FTS 3000. Split-ratios were then calculated by: 1) Converting FFA and TG from grams to moles (using the molecular weight of oleic acid for FFA (282.46g/

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mol) and three times the weight of oleic acid for TG (847.38g/mol)); 2) Calculating total fatty acids in feces by the sum of FFA and TG; and 3) obtaining the split-ratio by dividing FFA by total fatty acids.

Statistical Analyses Sample size calculations were based on estimates derived from a large cohort of children previously admitted to the NRU. That cohort showed a mean weight-gain of 13.9% ±11.0 which was calculated using the difference between the lowest weight recorded during hospital stay and a follow-up weight obtained after 28 days. The present study aimed to detect 10% difference in weight-gain between the intervention and control groups. Assuming a standard deviation of 11.0, 26 patients would be required in each arm of the study (α=0.05, β=0.1). As this study was designed to guide a future trial, we aimed to include 50 patients in each group to attain 99.5% power to detect a 10% effect of PERT on weight gain and insure against contingencies. For the calculation of weight change, weight after 28 days was compared to lowest weight during hospital stay instead of weight on admission since children with edematous malnutrition will initially lose their edema and therefore weight.Interim analysis was performed after 50% of patients were included in the study; mortal-ity between both arms was assessed by an independent monitor. Pearson’s chi-square test and a 1% significance threshold level were used. The detection of a statistically significant mortality increase in the intervention group would have led to the immediate termination of the trial. Data were collected on standardized forms, entered into an Access 2013 database and analyzed with Stata (Release 13)(42) and with R (Version 3.2.3) statistical software. The baseline characteristics of the study participants in both groups were compared as appropriate using Fisher exact test, two-way ANOVA or logistic regression. A two-way ANOVA with or without correcting for HIV status was used to test for group differences in % weight gain. Because non-edematous and edematous SAM display distinct clinical and biochemical characteristics, we also conducted sub-analysis for these groups. We used generalized linear models to analyze group differences and mixed effects models when needing to account for repeated measures. The competitive risk analysis was done to de-termine if time-to-discharge and time-to-death differed between treatment groups using the cmprsk R-package (43). This analysis produces an incidence function that indicates the cumulative probability of either being discharged or dying as treatment progresses.

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ReSUlTS

Baseline characteristicsBetween February 24th 2014 – September 30th 2014, a total of four hundred and thirty children were admitted to the NRU with SAM and ninety provided consent and were randomized to receive PERT as a supplement over standard care or to receive standard care only. Overall, twenty-five children died (27.8%) and fifty-nine (65.6%) completed the 28-day follow up (Figure 1). Patients’ characteristics on admission are described in Table 1. Despite randomization the PERT group had a higher percentage of children with edematous malnutrition than the control group (69% vs 44%, p=.03). The number of children (n=6, 6.7%) lost to follow-up did not differ between groups (Figure 1). The main comorbidities on admission were gastroenteritis and pneumonia and were evenly prevalent in both groups.

Figure 1. Flowchart of patient enrolment, randomization and follow-up for OPTIMISM study.

Percentage of weight change after 28 days of PeRTAfter 28 days, the control group showed an average weight gain of 13.7% ±9.0 which did not differ from the PERT group (15.3% ± 11.3, p=.56) (Figure 2). Edematous patients receiv-ing PERT did not lose weight faster (p=.2) than edematous patients in the control group. HIV status also did not influence weight change after 28-days of treatment. Changes in age- and sex-corrected Weight-for-Height z-scores also did not show any group differences.

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Table 1. Characteristics of patients randomized to receive standard treatment or PERT intervention

All Patients Control PERT

N=90 n=45 n=45 P

Male, n (%) 50 (56%) 25 (56%) 25 (56%) 1

Age, months Ϯ 21.3 ±11.8 20.0 ±12.2 22.7 ±11.4 0.3

HIV reactive, n (%) 41 (46%) 21 (47%) 20 (44%) 1

Edematous, n (%) 51 (57%) 20 (44%) 31 (69%) 0.03

MUAC, cm Ϯ 11.4 ±1.7 11.2 ±1.8 11.5 ±1.7 0.5

Non-edematous 10.2 ±1.1 10.2 ±1.1 10.2 ±1.1 0.9

Edematous 12.3 ±1.6 12.5 ±1.7 12.1 ±1.6 0.3

Weight-for-age, Z-score Ϯ -3.6 ±1.7 -3.6 ±1.8 -3.5 ±1.6 0.7

Non-edematous -4.6 ±1.0 -4.5 ±1.0 -4.8 ±1.0 0.5

Edematous -2.7 ±1.7 -2.5 ±2.0 -2.9 ±1.5 0.4

Weight-for-length, Z-score Ϯ -2.7 ±1.8 -2.9 ±1.8 -2.6 ±1.8 0.5

Non-edematous -3.9 ±1.2 -3.9 ±1.3 -3.9 ±1.0 1

Edematous -1.8 ±1.6 -1.5 ±1.4 -1.9 ±1.7 0.3

Breastfeeding, yes (%) 41 (46%) 24 (53%) 17 (38%) 0.2

Duration of illness before admission, days ¤ 7 (3.8 - 28) 7 (3.5 - 21) 14 (4 - 28) 0.4

Diarrhea, yes (%) 31 (34%) 14 (31%) 17 (38%) 0.7

Fever on admission (>37.5oC*), n (%) 9 (10%) 4 (9%) 5 (11%) 1

Hemoglobin, g/dl Ϯ 8.9 ±1.9 9.2 ±1.4 8.5 ±2.4 0.1

Values are presented as n (%), means and standard deviations(Ϯ) or median and interquartile range (¤). Fever cut-off for axillary temperature (*). Differences between groups were tested using either Fisher Exact test, two-way ANOVA or logistic regression. Significance threshold was considered to be p < .05. MUAC, mid upper arm circumference.

Figure 2. Percentage of weight change in children with severe acute malnutrition (SAM) after 28 days of PERT treatment (n=34) or standard care (n=25). Boxplots summarize the median (midline) and in-terquartile ranges (upper and lower box). Differ-ences in mean weight change was tested between groups using two-way ANOVA (p=.56).

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Fecal markers in children with or without edema on admission and after 28 days Overall levels of FE-1 were markedly reduced in children with SAM at hospital admis-sion (Supplemental Figure 1). Eighty-three % of admitted patients showed evidence of pancreatic insufficiency (FE-1 < 200 ug/g of stool), while 69 % showed severe pancreatic insufficiency (FE-1 < 100 ug/g of stool) (Supplemental Table 1). Children with edema had lower FE-1 levels with a median of 32 ug/g of stool, IQR (23 – 61) compared to 110 ug/g of stool, IQR (48 – 228, p=.002) in children without edema (Supplemental Figure 1). Consequently, pancreatic insufficiency was significantly more prevalent in patients with edematous malnutrition compared to those with the non-edematous form (EPI in edematous: n=36, 97% vs. non-edematous: n=22, 69%, p=.002; and severe EPI in edematous: n=33, 89% vs. non-edematous: n=15, 47%, p<.001) (Supplemental Table 1). These relationships were not modulated by HIV nor by the presence of diarrhea (which has been associated with misleadingly low FE-1 results (44)). After 28 days, overall FE-1 levels in children treated for SAM increased from 42 ug/g IQR (24 – 96) to 168 ug/g IQR (72 – 256, p<.0001). The prevalence of EPI in children that completed the study fell from 83% to 55% whereas severe EPI fell from 69% to 35%, irrespective of PERT (p<.6). When analyzing FE-1 levels by nutritional diagnosis, FE-1 level increased more in children with non-edematous SAM: 312.5 ug/g of stool IQR (223.8 – 371.2) compared to children with edema who only reached 102.5 ug/g of stool IQR (49.8 – 238, p<.002) (Supplemental Figure 1). Most children with edematous SAM still showed signs of EPI after 28 days of treatment (68%) and almost half (46%) had severe EPI.

Mortality and duration of admission in relation to PeRT treatmentAlthough this trial was not powered to detect differences in mortality between the treat-ment groups, mortality was significantly lower in the PERT treated group (PERT: n=8/43, 18.6% vs. Controls: n=17/45, 37.8%, p<.05) (Figure 3). The number of days between admission and death did not differ between the intervention and control groups (4.6±4.1 days vs. 4.9±3.5 days, p=.8); and neither did the number of days to discharge and death (6.7±2.6 days vs. 7.7±4.6, p=.14). The competitive risk analysis suggested that, compared to controls, children receiving PERT had a higher probability of being discharged on every passing day of treatment (p=.02). (Figure 3).

Mortality in relation to fecal markers and clinical characteristicsFE-1 levels at admission were not associated with mortality nor did they differ between the intervention and control group (Supplemental Figure 2). However, split-ratios of fatty acids measured in stool collected at admission, were significantly lower in children that died compared to those that survived with respective medians of 69% IQR (52 – 100) and 98% IQR (82 – 100, p=.002) (Supplemental Figure 2). Split-ratios did not differ with nutritional diagnosis, diarrhea or treatment groups as children that survived all showed

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split ratios of nearly 100% already (Supplemental Table 2). Mortality was not influenced by HIV status (p=0.72). Children that died were younger (15.5±9.3 months vs. 23.6±12.1 months) and had lower MUAC (10.5±1.7cm vs. 11.7±1.6cm).

Figure 3. Mortality in children treated with either PERT or standard of care.A) Percentage of mortality in each group. Black boxes indicate the percentage of children that died; white boxes indicate the percentage of those that survived. Group differences were tested with logistic regres-sion. B) Cumulative incidence curves representing the probability of discharge or death at any given day of hospitalization. Group differences were tested with competitive risk analysis which showed that the rate of discharge differed between controls and children receiving PERT (p=.02); whereas the rate of mortality at any given day was not significantly different (p=.051); difference in discharge rate was still significant between groups (p<.05) after accounting for edema status. The different line types indicate the cumulated incidence of discharge or death in the Control or PERT treated groups as detailed by the legend. *Significance code, p-value<.05.

DISCUSSIOn

This study shows that PERT treatment of EPI in children with complicated SAM does not improve weight gain after 28 days of treatment. Mortality in the intervention group was significantly lower although this trial was not powered to detect such a finding. Supple-mentation with PERT may be associated with an increased rate of hospital discharge. Malnourished children showed improvement of pancreatic function unrelated to PERT treatment but this was mostly seen in children with non-edematous SAM. Previous studies have demonstrated the high prevalence of EPI in children with SAM (12–24). However, this is the first large cohort interventional study examining PERT as a potential treatment for SAM. Previously, only Sauniere et al. (1988) reported on the use of pancreatic enzymes in children with edematous SAM but the study was very small (30). Their placebo controlled intervention consisted of giving porcine pancreatic powder three times daily for 5 days to 8 children in Ivory Coast and for 28 days to 8 Senegalese

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patients. No significant differences were found in concentrations of pancreatic enzymes in duodenal juice. This study was limited in its small sample size, short treatment of patients from the Ivory Coast and the inclusion of only children with edematous SAM. Also, clinical outcomes were not described. Our study emphasizes clinical outcomes relevant to daily practice, examines a larger cohort and includes both the edematous and non-edematous forms of SAM. Our primary outcome, weight change in children with SAM, did not differ between the intervention and control groups as described in patients with CF (11). This may be due to several reasons. The clinical use of PERT in children with CF aims to help maintain healthy nutritional status and growth whereas we aimed to use PERT as an aid-to-recovery from a severely malnourished state. Another issue may be compliance. Throughout hospital stay, PERT intake was closely monitored by clinical staff. However, PERT intake post-discharge was evaluated only through guardian reporting and counting of empty blister packets brought back on follow-up visits. Alternatively, we cannot rule out that twenty-eight days of PERT may not be long enough to affect weight change. The time frame was chosen as a ‘trade-off’ to avoid the loss to follow up frequently encountered in low resource settings. Finally, our study population are/were severely ill; children with complicated SAM suffer from severe acute illness and often present important co-morbidities such as pneumonia, tuberculosis or HIV. Focusing on impaired digestion to improve weight change may be too limited an approach to have a significant clinical impact in children with complicated SAM. Thus weight change is likely not the ideal primary outcome and short-term weight gain might not be realistic irrespective of the intervention. It is a het-erogenous parameter and weight might take longer to improve as many of the patients in our study were very wasted and severely ill.In this study, EPI was assessed by FE-1 as a marker of pancreatic function. EPI can be di-agnosed by ‘direct’ and/or ‘indirect’ tests of exocrine pancreatic function (27,44). Direct tests are not routine in clinical practice as they are invasive, require both exogenous hor-monal stimulation, and intubation of the pancreatic duct to measure the enzyme activity of pancreatic secretions (44). Less invasive, indirect tests measure pancreatic enzymes or their substrate/by-products in stool, serum, or breath (44). Measuring FE-1 in the stool is the most widely used indirect pancreatic function test; it has good specificity and sen-sitivity (86-100%) to diagnose severe EPI and is currently recommended as a screening tool (39,44,45). In line with previous studies that investigated EPI in SAM, we found that pancreatic function improved with nutritional rehabilitation (12,13,15,19–22,24,46). However, pancreatic function was not normalized even after 28 days of treatment, especially in the edematous group. Our study is the first to show that the recovery pat-tern of pancreatic function differs between children with edematous or non-edematous SAM. Children that presented with edema at hospital admission showed more severe EPI and only minimal improvements were achieved. These children may require specific and

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longer medical treatment to recover. Future treatment programs should consider having specific treatment modalities between the two different phenotypes of SAM.As a biomarker for gastro-intestinal digestion, we measured fecal FFA and TG to calculate the fatty acid split-ratio. However, the gold standard for diagnosing fat malabsorption (steatorrhea) is to quantitatively measure stool fat via a traditional biochemical assay (47). A coefficient of fat absorption can be calculated but this requires 3 days of feces col-lection with records of all dietary intake. A three-day collection of feces was unfeasible in our setting. This test is also known to lack specificity to differentiate between syndromes of malabsorption and maldigestion (48). Steatorrhea is not specific to pancreatic dysfunc-tion but can also reflect impaired digestion or absorption of dietary fats and is associated with multiple diseases including cystic fibrosis, chronic pancreatitis, cholestatic liver disease, celiac disease, and inflammatory bowel disease. FFA and TG and split-ratios on admission and after 28 days of treatment did not differ between groups. Split-ratios also did not vary with edema status, diarrhea or HIV reactivity. Most children showed very high splitting ratios, close to 100%. Split ratios were only found to be lower in children that died. However, based on split-ratios alone we cannot conclude failed absorption, since we have not taken into account the intake and output of fat and numerous other factors in SAM that influence this such as impaired bile homeostasis, enteropathy and small intestine bacterial overgrowth (49,50). Mortality was significantly lower in children that received PERT (17%) compared to 37% in children receiving standard of care. Our control and intervention group differed in the proportion of edematous and non-edematous malnutrition. In the past different mortality rates have been described between non-edematous malnutrition and edema-tous malnutrition, albeit not consistently (24,51). We therefore cannot clearly conclude that our finding for differences in mortality are explained a difference in phenotypical characteristics with more children with marasmus in the control group. The fact that the number of days between death in the two groups are similar also provides no insight into the mechanism behind the lower mortality in the PERT group. A future trial with a larger sample size and with mortality as a primary outcome should provide the answer to this question.Both the lower mortality as well as the significant increase in earlier hospital discharge rate in the intervention group stresses the possible beneficial effect of PERT early in the management of children with SAM, albeit not evident by our primary outcome. This could have important implications for the future management of SAM and therefore deserves further investigation in a larger cohort with stratification for SAM phenotype. Predictors of mortality were as previously described younger age, lower MUAC and lower W/H. However, a novel finding was that low split-ratios on admission were significantly associated with mortality and this was mostly driven by high levels of TG measured on admission in the stool of children that died. Since we have not taken into account the fat

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intake and output, we cannot confirm based on split-ratios alone that children who die are failing to digest and absorb fatty acids but this seems likely to be the case. Failing to process TG into FFAs that can be absorbed may be an acute marker of death and calculat-ing the split-ratio from levels of FFA and TG in a single stool sample may be a valuable marker of the digestive function. The breakdown of TG into FFAs and monoglycerides depends on several processes such as the emulsification of fats by bile acids produced by the liver and lipases secreted by the pancreas. Impaired bile acid homeostasis has been recently described in children with SAM by our group (49). Together with the high prevalence of EPI, this shows that the digestive system is severely impaired and likely contributes to mortality. The inpatient mortality rate for our NRU was high, but previous studies have reported similar rates around 20-30% (51–53). Since the development of the ‘CMAM guidelines’ (Community based Management of Acute Malnutrition), less acutely ill children now receive adequate management in district hospitals and are no longer referred to NRU’s like ours (4). Therefore, children admitted to hospital with malnutrition are those that are critically sick and at high risk of mortality.Our study has several strengths. First, the follow up rates were very high (93%). Secondly, we tracked weight, our primary outcome, on a daily basis during hospital admission. Thirdly, the study was single blinded as the nurse weighing the children was unaware of treatment allocation. Finally, our study examined the effects of PERT on relevant clinical outcomes that are routinely used in low resource settings. However, in addition to issues already discussed, our study would have gained from a longer follow-up which would have helped evaluate recovery of the pancreas function in children with and without edema. In conclusion, our study showed that 1) PERT does not improve weight gain in children with complicated SAM, 2) mortality is lower in the intervention group treated with pan-creatic enzymes, and that markers of maldigestion are associated with higher mortality, 3) that EPI shows modest improvement after 28 days of nutritional rehabilitation but this mostly in children with non-edematous SAM and this improvement was not related to PERT, and 4) that the rate of discharge from hospital may be influenced by PERT. A larger cohort is needed to confirm our findings focusing on the effect of PERT on mortality. If the current results are confirmed PERT should be considered as an additional treatment available for children with SAM worldwide.

ACKnOWleDGeMenTS

We would like to thank all study participants and their guardians for their participation and dedication to this study. We also would like to thank all members of our research and

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lab team (all clinicians, nurses, data managers, lab personnel and kitchen staff), located in Malawi, The Netherlands and Canada, who have worked hard to make this research possible. We thank Dr Alfred van Meurs for his contribution to the original idea for this trial.

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9. Attia S, Versloot CJ, Voskuijl W, van Vliet SJ, Di Giovanni V, Zhang L, et al. Mortality in children with com-plicated severe acute malnutrition is related to intestinal and systemic inflammation: an observational cohort study. Am J Clin Nutr 2016;104:1441–9.

10. Bandsma RHJ, Spoelstra MN, Mari A, Mendel M, van Rheenen PF, Senga E, et al. Impaired Glucose Absorption in Children with Severe Malnutrition. J Pediatr 2011;158:282–287.e1.

11. Littlewood JM, Wolfe SP, Conway SP. Diagnosis and treatment of intestinal malabsorption in cystic fibrosis. Pediatr Pulmonol 2006;41:35–49.

12. Veghelyi P V. Nutritional oedema. Ann Paediatr 1950;175:349–77. 13. Barbezat GO, Hansen JD. The exocrine pancreas and protein-calorie malnutrition. Pediatrics

1968;42:77–92. 14. Tarasov NI. [External secretion of the pancreas in chronic infant nutrition disorders]. Vopr Pediatr

1953;21:33–9. 15. Thompson MD, Trowell HC. Pancreatic enzyme activity in duodenal contents of children with a type of

kwashiorkor. Lancet 1952;1:1031–5. 16. Blackburn WR, Vinijchaikul K. The pancreas in kwashiorkor. An electron microscopic study. Lab Invest

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disease and the malabsorption syndrome in tropical Africa. Gut 1967;8:388–401. 18. Bras G, Waterlow JC, Depass E. Further observations on the liver pancreas and kidney in malnourished

infants and children: the relation of certain histopathological changes in the pancreas and those in liver and kidney. West Indian Med J 1957;6:33–42.

19. Sauniere JF, Sarles H, Attia Y, Lombardo A, Yoman TN, Laugier R, et al. Exocrine pancreatic function of children from the Ivory Coast compared to French children. Effect of kwashiorkor. Dig Dis Sci 1986;31:481–6.

20. Pitchumoni CS. Pancreas in primary malnutrition disorders. Am J Clin Nutr 1973;26:374–9.

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21. El-Hodhod MA, Nassar MF, Hetta OA, Gomaa SM. Pancreatic size in protein energy malnutrition: a predictor of nutritional recovery. Eur J Clin Nutr 2005;59:467–73.

22. Durie PR, Forstner GG, Gaskin KJ, Weizman Z, Kopelman HR, Ellis L, et al. Elevated serum immunoreac-tive pancreatic cationic trypsinogen in acute malnutrition: evidence of pancreatic damage. J Pediatr 1985;106:233–8.

23. Brooks SE, Golden MH. The exocrine pancreas in kwashiorkor and marasmus. Light and electron mi-croscopy. West Indian Med J 1992;41:56–60.

24. Bartels RH, Meyer SL, Stehmann TA, Bourdon C, Bandsma RHJ, Voskuijl WP. Both Exocrine Pancreatic Insufficiency and Signs of Pancreatic Inflammation Are Prevalent in Children with Complicated Severe Acute Malnutrition: An Observational Study. J Pediatr 2016;174:165–70.

25. Nousia-Arvanitakis S. Cystic fibrosis and the pancreas: recent scientific advances. J Clin Gastroenterol 1999;29:138–42.

26. Shwachman H, Diamond LK, Oski FA, Khaw KT. The Syndrome of Pancreatic Insufficiency and Bone Marrow Dysfunction. J Pediatr 1964;65:645–63.

27. Carroccio A, Fontana M, Spagnuolo MI, Zuin G, Montalto G, Canani RB, et al. Pancreatic dysfunction and its association with fat malabsorption in HIV infected children. Gut 1998;43:558–63.

28. Taylor JR, Gardner TB, Waljee AK, Dimagno MJ, Schoenfeld PS. Systematic review: efficacy and safety of pancreatic enzyme supplements for exocrine pancreatic insufficiency. Aliment Pharmacol Ther 2010;31:57–72.

29. Ferrone M, Raimondo M, Scolapio JS. Pancreatic enzyme pharmacotherapy. Pharmacotherapy 2007;27:910–20.

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32. WHO. Guideline: Updates on the management of severe acute malnutrition in infants and children. World Health Organization. Geneva: World Health Organization; 2013.


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36. Wardlaw T, Salama P, Brocklehurst C, Chopra M, Mason E. Diarrhoea: why children are still dying and what can be done. Lancet 2010;375:870–2.

37. Schibli S, Durie PR, Tullis ED. Proper usage of pancreatic enzymes. Curr Opin Pulm Med 2002;8:542–6. 38. Leeds JS, Oppong K, Sanders DS. The role of fecal elastase-1 in detecting exocrine pancreatic disease.

Nat Rev Gastroenterol Hepatol 2011;8:405–15. 39. Nandhakumar N, Green MRR. Interpretations: How to use faecal elastase testing. Arch Dis Child Educ

Pr Ed 2010;95:119–23. 40. Nakamura T, Takebe K, Tando Y, Arai Y, Yamada N, Ishii M, et al. Faecal triglycerides and fatty acids in the

differential diagnosis of pancreatic insufficiency and intestinal malabsorption in patients with low fat intakes. J Int Med Res 1995;23:48–55.

41. Voortman G, Gerrits J, Altavilla M, Henning M, van Bergeijk L, Hessels J. Quantitative determination of faecal fatty acids and triglycerides by Fourier transform infrared analysis with a sodium chloride transmission flow cell. Clin Chem Lab Med 2002;40:795–8.

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42. StataCorp. Stata Statistical Software: Release 13. College Station, TX: StataCorp LP; 2013. 43. Gray B. cmprsk: Subdistribution Analysis of Competing Risks. R package version 2.2-7. 2014. 44. Taylor CJ, Chen K, Horvath K, Hughes D, Lowe ME, Mehta D, et al. ESPGHAN and NASPGHAN Report on

the Assessment of Exocrine Pancreatic Function and Pancreatitis in Children. J Pediatr Gastroenterol Nutr 2015 Jul;61:144–53.

45. Walkowiak J, Nousia-Arvanitakis S, Henker J, Stern M, Sinaasappel M, Dodge JA. Indirect pancreatic function tests in children. J Pediatr Gastroenterol Nutr 2005;40:107–14.

46. Scrimshaw NS, Behar M, Arroyave G, Tejada C, Viteri F. Kwashiorkor in children and its response to protein therapy. J Am Med Assoc 1957;164:555–61.

47. Van de Kamer JH, Ten Bokkel Huinink H, Weyers HA. Rapid method for the determination of fat in feces. J Biol Chem 1949;177:347–55.

48. Khouri MR, Ng SN, Huang G, Shiau YF. Fecal triglyceride excretion is not excessive in pancreatic insuf-ficiency. Gastroenterology 1989;96:848–52.

49. Zhang L, Voskuijl W, Mouzaki M, Groen AK, Alexander J, Bourdon C, et al. Impaired Bile Acid Homeosta-sis in Children with Severe Acute Malnutrition. PLoS One. Public Library of Science; 2016;11:e0155143.

50. Grace E, Shaw C, Whelan K, Andreyev HJN. Review article: small intestinal bacterial overgrowth - preva-lence, clinical features, current and developing diagnostic tests, and treatment. Aliment Pharmacol Ther 2013;38:674–88.

51. Kerac M, Bunn J, Chagaluka G, Bahwere P, Tomkins A, Collins S, et al. Follow-up of post-discharge growth and mortality after treatment for severe acute malnutrition (FuSAM study): a prospective cohort study. PLoS One 2014;9:e96030.

52. Schofield C, Ashworth A. Why have mortality rates for severe malnutrition remained so high? Bull World Health Organ 1996;74:223–9.

53. Collins S. Treating severe acute malnutrition seriously. Arch Dis Child 2007;92:453–61.

118

Chapter 4

Supp

lem

enta

l Tab

le 1

. Clin

ical

gra

de o

f exo

crin

e pa

ncre

atic

insu

ffici

ency

as d

eter

min

ed b

y ab

norm

al fe

cal e

last

ase-

1 le

vels

mea

sure

d in

pati

ents

with

SAM

pre

sent

-in

g w

ith o

r with

out e

dem

a w

ho re

ceiv

ed P

ERT

trea

tmen

t for

28

days

or t

he st

anda

rd o

f car

e.

Feca

l ela

stas

e-1

n=

Clin

ical

Cut

-off

All p

atien

tsn=

Cont

rols

n=PE

RTP

At a

dmiss

ion

All

71<

200

ug/g

59/7

1 (8

3%)

3528

/35

(80%

)36

31/3

6 (8

6%)

0.5

<

100

ug/g

49/7

1 (6

9%)

22

/35

(63%

)

27/3

6 (7

5%)

0.3

Non

-ede

mat

ous

32<

200

ug/g

22/3

2 (6

9%)

2115

/21

(71%

)11

7/11

(64%

)0.

7

<

100

ug/g

15/3

2 (4

7%)

9/

21 (4

3%)

6/

11 (5

5%)

0.7

Edem

atou

s37

< 20

0 ug

/g36

/37

(97%

)14

13/1

4 (9

3%)

2524

/25

(96%

)1

<

100

ug/g

33/3

7 (8

9%)

13

/14

(93%

)

21/2

5 (8

4%)

0.6

n=Cl

inic

al C

ut-o

ffAl

l pati

ents

n=Co

ntro

lsn=

PERT

p

28 d

ays

Post

-Adm

issio

nAl

l40

< 20

0 ug

/g22

/40(

55%

)16

7/16

(44%

)24

15/2

4 (6

3%)

0.3

< 10

0 ug

/g14

/40(

35%

)

5/16

(31%

)

9/24

(38%

)0.

7

Non

-ede

mat

ous

12<

200

ug/g

3/12

(25%

)7

2/7

(29%

)5

1/5

(20%

)1

< 10

0 ug

/g1/

12(8

%)

1/

7 (1

4%)

0/

5 (0

%)

1

Edem

atou

s28

< 20

0 ug

/g19

/28(

68%

)9

5/9

(56%

)19

14/1

9 (7

4%)

0.4

<

100

ug/g

13/2

8(46

%)

4/

9 (4

4%)

9/

19 (4

7%)

1

Num

ber o

f pati

ents

with

or w

ithou

t ede

ma

that

sho

w e

ither

exo

crin

e pa

ncre

atic

insu

ffici

ency

or s

ever

e pa

ncre

atic

insu

ffici

ency

as

dete

rmin

ed b

y ab

norm

al le

vels

of fe

cal e

last

ase-

1. C

hild

ren

with

feca

l ela

stas

e-1

leve

ls be

low

the

clin

ical

cut

-off

of <

200

ug/g

of s

tool

sho

w s

igns

of p

ancr

eatic

insu

ffici

ency

, tho

se w

ith le

vels

<100

ug

/g h

ave

seve

re p

ancr

eatic

insu

ffici

ency

. Diff

eren

ces

in p

ropo

rtion

s be

twee

n tr

eatm

ent g

roup

s w

ere

test

ed b

y Fi

sher

’s ex

act t

est a

nd p

-val

ues

<0.0

5 w

as u

sed

as

the

signi

fican

ce th

resh

old.

119

4


Supp

lem

enta

l Tab

le 2

. Fre

e fa

tty

acid

and

trig

lyce

ride

leve

ls w

ith ca

lcul

ated

split

-rati

os o

n ad

miss

ion

and

after

28

days

of S

AM p

atien

ts w

ith o

r with

out o

edem

a th

at

eith

er re

ceiv

ed P

ERT

or th

e st

anda

rd o

f car

e

All p

atien

tsCo

ntro

lsPE

RT

n=M

edia

n (IQ

R)n=

Med

ian

(IQR)

n=M

edia

n (IQ

R)p-

valu

e

Adm

issio

nAl

l pati

ents

Free

Fatt

y Ac

ids

(g/k

g)78

16.2

(5.0

−34.

4)38

20.6

(10.

2−45

.8)

4012

.2 (4

.4−2

4.6)

0.2

Tr

igly

cerid

es (g

/kg)

0.6

(0−7

.6)

0.4

(0−5

.7)

1.1

(0−1

0.1)

1

Sp

lit-r

atio

(%)

94 (7

3−10

0)98

(78−

100)

87 (7

0−10

0)0.

4

N

on-e

dem

atou

sFr

ee F

atty

Acid

s (g

/kg)

3320

.6 (4

.5−4

7.8)

2225

.1 (1

3.3−

58.9

)11

14.4

(3.8

−24.

3)0.

2

Tr

igly

cerid

es (g

/kg)

0.6

(0−6

.7)

0.85

(0−1

0.4)

0 (0

−4.3

)0.

3

Sp

lit-r

atio

(%)

98 (8

1−10

0)94

(75−

100)

100

(84−

100)

0.6

Ed

emat

ous

Free

Fatt

y Ac

ids

(g/k

g)45

12.3

(5.1

−25.

7)16

16.3

(8.9

−28.

5)29

12.1

(5−2

4.5)

0.9

Tr

igly

cerid

es (g

/kg)

0.6

(0−7

.7)

0 (0

−2.6

)1.

5 (0

−15.

1)0.

7

Sp

lit-r

atio

(%)

90 (6

7−10

0)10

0 (8

0−10

0)85

(62−

100)

0.3

28 d

ays

post

-adm

issio

n

All p

atien

tsFr

ee F

atty

Acid

s (g

/kg)

4510

(2.7

−20)

2210

.8 (2

.7−3

2.6)

239.

4 (3

.2−1

5.1)

0.2

Trig

lyce

rides

(g/k

g)0.

4 (0

−0.6

)0

(0−0

.4)

0.4

(0−0

.9)

0.7

Sp

lit-r

atio

(%)

99 (8

8−10

0)10

0 (9

3−10

0)96

(84−

100)

0.2

N

on-e

dem

atou

sFr

ee F

atty

Acid

s (g

/kg)

1626

.2 (2

.9−4

4.1)

1117

.7 (2

.7−4

2.6)

534

.5 (1

7.8−

45.8

)0.

5

Tr

igly

cerid

es (g

/kg)

0 (0

−0.4

)0

(0−0

.4)

0 (0

−1.2

)0.

8

Sp

lit-r

atio

(%)

100

(94−

100)

100

(87−

100)

100

(97−

100)

0.3

Ed

emat

ous

Free

Fatt

y Ac

ids

(g/k

g)29

6.5

(2.5

−11.

9)11

10 (3

.4−1

8)18

5.4

(2.1

−10.

2)0.

1

Tr

igly

cerid

es (g

/kg)

0.4

(0−0

.8)

0 (0

−0.5

)0.

4 (0

−0.8

)0.

2

Sp

lit-r

atio

(%)

97 (8

4−10

0)10

0 (9

7−10

0)91

(83−

100)

0.07

Valu

es a

re p

rese

nted

as

med

ian

and

inte

rqua

rtile

rang

es (I

QR)

. Diff

eren

ces

betw

een

trea

tmen

t gro

ups

wer

e te

sted

with

gen

eral

ized

linea

r mod

els

with

a b

inom

ial

erro

r str

uctu

re.

120

Chapter 4

Supplemental Figure 1. Concentration levels of fecal elastase-1 in SAM patients with or without edema at A) Admission (non-edematous, n=32; edematous, n=39) and B) After 28 days of pancreatic enzyme replacement therapy (PERT: non-edematous, n=14; edematous, n=31) or standard care (Control: non-edematous, n=25; edematous, n=20). Boxplots summarize the median (midline) and interquartile rang-es (upper and lower box). Group differences were tested using generalized linear models with a gamma error structure. Significance code: *p-value<.05.

Supplemental Figure 2. The relationship between mortality and fecal markers in children with SAM. A) Concentration of fecal elastase-1 at admission in SAM patients that died or survived and B) split by PERT or standard of care treatment groups. C) Difference in split ratios at admission between children that died versus those that survived. Boxplots summarize the median (midline) and interquartile ranges (upper and lower box). Group differences in levels of fecal elastase-1 were tested using generalized lin-ear models with a gamma error structure; differences in split-ratios were tested using binomial logistic regression. Significance code: *p-value<.05.

Chapter 5Hypoallergenic and anti-inflammatory feeds in children with complicated severe acute malnutrition: an open randomized controlled 3-arm intervention trial in Malawi

Rosalie H. Bartels, Emmanuel Chimwezi, Vicky Watson, Leilei Pei, Isabel Potani, Benjamin Allubha, Katherine Chidzalo, Duolao Wang, Queen Dube, Macpherson Mallewa, Angela Allen, Robert H.J. Bandsma, Wieger P. Voskuijl, Stephen J Allen

Submitted

124

Chapter 5

ABSTRACT

Background: Intestinal pathology in children with complicated severe acute malnutrition (SAM) has similarities to intestinal inflammation in non-IgE mediated gastrointestinal food allergy and Crohn’s disease and persists despite standard management. Objective: We tested whether therapeutic feeds effective in treating intestinal inflamma-tion in food allergy and Crohn’s disease benefit children with SAM.Design: We recruited children aged 6-23 months with complicated SAM at Queen Eliza-beth Central Hospital, Blantyre, Malawi between January 1st and December 31st, 2016. After initial clinical stabilization, children were allocated randomly to either standard feeds (F-100 and/or ready-to-use therapeutic food), an elemental feed or a polymeric feed for 14 days. The primary outcome was change in fecal calprotectin as a marker of intestinal inflammation. Results: Thirty-one children received standard feeds, 32 elemental feed and 32 poly-meric feed. Overall, mean (SD) age was 15.6 (5.7) months, 46 (48.4%) were male and 38 (40.0%) children had edema. Change in fecal calprotectin was similar in each arm: elemental vs. standard 4.1 μg/mg stool/day (95% CI, -29.9, 38.15; P=.81) and polymeric vs. standard 10 (-23.96, 43.91; P=.56). In all children, mean (SD) fecal calprotectin con-centration was 547 μg/mg stool (744) at baseline and remained highly abnormal at the end of the intervention (697 (735); normal <50). Biomarkers of mucosal integrity and systemic inflammation were highly abnormal at baseline and generally persisted in all three arms. Weight gain and changes in growth biomarkers were similar in each arm. Two children died in the standard arm (6.5%), 2 in the elemental arm (6.3%) and 3 in the polymeric arm (9.4%; P=1.0).Conclusions: The enteropathy in complicated SAM did not respond to either standard feeds or novel therapeutic feeds administered for up to 14 days. A better understanding of the pathogenesis of the gut pathology in complicated SAM is an urgent priority to inform the development of improved therapeutic interventions.

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5

Hypoallergenic and anti-inflammatory feeds in children with complicated severe acute malnutrition

InTRODUCTIOn

In 2016, severe wasting (weight-for-height Z score (WHZ) <-3) occurred in 17 million under-fives (2.5% of all children) and was estimated to account for between 7.4–7.8% of all child deaths (1–3). About 20% of children with severe acute malnutrition (SAM) require in-patient care because of poor feeding, medical complications such as severe edema and infection, or failure to improve under community management(4). Despite following a well-established WHO protocol (4), case fatality in “complicated” SAM re-mains up to 35%(5–9) with additional deaths after hospital discharge (8,10).Intestinal pathology in SAM is thought to result from increased exposure to microbial pathogens and poor nutrition(11–15). A significant feature is gut inflammation that per-sists despite management (16,17). The similarity to gut inflammation in non-IgE mediated food allergy (hereafter “food allergy”; e.g. due to cow’s milk protein)(18,19) and Crohn’s disease(20) raises the intriguing possibility that treatments that reduce gut inflammation in these conditions may also be of benefit in SAM. Intestinal inflammation in food allergy responds well to exclusion of the offending dietary antigen or, if the offending antigen is not known, a hypoallergenic, elemental feed composed of single amino acids (20). In pediatric Crohn’s disease, first-line therapy is with exclusive enteral nutrition where all foods are replaced by an elemental formula or polymeric formula (20–23). In previ-ous research, hypoallergenic and elemental feeds were well tolerated in children with malnutrition but evidence of benefit was limited (24,25).The concentration of calprotectin in feces, a non-specific biomarker of intestinal inflam-mation, is validated in the diagnosis and management of inflammatory bowel disease (26). Fecal calprotectin (FC) is markedly increased in SAM and remains elevated despite standard WHO management (12,17). Our hypothesis was that a hypoallergenic and an anti-inflammatory therapeutic formula would improve enteropathy and be well tolerated and safe in children with complicated SAM. The primary outcome was change in FC concentration. We also assessed biomarkers of intestinal integrity and systemic inflammation, tolerability of feeds, clinical outcomes and serious adverse events. Children managed with therapeutic feeds recommended by WHO formed the comparison group.

SUBJeCTS AnD MeTHODS

Study design This randomized, open-label, 3-arm study was conducted at “Moyo” Nutritional Reha-bilitation Unit, Queen Elizabeth Central Hospital, Blantyre, Malawi in accordance with the principles of good clinical practice and the Declaration of Helsinki (27). The College

126

Chapter 5

of Medicine Research and Ethics Committee, Blantyre, Malawi (P.06/15/1745) and the Liverpool School of Tropical Medicine Ethics Committee, Liverpool, UK (15.048) gave approval.

Participant recruitmentChildren admitted from January 1st to December 31st, 2016 were screened for eligibility. Inclusion criteria were age 6-23 months, SAM (WHZ <-3 and/or mid-upper arm circum-ference (MUAC) <11.5 cms and/or nutritional edema)(28); completed the stabilization phase of management and willing to stay on the ward for 2 weeks. We excluded children with a specific identifiable cause of malnutrition (e.g. feeding difficulties due to cerebral palsy; treatment for tuberculosis), those participating in another study or those with a sibling admitted with SAM. Both HIV positive and negative children were included. Children were managed according to WHO guidelines using F75 therapeutic feeds during the stabilization phase (4). Legal guardians were provided with verbal and written information either in Chichewa or English by a member of the study team. Following witnessed signed or thumbprint informed consent, research staff allocated children according to a computer-generated random sequence with blocks of random size in equal numbers to the three study arms. The legal guardian opened the next in a series of sealed, opaque envelopes each labeled with a unique study number and containing a colored card indicating the intervention arm. Legal guardians were offered travel expenses.

InterventionsChildren were allocated to standard feeds (ready-to-use therapeutic food (RUTF) or, if not tolerated, F100), polymeric feed (Modulen IBD; Nestlé Health Science; York, UK) or elemental feed (PurAmino; Mead Johnson Nutrition; Chicago, 606 USA). Nutriset CMV (Malaunay, France) was added to both of the alternative feeds to approximate the nutri-ent content of F-100 (Supplemental Table 1). All feeds were introduced gradually over 2-3 days and provided 3-hourly for 14 days. Mothers were encouraged to re-establish or continue breastfeeding. No feeds other than breastfeeding and the intervention formulas were administered. Care was according to standard Malawian practice based on WHO guidelines which included a malaria slide, blood count and an HIV rapid antibody test, chloramphenicol and gentamicin as first line and ceftriaxone as second line antibiotic therapy and withholding oral iron until discharge.(4) All children received standard feeds after 14 days.

Clinical data and biological sample collectionDemographic, anthropometric, socioeconomic and clinical information was collected at recruitment. Children were reviewed daily by the research team and volume of feed

127

5


taken, symptoms reported by parents/caregivers, clinical signs and medications re-corded. Research samples were collected alongside clinical samples whenever possible at recruitment, 7 and 14 days or within 48 hours of these time points.

Biomarkers of systemic and mucosal inflammation, gut integrity and growth Stool samples were analyzed for calprotectin (Bühlmann fCAL® ELISA; www.buhlmann-labs.ch) as a biomarker of intestinal inflammation. Fecal α1-antitrypsin (ELISA, Immuno-diagnostik AG; www.IBL-International.com), plasma intestinal fatty acid binding protein (IFABP; Human FABP2/I-FABP Quantikine ELISA Kit; R&D Systems, Inc.; www.RnDSystems.com) and plasma IgG anti-endotoxin antibody concentration (EndoCab® Human, ELISA; Hycult biotech; www.hycultbiotech.com) were measured as markers of intestinal mu-cosal integrity. Plasma C-reactive protein and α1-acid glycoprotein (Quantikine® ELISA, R&D Systems, Inc.; www.RnDSystems.com) were measured as markers of acute and chronic systemic inflammation respectively. Plasma insulin like growth factor (IGF)-1 and IGF binding protein 3 (IGFBP3; Quantikine® ELISA, R&D Systems, Inc.; www.RnDSystems.com) were also measured. All analyses followed the manufacturer’s instructions. Labora-tory staff was blinded to treatment allocations.

OutcomesThe primary endpoint was the change in fecal calprotectin during the intervention pe-riod. Secondary outcomes were change in other biomarkers, gain in weight (g/kg/day after resolution of edema if present), diarrhea reported by caregivers and tolerance of feeds (vomiting reported by caregivers and requirement for naso-gastric tube feeds). Suspected sepsis was defined as clinical suspicion and the start or change in antibiotic therapy by the child’s clinical team. Treatment-emergent serious adverse events (SAEs), defined as events that commenced, or worsened, after the allocated feed had been administered, were reviewed by a senior pediatrician and reported to the Pharmaco-vigilance Pharmacist at LSTM. SAEs were reviewed by two blinded independent safety monitors and categorized according to Medical Dictionary for Regulatory Activities (Med-DRA®) Preferred term.

Participant withdrawalLegal guardians were free to withdraw their child at any time. Participants were asked to provide an additional stool and blood sample on withdrawal from the study.

Sample size calculationBased on fecal calprotectin concentration in children with SAM in Kenya (17), at the 5% significance level and with a coefficient of variation of 0.4, a total of 90 children (30 in each group) was needed to detect a 25% reduction in mean log calprotectin in each of

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the alternative feed arms compared with the standard arm with at least 81% power. We intended to recruit 120 children (40/group) to allow for deaths and dropouts.

Statistical analysisThe effects of the therapeutic feeds were assessed according to average change from baseline in laboratory and clinical variables. The primary endpoint was analyzed using a generalized linear model (GLM) with treatment as predictor and baseline fecal calpro-tectin as covariate. The model had Gaussian distribution and identity link function. The treatment difference in the mean of the primary outcome and its 95% CI were derived from the GLM model. Other biomarkers and clinical endpoints were analyzed similarly with distributions and link functions determined by type of data (continuous, binary and count). For repeated measurement outcomes, mixed models with treatment as fixed effect, baseline measurement as covariate, and subject as random effect were used to derive treatment differences and 95% CIs. The primary endpoint analysis was based on the intention to treat (ITT) population. Additional analysis in the ITT and per protocol population of the primary and secondary endpoints were also performed. No interim analysis was conducted. Analysis was performed using SAS v9.4.

ReSUlTS

Three hundred and sixty-seven children admitted with complicated SAM were screened. Of the 172 (46.9%) eligible children, 95 (55.2%) were randomly assigned to the three treatment arms (Figure 1). Excluding study deviations, the number of children withdrawn by families was similar in the standard (3 children), elemental (5 children) and polymeric arms (3; P=.31). Withdrawals were mostly related to family concerns with the child being enrolled in a research study and refusal to remain under admission.

Baseline demographic and clinical characteristics (Table 1) and socioeconomic factors (Supplemental Table 2) were similar in the three arms. Mean (SD) age was 15.6 (5.7) months and most children lived in either an urban or peri-urban setting. Thirty-five (36.8%) children were breastfed and all but one child was receiving complementary feeds. Mean (SD) MUAC was 11.15 cms (1.38) and WHZ in children without edema was -3.74 (1.25). Stunting was common (height-for-age z score -2.94; 2.06). Thirty-eight (40.0%) children with edematous malnutrition were significantly older (P<.001) and had a greater mean MUAC (P<.001) than those without edema (Supplemental Table 3). Thirty-four (36.2%) children positive for HIV had a lower mean MUAC than HIV negative children (P=.002; Supplemental Table 4). Children in the standard arm all received RUTF; only 6 required some F-100 during their care.

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Hypoallergenic and anti -infl ammatory feeds in children with complicated severe acute malnutriti on

Lost to follow-up (n=0)Discon�nued interven�on (n=4)

Died (n=21)Withdrawn (n=2)- Family disagreed with child being in a study (n=1)- Study devia�on: child already enrolled in another study (n=1)


Died (n=2)Withdrawn (n=7)- Mother refused inser�on of NGT(n=1)- Family disagreed with child being in a study (n=2)- Family felt childs condi�on was not improving and the treatment was not working (n=1)- Caregiver no longer agreed to stay on the ward for the full 14 study days (n=1)- Study devia�on: feed had expired (n=2)


Died (n=3)Withdrawn (n=2)- Family disagreed with child being in a study (n=1)- Caregiver no longer agreed to stay on the ward for the full 14 study days (n=1)

Completed 14 days (n=27)Analysed (n=31)



Analysis

Follow-Up

Alloca�on

Enrollment

Allocated to Standard group (n=31)Received allocated interven�on (n=31)Did not receive allocated interven�on (n=0)

Allocated to Elemental group (n=32)Received allocated interven�on (n=32)Did not receive allocated interven�on (n=0)

Allocated to Polymeric group (n=32)Received allocated interven�on (n=32)Did not receive allocated interven�on (n=0)

Randomized (n=95)

Excluded (n=272)Not mee�ng inclusion criteria (n=195)

Age above 23 months (n=158)Cerebral Palsy (n=12)Renal disease (n=9)On TB treatment (n=6)Enrolled in other study (n=5)Cardiac disease (n=2)Sibling enrolled in study (n=2)Age below 6 months (n=1)

Declined to par�cipate (n=74)Other reasons (n=3)

Assessed for eligibility (n=367)

Figure 1. Study fl ow diagram of SAM children assessed for eligibility and recruited for the study. 1 2 children were known to have died aft er the 14-day study period.

BiomarkersIn all children, fecal calprotecti n was markedly elevated both at baseline (mean (SD) 547 (744) μg/gm stool) and at 14 days (697 (735); P=.31). The earliest and latest stool and blood samples available during the 14-day interventi on period were used to assess change in biomarkers. Change in fecal calprotecti n was similar in all treatment arms (Figure 2a; Table 2; Supplemental Table 5). Similarly, biomarkers of mucosal integrity were elevated at recruitment and remained high during treatment (Figure 2b; Table 2; Supplemental Table 5). Fecal α1-anti trypsin increased signifi cantly in the polymeric versus the standard arm (P=.0013). IFABP fell in the elemental compared with the standard arm (P=.049).Platelet counts rose signifi cantly (P=.003) during treatment with similar changes in each of the three arms. C-reacti ve protein remained elevated in many children in each of the

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three arms. Plasma α1-acid glycoprotein fell significantly in the polymeric compared with the standard arm (P=.01; Figure 2c; Table 2; Supplemental Table 5).

Table 1. Baseline demographic and clinical characteristics according to intervention arm

Variable Standardn=31

Elementaln=32

Polymericn=32

Totaln=95

Demographic variables

Age in months (mean; SD) 16.6 (5.4) 14.8 (6.6) 15.5 (5.2) 15.6 (5.74)

Male (N; %) 14 (45.2) 15 (46.9) 17 (53.1) 46 (48.4)

Residence type (N; %)

Rural 10 (32.3) 9 (28.1) 10 (31.3) 29 (30.5)

Urban 14 (45.2) 19 (59.4) 17 (53.1) 50 (52.6)

Peri-urban 7 (22.6) 4 (12.5) 5 (15.6) 16 (16.8)

Mother main caregiver (N; %) 30 (96.8) 29 (90.6) 27 (84.4) 86 (90.5)

Mother HIV positive (N; %) 10 (37) 14 (43.8) 14 (43.8) 38 (40)

Mother died 1 (3.2) 1 (3.1) 2 (6.3) 4 (4.2)

Number of siblings alive (mean; SD) 1.87 (1.5) 1.78 (1.5) 1.13 (1.3) 1.59 (1.45)

Number of siblings died (mean; SD) 0.19 (0.5) 0.13 (0.42) 0.41 (1.0) 0.24 (0.70)

Clinical variables

Child HIV positive (N; %) 11 (35.5) 12 (37.5) 11 (34.4)1 34 (35.8)

Breastfed (N; %) 22 (71) 20 (62.5) 20 (62.5) 62 (65.3)

Receiving complementary feeds (N; %) 31 (100) 31 (96.9) 32 (100) 94 (98.9)

Edema (N; %)

None 17 (54.8) 18 (56.3) 22 (68.8) 57 (60)

+ 4 (12.9) 3 (9.38) 3 (9.38) 10 (10.5)

++ 5 (16.1) 9 (28.1) 6 (18.8) 20 (21.1)

+++ 5 (16.1) 2 (6.25) 1 (3.13) 8 (8.42)

MUAC (cms; mean; SD) 10.78 (1.50) 11.22 (1.59) 11.45 (0.95) 11.15 (1.38)

WHZ (mean; SD)2 -4.10 (1.34) -3.79 (1.28) -3.41 (1.13) -3.74 (1.25)

Length-for-age z score (mean; SD) -2.80 (1.49) -3.01 (2.85) -3.00 (1.60) -2.94 (2.06)

Notes1. HIV status not known for 1 child2. Only reported for children without edema

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IGF-1 and IGFBP3 were low at recruitment and increased significantly during treatment (P=<.0001 for both) and to a similar degree in all treatment arms (Figure 2d; Table 2; Supplemental Table 5). Tracking changes in biomarkers in individual children did not identify subgroups of children who responded either better or worse to the interventions (Supplemental Figure 1). Children with edema had a significantly higher mean plasma α1-acid glycoprotein at recruitment than those without (P=.024; Supplemental Table 3). HIV positive children had higher mean baseline values for fecal calprotectin (P=.005) and plasma C-reactive protein (P=.008) and lower hemoglobin (P=.007) than HIV negative children (Supplemen-tal Table 4).

Table 2. Change in laboratory biomarkers and clinical variables according to intervention arm

Variable Elemental vs Standard Polymeric vs Standard

Change/day (95% CI) P value Change/day (95% CI) P value

Intestinal inflammation

Fecal calprotectin (μg/g stool) 4.1 (-29.9,38.15) .81 10 (-23.96,43.91) .56

Intestinal integrity

Fecal α1-antitrypsin (mg/dl) 1.7 (-0.42,3.78) .12 3.5 (1.4,5.53) .0013

Plasma IgG anti-endotoxin antibodies (GMU/ml)

-1.8 (-11.83,8.26) .72 -2.1 (-11.72,7.48) .66

Plasma Intestinal fatty acid binding protein (pg/ml)

-194.3 (-387.6, -1.01) .05 -83.8 (-276.2,108.53) .38

Systemic inflammation

Platelets (x109/L blood) 7.4 (-9.08,23.94) .37 -7.1 (-23.03,8.87) .38

Plasma C-reactive protein (mg/L) -0.7 (-2.22,0.81) .35 -0.04 (-1.55,1.46) .95

Plasma α1-acid glycoprotein (μg/ml) -15.7 (-84.83,53.38) .65 -89.4 (-157.7,21.13) .01

Growth Factors

Insulin-like growth factor -1 (ng/ml) 4.4 (-2.06, 10.9) .17 4.3 (-1.32,9.91) .13

Insulin-like growth factor binding; protein 3 (ng/ml)

22.3 (-29.73, 74.37) .39 37.1 (-14.06,88.32) .15

Clinical

Weight (g/kg/day) 1.6 (-3.61, 6.8) .54 2.54 (-2.1, 7.7) .33

MUAC (cms/day) 0.025 (-0.025,0.07) .32 0.042 (-0.009,0.09) .1

Weight-for-length z-score 0.22 (-0.29,0.76) .39 0.19 (-0.31,0.7) .45

Generalized linear analysis of mean (95% CI) difference in change in variable/day in the novel feed com-pared to standard feed. A negative value signifies a fall in the variable with the novel therapeutic feed compared with standard feeds and vice versa for a positive value. Laboratory measurements were made in the first and last samples available and clinical measurements on recruitment and the last measure-ment available during the 14-day intervention period.

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C. Systemic inflammation

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nutriti onal recovery and clinical outcomes Weight gain and increase in MUAC and weight-for-length z score was similar in each arm (Figure 3; Table 2). The number of days to resoluti on of edema was also similar in the standard, elemental and polymeric arms (mean (SD): 2.38 (1.12); 2.42 (1.44); 2.0 (1.12) respecti vely). Loose or watery stools were common in all three arms. (Supplemental Table 6) The novel feeds were tolerated less well than standard feeds with greater requirement for NGT feeding and caregiver reporti ng of vomiti ng. In additi on, amongst those aff ected, vomiti ng occurred more frequently with the elemental compared with standard feeds

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(average difference =2.22/day, 95% CI=1.08, 4.58; P=.031). However, reverting back to F75 was not required with the polymeric feed (Supplemental Table 6). The number of days with loose stools and vomiting amongst children who experienced these symptoms was similar in each arm. When HIV status or edema at recruitment were included in the GLM, there was no evidence of interaction between treatment arms and changes in clinical or laboratory outcomes except that plasma α1-acid glycoprotein fell to greater degree in children with edema than those without (P=.025).

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Serious adverse events and deathsA total of 43 SAEs occurred in 25 (27.4%) children with a similar frequency in each arm (P=.5; Supplemental Table 7). The most frequent SAEs were gastroenteritis (13.7% children), dehydration (11.6%) and sepsis (6.3%). Gastroenteritis and dehydration in two children and an urticarial rash in one child were considered possibly related to the therapeutic feeds and all occurred in the standard arm. Seven children died (7.4%) within the 14 days study period; 5 deaths (71%) occurred in HIV positive children and 3 (43%) in children with edema. Another two children died on day 15 with death attributed to a SAE that started within the study period; both of these children were in the standard arm. The number of deaths was similar in each intervention arm.

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DISCUSSIOn

This study confirms previous observations that compromised gut integrity and mucosal and systemic inflammation occur in complicated SAM. These abnormalities did not im-prove during 14 days treatment either with standard or the novel therapeutic feeds. The persistence of gut pathology likely contributes to the 10-30% case-fatality(6,12,29–33), with even higher mortality in HIV positive children, (8,34) and also poor longer-term outcomes with mortality after hospital discharge typically around 25% (8,10). Small intestinal biopsy studies in SAM revealed Th-1 dominated mucosal inflammation with variable degrees of villous atrophy, crypt hyperplasia and increased inflammatory infiltrate (11,16,35). A recent biopsy and biomarker study in Zambian children with SAM and persistent diarrhea not responding to standard therapy reported severe enteropathy with intestinal barrier failure and immune dysregulation(14). These histological and im-mune features are similar to those that occur in children with Crohn’s disease (23) and also non-IgE-mediated gastrointestinal food allergies although the pathology of these latter disorders is poorly understood (18,19). The similarities between these conditions formed the rationale for evaluating the novel therapeutic feeds in this study.Fecal calprotectin concentration in complicated SAM was similar to that observed in Crohn’s disease (26). Severely impaired mucosal integrity was evidenced by marked ab-normalities in three biomarkers. Levels of plasma I-FABP, reflecting enterocyte destruc-tion(36), were similar to those in south African children with SAM (37), active Crohn’s disease(38) and greater than those reported in adults with acute mesenteric isch-emia(39,40). Fecal α1-antitrypsin, a biomarker of intestinal protein loss and moderately increased in our patients, is also elevated in Crohn’s disease(41). Finally, anti-endotoxin antibodies in plasma likely indicates translocation of gut bacteria(37).Significant systemic inflammation was evidenced by raised CRP, platelet count and plasma α1-acid glycoprotein consistent with findings in inflammatory bowel disease(42). In com-plicated SAM, systemic inflammation may result from infections, including translocation of gut bacteria (37), as well as intestinal inflammation (12).Despite moderate weight gain and a rise in growth factors in all three arms, intestinal pathology persisted during the intervention period consistent with the on-going or recurrent diarrhea in many children and the persistence of histological abnormalities despite treatment reported previously(16). The failure of the two novel feeds to improve intestinal pathology, systemic inflammation or clinical outcomes contrasts markedly with their clinical efficacy in non-IgE mediated food allergy(18,19) and Crohn’s disease. In the latter, exclusive enteral nutrition with either a polymeric or elemental formula reduces intestinal inflammation, promotes mucosal healing, down regulates pro-inflammatory cytokines and improves nutritional status. (16-19) Although we observed a greater fall in plasma α1-acid glycoprotein in the polymeric arm, possibly related to its content of trans-

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forming growth factor beta (43), in the absence of a similar effect on other biomarkers of systemic inflammation and an increase in fecal a1-antitrypsin with the polymeric feed, the clinical relevance of this finding is unclear. The greater fall in IFABP in the elemental compared with the standard group was of borderline statistical significance and, in the absence of associated clinical benefits, is unlikely to be of clinical significance. The dis-crepancy between our findings with older studies of elemental formula which reported improved weight gain in malnourished children with diarrhea (24,25) may be due to dif-ferences in the patient population or the use of RUTF as standard treatment in our study.The higher a1-acid glycoprotein at recruitment in edematous children may reflect greater bacterial translocation consistent with impaired intestinal integrity related to deficient glycosylation reported in kwashiorkor (35). However, anti-endotoxin antibodies and other markers of gut integrity did not differ significantly according to presence of edema. CRP and fecal calprotectin were particularly elevated in HIV positive children at recruitment, the latter consistent with findings in HIV infected Zambian children with SAM and persistent diarrhea (14). However, biomarkers of mucosal integrity did not differ significantly according to HIV status and this has also been reported in South African children with SAM in whom plasma IFABP and also plasma 16sDNA as a marker of microbial translocation were similar in those with and without HIV (37). The contribution of HIV to the enteropathy in complicated SAM requires further study.Our study had several limitations. We recruited only 55% of eligible children which limits the generalizability of our findings. Although exclusive enteral nutrition with a polymeric formula in Crohn’s disease reduced systemic inflammation within 3-14 days(44–46), and guidelines recommend that an alternative therapy should be considered if a clinical response has not occurred within 2 weeks (47), it is possible that feeding for longer than 14 days may have improved outcomes. However, longer periods of exclusive enteral nu-trition would be impractical given their poorer tolerance that RUTF, greater requirement for NGT feeding and likely need for prolonged hospital admission to supervise feeding which also carries the risk of increased exposure to hospital acquired infections.We are not aware of any previous studies of interventions for enteropathy in children with complicated SAM. In a previous study on the Moyo ward, a symbiotic supplement did not improve clinical outcomes (48). Our findings that neither the elemental nor polymeric feeds administered for up to 14 days resulted in any clear improvement in biomarkers of intestinal inflammation and integrity or clinical benefit in complicated SAM suggests multifactorial intestinal pathol-ogy. The enteropathy in complicated SAM may be a continuum of environmental enteric dysfunction (EED) that is ubiquitous in unhygienic settings (13). Recent studies of EED have highlighted the critical importance of sub-clinical infection with enteropathogens in compromising mucosal integrity and causing intestinal and systemic inflammation (49) and the high burden of gut pathogens in complicated SAM was associated with intestinal

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inflammation (12). Targeting enteropathogens in complicated SAM may be required to improve the enteropathy.

ConclusionsCurrent WHO management fails to improve intestinal pathology in complicated SAM. Elemental and polymeric feeds did not have the hoped anti-inflammatory or clinical ben-efits and proved to be poorly tolerated. Further research is needed to better understand the intestinal pathology in complicated SAM to help develop interventions that may address the unacceptably high case-fatality and poor long-term outcomes.

ACKnOWleDGeMenTS

We thank the staff of the Moyo NRU for their great support for the project. We thank Cheryl Pace, Pharmacovigilance Pharmacist at LSTM for reviewing and reporting SAE data and Julian Thomas and Anne Dale in their role as external safety monitors. We thank André Briend for advice on preparation of the alternative therapeutic feeds. We thank Mead Johnson Nutrition for donating the PurAmino Infant Formula and Nutriset for donating Therapeutic CMV. Finally, we thank the children and their families who agreed to participate in the study.

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20. Hartman C, Eliakim R, Shamir R. Nutritional status and nutritional therapy in inflammatory bowel diseases. World J Gastroenterol. 2009;15(21):2570-2578.

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22. Dziechciarz P, Horvath A, Shamir R, Szajewska H. Meta-analysis: Enteral nutrition in active Crohn’s disease in children. Aliment Pharmacol Ther. 2007;26(6):795-806.

23. Mayberry JF, Lobo A, Ford AC, Thomas A. NICE clinical guideline (CG152): the management of Crohn’s disease in adults, children and young people. Aliment Pharmacol Ther. 2013;37(2):195-203.

24. Amadi B, Mwiya M, Chomba E, et al. Improved nutritional recovery on an elemental diet in Zambian children with persistent diarrhoea and malnutrition. J Trop Pediatr. 2005;51(1):5-10.

25. Eichenberger JR, Hadorn B, Schmidt BJ. A semi-elemental diet with low osmolarity and high content of hydrolyzed lactalbumin in the treatment of acute diarrhea in malnourished children. Arq Gastroen-terol. 1984;21(3):130-135.

26. Henderson P, Anderson NH, Wilson DC. The diagnostic accuracy of fecal calprotectin during the investi-gation of suspected pediatric inflammatory bowel disease: a systematic review and meta-analysis. Am J Gastroenterol. 2014;109(5):637-645.

27. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013;310(20):2191-2194.

28. WHO, World Health Organization, United Nations Children’s Fund, et al. WHO Child Growth Standards and the Identification of Severe Acute Malnutrition in Infants and Children. Geneva: World Health Organization; 2009. http://www.who.int/nutrition/publications/severemalnutrition/9789241598163/en/. Accessed November 29, 2017.

29. Tickell KD, Denno DM. Inpatient management of children with severe acute malnutrition: a review of WHO guidelines. Bull World Health Organ. 2016;94(9):642-651.

30. Ahmed T, Ali M, Ullah MM, et al. Mortality in severely malnourished children with diarrhoea and use of a standardised management protocol. Lancet. 1999;353(9168):1919-1922.


32. Talbert A, Thuo N, Karisa J, et al. Diarrhoea complicating severe acute malnutrition in Kenyan children: a prospective descriptive study of risk factors and outcome. PLoS One. 2012;7(6):e38321.

33. Maitland K, Berkley JA, Shebbe M, Peshu N, English M, Newton CRJC. Children with severe malnutri-tion: can those at highest risk of death be identified with the WHO protocol? Molyneux E, ed. PLoS Med. 2006;3(12):e500.

34. Fergusson P, Tomkins A, Kerac M. Improving survival of children with severe acute malnutrition in HIV-prevalent settings. Int Health. 2009;1(1):10-16.

35. Amadi B, Fagbemi AO, Kelly P, et al. Reduced production of sulfated glycosaminoglycans occurs in Zambian children with kwashiorkor but not marasmus. Am J Clin Nutr. 2009;89(2):592-600.

36. Piton G, Capellier G. Biomarkers of gut barrier failure in the ICU. Curr Opin Crit Care. 2016;22(2):152-160.

37. Muenchhoff M, Healy M, Singh R, et al. Malnutrition in HIV-infected children is an indicator of severe disease with an impaired response to antiretroviral therapy. AIDS Res Hum Retroviruses. 2017:AID.2016.0261.

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38. Sarikaya M, Ergül B, Doğan Z, Filik L, Can M, Arslan L. Intestinal fatty acid binding protein (I-FABP) as a promising test for Crohn’s disease: a preliminary study. Clin Lab. 2015;61(1-2):87-91.

39. Kanda T, Tsukahara A, Ueki K, et al. Diagnosis of ischemic small bowel disease by measurement of serum intestinal fatty acid-binding protein in patients with acute abdomen: a multicenter, observer-blinded validation study. J Gastroenterol. 2011;46(4):492-500.

40. Güzel M, Sözüer EM, Salt Ö, İkizceli İ, Akdur O, Yazıcı C. The value of the serum I-FABP level for diagnos-ing acute mesenteric ischemia. Surg Today. 2014;44(11):2072-2076.

41. Sutherland AD, Gearry RB, Frizelle FA. Review of fecal biomarkers in inflammatory bowel disease. Dis Colon Rectum. 2008;51(8):1283-1291.

42. Fengming Y, Jianbing W. Biomarkers of inflammatory bowel disease. Dis Markers. 2014;2014:710915. 43. Hartman C, Berkowitz D, Weiss B, et al. Nutritional supplementation with polymeric diet enriched with

transforming growth factor-beta 2 for children with Crohn’s disease. Isr Med Assoc J. 2008;10(7):503-507.

44. Fell JM, Paintin M, Arnaud-Battandier F, et al. Mucosal healing and a fall in mucosal pro-inflammatory cytokine mRNA induced by a specific oral polymeric diet in paediatric Crohn’s disease. Aliment Phar-macol Ther. 2000;14(3):281-289.

45. Bannerjee K, Camacho-Hübner C, Babinska K, et al. Anti-inflammatory and growth-stimulating effects precede nutritional restitution during enteral feeding in Crohn disease. J Pediatr Gastroenterol Nutr. 2004;38(3):270-275.

46. Borrelli O, Cordischi L, Cirulli M, et al. Polymeric diet alone versus corticosteroids in the treatment of active pediatric Crohn’s disease: a randomized controlled open-label trial. Clin Gastroenterol Hepatol. 2006;4(6):744-753.

47. Ruemmele FM, Veres G, Kolho KL, et al. Consensus guidelines of ECCO/ESPGHAN on the medical management of pediatric Crohn’s disease. J Crohn’s Colitis. 2014;8(10):1179-1207.

48. Kerac M, Bunn J, Seal A, et al. Probiotics and prebiotics for severe acute malnutrition (PRONUT study): a double-blind efficacy randomised controlled trial in Malawi. Lancet. 2009;374(9684):136-144.

49. Kosek M, Guerrant RL, Kang G, et al. Assessment of environmental enteropathy in the MAL-ED cohort study: Theoretical and analytic framework. Clin Infect Dis. 2014;59.

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Chapter 5

Supplemental Table 1. Composition of therapeutic feeds per liter

nutrient F-1001 Modulen IBD2,3 with 1.7g CMV/l PurAmino2,4,5 with 1.7g CMV/lEnergy (kcal) 998 1000 1000Protein (g) 29 36 27.8Carbohydrates (g) 50 110 105.9Fat (g) -6 47 52.9Osmolality (mOsmol/l) 419 290 450.6MineralsSodium (mg) 560 350.0 470.6Potassium (mg) 2300 1782.3 1670.5Calcium (mg) 850 910.0 941.2Phosphorus (mg) 825 610.0 514.7Magnesium (mg) 154 263.3 172.1Zinc (mg) 21.2 21.6 22.0Copper (mg) 3 2.5 2.3Iron (mg) 0.4 11.0 17.9Manganese (mg) - 2 -Fluoride (μg) - <20 -Chromium (μg) - 51 -Molybdenum (μg) - 75 -Selenium (μg) 57 35.0 27.8Iodine (μg) 225 100.1 148.6VitaminsA μg RE 1544 1911.0 1968.1D μg 29 27.0 29.5E mg α-TE 38.6 32.4 32.8K μg 29 76.3 111.0C mg 96.5 145.5 167.6B1 (Thiamin) mg 0.97 1.7 1.3B2 (Riboflavin) mg 3.1 2.8 2.4Pantothenic acid mg 5.8 8.0 8.0B6 mg 1.2 2.3 1.2B12 μg 3.1 4.7 4.5Niacin mg 9.7 16.8 14.8Folic acid μg 386 435.5 354.3Biotin μg 116 89.8 87.2

1. For composition, see: http://apps.who.int/iris/bitstream/10665/41999/1/a57361.pdf2. Both feeds were prepared by adding cooled, boiled water to 400g to make-up to 2L and then adding

1.7g CMV/L3. For composition, see: https://www.nestlehealthscience.co.uk/asset-library/documents/data%20

card%20modulen%20ibd.pdf4. For composition, see: http://www.nutramigen.co.uk/files/5114/2565/5560/Filofax_Purami-

no_01_2015_5.pdf5. Prepared as described results in a formula with approx. 1kcal/ml (http://www.meadjohnson.com/pe-

diatrics/us-en/sites/hcp-usa/files/345%20PurAmino%20Scoop%20Dilution.pdf)6. Fat content of F-100 equates to 53% of total energy

141

5


Supplemental Table 2. Socioeconomic variables at recruitment according to intervention arm

Variable n (%) Standard (n=31)

Elemental (n=32)

Polymeric (n=32)

Total (n=95)

House type 31 32 32 95

• Owned 12 (38.7) 19 (59.4) 13 (40.6) 44 (46.3)

• Rented 19 (61.3) 12 (37.5) 18 (56.3) 49 (51.6)

• Other 0 (0.0) 1 (3.13) 1 (3.13) 2 (2.11)

Number sleeping rooms (mean; SD) 1.97(0.71) 2.56(1.29) 2.13(0.91) 2.22(1.02)

Number of people usually sleeping in the house (mean; SD) 4.61(1.84) 4.66(1.77) 4.81(2.05) 4.69(1.87)

Electricity supply 31 32 32 95

8 (25.8) 10 (31.3) 5 (15.6) 23 (24.2)

The family owns: 31 32 32 95

• A radio 11 (35.5) 16 (50) 10 (31.3) 37 (38.9)

• A bicycle 2 (6.45) 5 (15.6) 4 (12.5) 11 (11.6)

• A motorbike 0 (0.0) 1 (3.13) 1 (3.13) 2 (2.11)

• A car or truck 1 (3.23) 0 (0.0) 0 (0.0) 1 (1.05)

• A paraffin lamp 7 (22.6) 10 (31.3) 8 (25) 25 (26.3)

• A koloboyi 10 (32.3) 9 (28.1) 18 (56.3) 37 (38.9)

• An oxcart 0 (0.0) 1 (3.13) 0 (0.0) 1 (1.05)

• A television 4 (12.9) 10 (31.3) 3 (9.38) 17 (17.9)

• A cell phone 12 (38.7) 14 (43.8) 15 (46.9) 41 (43.2)

• A telephone (landline) 0 (0.0) 1 (3.13) 1 (3.13) 2 (2.11)

• A bed with mattress 12 (38.7) 20 (62.5) 7 (21.9) 39 (41.1)

• A sofa set 4 (12.9) 10 (31.3) 3 (9.38) 17 (17.9)

• A table and chair 16 (51.6) 23 (71.9) 16 (50) 55 (57.9)

• A refrigerator 2 (6.45) 4 (12.5) 2 (6.25) 8 (8.42)

• A watch 5 (16.1) 10 (31.3) 4 (12.5) 19 (20)

The family owns agricultural land 31 32 32 95

21 (67.7) 25 (78.1) 19 (59.4) 65 (68.4)

The principal households source of drinking water is (N; %): 31 32 32 95

• Pipe inside dwelling 1 (3.23) 0 (0.0) 0 (0.0) 1 (1.05)

• Pipe outside dwelling/ to yard 1 (3.23) 3 (9.38) 1 (3.13) 5 (5.26)

• Protected borehole/ well 9 (29) 15 (46.9) 9 (28.1) 33 (34.7)

• Public tap 16 (51.6) 11 (34.4) 18 (56.3) 45 (47.4)

• Traditional public well 4 (12.9) 3 (9.38) 4 (12.5) 11 (11.6)

• River/ canal/ lake 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Tanker truck 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Bottled water 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Rain water 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Cart with small tank 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

142

Chapter 5


Elemental (n=32)

Polymeric (n=32)

Total (n=95)

To make the water safe to drink, the water is:

• Boiled 7 5 7 19

0 (0.0) 1 (20) 2 (28.6) 3 (15.8)

• Mixed with bleach/chlorine/water guard 7 5 7 19

5 (71.4) 5 (100) 5 (71.4) 15 (78.9)

• Strained through a cloth 7 5 7 19

1 (14.3) 0 (0.0) 0 (0.0) 1 (5.26)

• Filtered (ceramic/sand/etc.) 7 5 7 19

0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Standing to settle 7 5 7 19

0 (0.0) 1 (20) 1 (14.3) 2 (10.5)

• Not changed. Nothing has been done with it 31 32 32 95

7 (22.6) 5 (15.6) 7 (21.9) 19 (20)

The principal type of toilet facility used by the household members is:

31 32 32 95

• Own (exclusive) flush 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Shared flush toilet 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Ventilated latrine 6 (19.4) 8 (25) 6 (18.8) 20 (21.1)

• Pit latrine with slab 9 (29) 13 (40.6) 11 (34.4) 33 (34.7)

• Pit latrine without slab/ open pit 16 (51.6) 11 (34.4) 15 (46.9) 42 (44.2)

• Bush or field 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

The number of households using this toilet facility (mean; SD)

2.55(1.43) 2.56(2.06) 2.75(2.49) 2.62(2.03)

The principal type of flooring of the house: 31 32 32 95

• Tiles/cement/vinyl 18 (58.1) 20 (62.5) 21 (65.6) 59 (62.1)

• Wood/planks/broken bricks 1 (3.23) 0 (0.0) 0 (0.0) 1 (1.05)

• Dirt/sand/dung 12 (38.7) 12 (37.5) 11 (34.4) 35 (36.8)

The principal type of roofing of the house: 31 32 32 95

• Cement 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Wood planks/cardboard 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Iron and tiles 2 (6.45) 0 (0.0) 1 (3.13) 3 (3.16)

• Iron sheets 23 (74.2) 21 (65.6) 23 (71.9) 67 (70.5)

• Natural materials 6 (19.4) 11 (34.4) 8 (25) 25 (26.3)

A member of the household owns a bank account: 31 32 32 95

8 (25.8) 8 (25) 7 (21.9) 23 (24.2)

The usual daily income for the family is: 31 32 32 95

• <500 MWK 3 (9.68) 2 (6.25) 1 (3.13) 6 (6.32)

• 500 - 1000 MWK 23 (74.2) 19 (59.4) 23 (71.9) 65 (68.4)

143

5



Elemental (n=32)

Polymeric (n=32)

Total (n=95)

• >1000 MWK 5 (16.1) 11 (34.4) 8 (25) 24 (25.3)

In the past 12 months, the inside walls of the house have been treated against mosquitoes

31 32 32 95

2 (6.45) 3 (9.38) 1 (3.13) 6 (6.32)

The number of mosquito nets used for sleeping in the household (mean; SD)

2(1.24) 2.41(1.88) 2.13(1.84) 2.18(1.68)

Type of fuel mainly used for cooking: 31 32 32 95

• Electricity 0 (0.0) 1 (3.13) 0 (0.0) 1 (1.05)

• Charcoal 22 (71) 15 (46.9) 20 (62.5) 57 (60)

• Wood 9 (29) 16 (50) 12 (37.5) 37 (38.9)

• Natural gas 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Biogas 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Kerosene 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

• Straw/shrubs/grass 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

Type of stove/fire used for cooking: 31 32 32 95

• Open fire 29 (93.5) 31 (96.9) 30 (93.8) 90 (94.7)

• Stove without chimney/ flute 2 (6.45) 1 (3.13) 2 (6.25) 5 (5.26)

• Stove with chimney/ flute 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

Location where cooking is usually done: 31 32 32 95

• In the house 8 (25.8) 7 (21.9) 12 (37.5) 27 (28.4)

• In a separate building 4 (12.9) 7 (21.9) 4 (12.5) 15 (15.8)

• Outside 19 (61.3) 18 (56.3) 16 (50) 53 (55.8)

The highest education level of the main care giver is: 31 32 32 95

• No education 3 (9.68) 2 (6.25) 1 (3.13) 6 (6.32)

• some primary education 17 (54.8) 12 (37.5) 21 (65.6) 50 (52.6)

• Completed primary education 3 (9.68) 5 (15.6) 2 (6.25) 10 (10.5)

• some secondary education 6 (19.4) 11 (34.4) 5 (15.6) 22 (23.2)

• Completed secondary education 2 (6.45) 2 (6.25) 3 (9.38) 7 (7.37)

• More than secondary education 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

144

Chapter 5

Supplemental Table 3. Baseline characteristics according to presence of edema

Variable (n, mean, ± SD)Without edema(n=57)

With edema(n=38) P-value

Demographic

Male N(%) 26/57 (46.7 %) 20/38 (52.7 %) .50

Age in months 57, 12.8 ± 4.8 38, 17.9 ± 5.4 <.001

Anthropometry

Mid-Upper-Arm-Circumference (cm) 57, 10.6 ± 1.2 38, 14.3 ± 14.2 <.001

laboratory

Fecal calprotectin (μg/mg) 48, 660.8 ± 1040.7 30, 496.8 ± 545.2 .885

Fecal α1-antitrypsin (mg/dL) 49, 8.4 ± 24.6 29, 4.7 ± 5.9 .842

Plasma IgG anti-endotoxin antibodies (GMU/ml) 34, 94.3 ± 101.9 27, 96 ± 144.7 .689

Plasma intestinal fatty acid binding protein (pg/ml) 34, 4310.6 ± 2652.2 26, 4862.8 ± 3150.9 .464

Platelets (x109/L) 33, 445.8 ± 290.8 26, 475.3 ± 234.8 .676

Plasma C-reactive protein (mg/L) 33, 28.2 ± 50 27, 18 ± 21.8 .768

Plasma α1-acid glycoprotein (μg/ml) 34, 2864.4 ± 993.3 27, 3403.9 ± 780.2 .024

Insulin-like growth factor-1 (ng/ml) 22, 51.9 ± 27.5 15, 46.7 ± 27.4 .687

Insulin-like growth factor binding protein 3 (ng/ml) 34, 785.3 ± 354.5 26, 888.2 ± 349 .267

Hemoglobin (g/dL) 33, 9.6 ± 1.1 26, 9.2 ± 1.4 .198

White cell count (x109/L) 33, 12.5 ± 5 26, 12.3 ± 5.2 .906

Data was analyzed using a one-way ANOVA, and variables which were not normally distributed were logarithmically transformed to detect any overall differences in group means according to edema status

145

5


Supplemental Table 4. Baseline characteristics according to HIV status

Variable (n, mean, ± SD)HIV negative(n=60)

HIV Positive(n=34) P-value

Demographic

Male N(%) 28 (29.8 %) 18 (19.1 %) .56

Age in months 60, 14.9 ± 5.8 34, 14.9 ± 5.3 .805

Clinical

Mid-Upper-Arm-Circumference (cm), 59, 11.5 ± 1.3 34, 10.6 ± 1.4 .002

Weight-for-length Z-score1 32, -3.5 ± 1.2 24, -3.9 ± 1.2 .254

With Edema N(%) 28 (29.8 %) 10 (10.6 %) .10

laboratory

Fecal calprotectin (μg/mg stool) 49, 423.5 ± 712.6 29, 892.1 ± 1063 .005

Fecal a1-antitrypsin (mg/dL) 50, 4.6 ± 5.2 28, 11.3 ± 32.3 .368

Plasma IgG anti-endotoxin antibodies (GMU/ml)

41, 84.5 ± 112.9 20, 116.8 ± 138.3 .090

Plasma Intestinal fatty acid binding protein (pg/ml)

40, 4760 ± 2991.7 20, 4129.7 ± 2620.5 .427

Platelets (x109/L) 39, 493.9 ± 259.8 20, 390.3 ± 270.5 .158

Plasma C-reactive protein (mg/L) 40, 16.2 ± 23.6 20, 38.4 ± 58.9 .008

Plasma a1-acid glycoprotein (μg/ml) 41, 2945.4 ± 941.2 20, 3426.7 ± 865 .059

Insulin-like growth factor -1 (ng/ml) 29, 49.2 ± 25.5 8, 51.7 ± 34.4 .854

Insulin-like growth factor binding protein 3 (ng/ml)

40, 896.5 ± 381.9 20, 696.7 ± 243.7 .038

Hemoglobin (g/dL) 39, 9.8 ± 1.1 20, 8.9 ± 1.3 .007

White cell count (x109/L) 39, 13 ± 4.7 20, 11.3 ± 5.4 .207

Data was analyzed using a one-way ANOVA, and variables which were not normally distributed were logarithmically transformed to detect any overall differences in group means according to HIV status. 1In children without edema

146

Chapter 5

Supp

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. Bio

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and

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and

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6 (7

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23 433

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7 (7

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23 538

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3 (7

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25 763

(777

)

Muc

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inte

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Feca

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(mg/

dl)

Nor

mal

<26

.8 m

g/dl

Day

064 25

.4(3

1.1)

.004

6

23 26.9

(38)

21 18.4

(12.

7)20 31

(35.

8)

Day

1458 46

.9(4

4.2)

19 30.8

(33.

6)19 45

.3(3

7.2)

20 63.8

(54.

1)

Plas

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IgG

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.6(1

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)

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15 73.4

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1.6(

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2.7(

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4

15 3487

.6(1

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15 5284

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1445 42

20.4

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13 4234

.9(2

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81.6

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17 463.

8(30

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13 466.

2(33

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16 490.

9(18

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3.8(

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9.4(

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14 646(

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6)16 54

2.4(

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5)

147

5


All c

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Inte

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(n=3

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Plas

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g/L)

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Day

044 26

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.07

14 19.7

(17.

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.7(2

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15 37.3

(70.

5)

Day

1445 12

.2(2

8.1)

16 13.1

(29.

8)13 6.

8(12

.7)

16 15.7

(35.

6)

Plas

ma

α 1-a

cid

glyc

opro

tein

(μg/

ml)

Nor

mal

322

-114

3 μg

/ml

Day

045 32

56.2

(908

.6)

<.00

01

14 3314

.9(8

55.3

)16 32

91.7

(950

.2)

15 3163

.5(9

66.4

)

Day

1443 19

69.4

(100

1.1)

15 2530

.1(1

036.

1)12 20

85.9

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Supplemental Table 6. Feeding and clinical symptoms according to intervention arm

Variable1 Standard(n=31)

Elemental(n=32)

Polymeric(n=32)

Elemental vs. standardP value2

Polymeric vs. standardP value2

Feeding

• Required NGT feeds 6 (19.4) 15 (46.9) 11 (34.4) .021 .18

• Required F75 8 (25.8) 6 (18.8) 0 (0) .50 .002

No. days completed feed: mean (SD) 12.0 (4.1) 11.0 (4.3) 12.1 (3.5) .32 .98

Symptoms and signs

Stool frequency/day 3.6 (1.93) 3.9 (2.0) 3.4 (1.9) .047 0.13

Children who experienced between day 0 and 14

• Vomiting 10 (32.3) 19 (59.4) 19 (59.4) .031 .031

• Loose stools 22 (71) 26 (81.3) 26 (81.3) .34 .34

• Watery stools 5 (16.1) 9 (28.1) 7 (21.9) .25 .56

• Mucus in stools 0 (0) 2 (6.3) 0 (0) .49 -

• Blood in stools 1 (3.23) 0 (0) 2 (6.25) .49 1.0

On day 14, children with

• Loose/watery stools 6 (26.1) 5 (19.2) 6 (18.8) .97 1.0

• Any edema 2 (6.5) 3 (9.4) 1 (3.1) - -

1Data are n (%) unless shown otherwise. 2 Data was analyzed using a Chi-Square test for n (%) data, a t-test for stool frequency/day and Generalized Linear Analysis for number of days completed feed with Poisson distribution and a link log. NGT=Naso-gastric tube

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Supplemental Table 7. Severe adverse events according to MedDRA preferred term, intervention group and outcome

number (%) of children

Total Standard Elemental Polymeric

n=31 n=32 n=32

25 9b (29.0) 9b (28.1) 7 (21.9)

MedDRA preferred term

• Gastroenteritis 13 4 8 1

• Dehydration 11 5 5 1

• Sepsis 6 - 3 3

• Metabolic acidosis 3 - 2 1

• Pulmonary tuberculosisa 3 1 - 2

• Pneumonia 1 1 - -

• Septic shock 1 - 1 -

• Hypoglycemia 1 - - 1

• Urticaria drug-induced 1 1 - -

• Hypovolemic shock 1 1 - -

• Acute kidney injury 1 - 1 -

• Unknownc 1 - - 1

Total number SAEs 43 13 20 10

A total of 43 SAEs occurred in 25 children. aTreatment for TB was on-going in these 3 cases at the end of the study. bTwo patients (one in the control group and one in the elemental group) had an SAE onset within the study period, but died one day after the 14th day. c20-month-old boy, HIV positive, malaria slide negative, admitted with edematous SAM, gastroenteritis, oral thrush and septicemia. He was re-ported to be well on review on the third study day but then died suddenly and the cause was unclear. The results of an initial blood culture were not available.

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Hypoallergenic and anti -infl ammatory feeds in children with complicated severe acute malnutriti on


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Chapter 6The clinical use of longitudinal bio-electrical impedance analysis in children with severe acute malnutrition

Rosalie H. Bartels*, Céline Bourdon*, Emmanuel Chimwezi, Jacintha Kool, Katherine Chidzalo, Robert H.J. Bandsma, Michael Boele van Hensbroek, Wieger P. Voskuijl

*These authors contributed equally

Manuscript in preparation

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ABSTRACT

Background & Aims: The severity of malnutrition in children is normally assessed us-ing anthropometry: weight for height (W/H), height for age (W/A), and mid upper arm circumference (MUAC) which measure body composition indirectly. Altered body com-position (loss of fat-free mass) is linked to poor clinical outcome and can be estimated by bio-electrical impedance analysis (BIA). We aimed to assess how BIA parameters in children with severe acute malnutrition (SAM) with or without edema: 1) change during clinical stabilization; or 2) add a prognostic value to clinical outcome when combined with ‘classic’ anthropometry.Methods: For this prospective, observational study, we enrolled children, aged 6-60 months, that were admitted for complicated SAM at the Queen Elizabeth Central Hospital in Blantyre, Malawi. Height, weight, MUAC and bio-electrical impedance were measured on admission and after clinical stabilization. Resistance (R) was considered to reflect body fluids, reactance (Xc) soft tissue, and phase angle (PA) the arc tangent relation of Xc/R. The R and Xc values were divided by height to correct for body size (R/H and Xc/H).Results: We studied 183 SAM children (edematous 53%; age 23.0±12.0 months; male 54%) and 11 healthy controls (age 14.6±5.3 months; male 73%). On admission, edematous SAM children had lower bio-electrical impedance parameters (R, Xc, PA) than non-edematous children (p<0.0001); and after clinical stabilization BIA increased only in children with edema (p<0.0001). MUAC and W/H z-scores were negatively correlated with R/H (-0,5967, p<0.0001 and -0.6786, p<0.0001) and this relationship was stronger in edematous cases. Children that died had lower W/H z-scores (-2.7 ± 1.8 vs -4.1 ± 1.8, p=0.0002) and lower MUAC (11.6 ± 1.6 vs 10.3 ± 1.8, p < 0.0001). Combining BIA and anthropometric variables did not improve classification error rates or sensitivity and specificity in predicting clinical groups or outcomes compared to using anthropometric variables alone. However, BIA did help better distinguish children with edematous SAM from community children. Overall, the variability in BIA measures was high and their added predictive value low. Conclusions: During clinical stabilization, BIA parameters increase in children with edematous SAM, which likely reflects fluid loss. BIA is correlated with classic anthropom-etry, and this more so in children with edema. Classic anthropometry is associated with mortality but BIA, as currently implemented, does not add prognostic value in predicting the clinical outcome of children with SAM. Unless the method is improved, our data does not support the clinical use of BIA in low-resource settings for care of children with complicated SAM.

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Bio-electrical impedance analysis in children with severe acute malnutrition

InTRODUCTIOn

Severe acute malnutrition (SAM) in children remains a major global health problem, hence its inclusion in the Sustainable Development Goals.(1,2) Nearly half (45%) of global under-five mortality is related to poor nutrition and the vast majority of these children live in Sub Saharan Africa and South east Asia.(3) Despite adherence to WHO protocols the case fatality rate in children with SAM remains unacceptably high (up to 35%).(4) Thus, current SAM management and identification of children at risk of clinical deterioration needs to be urgently improved.(1,5–7)SAM is defined by WHO standards, as any of the following: for non-edematous SAM/marasmus, a weight for height (W/H) below -3 standard deviation (SD), a mid-upper arm circumference (MUAC) of less than 115mm, or, for edematous SAM/kwashiorkor, the presence of bilateral edema.(8) Current management strategies are ‘blanket approaches’ that disregard the different presentations of SAM(4) and their associated clinical risk. Several recent studies show higher mortality in children with non-edematous SAM.(9–11)In the 1960s, the four-surface electrode technique of bioelectrical impedance analysis (BIA) was introduced and several multi-frequency analyzers have since become available.(12) Over the past two decades, BIA has proven to be non-invasive, inexpensive, and easy to implement. This method of estimating body composition is now widely used for clini-cal assessment in high resource settings since it is non-invasive, portable and relatively inexpensive.(12–17) Body composition is not quantified by BIA directly but is calculated from body reactance (Xc) and resistance (R) which are measured by changes that occur in a small alternating electrical current when it passes through the body.(18,19) Reac-tance arises from cell membranes, and resistance from extra- and intracellular fluid and electrolytes, and their combination is termed ‘(body-)impedance’.(12) It can provide a reliable estimate of total body water (TBW) and fat free mass (FFM) in healthy individu-als but requires population and disease-specific equations.(20) The phase angle (PA) is the arc tangent relation of Xc/R converted to degrees.(21) It describes the phase shift between the current and voltage that results from the electrochemical membrane.(19) It is considered to be an indicator of membrane integrity and body cell mass. In several diseases a reduced PA is associated with reduced survival.(14) BIA measurements can also be interpreted using bioelectrical impedance vector analysis (BIVA) as developed by Piccoli et al.(22,23) This method plots the raw impedance parameters R and Xc normal-ized by height as a bivariate vector in the ‘R-Xc graph’(24) and differences in these R-Xc vectors can be useful (e.g. to monitor hydration status or muscle mass changes).(25)In addition to estimating body composition or hydration status, components of BIA have been proposed to be useful prognostic markers and shown to correlate with clini-cal outcome in two high-resource studies in children with renal disease or undergoing hematopoietic stem cell transplantation.(26,27) Girma et al. recently published a cross-

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sectional paper on BIA in malnourished children and showed that Xc was associated with serum calcium and chloride while R correlated with serum albumin. The authors suggested that R could be used to monitor nutritional recovery.(16) However, the study was cross-sectional with a small sample size. Our prospective study aimed to assess whether BIA parameters: 1) change during hospitalization in children with non-edematous or edematous complicated SAM and 2) whether they add a prognostic value to predict clinical outcome compared to using only classic anthropometry.

MATERIAlS AnD METHODS

Study design and settingThis prospective, observational study was conducted within the framework of the “F75 trial”, a multicenter, randomized, double-blind intervention trial (ClinicalTrials.gov: NCT02246296). Briefly, the study aimed to determine whether stabilization of mal-nourished children is improved by reducing carbohydrates and removing lactose in F75, the standard milk formula recommended by the WHO. The trial randomized children with SAM to either receive the standard F75 milk or a modified formulation which was iso-caloric but containing more triglycerides and less carbohydrates. In Malawi, the trial was conducted at the Nutrition Rehabilitation Unit (NRU) in the Pediatric Department of Queen Elizabeth Central Hospital in Blantyre and enrolled 320 patients between December 2014 and December 2015 (manuscript in preparation). Our BIA sub-study started recruiting patients later from February 2015 until the trial stopped. The study was approved by the Malawi College of Medicine Research and Ethics Committee (COMREC nr P.03/14/1540) and conducted according to guidelines of Good Clinical Practice which are based on the principles of the Declaration of Helsinki.(28)

ParticipantsChildren aged 6 – 96 months admitted with complicated SAM (i.e. those with signs of severe systemic illness and/or poor appetite)(4) were screened for eligibility for the F75 trial. SAM was defined according to WHO standards (see above).(29) Children were excluded if parental consent was not obtained or if the child had known allergies to milk products. For the BIA study, only children between 6-60 months were included and additional exclusion criteria were: 1) the presence of open skin lesions on hands or feet that would impede the positioning of BIA electrode stickers, 2) inability to stretch the limbs due to cerebral palsy (CP), and 3) significant body asymmetry such as amputations, unilateral hemiparesis, and neuromuscular conditions causing localized changes in perfusion or

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tissue atrophy.(13) Also, for the BIA study, we enrolled children during weekdays and office hours only. For comparison, healthy children (n=11) were later recruited for BIA analysis. These children were measured using the same equipment between January and June 2017 within the framework of another observational study conducted by The Childhood Acute Illness and Nutrition (‘CHAIN’) Network (ClinicalTrials.gov Identifier: NCT03208725). These healthy controls were recruited from the community to establish expected norms for demographic and biological factors.

Inpatient careThe standard clinical care for children with SAM consists of three distinct phases. In ‘phase 1’ or ‘stabilization phase’, F75, a low protein milk with reduced caloric energy (80-100 kcal/kg/day) is given. Once stabilized, a child is moved to the ‘transition phase’ and receives either ready-to-use therapeutic foods (RUTF) or a milk formula known as F100. Compared to F75, RUTF and F100 have higher energy density and protein content. A child is discharged when clinically stable and able to finish their RUTF feeds (‘rehabilita-tion phase’/‘phase 2’). BIA measurements were taken at hospital admission and after stabilization (i.e. on the first day of transition). All children admitted to the NRU had a thick blood film examined for parasitemia, hematocrit counts and were offered a rapid Human Immunodeficiency Virus (HIV) antibody test with appropriate pre- and post-counselling. These tests were also performed in the recruited community children.

Data collectionWeight was measured using a digital scale (Marsden Portable Digital Baby Scale - Class III MS-4101). The height of children under 24 months was measured in supine position using length boards, whereas stadiometers were used for those aged 24 months and older. Edema was scored based on the WHO grading system.(8)BIA output variables (Xc, R and PA) were measured using a Bioelectrical Impedance Analyzer (Bodystat QuadScan4000) at 50 kHz. Self-adhesive disposable electrodes were attached at the right hand and foot just proximal to the fingers and toes, injecting leads were connected to both electrodes and the measuring leads to those on the right wrist and right ankle. Measurements were taken in triplicates but tests were repeated up to 5 times if the variance in R or Xc was above 5%.

Statistical methodsData were collected on standardized proforma’s, entered into a database and analyzed with Stata (Release 13) (30) and R (Version 3.4.0). Differences in baseline characteristics of participants were assessed using Fisher exact test, or logistic regression. As children

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with non-edematous and edematous SAM display different clinical and biochemical char-acteristics, we conducted sub-analyses. For BIA, we used logistic regression to analyze group differences at admission and logistic mixed effects models to evaluate changes between admission and transition phase while accounting for repeated measures within subjects. The sample size was based on a convenience sample. To compare the prognostic value of using anthropometrics alone or anthropometrics complemented by BIA, we conducted Partial Least Squares discriminant analysis (PLS-DA) using the mixOmics package.(31) Values were offset by 10, log transformed, mean centered and scaled. Multilevel PLS-DA was used to compare admission and nutritional stabilization to account for repeated measures. The discriminative power of the PLS-DA models to classify groups was assessed using the balanced error rate (BER) based on centroid distance obtained from leave-one-out cross-validation. BIVA was performed as described by Piccoli to test for differences in BIA between groups of: 1) children with or without edema on admission; 2) between patients at admission and after clinical stabilization.(32) The height-corrected values of R and Xc were plotted with ellipses indicating the 95% confidence area of the means as previously described.(32) Differences in independent multivariate means were tested using Hotelling’s T2 test (e.g. between edema and non-edema or survival and mortality) whereas paired Hotelling’s T2 test was used to evaluate changes between patients at admission and after stabilization. Hotelling’s T-test is a multivariate extension of the Student-T test and p<0.05 where considered to be significant. Shifts in BIVA were interpreted as previously described and illustrated in Supplemental Figure 1.(32)Spearman’s correlation was used to relate log-transformed height-corrected BIVA param-eters (i.e. R, Xc) and classic measures of anthropometry (i.e. W/H, W/A, MUAC).

ReSUlTS

Between December 2014 and December 2015, 183 patients were recruited for our BIA sub-study. Anthropometry and BIA measurements were successfully conducted on admission in 175 children (non-edematous n=81, edematous n=94), and in 155 patients after clinical stabilization (non-edematous n=73, edematous n=82); with 147 children (non-edematous n=68, edematous n=79) having both time points (Supplemental Figure 2. Flow chart). Overall mortality in SAM patients was 17% (total deaths, n=31; non-edem-atous, n=17; edematous, n=14). Of these children, some passed away before clinical stabilization (n=13) while others died after (n=18). Baseline characteristics are detailed in Table 1 and separated by nutritional status and clinical outcome in Supplemental Table 1. Children with edematous SAM were older (p=0.002), and less likely to be HIV positive (16% vs. 38% in the non-edematous group, p=<0.001).

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We measured anthropometry and BIA in 11 children recruited from the community. These controls were younger than our patient group with SAM (14.6 ± 5.3 months versus 23.0 ± 12.0 months, p=0.01) but their anthropometric measures tended to be normal for their age; with their lowest measurement being H/A Z-score of -1.1 ± 1.6.

Table 1 – Characteristics of community children and patients with severe acute malnutrition.

SAM

Control SAM pNon-

edematous Edematous p

n=11 n=183 n = 86 n = 97

Male sex, n (%) 8 (73) 99 (54) .4 46 (53) 53 (55) .9

HIV reactive*, n (%) 0 (0) 47 (26) .07 32 (38) 15 (16) < .001

Age, mon 14.6 ± 5.3 23.0 ±12.0 .01 20.0 ± 12.5 25.6 ± 11.0 .002

Height-for-age, z-score -1.1 ± 1.6 -3.4 ±1.5 < .0001 -3.7 ± 1.5 -3.2 ± 1.5 .02

Weight-for-age, z-score -0.7 ± 1.1 -3.8 ±1.6 < .0001 -4.7 ±1.1 -3.1 ± 1.6 < .0001

Weight-for-height, z-score -0.2 ± 1.3 -3.0 ±1.8 < .0001 -4.0 ±1.1 -2.0 ± 1.8 < .0001

MUAC, cm 14.0 ± 1.1 11.4±1.7 < .0001 10.4 ± 1.2 12.2 ± 1.7 < .0001

Time to stabilization, days - 3.3 ± 2.0 - 3.2 ± 1.8 3.5 ± 2.2 .3

Duration of admission, days** - 5.6 ± 3.3 - 5.3 ± 3.0 5.9 ± 3.5 .3

Time to death, days - 6.5 ± 4.2 - 7.1 ± 5.1 5.8 ± 3.0 .4

Death, n (%) - 31 (17) - 17 (16) 14 (14) .4

Data in cell are mean +/- SD or n (%), *3 SAM patients with unknown HIV status; ** Duration of admission calculated in only patients that survived. Significance test performed with either Fisher exact or logistic regression as appropriate. Significance threshold, p < 0.5. MUAC, mid upper arm circumference; SAM, severe acute malnutrition.

BIA in children with or without edematous SAM on admission and after clinical stabilization Bio-electrical impedance values are shown in Table 2 and BIVA plots of height adjusted R and Xc values are presented in Figure 1. SAM children without edema had higher resis-tance index than controls (1241 ± 250 ohms/m vs. 993 ± 188 ohms/m, p<.0001), whereas children with edema had lower PA (2.3 ± 1.3 vs. 3.2 ± 0.5 degree, p=.04) and lower indices of both resistance and reactance (respectively, 803 ± 272 vs. 993 ± 188, p=.03; 54 ± 9 ohms/m and 33 ± 20 ohms/m, p=.005). Compared to children without edema, those with edematous SAM had lower BIA values (PA, R and Xc indices) both on admission and after stabilization; BIVA vectors of edematous and non-edematous SAM patients differed between the two time points (p<0.001 and p<0.001 respectively, Figure 1. In children with edematous SAM, BIA values increased between admission and stabilization (PA 2.3 ± 1.3 degree vs. 2.9 ± 2.1 degree, p=.006; Resistance index 803 ± 272 ohm/m vs. 939 ±

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297 ohm/m, p=.002; Reactance index 33 ± 20 ohm/m vs. 48 ± 35 ohm/m, p=.0007) and stabilizati on values of all BIA parameters for children with edematous SAM no longer diff ered from those of controls. Concordantly, the BIVA vector of children with edema-tous SAM showed a shift along the major hydrati on axis aft er stabilizati on (p=0.001), which likely refl ects loss of edema in these pati ents (explained in Supplemental Figure 1). In contrast, the BIVA parameters of children with non-edematous SAM did not diff er signifi cantly between admission and stabilizati on, while their resistance index remained diff erent from controls.

R /H , O h m /m

0 500 1000 1500 2000

Xc/

H,O

hm/m

0

50

100

150

200

nE nE'E E'

CP

Legend

E

E'

nEnE'

CP

nE nE', p=0.2

E E', p=0.001

Figure 1 BIVA vector analysis.Bio-electrical vector analyses (BIVA) of healthy controls, SAM pati ents with and without edema on ad-mission and aft er stabilizati on. CP, community parti cipants (n=11); nE, Non-edematous children with SAM on admission (n=81); nE’, Non-edematous children aft er stabilizati on (n=73); E, Edematous children on admission (n=94); E’ Edematous children aft er stabilizati on (n=82). Black dots represent the centroids and ellipses indicate the 95% confi dence interval of the mean for each group as indicated by correspond-ing label. The mean shift between admission and stabilizati on of children with or without edema was tested using paired Hotelling’s T2 test, p<0.05 was considered signifi cant.

163

6


Tabl

e 2

– Bi

o-el

ectr

ical

impe

danc

e va

lues

of c

omm

unity

con

trol

chi

ldre

n an

d pa

tient

s with

eith

er e

dem

atou

s or n

on-e

dem

atou

s SAM

on

adm

issio

n an

d st

abili

za-

tion

Adm

issio

nSt

abili

zatio

n

Co

ntro

lsaSA

Mb

pa,b

Non

-ed

emat

ousc

Edem

atou

sdpc,

dpa,

cpa,

dSA

Me

Non

-ed

em-

atou

sfEd

em-

atou

sgpf,g

pa,f

pa,g

pc,f

pd,g

n=

11n=

175

n

= 81

n =

94

n= 1

55n

= 73

n =

82

Resis

tanc

e, O

hm73

6 ±

9373

5 ±

236

188

0 ±

188

611

± 20

1<

.000

1.0

1<.

0578

5 ±

211

865

± 18

071

3 ±

212

< .0

001

.02

.7.6

.001

Reac

tanc

e, O

hm41

± 6

35 ±

18

.345

± 1

525

± 1

6<

.000

1.3

.01

42 ±

25

49 ±

19

36 ±

28

.003

.2.1

.4.0

006

Phas

e an

gle,

deg

ree

3.2

± 0.

52.

6 ±

1.2

.13.

0 ±

1.0

2.3

± 1.

3 .0

007

.4.0

43.

0 ±

1.7

3.2

± 1.

02.

9 ±

2.1

.3.9

.6.1

.006

Resis

tanc

e in

dex

993

± 18

810

05 ±

34

1.9

1241

± 2

5080

3 ±

272

< .0

001

.004

.03

1072

±

299

1221

±

221

939

± 29

7<

.000

1.0

05.6

.7.0

02

Reac

tanc

e in

dex

54 ±

947

± 2

6.4

64 ±

21

33 ±

20

< .0

001

.1.0

0558

± 3

369

±

2548

±

35.0

002

.07

.5.3

.000

7

Bio-

elec

tric

al im

peda

nce

valu

es w

ere

mea

sure

d at

50

kHz,

resis

tanc

e an

d re

acta

nce

inde

x =

valu

e/he

ight

in m

. Dat

a ar

e pr

esen

ted

as m

ean

+/- S

D an

d lo

gisti

c re

-gr

essio

n w

as u

sed

to a

sses

s gr

oup

diffe

renc

es a

nd m

ixed

effe

ct lo

gisti

c re

gres

sion

to c

ompa

re a

dmiss

ion

and

stab

iliza

tion

whi

le a

ccou

nting

for r

epea

ted

mea

sure

s w

ithin

sub

ject

s. S

igni

fican

ce, p

< 0

.5.

164

Chapter 6

Association of BIA and classic anthropometry with clinical outcomesOverall, BIA values negatively correlated with anthropometric measures, but this re-lationship was mostly driven by children with edematous SAM. Correlations between bio-electrical impedance and anthropometry among children with SAM are shown on admission in Table 3 and after stabilization in Supplemental Table 2 and Supplemental Figure 3. In the edematous group, significant negative correlations were found between both resistance index, reactance index and all anthropometry both on admission and after stabilization. In the non-edematous group, the correlation between BIA and anthro-pometry was weak both on admission and after clinical stabilization.We then used PLS-DA models to evaluate the relationships between variables and their collective capacity to distinguish groups of children with edematous SAM, non-edematous SAM, or community participants (Figure 2). The anthropometric variables of W/H, W/A and MUAC were highly correlated as indicated by their parallel directional arrows (Figure 2A). Using only anthropometric measures (W/H, W/A, H/A, MUAC), groups separated mainly along Variate 1, i.e., the first composite variable that summarizes 75% of the vari-ance of the 4 anthropometric measures. Unsurprisingly, the PLS-DA ROC curves indicate that anthropometric measures have predictive value and can classify children into their respective nutritional groups (AUC values: Controls vs. others, 0.94; non-edema vs. oth-ers 0.83; edema vs. others, 0.74). However, combining anthropometric measures with BIA values only marginally improved classification of non-edematous and edematous children as evaluated by the distance between group centroids but did improve differ-entiation of community children from those with SAM (i.e. community children scored higher along composite Variate 2). Also, the Balanced Error Rate (BER) based on centroid distance was 0.27 for the PLS-DA model using only anthropometric variables, and was 0.25 when using both anthropometric measurements and BIA values. The specific class error rates based on centroid distance also did not significantly improve as they were 0.56 vs. 0.48 for edematous SAM, 0.27 vs. 0.26 for non-edematous SAM, and 0 vs. 0 for community patients. Thus, using more complex composite measures that include both anthropometry and BIA does not improve the group classification of children with SAM compared to using only anthropometry. Also, these error rates are likely underestimated as we used a leave one out cross-validation approach due to the small sample size of the sub analyses. Overall, bio-electrical impedance may help to differentiate children with edematous SAM from controls who may have similar anthropometric measures. The BIVA values of children that died in the edematous group did significantly differ from those that survived (p=0.004) but this subgroup of children was small (n=14) and the difference was driven by 3 outliers which suggests that this relationship may be spuri-ous (Supplemental Figure 4A shows BIVA on admission of children with edematous SAM who survived or died). BIVA on admission of SAM children that died before stabilization did not differ from those that died after stabilization (p=0.8); furthermore, BIVA did not

165

6


Tabl

e 3

– Co

rrel

ation

s bet

wee

n bi

o-el

ectr

ical

impe

danc

e in

dice

s and

ant

hrop

omet

ry a

t hos

pita

l adm

issio

n of

chi

ldre

n w

ith se

vere

acu

te m

alnu

triti

on

Adm

issio

nSA

M p

atien

tsN

on-E

dem

atou

sEd

emat

ous

Cont

rols

r (95

% C

I)df

R2p

r (95

% C

I)df

R2p

r (95

% C

I)df

R2p

r (95

% C

I)df

R2p

Resis

tanc

e in

dex

Wei

ght-f

or-h

eigh

t-0

.63

(-0.7

1, -0

.53)

173

.4<.

001

-0.1

8 (-0

.39,

0.0

4)79

.03

.1-0

.54

(-0.6

7,

-0.3

8)92

.3<.

001

-0.5

8 (-0

.87,

0.03

)9

.3.0

6

Wei

ght-f

or-a

ge-0

.50

(-0.6

1, -0

.38)

173

.3<.

001

-0.0

4 (-0

.25,

0.1

8)79

.0.7

-0.4

2 (-0

.57,

-0

.24)

92.2

<.00

1-0

.49

(-0.8

4,

0.16

)9

.2.1

MU

AC, c

m-0

.47

(-0.5

8, -0

.35)

173

.2<.

001

0.03

(-0.

25, 0

.19)

79.0

.76

-0.3

9 (-0

.55,

-0

.20)

92.2

<.00

1-0

.70

(-0.9

2,-

0.17

)9

.5.0

2

Reac

tanc

e in

dex

Wei

ght-f

or-h

eigh

t-0

.52

(-0.6

2, -0

.40)

173

.3<.

001

0.06

(-0.

16, 0

.27)

79.0

.6-0

.39

(-0.5

5,

-0.2

0)92

.2.0

010.

01 (-

0.59

, 0.

60)

9.0

1

Wei

ght-f

or-a

ge-0

.45

(-0.5

6, -0

.33)

173

.2<

.001

0.07

(-0.

15, 0

.28)

79.0

.5-0

.33

(-0.5

0,

-0.1

4)92

.1.0

01-0

.32

(-0.7

7,

0.35

)9

.1.3

MU

AC, c

m-0

.41

(-0.5

3, -0

.28)

17

3.2

< .0

010.

13 (-

0.10

, 0.3

4)79

.0.3

-0.2

9 (-0

.46,

-0

.09)

92.0

8.0

050.

05 (-

0.56

, 0.

63)

9.0

.9

Phas

e an

gle

Wei

ght-f

or-h

eigh

t-0

.23

(-0.3

7, -0

.09)

173

.05

.002

0.20

(-0.

02, 0

.40)

79

.04

.08

-0.1

2 (-0

.31,

0.

09)

920

.26

0.32

(-0.

35,

0.77

)9

.1.3

Wei

ght-f

or-a

ge-0

.25

(-0.3

8, -0

.10)

173

.06

.001

0.1

(-0.1

2, 0

.31)

79.0

.4-0

.15

(-0.3

5,

0.05

)92

0.1

-0.0

4 (-0

.63,

0.

57)

9.0

.9

MU

AC, c

m-0

.21

(-0.3

5, -0

.07)

173

.04

.005

0.15

(-0.

07, 0

.35)

79.0

.2-0

.11

(-0.3

1,

0.09

)92

0.3

-0.4

5 (-0

.20,

0.

83)

90.

2.2

Bio-

elec

tric

al im

peda

nce

valu

es w

ere

mea

sure

d at

50

kHz,

resis

tanc

e an

d re

acta

nce

inde

x =

valu

e/he

ight

in m

. Cor

rela

tion

(r) w

as a

sses

sed

with

Pea

rson

pro

duct

-m

omen

t cor

rela

tion;

for

this

resis

tanc

e, re

acta

nce

and

phas

e va

lues

wer

e lo

g tr

ansf

orm

ed. C

I is

95%

con

fiden

ce in

terv

al; d

f is

degr

ees

of fr

eedo

m (n

-2);

R2 is th

e co

effici

ent o

f det

erm

inati

on. p

-val

ues

less

than

.05

wer

e co

nsid

ered

sig

nific

ant.

166

Chapter 6

Comparisons

B)

C)

D)

H/A

W/A

MUACW/H

Component 1

Com

pone

nt 2

R

XcH/A

W/AMUACW/H

Component 1

Com

pone

nt 2

Variate 1 (75% explained variance)

Varia

te 2

(19%

exp

lain

ed v

aria

nce)


Varia

te 2

(20%

exp

lain

ed v

aria

nce)

A) B) C) D)

non-edema vs. others: 0.83edema vs. others: 0.74

CP vs. others: 0.94


CP vs. others: 0.95


CP vs. others: 0.79


CP vs. others: 0.99

non-edema vs. others edema vs. others CP vs. others

A)

Comparisons

B)

C)

D)

H/A

W/A

MUACW/H

Component 1

Com

pone

nt 2

R

XcH/A

W/AMUACW/H

Component 1

Com

pone

nt 2


Varia

te 2

(19%

exp

lain

ed v

aria

nce)


Varia

te 2

(20%

exp

lain

ed v

aria

nce)

A) B) C) D)


CP vs. others: 0.94


CP vs. others: 0.95


CP vs. others: 0.79


CP vs. others: 0.99

non-edema vs. others edema vs. others CP vs. others

A)

Figure 2 Parti al Least Squares Discriminant Analysis and ROC curves.Parti al Least Squares Discriminant Analysis (PLS-DA models based on distance matrix as a functi on of nutriti onal group. A) Correlati on plots of anthropometric variables, and C) of anthropometric variables together with bio-electrical impedance variables. B) Individual score plot based only on anthropometric values or on D) both anthropometric and bio-electrical impedance variables; colors indicate the nutri-ti onal group of each individual: non-edematous SAM (grey), edematous SAM (salmon), and community children (blue); group centroids are indicated by stars. H/A, height-for-age z-score; W/A, weight-for-age z-score; MUAC, mid upper arm circumference; W/H, weight-for-length z-score; R, resistance; Xc, reac-tance. ROC curves are generated from leave-one-out cross-validati on of predicted classes and associated aver-age AUC value for the multi variate analysis on A) component 1 and B) component 2 of the PLS-DA mod-els including only anthropometric measures; and on C) component 1 and D) component 2 of the PLS-DA model including both anthropometric and bio-electrical impedance variables.

167

6


differ between SAM children that survived until discharge versus of those that died after stabilization (p=0.3). We also ran PLS-DA models to assess whether admission BIA values can be combined to classical anthropometric measurements to help identify children with diarrhea or those at high risk of mortality. For diarrhea, the classification error rates were high, and AUC values was 0.55, i.e. very low and not better than random chance, suggesting that BIA would not be helpful in identifying children with or without diarrhea or grading its’ severity. For mortality, adding the BIA values to anthropometric measures did not improve classification of children with edematous SAM that died versus survived (BER, 0.31 with only anthropometry vs. 0.31 with both anthropometry and BIA); and this was also the case for children with non-edematous SAM that died or survived (BER, 0.31 with only anthropometry vs. 0.31 with both anthropometry and BIA). This suggests that BIA as performed here would not improve the identification of children at high risk of mortality beyond the information captured using classic anthropometry.

DISCUSSIOn

This study is the first to show how BIA parameters change together with ‘classic’ an-thropometry throughout nutritional recovery of children with complicated SAM. As ex-pected, BIA was correlated with measures of anthropometry, however BIA, as currently implemented, did not add significant clinical prognostic value in predicting the clinical outcome of children with complicated malnutrition. BIA and its association with anthropometric and biochemical markers has been recently described in children with malnutrition by Girma et al.(16) Our results corroborate their findings in a larger sample size but also show how BIA parameters change with nutritional and clinical rehabilitation in children with edematous or non-edematous SAM. Similarly, to Girma et al, we found lower BIA parameters (PA, R and Xc) in children with edematous SAM compared to those without edema.(20) Interestingly, control children had interme-diate BIA parameters having higher values than children with edematous SAM but lower values than those with non-edematous SAM. This likely reflects differences in hydration status between the two phenotypes, but it is unclear if, upon full loss of edema, the BIA parameters of edematous children will stabilize to control levels or continue to increase and resemble those of SAM children without edema. Overall, the resistance and reactance indices of children with SAM negatively correlated with anthropometric measures but this relationship was mainly driven by children with edematous SAM. In these children, MUAC and weight reflect not only lean and fat mass but also edema; whereas BIA parameters may better reflect the separate components of fluid, fat and lean body mass; thus, better able to distinguish SAM patients from controls when compared to using anthropometry alone.

168

Chapter 6

In children with non-edematous SAM, anthropometric measures did not correlate strongly with BIA parameters; thus, it becomes unclear if BIA is still able to reflect body composition in severely wasted children that are ill. We conducted different analyses to draw firm conclusions on whether BIA could have a role in the diagnosis and management of children with complicated SAM. First, we used Piccolis’ BIVA method(32) and found that vectors from children with edematous SAM at admission were shifted after stabilization along the major axis representing changes in tissue hydration (Supplemental Figure 1). This likely reflects the general fluid loss that occurs in children with edematous SAM following nutritional recovery. This shift in multivariate mean was significant, but the variability of BIA parameters was high which reduces the usefulness of BIA in estimating the clinical risk of individual children as evalu-ated using PLS-DA models. We compared the classification performance of PLS-DA models that combined all an-thropometric variables with or without additional BIA parameters. This showed that as expected anthropometry alone had predictive value to classify children into nutritional groups but that adding BIA only marginally improved model performance. Also, the improvements were mostly in separating controls from children with edematous SAM. Therefore, in theory, BIA could help identify children with nutritional edema that present with subclinical symptoms; however, the robustness of BIA for this task would require further evaluation. Finally, in our study, BIA did not show clear prognostic value with regards to other clinical outcomes (i.e. diarrhea or mortality). Although BIA has been in use for over 4 decades, the required population and disease-specific equations to estimate fat mass and lean body mass have not yet been developed (20). Currently, the available equations for young children are based on populations living in developed countries; and appropriate equations to estimate fat/muscle mass from BIA parameters are lacking for children with SAM. Also, the interpretation of BIA parameters and their changes differ between children with edematous or non-edematous SAM. Therefore, pediatric BIA data over a spectrum of nutritional status would be required to facilitate the clinical interpretation of BIA in children with SAM living in low resource settings. Recently, an analytical variant of BIVA has been proposed: specific BIVA.(33) This method has been shown to be more accurate in estimating the relative proportion of fat mass in adults.(33,34) Based on the assumption of Ohm’s law that body impedance is affected by cross-sectional area, specific BIVA is thought to improve the normalization of BIA parameters for differences in body size by using not only height but also cross-sectional measures of the arm, waist, and calf. (34) This approach may be particularly relevant to use in children with SAM since both wasting (narrow limbs) and stunting (altered body fat distribution) are thought to contribute disproportionally to body impedance.(35,36)

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6


The need to normalize BIA values by height likely adds significant variability to BIVA since in young children height is challenging to measure accurately. Using specific BIVA together with a robust approach to measuring pediatric height could improve the clinical prognostic value of BIA in children with SAM.

Strengths and limitationsThis is the first large prospective longitudinal study reporting BIA changes in children hospitalized for complicated SAM. BIA has inherent limitations that become more prob-lematic when being applied to young, ill, malnourished and stunted children. BIA is also influenced by movement of the child during measurements, feeding status, hydration status and/or urine volume in the bladder. These external factors can be, at least in part, mitigated by performing BIA at a particular time to standardize measurement conditions but this was very challenging to achieve in our low-resource setting. Also, our control participants were obtained from a study that recruited children of a younger age range, however, we found that including their data added value to the interpretation of the BIA values obtained from children with SAM. Finally, it would have be useful to follow changes in BIA parameters throughout the duration of hospitalization to assess the point of stabilization of BIA in children with edematous SAM. If the clinical use of BIA is to be pursued, height measures and robust body size correction methods are to be considered to better establish the specific range of BIA values for each patient groups.

ConclusionIn conclusion BIA parameters correlated with anthropometry but this mainly in children with edematous SAM. Also, BIA values differed significantly between children with edem-atous or non-edematous SAM. In those with edema, BIA values shifted towards control values after clinical stabilization and this may reflect receding edema. However, this process can be followed with physical examination, and BIA, as currently implemented, is likely too variable to reliably quantify fluid loss, therefore, clinical scoring continues to be more practical and likely just as informative. Based on our results, the current implemen-tation of BIA would not justify its clinical use in the care of ill hospitalized children with SAM and does not add prognostic value beyond what is achieved by clinical examination together with classic anthropometry.

ACKnOWleDGeMenTS

We would like to thank all study participants and their guardians for taking part in this study and the staff of the nutritional rehabilitation unit “Moyo” of the Queen Elizabeth Central Hospital in Blantyre, for their very hard work.

170

Chapter 6

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25. Norman K, Smoliner C, Kilbert A, Valentini L, Lochs H, Pirlich M. Disease-related malnutrition but not underweight by BMI is reflected by disturbed electric tissue properties in the bioelectrical impedance vector analysis. Br J Nutr. 2008 Sep;100(3):590–5.

26. Bozzetto S, Piccoli A, Montini G. Bioelectrical impedance vector analysis to evaluate relative hydration status. Pediatr Nephrol. 2010;25(2):329–34.

27. Farias CLA, Campos DJ, Bonfin CMS, Vilela RM. Phase angle from BIA as a prognostic and nutritional status tool for children and adolescents undergoing hematopoietic stem cell transplantation. Clin Nutr. 2013;32(3):420–5.

28. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013;310(20):2191–4.

29. World Health Organization, United Nations Children’s Fund, Organization WH, Fund UNC, et al. WHO child growth standards and the identification of severe acute malnutrition in infants and children [Internet]. 2009. Available from: http://www.who.int/nutrition/publications/severemalnutri-tion/9789241598163/en/. Accessed December 3, 2017.

30. StataCorp. Stata Statistical Software: Release 13. College Station, TX: StataCorp LP; 2013. 31. Kim-Anh Le Cao, Ignacio Gonzalez, Sebastien Dejean with kKim-Anh Le Cao, Ignacio Gonzalez, Sebastien

Dejean with key contributors Florian Rohart, Benoit Gautier, Pierre Monget, Jeff Coquery, FangZou Yao, Benoit Liquetey contributors Florian Rohart, Benoit BL. CRAN - Package mixOmics. Toulouse, France; 2015.

32. Piccoli A, Pastori G. BIVA SOFTWARE. Padova, Italy: Department of Medical and Surgical Sciences, University of Padova; 2002.

33. Marini E, Sergi G, Succa V, Saragat B, Sarti S, Coin A, et al. Efficacy of specific bioelectrical imped-ance vector analysis (BIVA) for assessing body composition in the elderly. J Nutr Health Aging. 2013;17(6):515–21.

34. Buffa R, Saragat B, Cabras S, Rinaldi AC, Marini E. Accuracy of Specific BIVA for the Assessment of Body Composition in the United States Population. Bacurau RF, editor. PLoS One. 2013;8(3):e58533.

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35. Fuller NJ, Fewtrell MS, Dewit O, Elia M, Wells JCK. Segmental bioelectrical impedance analysis in children aged 8-12 y: 1. The assessment of whole-body composition. Int J Obes Relat Metab Disord. 2002;26(5):684–91.

36. Martins PA, Hoffman DJ, Fernandes MTB, Nascimento CR, Roberts SB, Sesso R, et al. Stunted children gain less lean body mass and more fat mass than their non-stunted counterparts: a prospective study. Br J Nutr. 2004;92(5):819–25.

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6


Supp

lem

enta

l Tab

le 1

– P

atien

t cha

ract

eristi

cs a

t adm

issio

n of

chi

ldre

n w

ith e

dem

atou

s or

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that

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Mal

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x, n

(%)

8 (7

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(48)

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7).6

46

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0).8

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, n (%

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1.2

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D or

n (%

), *3

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with

unk

now

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ing

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n=2

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nific

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test

per

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xact

or

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stic

regr

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n as

app

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upp

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rm c

ircum

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nce;

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ere

acut

e m

alnu

triti

on.

174

Chapter 6

Supp

lem

enta

l Tab

le 2

– C

orre

latio

ns b

etw

een

bio-

elec

tric

al im

peda

nce

indi

ces a

nd a

nthr

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in c

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with

seve

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cute

mal

nutr

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afte

r sta

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stab

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tion

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mat

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r (95

% C

I)df

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r (95

% C

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r (95

% C

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tanc

e in

dex

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ght-f

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eigh

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(-0.5

9, -0

.34)

150

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001

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5 (-0

.46,

0.0

2)69

.06

.03

-0.4

7 (-0

.62,

-0.2

8)79

.2<.

001

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ght-f

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ge-0

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4, -0

.28)

150

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001

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8 (-0

.31,

0.1

5)69

.01

.5-0

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(-0.6

0, -0

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<.00

1

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m-0

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(-0.4

6, -0

.18)

149

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001

0.12

(-0.

12,0

.34)

68.0

1.3

-0.3

3 (-0

.51,

-0.1

2)79

.1.0

02

Reac

tanc

e in

dex

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eigh

t-0

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7, -0

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150

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001

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1 (-0

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0.2

3)69

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-0.3

4 (-0

.52,

-0.1

3)79

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7, -0

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150

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001

0.0

(-0.2

4, 0

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(-0.5

1, -0

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79.1

.003

MU

AC, c

m-0

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1, -0

.11)

149

.07

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24 (0

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0.4

4)68

.05

.06

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7 (-0

.46,

0.0

5)79

.07

.02

Phas

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gle

Wei

ght-f

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0.27

(0.0

5, 0

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69.0

7.0

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6, 0

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1, 0

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150

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69.0

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5 (-0

.36,

0.0

7)79

.02

.2

MU

AC, c

m-0

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1, 0

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149

.03

>.05

0.22

(-0.

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68.0

5.0

6-0

.14

(-0.3

5, 0

.08)

79.0

2.2

Bio-

elec

tric

al im

peda

nce

valu

es w

ere

mea

sure

d at

50

kHz,

resis

tanc

e an

d re

acta

nce

inde

x =

valu

e/he

ight

in m

. Cor

rela

tion

(r) w

as a

sses

sed

with

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rson

pro

duct

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omen

t cor

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tion;

for

this

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and

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ansf

orm

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I is

95%

con

fiden

ce in

terv

al; d

f is

degr

ees

of fr

eedo

m (n

-2);

R2 is th

e co

effici

ent o

f det

erm

inati

on. p

-val

ues

less

than

.05

wer

e co

nsid

ered

sig

nific

ant.

175

6


Supplemental Figure 1.

Interpretation of individual vector position on the RXc graph

Vector BIA with the RXc graph method, allows an evaluation of soft tissues through patterns based on percentiles of their electrical properties without prior knowledge of body weight. From clinical validation studies in adults, vectors falling out of the 75% tolerance ellipse indicate an abnormal tissue impedance, which is interpreted and ranked following the two directions of major and minor axis of tolerance ellipses:

1. Vector displacements parallel to the major axis of tolerance ellipses indicate progressive changes in tissue hydration (dehydration with long vectors, out of the upper pole, and hyperhydration with apparent edema with short vectors, out of the lower pole);

2. Vectors falling (steady state) or migrating (dynamic state) parallel to the minor axis, above (left) or below (right) the major axis of tolerance ellipses indicate more or less cell mass, respectively, contained in soft tissues (i.e. vectors with a comparable R value and a higher or lower Xc value, respectively).

3. Different trajectories indicate combined changes in both hydration and tissue mass.

Piccoli A, Pastori G: BIVA software. Department of Medical and Surgical Sciences, University of Padova, Padova, Italy, 2002 (available at E-mail:[email protected])

Supplemental Figure 1 Interpretation of individual vector position on the RXc graph. Vector BIA with the RXc graph method, allows an evaluation of soft tissues through patterns based on percentiles of their electrical properties without prior knowledge of body weight. From clinical validation studies in adults, vectors falling out of the 75% tolerance ellipse indicate an abnormal tissue impedance, which is interpreted and ranked following the two directions of major and minor axis of tolerance el-lipses: 1. Vector displacements parallel to the major axis of tolerance ellipses indicate progressive changes in

tissue hydration (dehydration with long vectors, out of the upper pole, and hyperhydration with ap-parent edema with short vectors, out of the lower pole);

2. Vectors falling (steady state) or migrating (dynamic state) parallel to the minor axis, above (left) or be-low (right) the major axis of tolerance ellipses indicate more or less cell mass, respectively, contained in soft tissues (i.e. vectors with a comparable R value and a higher or lower Xc value, respectively).

3. Different trajectories indicate combined changes in both hydration and tissue mass.Piccoli A, Pastori G: BIVA software. Department of Medical and Surgical Sciences, University of Padova, Padova, Italy, 2002 (available at E-mail:[email protected])

176

Chapter 6

Test 1 - BIA on admission n=175

Enrolled for BIA StudySAM, n=183

Community Par�cipants, n=11

Excluded, n=137Out of office hours

Unable to stretch limbs Open skin lesionsToo sickWithdrew from F75 study Both Limbs tapedDied before Test 1

Recruited into F75 trialn=320

Test 2 - BIA a�er stabiliza�onn=155

Died before stabiliza�on, n=13BIA a�er stabiliza�on failed, n=15

Controlsn=11

n=8*

Supplemental Figure 2 Flowchart

Edema levelSurvival

Death

R /H , O h m /m

Xc/

H, O

hm/m

R /H , O h m /m

Xc/

H, O

hm/m

A) B)

Supplemental Figure 4 BIVA of children with diff erent grades of edema who died and survived.A: BIVA on admission of children with edematous SAM who survived (n=83) or died (n=14). Black dots represent the centroids and ellipses indicate the 95% confi dence interval of the mean for each group as indicated by corresponding label. B: BIVA on admission of children with edematous SAM, separated by degree of edema. 1 = +, 2 = ++, 3 = +++.

177

6

Bio-electrical impedance analysis in children with severe acute malnutriti on

Admission After-stabilizationlo

g PA

, deg

ree

log

Xc, o

hmlo

g R

, ohm

W/H, z-score W/H, z-score

W/H, z-score

W/H, z-score

W/H, z-score

W/H, z-score

Control

Edematous SAM, survived

Non-edematous SAM, survived

A)

B)

C)

log

PA (d

egre

e)lo

g Xc

(ohm

)lo

g R

(ohm

)

Edematous SAM, died

Non-edematous SAM, died

ρ=-0.58 (95%CI: -0.47, -0.67) R2=0.3; p<0.0001

ρ=-0.45 (95%CI: -0.31, -0.56) R2=0.2; p<0.0001

ρ=-0.43 (95%CI: -0.31, -0.54) R2=0.19; p<0.0001

ρ=-0.15 (95%CI: -0.001, -0.28) R2=0.02; p<0.05

ρ=-0.28 (95%CI: -0.13, -0.41) R2=0.08; p<0.0003

ρ=-0.06 (95%CI: 0.09, -0.21) R2=0.004; p<0.4

Supplemental Figure 3 Individual correlati ons between bio-electrical impedance and anthropometry on admission and aft er stabilizati on

Chapter 7Summary, General Discussion and Conclusions

Rosalie H. Bartels

181

7

Summary, General Discussion and ConclusionsSummary, General Discussion and Conclusions

SUMMARy AnD GeneRAl DISCUSSIOn

Forty-five percent of worldwide deaths in children under-5 years of age is directly or indi-rectly attributable to poor nutrition.(1,2) Tackling the global problem of malnutrition and of severe acute malnutrition (SAM) in particular, to increase health, quality of life, and to reduce under-5 mortality, is now receiving much greater attention from the international community. This is demonstrated, among others, by its inclusion in the United Nations’ Sustainable Development Goals.(2)The aim of this thesis is to gain further insight into how to improve diagnosis and treat-ment of children with complicated SAM. In this thesis we described the results of two randomized controlled intervention trials, two observational studies and one systematic review.

Severe Acute Malnutrition and the exocrine pancreasIn the first three chapters we explored the relationship between exocrine pancreatic insufficiency (EPI) and SAM, and the prevalence and treatment of EPI in children with complicated SAM.(3–5) EPI results in impaired digestion because of the central role of the exocrine pancreas in nutrient digestion. EPI can cause diarrhea; one of the common complications in children with SAM, which can greatly increase mortality.(6–8) EPI can be treated by supplying the pancreatic enzymes through pancreatic enzyme replacement therapy (PERT).(9,10) The relationship between EPI in malnutrition in children had been described previously, but it was unclear if malnutrition led to EPI, or vice versa. Previous studies, mostly performed between 1940 and 1980, found that children with SAM also have EPI.(11–22) While it was also known that children with EPI as a complication of an underlying illness, are at risk of becoming malnourished.(23–25)To find out whether malnutrition led to EPI, or vice versa, we systematically assessed the evidence concerning the relation between EPI and malnutrition in children, as presented in chapter 2. We performed database searches to find studies reporting on prevalence or incidence of EPI and malnutrition in children. Nineteen studies were included and divided into two groups: ten studies reporting on patients diagnosed with EPI who were later found to be malnourished, all conducted in cohorts of patients with an underlying disease leading to EPI (i.e. Cystic Fibrosis (CF), Chronic Pancreatitis, Human Immuno-deficiency Virus (HIV), Shwachmann-Diamond Syndrome (SDS) and celiac disease), and nine studies reporting on patients diagnosed with malnutrition who were later found to have EPI. This last group mostly performed in a low resource setting. Studies included were published between 1952 and 2016, and showed large heterogeneity in: quality (i.e. mainly small sample sizes), design, definitions used and outcome measures (using techniques that are not up to current gold standard). This heterogeneity inhibited us to conduct a quantitative analysis, and limited us in drawing firm conclusions. Although EPI

182

Chapter 7

was linked to decreased nutritional status, this link was not specified properly in most ar-ticles. We concluded that there is sufficient evidence for an association between EPI and malnutrition, but could not confirm whether there is a causal relationship between the two. More longitudinal clinical trials, using standardized definitions and (current) gold standard techniques are needed to explore this relationship further. A problem however, in exploring this relation in future research, is that a placebo arm (withholding treatment of EPI in children with underlying diseases) would not be ethical, because treatment for this group is part of standard clinical practice. Because children with SAM (and EPI) are very vulnerable patients, we decided to focus on SAM patients with EPI and explore whether treatment of EPI could improve their clinical outcome. Before we could start this investigation, it was necessary to determine the prevalence of EPI amongst children with complicated SAM. Therefore, we studied the pancreatic function in Malawian children with complicated SAM, as described in chapter 3. We recruited 89 children admitted to Queen Elizabeth Central Hospital in Blantyre, Malawi with complicated SAM, and tested their pancreas function, using the sensitive and spe-cific EPI-marker Fecal Elastase-1 (FE-1). In addition to this, we measured their serum trypsinogen and amylase levels, which are released by damaged pancreatic cells, and are used as markers of pancreas inflammation.(26,27) We found that 92.2% of patients showed evidence of EPI on admission, and 76.6% showed evidence of severe EPI. Prevalence of EPI was significantly higher and more severe in children with edematous SAM compared with those with non-edematous SAM. Though we found some improve-ment of the pancreas function during admission, with increasing FE-1 levels, these values did not normalize. Also, we found elevated levels of trypsinogen and amylase, suggesting pancreatic inflammation. Mortality in the entire study cohort was 15.7%, with a signifi-cantly higher number of children dying with non-edematous SAM than edematous SAM. However, no differences in mortality were found between pancreatic sufficient versus insufficient patient groups.We concluded that EPI is highly prevalent in children with SAM, especially in children with edematous SAM, and that biochemical signs suggestive of pancreatitis are common in children with SAM. We cannot clarify but only speculate about the interesting difference in the prevalence of EPI between the two phenotypes in malnutrition (edematous vs. non-edematous). One of the potential explanations could be that edematous malnutrition or kwashiorkor is a different entity than non-edematous malnutrition or marasmus. The fact that WHO advises to treat the two phenotypes with one blanket approach might at some point change when more evidence is found that the two phenotypes are indeed also pathophysiologically dif-ferent than is currently thought. In the 1980’s post-mortem analyses showed differences in liver pathology between the two phenotypical groups.(22) It would be of great interest to repeat these post-mortem studies, using modern techniques investigating pancreatic

183

7


architecture, organelle function, and inflammation makers. This might improve our un-derstanding of the differences found in chapter 3 and hopefully lead to new, phenotype-tailored treatment options. The reason why we did not find differences in clinical outcomes in our cohort was likely due the small number of children without EPI in our cohort (n=6). It would be interesting to assess these differences in clinical outcome in malnourished children with and without EPI, both in the short and in the long-term, and to compare them with a control group by collecting data from healthy Malawian children. This could provide more in-depth insight in the underlying pathophysiology and the ‘pathways’ in which the exocrine pancreas is related to outcome. As a next step we wanted to explore the possible benefits of PERT in children with com-plicated SAM. In chapter 4, we presented our findings of a randomized controlled trial in 90 children with complicated SAM. All children received standard care as per WHO and Malawian guidelines, while the intervention group also received PERT for 28 days.(28,29) The primary outcome was weight gain (%), while secondary outcomes were pancreatic function (assessed by measuring FE-1), duration of hospital stay, mortality, and digestive function reflected by fecal fatty acid split-ratios. This ratio can reflect failed fatty acid breakdown (i.e., a high proportion of triglycerides in total fecal fatty acids) and/or failed absorption (i.e., a high proportion of free fatty acids in total fecal fatty acids).(30)Children treated with PERT did not gain more weight than controls. Similar to our findings in chapter 3, we found a high prevalence of EPI (83%) and severe EPI (69%) on admission. During the 28 days of the intervention, there was an improvement but no normaliza-tion of the pancreas function, irrespective of the treatment group. Again, children with edematous SAM showed more severe EPI and showed less improvement of FE-1 levels over time than children with non-edematous SAM. After 28 days of nutritional rehabilita-tion, irrespective of PERT, 68% of the study patients still showed EPI, and 46% still showed severe EPI. Duration of hospital stay was not found to be different between the groups. However, competitive risk analysis showed that, compared with controls, children receiving PERT had a significantly higher probability of being discharged on every passing day of treatment. An additional intriguing finding was that mortality overall was 27.8%, and was significantly lower in the intervention group. Eight children (18.6%) in the PERT group died versus seventeen (37.8%) in the control group. Finally, we found significantly lower fecal fatty acid split-ratios at admission in children that died compared to those that survived. This implies that a marker of poor fat digestion is associated with mortality. We concluded that PERT does not improve weight gain in children with complicated SAM, but does increase their rate of hospital discharge and is associated with lower mortality. These chapters together provided new and thorough insight into the role of the pancreas in children with complicated SAM. We showed that despite treating EPI with PERT, our patients did not show improved weight gain. One of the explanations that an effect in weight gain is seen in children that are treated in the West with, for example, CF, is that

184

Chapter 7

these children tend to be in a much better nutritional status than the SAM children in our study. Also, the known malabsorption of the malnourished gut may inhibit optimal effect of the treatment. Giving PERT on its own to improve digestion does not have any beneficial effect when the next step, absorption, is dysfunctional as well. Several limitations need mentioning, such as the possibility of a too short duration of treatment and follow-up. We cannot rule out that treating longer than 28 days might result in improved weight gain. As a trend of improvement of pancreas function has been found in both chapter 2 and 3, it is of great interest to follow these patients over a longer time. With more intense follow-up we could observe if pancreas function eventually also normalizes with nutritional rehabilitation only, or whether this improvement stagnates after some time. If pancreas function normalizes just by nutritional rehabilitation alone, this would lead to the recommendation of not treating this specific patient group with PERT. Another limitation may have been weight gain as a primary outcome. Although a clinically important variable, it is both a noninvasive measuring tool and relevant for measuring nutritional status and improvement, it is also a less relevant variable in the sense that it may not represent the clinical status of a child in the acute setting, but has more value over longer periods of time. In the acute setting, the weight of children with SAM tends to fluctuate, and although these children improve clinically, they do not necessarily gain weight in those first days of nutritional rehabilitation. Also, there is an important difference in the course of weight gain throughout clinical stabilization and nutritional rehabilitation, between children with non-edematous and edematous SAM. Children with non-edematous SAM will normally show a slow increase of weight over time. In children with edematous SAM however, there is much more variation throughout admission. These children first lose their edema, and thus weight, before gaining weight. We corrected for this in our study by using their lowest weight throughout admission as their baseline weight. However, in some children their lowest weight was only reached a few days before discharge. So, for these edematous children, their weight gain was mea-sured over a very short time period. Weight gain could be a good outcome in children with non-edematous SAM, but may not be ideal for those with edema. We found significant differences in mortality amongst the groups, but had not powered the study for this variable. However, this secondary outcome is literally of vital impor-tance, as the findings were significant and request further exploration. Before we can recommend that PERT has no importance in the treatment of children with complicated SAM, the influence of PERT on the mortality rate and discharge rate deserve to be studied in more detail. This study had not stratified for edematous status, and a higher portion of children with edematous SAM were found to be in the PERT intervention group. Recent studies have shown inconsistent findings of which phenotype of SAM is associated with higher mortality risk.(31,5,32) Even after correcting for edematous status, significant difference in mortality remained, so this cannot (fully) explain the lower mortality rate in

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the PERT intervention group. The number of days until death were similar in both groups, around 4-5 days into admission. As such, the following questions need answers: Why would a SAM child treated with PERT have an improved survival risk? Does treatment with pancreatic enzymes improve their clinical recovery, and somehow protect some children with SAM from dying? Should we focus on subgroups to find these benefits? Would children with edematous SAM who have a higher prevalence and more severe degree of EPI, benefit from this treatment more than those without edema? There is a need for additional investigating into the potential benefits of PERT in a larger cohort, with stratification for edematous and non-edematous malnutrition, and with mortality as primary outcome. Future research should also focus on clarifying the exact ‘damage’ to the exocrine pancreas, or on more tailored diets that specifically address the impaired digestive function in SAM children. This could be done through post-mortem sampling of the pancreas, and by using diets used in pancreatitis; a state of increased inflammation of the pancreas.

Severe Acute Malnutrition and gut inflammation In chapter 5 we focused on gut enteropathy in children with complicated SAM. Children with SAM have gut inflammation as a feature of intestinal pathology.(31) This inflamma-tion has similarities to that which occurs in non-IgE mediated food allergy and Crohn’s disease, which are treated with elemental and polymeric feeds respectively.(33–35) As such, it may be possible that these feeds could also benefit children with SAM. To investigate this, we designed a randomized, controlled, three-arm intervention trial, recruited 95 children with complicated SAM, who were allocated randomly to receive, for two weeks, either standard dietary management with ready-to-use therapeutic food (RUTF), an elemental feed, or a polymeric feed. Primary outcome was fecal calprotectin, a non-specific biomarker of intestinal inflammation. Some of the secondary outcomes were other biomarkers of intestinal inflammation, mucosal integrity, systemic inflamma-tion, and tolerability of feeds. In all children, we found highly abnormal levels of fecal calprotectin and other biomarkers at baseline, and these generally persisted in all three treatment arms. In addition, no clinical benefits were found in the two intervention groups, while the novel feeds were actually poorly tolerated by the children with SAM. In short, we concluded that both elemental and polymeric feeds did not have the antici-pated anti-inflammatory or clinical benefits. The most important limitations of this study were the duration of administration of the novel feeds and the smaller number of patients recruited than was the target (95 instead of 120). As for the duration of administration: although a longer period of feeding (4-8 weeks) is recommended to achieve clinical remission and mucosal healing, this was not feasible in our setting.(36,37) WHO and Malawian malnutrition guidelines recommend discharge once children are improving and tolerating RUTF, and on average this is before two weeks

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of admission.(28,29,38) Requesting for patients and their guardians to stay in hospital for several additional weeks would result in lower consent rates, and would put them at higher risk of hospital-acquired infections, and therefore should be considered unethical. A solution could be to treat them as outpatients, but this would require intensive follow-up to make sure children are compliant to their diets, which is challenging in any setting, but even more so in a low resource setting with limited logistics and infrastructure. As for the recruitment below target: this was mainly caused by the lower number of admissions than in previous years. We managed to recruit 55% of eligible patients. The primary reason guardians gave for not giving consent was the prolonged hospital duration. With this study we again demonstrated the complexity of the pathophysiology in children with complicated SAM, which has recently been described and summarized by Bhutta et al.(31) Clearly, in children with complicated SAM, the treatment of intestinal pathology is more complex than in populations with enteropathies alone, such as in non-IgE mediated food allergy and Crohn’s disease, in which used treatment feeds have a demonstrated beneficiary effect. This is similar to our findings in chapter 4, when PERT did not have the anticipated beneficial effect that is found when administered to children suffering from EPI secondary to an underlying illness that is more straightforward. The persistent inflammation of the malnourished gut despite receiving nutritional rehabilitation needs further exploration. It is important to know whether the cause of inflammation persists throughout nutritional rehabilitation, or whether it is due to hospital acquired infections. A confounder could be HIV enteropathy, which has also been previously described in children with SAM.(39) However, we found no differences when analyzing children with and without HIV separately. Another important finding from our study is the lack of improvement in intestinal pathol-ogy, following current recommended WHO treatment for children with complicated SAM. A consequence of gut enteropathy is malabsorption, which means WHO recommended supply with RUTF does not currently have its optimal effect. To fill the current knowledge gap on pathophysiology should be a priority in order to better remedy enteropathy in children with SAM and optimize recovery.

Severe Acute Malnutrition and Bio-electrical Impedance AnalysisFinally, we were interested in finding new measurement tools for children with compli-cated SAM that would have a better prognostic value than classic anthropometry alone. It would be of interest to find new, noninvasive, and low-cost strategies that help in bet-ter predicting the severity and outcome of the illness of a child with complicated SAM. Bioelectrical impedance analysis (BIA) is an economical, noninvasive, safe, and easy to use technique for the assessment of body composition.(40) The technology determines the electrical impedance of body tissues, which provides an estimate of total body water as well as estimates of fat-free mass and body fat.(40)

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Several studies had looked at BIA in children with SAM, but no longitudinal data were available yet.(41,42) We developed a prospective study to explore the change of BIA throughout clinical stabilization in children with complicated SAM, and to investigate whether BIA would have added prognostic value to clinical outcome, compared to using classic anthropometry alone, which is reported in chapter 6. We studied 183 children with complicated SAM admitted to the ‘Moyo’ NRU, 11 healthy Malawian children, and measured their bio-electrical impedance parameters on admission and after clinical stabilization. We found that children with edematous SAM had significantly lower bio-electrical impedance parameters than non-edematous children and control subjects, and that only in the edematous group a significant change of BIA was measured over time, which reflects fluid loss. BIA parameters were not significantly associated with mortality, and had no added predictive value over classic anthropometry alone. With our PLS-DA models we showed that BIA helps marginally in separating the different nu-tritional groups. This means that the additional value of BIA may only be of significance when there is clinical doubt whether the (malnourished) child has edema.We concluded that throughout clinical stabilization, BIA showed changes in children with edematous SAM, but that BIA did not provide any significant contribution to predict clini-cal outcome than classic anthropometry. This study did not clearly identify a role for the clinical use of BIA for children with complicated SAM.We analyzed our data in multiple ways in order to be able to give firm advice on whether or not BIA in children with SAM should have a role or deserves further exploration. We did not look at long-term follow up and BIA in relation to clinical outcome later in time. For example, it could still be of interest to find out how body composition of children recovered from complicated SAM adjusts. Do they gain fat mass or lean mass? These questions may deserve further exploring in future research. However, we know that in a NRU with low resource setting, BIA is of no added value and does not need further exploration.

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COnClUSIOnS AnD ReCOMMenDATIOnS

In conclusion, in children with complicated SAM we found- an extremely high prevalence of EPI, but treatment with PERT for 28 days did not

improve EPI or clinical status of the child with SAM. Nutritional rehabilitation on itself did significantly improve the EPI, however, pancreatic function had not normalized after 28 days

- enteropathy, but treatment with elemental and polymeric feeds did not significantly improve the enteropathy, and neither did it contribute to faster clinical recovery of the child with SAM

- that bio-electrical impedance analysis does not add any prognostic or clinical value to classic anthropometry, and hence does not seem a useful tool in this specific patient population.

This thesis has increased our insight into potential new diagnostics and treatment strate-gies in children with complicated SAM. Intervention studies in low resource settings, and in children with complicated SAM in particular, are extremely challenging, which likely explains the few existing publications despite the scale of the problem. Therefore, this thesis makes a significant contribution to the limited published research: apart from the main conclusions on the different specific topics, it has underlined the complexity of this disease, the large deficit in current knowledge and management, and the urgent need for more research to better understand the pathophysiology of this disease in order to stimulate development of interventions that address the unacceptably high case-fatality and poor long-term outcomes of children with complicated SAM. Despite the logistical difficulties and challenges in performing high quality research inherent to studies on this subject, it is of vital importance that we prioritize this research with funds and other re-sources - for the difficulties in finding scientific significance are trumped a thousand-fold by the ethical relevance of the suggested research: our children depend on us to tackle this problem now, so that they may live a healthy life. In Malawi, two examples of great projects focusing on the problem of childhood malnutrition are CHAIN (‘CHildhood Acute Illness and Nutrition Network’ http://www.chainnetwork.org/) and Project Peanut Butter (http://www.projectpeanutbutter.org/). CHAIN was started recently, and is funded by the Bill & Melinda Gates Foundation. It is a global research network focusing on “optimizing the management and care of highly vulnerable children in resource-limited settings to improve survival, growth and development”.(43) Moyo NRU is included in this network as one of their many research sites. Project Peanut Butter has its focus on community management of malnutrition in several African countries and “seeks to advance the treat-ment of severe malnutrition, the single largest cause of child death in the world today, using effective, locally produced ready-to-use therapeutic foods”. They are responsible

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for the first factory in Malawi to produce RUTF with local product, and which is run by local staff. It is projects like these that need our ongoing (financial) support, as they make a substantial difference for the malnourished child and their community.With more funds and more researchers to perform more observational and interven-tional research, we will be able to succeed in tackling this global burden of childhood malnutrition, and to contribute to the nutritionally healthy future of children recovering from SAM and of those yet to be born. Our children’s futures are not to be wasted.

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REFEREnCES




4. Bartels RH, van den Brink DA, Bandsma RH, van Hensbroek MB, Tabbers MM, Voskuijl WP. The Relation Between Malnutrition and the Exocrine Pancreas. J Pediatr Gastroenterol Nutr [Internet]. 2017;1. Avail-able from: http://www.ncbi.nlm.nih.gov/pubmed/28991838. Accessed December 3, 2017.





9. Taylor JR, Gardner TB, Waljee AK, Dimagno MJ, Schoenfeld PS. Systematic review: efficacy and safety of pancreatic enzyme supplements for exocrine pancreatic insufficiency. Aliment Pharmacol Ther. 2010 Jan;31(1):57–72.


11. Veghelyi P V. Nutritional oedema. Ann Paediatr. 1950;175(5):349–77. 12. Barbezat GO, Hansen JD. The exocrine pancreas and protein-calorie malnutrition. Pediat-

rics.1968;42(1):77–92. 13. Tarasov NI. [External secretion of the pancreas in chronic infant nutrition disorders]. Vopr Pediatr.

1953;21(4):33–9. 14. Thompson MD, Trowell HC. Pancreatic enzyme activity in duodenal contents of children with a type of

kwashiorkor. Lancet. 1952;1(6717):1031–5. 15. Blackburn WR, Vinijchaikul K. The pancreas in kwashiorkor. An electron microscopic study. Lab Invest.

1969;20(4):305–18. 16. Banwell JG, Hutt MR, Leonard PJ, Blackman V, Connor DW, Marsden PD, et al. Exocrine pancreatic

disease and the malabsorption syndrome in tropical Africa. Gut. 1967;8(4):388–401. 17. Bras G, Waterlow JC, Depass E. Further observations on the liver pancreas and kidney in malnourished

infants and children: the relation of certain histopathological changes in the pancreas and those in liver and kidney. West Indian Med J. 1957;6(1):33–42.

18. Sauniere JF, Sarles H, Attia Y, Lombardo A, Yoman TN, Laugier R, et al. Exocrine pancreatic function of children from the Ivory Coast compared to French children. Effect of kwashiorkor. Dig Dis Sci. 1986;31(5):481–6.

19. Pitchumoni CS. Pancreas in primary malnutrition disorders. Am J Clin Nutr. 1973 Mar;26(3):374–9.

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20. El-Hodhod MA, Nassar MF, Hetta OA, Gomaa SM. Pancreatic size in protein energy malnutrition: a predictor of nutritional recovery. Eur J Clin Nutr. 2005;59(4):467–73.

21. Durie PR, Forstner GG, Gaskin KJ, Weizman Z, Kopelman HR, Ellis L, et al. Elevated serum immunoreac-tive pancreatic cationic trypsinogen in acute malnutrition: evidence of pancreatic damage. J Pediatr. 1985;106(2):233–8.


23. Nousia-Arvanitakis S. Cystic fibrosis and the pancreas: recent scientific advances. J Clin Gastroen-terol.1999;29(2):138–42.

24. Shwachman H, Diamond LK, Oski FA, Khaw KT. The Syndrome of Pancreatic Insufficiency and Bone Marrow Dysfunction. J Pediatr. 1964;65:645–63.

25. Carroccio A, Fontana M, Spagnuolo MI, Zuin G, Montalto G, Canani RB, et al. Pancreatic dysfunction and its association with fat malabsorption in HIV infected children. Gut.1998;43(4):558–63.


27. Al-Bahrani AZ, Ammori BJ. Clinical laboratory assessment of acute pancreatitis. Clin Chim Acta. 2005;362(1–2):26–48.

28. WHO. Guideline: Updates on the management of severe acute malnutrition in infants and chil-dren [Internet]. World Health Organization. 2013. Available from: http://apps.who.int/iris/bitstream/10665/95584/1/9789241506328_eng.pdf. Accessed December 3, 2017.

29. Ministry of Health (MOH) of Malawi. Guidelines for the management of severe acute malnutrition [Internet]. UNICEF; 2006. Available from: https://www.unicef.org/malawi/resources_9776.html. Ac-cessed December 3, 2017.

30. Nakamura T, Takebe K, Tando Y, Arai Y, Yamada N, Ishii M, et al. Faecal triglycerides and fatty acids in the differential diagnosis of pancreatic insufficiency and intestinal malabsorption in patients with low fat intakes. J Int Med Res. 1995;23(1):48–55.







37. Cameron FL, Gerasimidis K, Papangelou A, Missiou D, Garrick V, Cardigan T, et al. Clinical progress in the two years following a course of exclusive enteral nutrition in 109 paediatric patients with Crohn’s disease. Aliment Pharmacol Ther. 2013;37(6):622–9.


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41. Girma T, Kæstel P, Mølgaard C, Michaelsen KF, Hother A-L, Friis H. Predictors of oedema among children hospitalized with severe acute malnutrition in Jimma University Hospital, Ethiopia: a cross sectional study. BMC Pediatr. 2013;13.

42. Girma T, Hother Nielsen A-L, Kæstel P, Abdissa A, Michaelsen KF, Friis H, et al. Biochemical and an-thropometric correlates of bio-electrical impedance parameters in severely malnourished children: A cross-sectional study. Clin Nutr [Internet]. 2017 Feb 24 [cited 2017 Oct 19]; Available from: http://linkinghub.elsevier.com/retrieve/pii/S0261561417300675. Accessed December 3, 2017.

43. CHAIN Network | The Childhood Acute Illness & Nutrition Network. [Internet]. [cited 2017 Nov 23]. Available from: http://www.chainnetwork.org/. Accessed December 3, 2017.

Appendices

Rosalie H. Bartels

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neDeRlAnDSe SAMenVATTInG

Wereldwijd stierven er het afgelopen jaar dagelijks 15000 kinderen onder de vijf jaar oud.(1) Bijna de helft (45%) van dat aantal sterfgevallen was gerelateerd aan of veroorzaakt door ondervoeding.(2,3) Er bestaan verschillende vormen van ondervoeding, maar de ernstigste vorm is ‘acute, ernstige ondervoeding’; ‘severe acute malnutrition’, in de rest van dit stuk aangeduid als ‘SAM’. Wereldwijd zijn er 17 miljoen kinderen van onder de vijf jaar oud met deze vorm van ondervoeding.(4) Er bestaan twee fenotypisch verschillende vormen van SAM; met en zonder oedeem (zwelling):- Niet-oedemateuze SAM/marasmus: een gewicht naar lengte <-3 standaard deviatie

(SD), of een bovenarmomtrek van <115mm.- Oedemateuze SAM/kwashiorkor: de aanwezigheid van bilateraal oedeem.- Marasmic kwashiorkor: een combinatie van de twee.

Kinderen met SAM worden in principe buiten het ziekenhuis behandeld, tenzij ze ‘complicated SAM’ hebben. Dat betekent dat ze, behalve ernstig ondervoed, acuut ziek zijn, geen voeding tolereren, vaak ook andere ziektes hebben (bijv. Humaan Immuun-deficiëntie Virus (HIV), Tuberculose, diarree etc.) en opgenomen moeten worden in het ziekenhuis op een voedingsrehabilitatie unit (NRU) voor intensieve behandeling. Deze bestaat o.a. uit het stabiliseren van het kind, het behandelen van infecties en het heel voorzichtig aanbieden van voeding in aangepaste laag-calorische vorm. Op dit moment bestaan er wel richtlijnen, opgesteld door de wereldgezondheidsorgani-satie (WHO), hoe kinderen met (complicated) SAM het beste kunnen worden behandeld, maar ondanks het naleven van deze richtlijnen, sterft nog steeds een onacceptabel hoog aantal kinderen (tot wel 35%) met complicated SAM.(2,3,5–8) Het ziektebeeld van het kind met SAM is enorm complex, betreft meerdere organen en mechanismen en is in zijn geheel nog altijd niet compleet ontrafeld. Dat belemmert het ontwikkelen van de ideale behandeling van kinderen met SAM.Het doel van dit proefschrift is om diagnostiek en behandeling van kinderen met compli-cated SAM te verbeteren, middels het verkrijgen van beter inzicht in dit ziektebeeld en het exploreren van nieuwe behandelingsstrategieën.In dit proefschrift zijn de resultaten beschreven van twee interventiestudies en twee ob-servatiestudies, uitgevoerd op de ‘Moyo’ NRU van het Queen Elizabeth Central Hospital in Blantyre, Malawi, en een systematisch literatuuronderzoek.

SAM en de exocriene PancreasIn de eerste drie hoofdstukken onderzochten wij de relatie tussen exocriene pancre-asinsufficientie (EPI) en SAM in kinderen en de prevalentie en behandeling van EPI in kinderen met complicated SAM.(6,9,10)

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EPI leidt tot verstoorde digestie (vertering) vanwege de centrale rol die de exocrience pancreas (alvleesklier) speelt bij de vertering van voeding. EPI kan diarree veroorzaken, een complicatie die veelvuldig voor komt bij kinderen met SAM en die de kans op sterven verhoogt.(11–13) EPI kan worden behandeld door het suppleren van pancreasenzymen middels pancreatic enzyme replacement therapy (PERT).(14,15) De relatie tussen EPI en ondervoeding bij kinderen was al eerder beschreven, maar het was nooit duidelijk of EPI nu leidt tot ondervoeding of andersom. Eerdere studies, voornamelijk uitgevoerd tussen 1940 en 1980, suggereerden dat kinderen met SAM daarbij ook EPI hadden.(16–27) Tegelijkertijd was het ook al bekend dat kinderen met EPI als gevolg van een andere onderliggende ziekte (bijv. Cystische fibrose (CF)), een toegenomen risico hebben op on-dervoeding.(28–30) Om erachter te komen of EPI nou leidt tot ondervoeding of anders-om, hebben wij een systematisch literatuuronderzoek uitgevoerd. Dit staat beschreven in hoofdstuk 2. We hebben meerdere wetenschappelijke databanken doorzocht naar artikelen die over EPI en ondervoeding bij kinderen rapporteerden. Negentien studies werden er geïncludeerd en vervolgens onderverdeeld in twee groepen: tien studies die patiënten met EPI beschreven, waarbij ondervoeding werd geconstateerd. Deze studies waren allemaal uitgevoerd in cohorten met patiënten met een onderliggende ziekte die leidt tot EPI (nl. CF, chronische pancreatitis, HIV, Shwachman-Diamond syndroom en coeliakie). De andere negen studies betreffen studies over ondervoede kinderen die ook EPI bleken te hebben. Deze studies zijn bijna allemaal uitgevoerd in ontwikkelingslanden/landen met beperkte middelen. De geïncludeerde studies werden gepubliceerd tussen 1952 en 2016 en zijn enorm heterogeen wat betreft kwaliteit, onderzoeksdesign, defini-ties voor EPI en ondervoeding en uitkomstmaten (waarbij technieken werden gebruikt die niet volgens de huidige Gouden Standaard zijn). Deze heterogeniteit belemmerde ons in het uitvoeren van kwantitatieve analyses en beperkte ons in het trekken van sterke conclusies. EPI werd gelinkt aan ondervoeding, maar deze link werd in het merendeel van de studies niet verder onderzocht of verklaard. We hebben geconcludeerd dat er voldoende bewijs is van een associatie tussen EPI en ondervoeding. Maar we konden niet bevestigen of er ook een causaal verband bestaat tussen de twee. Om dit verband beter te kunnen onderzoeken in de toekomst, zijn er meer longitudinale studies nodig, die de standaard definities hanteren en de huidige Gouden Standaard-technieken gebruiken. Echter, een probleem van nieuw onderzoek zal zijn, dat het niet mogelijk is een placebo-groep te gebruiken, waarbij kinderen met EPI als gevolg van onderliggend lijden geen medicamenteuze behandeling ontvangen. Deze behandeling behoort tot de standaardbehandeling van deze patiënten en het zou onethisch zijn om ze die te onthouden. Omdat kinderen met complicated SAM (en daarbij EPI) een enorm kwetsbare groep vormen, waarvoor huidige behandeling tekortschiet, wilden wij ons focussen op deze groep en onderzoeken of de medicamenteuze behandeling van EPI met PERT ook bij deze patiëntengroep klinisch effect zou hebben.

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Voordat we dit konden onderzoeken moesten we vaststellen wat de prevalentie van EPI is bij kinderen met complicated SAM. We hebben de pancreasfunctie onderzocht bij Malawiaanse kinderen met complicated SAM, dit staat beschreven in hoofdstuk 3. We hebben 89 kinderen, die opgenomen lagen met complicated SAM, geïncludeerd in deze studie en hun pancreasfunctie bepaald aan de hand van een specifieke en sensitieve pancreasmarker, Fecal Elastase-1 (FE-1). Daarnaast hebben we ook hun serum trypsi-nogeen en amylase levels gemeten. Deze enzymen komen vrij bij beschadiging van de pancreascel en dienden als markers voor pancreasinflammatie.(31,32) Bij opname vonden we EPI bij 92.2% van de patiënten en ernstige EPI bij 76.6%. De prevalentie van EPI was significant hoger en ernstiger bij kinderen met kwashiorkor, vergeleken met kinderen met marasmus. Ondanks dat we gedurende de opname enige verbetering van de pancreasfunctie constateerden aan de hand van stijgende FE-1 le-vels, normaliseerde deze niet. Hiernaast vonden we verhoogde levels van trypsinogeen en amylase, wat suggereert dat kinderen met SAM ook pancreasinflammatie hebben. Mortaliteit in het hele studiecohort was 15.7%, waarbij er een significant groter aantal kinderen met marasmus stierf dan met kwashiorkor. Daarentegen vonden we geen ver-schil in mortaliteit tussen de kinderen met en de kinderen zonder EPI. We concludeerden dat de prevalentie van EPI bij kinderen met complicated SAM aanzienlijk hoog is, vooral bij kinderen met kwashiorkor en dat bij deze kinderen ook biochemische tekenen van pancreasinflammatie, zoals wordt gezien bij pancreatitis, veel voorkomen. We kunnen het interessante verschil dat we vonden in de prevalentie en mate van EPI tussen de twee verschillende fenotypes van complicated SAM (oedemateus en niet-oe-demateus), niet uitleggen, maar er wel over speculeren. Eén van de mogelijke verklarin-gen zou kunnen zijn dat deze twee verschillende fenotypes ook compleet verschillende entiteiten zijn. Het is goed mogelijk dat het advies van de WHO om deze twee verschil-lende vormen van SAM op eenzelfde manier te behandelen, in de toekomst verandert wanneer er meer bewijs is dat deze twee vormen inderdaad ook pathofysiologisch van elkaar verschillen. Vorige eeuw, in de jaren tachtig, werd er middels post-mortem ana-lyses al aangetoond dat leverpathologie van de twee fenotypische vormen van elkaar verschilt.(27) Het zou zeer interessant zijn om deze post-mortem studies te hervatten en om, gebruikmakend van de moderne technieken, de bouw en functie van de pancreas te onderzoeken. Hierdoor zouden we de in hoofdstuk 3 gevonden verschillen mogelijk kun-nen begrijpen en daardoor hopelijk nieuwe, fenotype-specifieke behandelingen kunnen ontwikkelen. De reden dat we geen verschil vonden in klinische uitkomst tussen kinderen mét en zonder EPI in ons cohort, is zeer waarschijnlijk het kleine aantal kinderen zonder EPI (n=6). Het zou interessant zijn om de verschillen in klinische uitkomst, zowel op korte als op lange termijn, van ondervoede kinderen mét en zonder EPI verder te onderzoe-ken en ze te vergelijken met een controlegroep, door middel van het onderzoeken van

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gezonde Malawiaanse kinderen. Dit zou beter inzicht kunnen geven in de onderliggende pathofysiologie en in de wijze waarop de exocriene pancreas is gerelateerd aan klinische uitkomst.Vervolgens wilden we de mogelijke voordelen van behandeling met PERT bij kinderen met complicated SAM onderzoeken. In hoofdstuk 4 presenteren we onze bevindingen van een gerandomiseerde gecontroleerde studie van 90 kinderen met complicated SAM. Alle kinderen in de studie kregen standaard zorg, volgens de WHO en Malawiaanse richtlijnen.(33,34) Kinderen in de interventiegroep, kregen daarnaast ook PERT gedu-rende 28 dagen. De primaire uitkomstmaat was gewichtstoename (%) en de secundaire uitkomstmaten waren pancreasfunctie (bepaald door FE-1 levels), duur van de zieken-huisopname, mortaliteit en mate van verteringsfunctie, gemeten middels het bepalen van fecale vetzuur ‘split-ratios’. Deze split-ratios kunnen weergeven of er sprake is van falende vetzuurafbraak (hoge proportie van triglyceriden in het totaal aantal vetzuren) en/of falende absorptie (hoge proportie van vrije vetzuren in het totaal aantal vetzuren).(35) Het gewicht van de kinderen die PERT kregen, nam niet méér toe dan dat van de kinderen die dat niet kregen. Overeenkomstig met onze bevindingen in hoofdstuk 3 vonden we bij opname een hoge prevalentie van zowel EPI (83%) als van ernstige EPI (69%) in het hele cohort. Gedurende de 28 dagen van de studie, was er wel verbetering van de pancreas-functie maar geen normalisatie, ongeacht de behandelgroep (PERT-groep of controle-groep). Ook nu vonden we, vergeleken met kinderen met marasmus, bij kinderen met kwashiorkor een hogere prevalentie en ernstigere mate van EPI en een verminderde verbetering van pancreasfunctie. Na 28 dagen van voedingsrehabilitatie had, ongeacht de behandelgroep, 68% van de studiepatiënten nog steeds EPI en 46% ernstige EPI. Op-nameduur was niet verschillend tussen de behandelgroepen, alhoewel een competitieve risicoanalyse liet zien dat op elke opnamedag kinderen die PERT kregen een significant hogere kans hadden om ontslagen te worden dan de kinderen in de controle-groep. Een andere belangrijke bevinding was dat de mortaliteit, die in het gehele cohort 27.8% was, significant lager was in de PERT-groep. In de PERT-groep stierven 8 kinderen (18.6%) en in de controle-groep 17 (37.8%). Tenslotte stelden we vast dat kinderen die gedurende de opname dood gingen, bij opname significant lagere split-ratios hadden dan kinderen die overleefden. Dit impliceert dat een marker van verminderde vetdigestie is geassocieerd met mortaliteit. We concludeerden dat PERT de gewichtstoename van kinderen met complicated SAM niet verbetert, maar wel de ontslagsnelheid verbetert en geassocieerd is met lagere mortaliteit.Deze drie hoofdstukken samen, leverden nieuwe, diepgaande inzichten op in de rol van de pancreas in kinderen met complicated SAM. We hebben aangetoond dat ondanks het behandelen van EPI met PERT, onze patiënten geen verbeterde gewichtstoename

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lieten zien. Eén van de verklaringen dat een effect in gewichtstoename wel te zien is bij kinderen (met bijvoorbeeld CF) die met PERT worden behandeld in de westerse wereld, is dat deze kinderen over het algemeen in een veel betere voedingstoestand verkeren op het moment dat ze met de therapie starten dan de kinderen met SAM in onze studie. Daarnaast is het mogelijk dat een optimale werking van de therapie wordt belemmerd door de malabsorptie van de SAM darmen. Het geven van PERT om vertering te verbete-ren, heeft natuurlijk geen enkel effect wanneer het volgende proces, de absorptie van de verteerde voedingsstoffen, ook disfunctioneel is. Enkele beperkingen van de studie behoeven hier te worden genoemd en besproken. Mogelijk was de PERT behandelduur van 28 dagen en de follow-up te kort. We kunnen niet uitsluiten dat langere behandelduur mogelijk wel tot verbeterde gewichtstoename zou hebben geleid. Aangezien we in hoofdstuk 3 en 4 een trend konden vaststellen van verbetering van de pancreasfunctie, zou het heel interessant zijn om deze kinderen een langere tijd te volgen. Dan zouden we kunnen zien of de pancreasfunctie uiteindelijk volledig normaliseert na voedingsrehabilitatie, of dat de functieverbetering uiteindelijk stagneert. Als de pancreasfunctie herstelt, puur door voedingsrehabilitatie, dan zouden we het gebruik van PERT kunnen afraden in deze patiëntengroep. Een andere beperking van deze studie is mogelijk het gebruik van gewichtstoename als primaire uitkomstmaat. Ondanks het feit dat dit klinisch een belangrijke variabele is, noninvasief én relevant voor het meten van de mate van ondervoeding en herstel, is het wellicht een minder relevante variabele als het gaat om herstel in de acute setting. In de acute setting neigt het gewicht van kinderen met SAM nogal te fluctueren. Ook al gaan ze vooruit in klinisch oogpunt, ze komen niet altijd meteen aan in gewicht gedurende de eerste dagen van voedingsrehabilitatie. Ook is er tussen kinderen met kwashiorkor en marasmus een belangrijk verschil in het beloop van het gewicht gedurende klinische stabilisatie en voedingsrehabilitatie. Normaal gesproken laten kinderen met marasmus een langzame toename zien in gewicht. Kinderen met kwashiorkor echter, laten veel meer variatie in gewicht zien gedurende de opname. Zij verliezen eerst het oedeem, en dus gewicht, om vervolgens, net als de kinderen met marasmus, langzaam in gewicht toe te nemen. Bij het analyseren van de data hebben we hiervoor gecorrigeerd door het laagste gewicht gemeten tijdens de opname te gebruiken als basisgewicht. Echter, in enkele gevallen werd dit laagste gewicht pas bereikt vlak voor ontslag. Dus voor een aantal kinderen met kwashiorkor is de gewichtstoename gemeten over een hele korte periode. Gewichtstoe-name is wellicht een goede uitkomstmaat voor kinderen met marasmus, maar niet voor kinderen met kwashiorkor. We vonden een significant lagere mortaliteit in de PERT-groep, maar de studie was niet ‘gepowered’ voor mortaliteit. Toch is deze secundaire uitkomstmaat letterlijk van levensbelang, aangezien de verschillen significant waren en verder onderzoek behoeven. Voordat we kunnen concluderen dat PERT geen enkele rol verdient in de behandeling

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van kinderen met complicated SAM, moet de invloed van PERT op ontslagsnelheid en mortaliteit verder worden onderzocht. Er was niet gestratificeerd voor het fenotype van SAM en in de PERT-groep bleken er significant meer kinderen met kwashiorkor te zitten dan in de controle-groep. Recente studies hebben inconsistente bevindingen gerap-porteerd als het gaat om welk fenotype SAM geassocieerd is met hogere mortaliteit.(7,10,36) Maar ook na het corrigeren voor fenotype SAM, bleef het verschil in mortaliteit tussen de PERT-groep en controle-groep significant, dus dit kan de lagere mortaliteit in de PERT-groep niet (volledig) verklaren. Het aantal dagen tot sterven was vergelijkbaar in de twee groepen: rond de 4-5 dagen na opname. De volgende vraag moet worden beantwoord: Waarom heeft een kind met SAM dat PERT krijgt een verbeterde overle-vingskans? Leidt het suppleren van pancreasenzymen tot verbeterd klinisch herstel en beschermt dit het kind met SAM tegen sterven? Moeten we ons richtten op subgroepen om deze voordelen op te sporen? Hebben kinderen met kwashiorkor, waarbij EPI meer voorkomt en in ernstigere mate, mogelijk een groter voordeel van PERT dan kinderen met marasmus?Het is van belang dat er verder onderzoek wordt gedaan naar de mogelijke voordelen van PERT in een groter cohort, waarbij er gestratificeerd is voor fenotype en waarbij de primaire uitkomstmaat mortaliteit is. Onderzoek zou zich ook moeten richten op het ophelderen van wat voor exacte ‘schade’ er plaatsvindt aan de pancreas bij kinderen met SAM en op het ontwikkelen van voedingsdiëten die specifiek zijn aangepast aan de maldigestie. Dit zou gedaan kunnen worden door het uitvoeren van post-mortem studies en door het toepassen van voedingsdiëten zoals we die kennen voor patiënten met pancreatitis: een staat waarin er toegenomen inflammatie van de pancreas bestaat.

SAM en DarminflammatieIn hoofdstuk 5 richtten we ons op de zieke darm, enteropathie, bij kinderen met com-plicated SAM. De darmpathologie bij kinderen met complicated SAM wordt gekenmerkt door darminflammatie.(7) Deze toont overeenkomsten met de darminflammatie zoals die wordt gezien bij niet-IgE-gemedieerde voedselallergie (zoals koemelkeiwitallergie) en bij de ziekte van Crohn, welke worden behandeld met respectievelijk een hypoal-lergeen dieet (elementair) en anti-inflammatoir dieet (polymeer).(37–39) Het zou dan ook mogelijk zijn dat deze diëten ook een gunstig effect zouden hebben bij kinderen met complicated SAM. Om dit te onderzoeken, ontwierpen we een gerandomiseerde gecon-troleerde 3-arm interventiestudie, includeerden 95 kinderen met complicated SAM, die willekeurig werden onderverdeeld in 3 groepen en gedurende twee weken ofwel het WHO standaarddieet kregen bestaande uit RUTF, ofwel een elementair dieet, ofwel een polymeer dieet. De primaire uitkomstmaat was fecale calprotectine, een niet-specifieke biomarker van darminflammatie. Enkele secundaire uitkomstmaten waren andere bio-markers van darminflammatie, mucosale integriteit (de doorlaatbaarheid van het

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darmslijmvlies), systemische inflammatie en de tolerantie van de verschillende diëten. In alle studiekinderen troffen we bij aanvang van de studie uiterst abnormale levels van calprotectine en de andere biomarkers aan, welke over het algemeen afwijkend bleven in alledrie de studiearmen gedurende de studieperiode. Daarnaast vonden we in de twee interventiearmen geen klinische toegevoegde waarde ten opzichte van de standaardarm en werden de twee interventiediëten zelfs slecht getolereerd door de patiënten.Kortom, we concludeerden dat zowel de hypoallergene als de anti-inflammatoire diëten niet de verwachte anti-inflammatoire en klinische gunstige effecten hadden.De belangrijkste beperkingen van deze studie waren de behandelduur en het lagere aantal geïncludeerde patiënten dan waarnaar werd gestreefd (95 in plaats van 120). Wat betreft de duur van de behandeling met de verschillende diëten: ondanks het feit dat het wordt aanbevolen om de interventiediëten ten minste 4-8 weken vol te houden om heling van het darmslijmvlies te bewerkstelligen en klinische remissie te zien, was dit in onze setting niet haalbaar.(40,41) Zowel WHO richtlijnen als Malawiaanse richtlijnen bevelen aan om kinderen met complicated SAM naar huis te ontslaan zodra ze klinisch herstellen en de RUTF goed tolereren. Gemiddeld is dat binnen twee weken vanaf opname.(33,34,42) Het zou zeker resulteren in minder deelname aan de studie om kinderen en hun ouders te vragen om nog enkele weken langer in het ziekenhuis te blijven. Daarbij zouden de pati-enten langer worden blootgesteld aan het risico van oplopen van ziekenhuisgerelateerde infecties, wat onethisch is. Een oplossing zou zijn om de patiënten thuis te behandelen, maar dit vergt intensieve follow-up en begeleiding om zich ervan te kunnen verzekeren dat de patiënten het dieet strak volgen. Dat is in elke setting al een uitdaging, maar helemaal in een setting met beperkte middelen, infrastructuur en logistiek. Wat betreft het lagere aantal geïncludeerde patiënten dan waarnaar werd gestreefd: dit werd voornamelijk veroorzaakt door het lagere aantal opnames op de ondervoedings-afdeling dan voorgaande jaren. 55% van het aantal kinderen met complicated SAM dat voldeed aan de inclusiecriteria, werd geïncludeerd. De voornaamste reden die ouders opgaven voor het weigeren van deelname aan de studie was de langere opnameduur. Ook met deze studie hebben we de complexe pathofysiologie aangetoond van kinderen met complicated SAM, zoals ook recent werd beschreven en samengevat door Bhutta et al.(7) Het is duidelijk dat de behandeling van darmpathologie in deze patiëntengroep, complexer is dan in patiëntengroepen die puur enteropathie hebben, zoals bij niet-IgE-gemedieerde voedselallergie en bij de ziekte van Crohn, bij wie de voor deze studie gebruikte diëten een bewezen gunstig klinisch effect hebben. Dit komt overeen met de bevindingen in hoofdstuk 4, waar PERT niet het verwachte gunstige effect had dat wel wordt gezien bij patiënten die aan EPI lijden door een andere onderliggende ziekte. De persisterende inflammatie van de ondervoede darm, ondanks nutritionele rehabilitatie, dient verder te worden onderzocht. Het is belangrijk of de oorzaak van de inflammatie gedurende nutritionele rehabilitatie dezelfde blijft of wordt vervangen door ziekenhuis-

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gerelateerde infecties. Een confounder (een factor die verwarring kan veroorzaken) waarmee mogelijk rekening gehouden zou moeten worden is HIV-enteropathie, die ook voorkomt bij kinderen met complicated SAM.(43) Echter hebben we geen verschillen gevonden bij het apart analyseren van kinderen met en zonder HIV. Een andere belangrijke bevinding van deze studie was het gebrek aan verbetering van de darminflammatie gedurende het volgen van de WHO richtlijnen. Een van de gevolgen van enteropathie is malabsorptie en dan kan het geven van WHO-aanbevolen RUTF geen optimaal effect hebben. Het vullen van de kenniskloof over de pathofysiologie van kinde-ren met complicated SAM, moet prioriteit krijgen om de enteropathie bij deze kinderen te kunnen behandelen en herstel te optimaliseren.

SAM en Bio-elektrische Impedantie AnalyseTot slot waren we geïnteresseerd in het vinden van nieuwe meetdiagnostiek voor kinde-ren met complicated SAM, die een betere prognostische waarde zou hebben dan enkel de klassieke antropometrie (het meten van gewicht, lengte en omtrek van de bovenarm). Het is van belang om nieuwe niet-invasieve, goedkope manieren te vinden om de ernst en ziekteuitkomst van deze kinderen te voorspellen. Bio-elektrische Impedantie Analyse (BIA) is een niet-invasieve, goedkope en makkelijk te gebruiken techniek om lichaams-samenstelling te meten.(44) Het meet de geleiding van stroom door het lichaam. Via elektroden op handen en voeten wordt een wisselstroom met verschillende frequenties door het lichaam gestuurd. Weefsels met veel water en elektrolyten, zoals bloed en spieren, geleiden goed. Vetmassa, lucht of bot daarentegen geleiden nauwelijks stroom. Dus hoe groter de vetvrije massa, des te groter het geleidingsvermogen van het lichaam. Met behulp van de BIA-meting kunnen de watercompartimenten in het lichaam worden bepaald. Het intracellulaire en het extracellulaire water vormen samen het totaal aan lichaamswater.(44,45)Verschillende studies hebben gekeken naar het gebruik van BIA bij kinderen met SAM, maar er bestonden nog geen longitudinale data.(46,47) We hebben een prospectieve studie opgezet om de verandering van BIA gedurende klinische stabilisatie van kinderen met SAM te meten en om te onderzoeken of BIA toegevoegde prognostische waarde heeft ten opzichte van klassieke antropometrie en dit beschreven in hoofdstuk 6. We bestudeerden 183 kinderen met complicated SAM, 11 niet ondervoede, gezonde Ma-lawiaanse kinderen en maten hun bio-elektrische impedantie parameters bij opname en na klinische stabilisatie. Kinderen met kwashiorkor hadden significant lagere BIA parameters dan kinderen met marasmus en gezonde kinderen en alleen bij kinderen met kwashiorkor werd een significante verandering waargenomen gedurende stabilisatie, die kan worden verklaard door het verlies van oedeem/vocht. BIA parameters waren niet significant geassocieerd met mortaliteit en hadden geen toegevoegde voorspellende waarde ten opzichte van klassieke antropometrie. Met onze PLS-DA modellen toonden

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we aan dat BIA in enige mate helpt bij het differentiëren van de verschillende groepen (marasmus, kwashiorkor of niet ondervoed). Dit betekent dat BIA mogelijk alleen iets van waarde zou hebben als er twijfel is over of een kind wel of geen oedeem heeft.We concludeerden dat gedurende klinische stabilisatie BIA alleen verandert bij kinderen met kwashiorkor, maar dat BIA geen significante bijdrage levert aan het voorspellen van klinische uitkomst. Er lijkt geen rol weggelegd voor het gebruik van BIA bij kinderen met complicated SAM.We hebben meerdere analysemethoden toegepast op onze data om duidelijke conclu-sies te kunnen trekken en ondubbelzinnig advies te kunnen geven over het gebruik van BIA bij deze patiëntengroep. Hierbij hebben wij niet gekeken naar BIA bij latere follow-up na ontslag en de relatie van BIA met lange-termijnuitkomst. Het zou bijvoorbeeld nog steeds interessant kunnen zijn om te onderzoeken hoe de lichaamssamenstelling van deze kinderen verandert als zij hersteld zijn van ondervoeding. Komen ze alleen aan in vetmassa of ook in vetvrije massa? We weten nu in ieder geval dat BIA geen toegevoegde waarde heeft op een NRU met beperkte middelen en niet verder hoeft te worden onderzocht.

COnClUSIeS en AAnBeVelInGen

Concluderend, vonden wij bij kinderen met complicated SAM:- een uiterst hoge prevalentie van exocriene pancreasinsufficientie. Behandeling met

pancreasenzymen gedurende 28 dagen had geen effect op de EPI en ook geen klinisch effect. Nutritionele rehabilitatie op zich verbeterde de EPI wel significant, alhoewel de pancreasfunctie niet genormaliseerd was na 28 dagen;

- enteropathie, maar geen significante verbetering na een hypoallergeen of anti-inflammatoir dieet. Evenmin droeg dit bij aan een sneller klinisch herstel;

- dat bio-elektrische impedantie analyse geen toegevoegde prognostische dan wel klinische waarde heeft ten opzichte van klassieke antropometrie. Daarom is dit geen bruikbaar hulpmiddel in deze specifieke patiëntenpopulatie.

Dit proefschrift vergroot het inzicht in potentiële nieuwe diagnostische en behandelstra-tegieën bij kinderen met complicated SAM. In landen en situaties met beperkte middelen, en vooral bij kinderen met complicated SAM, zijn interventiestudies een grote uitdaging. Mogelijk is dat een verklaring voor het kleine aantal publicaties over een dermate ernstig probleem dat op wereldwijde schaal bestaat. Dit proefschrift levert een significante bijdrage aan het beperkt gepubliceerde onderzoek: los van de hoofdconclusies van de verschillende specifieke onderwerpen, benadrukt het de complexiteit van de ziekte, het grote gebrek aan huidige kennis over de pathofysiologie en optimale behandeling van dit

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ziektebeeld, en de dringende behoefte aan verder onderzoek, opdat nieuwe interventies worden ontwikkeld die de hoge mortaliteit en slechte lange-termijnuitkomst voor deze kinderen aanpakken.Ondanks de logistieke uitdagingen die inherent zijn aan het uitvoeren van hoog kwalita-tief onderzoek naar dit onderwerp, is het letterlijk van levensbelang dat we dit prioriteit geven door het toekennen en inzetten van meer financiering en onderzoekers. Immers, de uitdaging van dergelijk onderzoek en het vinden van wetenschappelijke significantie wordt volledig gerechtvaardigd door de ethische relevantie ervan: het is noodzakelijk dat we dit probleem nu bestrijden als we de gezondheid van kinderen in de toekomst willen garanderen. Twee voorbeelden van geweldige projecten, gericht op kinderen met ondervoeding, die deels in Malawi gevestigd zijn: CHAIN (‘CHildhood Acute Illness and Nutrition Network’ http://www.chainnetwork.org) en Project Peanut Butter (http://www.projectpeanutbutter.org). CHAIN is een recent gestart wereldwijd onderzoeksnetwerk, dat wordt gefinancieerd door de Bill & Melinda Gates Foundation, en als doel heeft: “het optimaliseren van de behandeling van en de zorg voor zeer kwetsbare kinderen in settings met beperkte middelen, om hun overlevingskans, groei en ontwikkeling te verbeteren”. Hun onderzoek wordt onder andere op de Moyo NRU uitgevoerd. Poject Peanut Butter is gericht op de behandeling van ondervoeding buiten het ziekenhuis, in verschillende landen in Afrika, en “streeft naar het verbeteren van de behandeling van ernstige ondervoeding, die momenteel de grootste oorzaak is van kindersterfte wereldwijd, middels het gebruik van effectief, lokaal geproduceerd RUTF”. Ze zijn ver-antwoordelijk voor de eerste fabriek in Malawi die RUTF produceert, gemaakt van lokale producten en die gerund wordt door de lokale bevolking. Projecten als deze maken een substantieel verschil voor ondervoede kinderen en hun omgeving en behoeven onze volste (financiële) steun. Met méér fondsen en méér onderzoekers die méér observatie- en interventiestudies kunnen uitvoeren, kunnen we dit wereldwijde probleem van ondervoeding bij kinderen aanpakken en bestrijden, om zo een gezonde voedingstoestand van kinderen herstellend van ondervoeding en van toekomstige kinderen te kunnen bewerkstelligen.Our children’s futures are not to be wasted.

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ABBReVIATIOnS

AIDS Acquired Immune Deficiency SyndromeCF Cystic FibrosisCMT Chymotrypsin; CP Chronic PancreatitisCRF Case Report FormEPI Exocrine Pancreatic InsufficiencyFE-1 Fecal Elastase-1FFA Free Fatty AcidsHAZ Height-for-Age Z-scoreHIV Human Immunodeficiency VirusIFABP plasma Intestinal Fatty Acid Binding ProteinIRT Immunoreactive TrypsinogenITT Intention To TreatMUAC Mid Upper Arm CircumferenceNRU Nutritional Rehabilitation UnitPERT Pancreatic Enzyme Replacement TherapyPST Pancreatic Stimulation TestRUTF Ready-to-Use Therapeutic FoodSAE Severe Adverse EventSAM Severe Acute MalnutritionSD Standard DeviationSDS Shwachman-Diamond syndromeTG TriglyceridesW/H Weight for Height WHO World Health OrganizationWHZ Weight-for-height Z-score

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COnTRIBUTInG AUTHORS

Angela AllenCentre for Tropical Infectious Diseases, Liverpool School of Tropical Medicine, Liverpool, UK

Stephen J AllenCentre for Tropical Infectious Diseases, Liverpool School of Tropical Medicine, Liverpool, UK

Benjamin AllubhaDepartment of Pediatrics and Child Health, Queen Elizabeth Central Hospital, Blantyre, Malawi

Robert H.J. BandsmaDivision of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, Toronto, Ontario, CanadaCenter for Global Child Health, The Hospital for Sick Children, Toronto, CanadaDepartment of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, University of Groningen, University Medical Center Groningen, Groningen, The NetherlandsDepartment of Biomedical Sciences, College of Medicine, University of Malawi, Blantyre MalawiThe Childhood Acute Illness & Nutrition Network

Michael Boele van HensbroekGlobal Child Health Group, Emma Children’s Hospital, Academic Medical Center, Amster-dam, the Netherlands

Deborah A. van den Brink Department of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands

Céline BourdonDivision of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, Toronto, University of Toronto, Toronto, CanadaDepartment of Physiology and Experimental Medicine, The Hospital for Sick Children, Toronto, Canada The Childhood Acute Illness & Nutrition Network

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Kate ChidzaloDepartment of Pediatrics and Child Health, Queen Elizabeth Central Hospital, Blantyre, MalawiThe Childhood Acute Illness & Nutrition Network

emmanuel ChimweziDepartment of Pediatrics and Child Health, Queen Elizabeth Central Hospital, Blantyre, MalawiThe Childhood Acute Illness & Nutrition Network

Queen DubeCollege of Medicine, University of Malawi, Blantyre, Malawi

Jacintha KoolDepartment of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

Macpherson MallewaCollege of Medicine, University of Malawi, Blantyre, Malawi

Sophie l. MeyerUniversity Medical Center Groningen, University of Groningen, The Netherlands

Brian MhangoDepartment of Pediatrics and Child Health, College of Medicine, University of Malawi, Blantyre, Malawi

John S. MpondaDepartment of Pharmacy, College of Medicine, University of Malawi, Blantyre, Malawi

Anneke C. Muller KoboldDepartment of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

leilei PeiDepartment of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, UK

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Isabel Potani Department of Pediatrics and Child Health, College of Medicine, University of Malawi, Blantyre, Malawi

Tijs A. StehmannUniversity Medical Center Groningen, University of Groningen, The Netherlands

Merit M. TabbersDepartment of Pediatric Gastroenterology and Nutrition, Emma Children’s Hospital, Academic Medical Centre, Amsterdam, the Netherlands

Wieger P. VoskuijlGlobal Child Health Group, Emma Children’s Hospital, Academic Medical Center, Amster-dam, the NetherlandsDepartment of Pediatrics and Child Health, College of Medicine, University of Malawi, Blantyre, MalawiThe Childhood Acute Illness & Nutrition Network

Duolao WangDepartment of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, UK

Vicky Watson Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, UK

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ABOUT THe AUTHOR

Rosalie Bartels was born in Amsterdam on December 8, 1983. She grew up in Bussum, and obtained her secondary school degree at the Gemeentelijk Gymnasium in Hilver-sum. After finishing secondary school in 2002, she spent three months in Rome, Italy, to learn the Italian language. Hereafter, she lived in Syracuse, upstate New York, USA, for six months, to live in a community for people with and without mental and physical disabilities. While waiting for a spot in medical school, she studied French language and culture for a year at the University of Amsterdam. In 2004, she finally started medical school to become a pediatrician; something she had already decided on in her early teenage years. She returned to Italy in the summer of 2005, for a medical internship at the Ospedale di Vicenza, in Vicenza. For her scientific internship in 2008, she traveled for eight months to Melbourne, Australia, and contributed to research on obesity in children at the Murdochs Childrens Research Institute of the Royal Childrens Hospital. During a pediatric internship in northern Namibia in 2011, she worked in a pediatric malnutrition ward for the first time, and fell in love with this patient population and Southern Africa. When she graduated as a medical doctor in the summer of 2011, she started working on the pediatric department of the Kennemer Gasthuis (currently Spaarne Gasthuis) in Haarlem. After six months, she moved on to working on the pediatric department of the Medisch Centrum Alkmaar (currently Noordwest Ziekenhuisgroep) in Alkmaar. She worked here for eighteen months, after which she decided to invest in her development as a researcher in pediatrics. It was obvious to her to apply for a project in a low resource setting and joined the research team of dr. Wieger Voskuijl on ‘Moyo’, the pediatric Nutritional Rehabilitation Unit of Queen Elizabeth Central Hospital in Blantyre, Malawi, at the end of 2013. The initial plan was to work on the OPTIMISM trial (Chapter 4) for six months, and then return to the Netherlands to apply for a position as a pediatric registrar. However, one study lead to many others, and resulted in her staying in Malawi until the end of 2016, where she worked about two thirds of her time as a clinician at both the NRU and the general pediatric department, and the rest as a PhD student under the supervision of professor Michael Boele van Hensbroek from the University of Amsterdam, in addition to working as a local trial coordinator of several trials. She moved back to Amsterdam in early 2017 to write her thesis and finally apply for a posi-tion as pediatric registrar. In January 2018, she has started her pediatric residency at the Wilhelmina Kinderziekenhuis of the University of Utrecht.

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