Biomedical Investigations for Optimized Diagnosis and Monitoring of Severe Acute Malnutrition (SAM): Elucidating the Heterogeneous Diagnosis of SAM by Current Anthropometric Criteria and Moving Beyond
Condition: Severe Acute Malnutrition
MeSH terms: Malnutrition; Severe Acute Malnutrition
Study type: Observational

Summary: 
      INTRODUCTION

      In 2014, 50 million children under 5 suffered from acute malnutrition, of which 16 million
      suffered from SAM, most of them living in sub-Saharan Africa and Southeast Asia. SAM children
      have higher risk of mortality (relative risk between 5 and 20). It is an underlying factor in
      over 50% of the 10 - 11 million preventable deaths per year among children under five. At
      present, 65 countries have implemented WHO recommendations for SAM treatment (both in-patient
      for complicated cases and outpatient for uncomplicated cases) but these programs have very
      low coverage, reaching only around 10 - 15 % of SAM children.

      In 2009 the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF)
      issued a joint statement in an effort to harmonize the application of anthropometric criteria
      for SAM diagnosis and monitoring in child aged 6 - 59 months; the statement presents
      recommended cut-offs, and summarizes the rational for the adoption, of the following two
      anthropometric criteria:

        1. Weight-for-Height Z-Score (WHZ): "WHO and UNICEF recommend the use of a cut-off for
           weight-for-height of below -3 standard deviations (SD) of the WHO standards to identify
           infants and children as having SAM." Additionally, analysis of existing data show that
           children with a WHZ < -3 have a highly elevated risk of death.

        2. Mid-Upper Arm Circumference (MUAC): "WHO standards for the MUAC-for-age show that in a
           well-nourished population there are very few children aged 6 - 59 months with a MUAC
           less than 115 mm. Children with a MUAC less than 115 mm have a highly elevated risk of
           death compared to those who are above. Thus it is recommended to [use] the cut-off point
           [of] 115 mm to define SAM with MUAC."

      GENERAL OBJECTIVE

      To generate new evidence on pathophysiological process, nutritional needs and risks
      associated with different types of anthropometric deficits in children under 5, in order to
      optimize the diagnosis and treatment of SAM.

      SPECIFIC OBJECTIVES

        -  To compare nutritional status, metabolism, pathophysiological process and risks in
           different types of SAM anthropometric diagnosis, with or without concomitant stunting
           (growth retardation).

        -  To analyze the extent to which current SAM treatment is promoting recovery and healthy
           growth in different categories of children.

        -  To evaluate the relevance of current discharge criteria used in nutrition programs and
           their association with metabolic recovery, in different age groups and among those who
           are stunted.

        -  To test novel rapid tests of emerging biomarkers predicting long-term outcomes and
           mortality risk in the field.

      METHODOLOGY

      A wide range of supplementary information related to nutritional status, body composition,
      metabolic and immune status, including emerging biomarkers of metabolic deprivation and
      vulnerability, will be collected besides anthropometry during prospective observational
      studies. They will be collected with minimum level of invasiveness, compatible with field
      work requirements in the humanitarian context.

      Phase 1: Cross-sectional surveys. Phase 2: Prospective cohort studies involving SAM children
      between 6 months and 5 years old.

      Children admitted as SAM at the nutrition centers will be enrolled into the cohort. The
      follow up duration will be at least three months.

      EXPECTED OUTCOMES

        -  Confirmation of current hypotheses related to:

             1. possible misdiagnosis of SAM made by MUAC or WHZ criteria,

             2. varying degree of severity and need for admission to treatment of the different
                types of diagnosis,

             3. underlying heterogeneity of the pathophysiology.

        -  Generation of new algorithms for the assessment and classification of malnourished
           children, based on the combined use of emerging biomarkers and anthropometric measures,
           or on the modification of anthropometric criteria.

        -  Generation of new treatment paradigms based on the predictive value of biomarkers in
           combination with traditional anthropometric measures. This will enable us to assess the
           power of current treatment regimens to promote long-term weight gain and growth and will
           allow us to tailor treatment to the physiological needs of the child.
    

Detailed description: 
      DIAGNOSTIC DISCREPANCY

      According to WHO experts, WHZ and MUAC can be used independently to indicate severe acute
      malnutrition (WH. There is however a significant and sometimes huge discrepancy between these
      two criteria: they do not usually identify the same children as acutely malnourished;
      moreover when used as proxy indicator to assess a deteriorating nutritional situation at a
      population level, these criteria do not report the same level of global acute malnutrition in
      the same zone.

      It was reported that only about 40% of SAM cases identified by one indicator are also
      diagnosed as such by the other. For example, among severely malnourished children
      hospitalized in rural Kenya, 65.1% (486/746) of the WHZ -3 cases also had a MUAC < 115 mm,
      whereas 56% (489/873) of the MUAC < 115 mm cases were also identified by WHZ < -3. In that
      study, 42.9% (489/1140) of the SAM cases were identified by both indicators. The discrepancy
      between the two indicators can be even more extreme.

      Fernandez et al. reported that among 34,937 children between the ages of 6 - 59 months from
      39 nutritional surveys, 75% of the children with a WHZ < -3 were not identified by a MUAC <
      115 mm. In Cambodia, this proportion was above 90%, whereas 80% of MUAC < 115 mm were not
      detected by WHZ < - 3.

      Most of the time, caseloads defined by WHZ are much larger than by MUAC, but the contrary may
      happen as well, especially in the younger age groups.

      PROGRAMMATIC CONFUSION

      Such discrepancy generates important programmatic challenges and confusion. On the one hand,
      a strategy where the diagnosis can be based on either indicator, as recommended by some
      authors may unduly inflate the workload of nutritional rehabilitation programs, as the most
      appropriate management of children identified by one indicator and not by the other is
      uncertain. On the other hand, relying on only one of these indicators, e.g. using only MUAC <
      115 mm in community-based programs, may under-detect true acute malnutrition cases and result
      in missed opportunities to treat a severe condition.

      In recent years, however, the use of MUAC alone for admission has been discussed, and is
      increasingly applied in a variety of contexts. In particular, more and more national
      protocols for SAM management consider MUAC only management as programmatic option. The
      national guidelines in Bangladesh, for example consider only low MUAC as an admission
      criterion for uncomplicated SAM management, which by de facto excludes a vast majority of the
      SAM children, those who have WHZ < - 3 and MUAC ≥ 115mm.

      Many benefits of using MUAC exist: MUAC is predictive of death, easy to use, acceptable, and
      favors community-based screening methods. Yet, as these two anthropometric tools select
      different children for treatment, as outlined above, this complicates the programmatic
      paradigm shift from admitting children using MUAC < 115 mm and/or WHZ < -3 to a new model
      admitting children using MUAC < 115 mm only.

      Depending on context, up to 63-79% of children currently recommended for therapeutic feeding
      with WHZ < -3 and/or MUAC < 115 mm would not be eligible if using MUAC < 115 mm alone for
      admission.

      RATIONALE

      To inform decision making regarding the use of MUAC as a standalone admission criterion in
      nutrition programming, more information on the programmatic and clinical implications of
      using MUAC alone is urgently needed. Despite WHO clearly highlighting the importance of this
      anthropometric diagnostic heterogeneity, and requesting more investigation, very little has
      been done so far.

      Preliminary reports demonstrate demographic and anthropometric differences among children
      identified by WHZ and MUAC: MUAC is more likely to identify children that are younger, female
      and with concomitant stunting. These data have been used to suggest a role for MUAC to
      identify children that are potentially more vulnerable or at a higher risk of death,
      supporting the transition to a MUAC-only based admission criterion. Recent secondary analysis
      of the clinical profile and outcomes of SAM children admitted to an outpatient SAM program in
      Niger infirmed this hypothesis, by showing a similar vulnerability profile in SAM children
      presenting with a MUAC < 115 mm (with or without concomitant WHZ <- 3) and in SAM children
      with a MUAC ≥ 115mm, i.e. only with WHZ < -3, who would not be considered for treatment in
      case of MUAC-only programming. Furthermore, according to this study, and a similar one from a
      SAM management program in South-Sudan, the anthropometric category of SAM children displaying
      the highest vulnerability at admission and the worse treatment outcomes are those combining
      MUAC < 115mm and WHZ < -3. These results are in line with previous observations from an
      in-patient SAM management program in Kenya.

      Beyond the investigation of possible variations in mortality risks, all reviews of available
      evidence on this issue highlight the need for robust research to further investigate the
      physiological significance of the different anthropometric criteria and to better understand
      how the clinical status and nutritional needs of the children are addressed over the course
      of nutritional rehabilitation. A key issue is indeed that these two different indicators
      identify different populations of children, the reason for which is unknown due to the lack
      of a gold standard.

      Current hypotheses to explain the diagnosis discrepancy are that:

        -  WHZ<-3 overestimates the diagnosis of acute malnutrition in populations with a slender
           morphology (i.e. with a low sitting-to-standing-height ratio; SSR) as observed in
           pastoralists.

        -  MUAC at a fixed cut-off overestimate SAM in the younger children, in girls and in the
           stunted children, and on the contrary underestimates SAM in older, male, and non-stunted
           children. Young age, being a girl and stunting are indeed factors known to be associated
           with lower MUAC measurements and were already shown to be independently associated with
           MUAC diagnosis: lesser levels of acute nutritional deficits and wasting might thus be
           necessary to reach the 115mm cut-off in these children.

      These hypotheses have recently been supported by the analysis of the strength of the
      association between these factors and the diagnosis discrepancy in nutritional
      cross-sectional surveys.

      WHZ and MUAC criteria also may identify a separate kind of physiological deficit. It has been
      hypothesized that this might be related to differing impairments of fat and muscle mass
      stores, with MUAC reflecting preferentially fat mass for some authors and muscle mass for
      others. An analysis of body composition in a cohort of Ethiopian infants recently confirmed
      WHZ as a good marker of tissue masses independent of length, while MUAC appeared more as a
      composite index of poor growth indexing jointly tissue masses and length.

      Children identified by different criteria may thus require different treatment, one that is
      tailored to nutritional deficit. For instance, lower anthropometric response to treatment
      (lower MUAC gain and weight gain, longer treatment duration and higher proportion of
      non-responders) has already been observed in younger, stunted girls identified by MUAC. This
      might be linked to a suboptimal response in less severely wasted children, or might be due to
      a higher proportion of false positives in this sub-population, or be an indicator that the
      treatment is less effective or required in such children. Also, a recent meta-analysis of
      follow-up datasets evidenced a dramatic increase in mortality risk in children combining low
      WHZ and stunting (MUAC was not factored in).

      Today, in the absence of a gold standard for SAM, it is difficult to interpret different and
      often divergent anthropometric diagnoses. Additionally, there is a vital need to better
      understand if and how far physiological recovery, beyond anthropometric growth (which might
      be transient or sub-optimal) is achieved under the current SAM management strategy. Moreover,
      this understanding should encompass the whole population of children affected by
      anthropometric deficit, beyond just those few complicated cases that reach the hospital for
      inpatient nutritional rehabilitation. It should also account for potential contextual
      variation in the link between anthropometry and nutritional status.

      In order to describe and compare nutritional needs and risks associated with the different
      types of diagnosis as they are present in the community, we propose to conduct prospective
      cohort studies of SAM children who will be detected and referred to treatment in the
      catchment areas of community-based acute malnutrition management programs. Such programs
      combine both outpatient and inpatient nutritional rehabilitation, and an effective community
      outreach component. Nutritional needs and risks will be evaluated using a range of
      indicators:

        -  proxy indicators of nutritional, metabolic and immune status, among which several
           biomarkers whose association with risk of death has been recently evidenced in SAM
           children;

        -  clinical characteristics; and,

        -  response to treatment in terms of cure rates, recovery speed, relapse. The indicators
           necessary to do so must be easy to collect with low invasiveness and should provide
           reliable information regarding the severity of nutritional status.

      ISOTOPIC EVALUATION OF HAIR

      Isotopic analysis of stable carbon and nitrogen in human hair can be investigated and
      measured throughout the course of nutritional deprivation to reconstruct the onset and
      duration of undernourishment as well as tracing the temporal evolution of nutritional status.

      Several studies have revealed that factors like diet, disease and injury can influence
      nitrogen isotope ratios (d15N) in human tissues. Specifically, d15N values reflect the
      nitrogen balance of an organism in that during a catabolic state (tissue breakdown) d15N
      values increase while during an anabolic state (tissue buildup) d15N values decrease.

      In contrast, carbon isotope ratios (d13C) are shown to decrease during catabolism and
      increase during anabolism. Thus, during starvation the body becomes enriched in 15N and
      depleted in 13C at the same time.

      Since keratin remains unchanged after synthesis, and the speed of hair growth is constant
      (around 2.5 mm per week), weekly information on protein-energy metabolism can be traced back
      along the hair follicle, thereby indicating not only the severity of the episode of wasting
      but also the metabolic effects of the nutritional rehabilitation (on both lipid and protein
      anabolism). Isotopic evaluation of stable carbon and nitrogen in hair will therefore be used
      to create a retrospective timeframe of nutritional status and trace the physiological
      recovery of children during SAM management.

      LEPTIN AND IL-6

      A recent study using non-targeted metabolomics analysis to characterize changes a broad array
      of hormones, cytokines, growth factors and metabolites during the treatment of SAM has
      revealed that a major biochemical predictive factor for mortality is low-level leptin. Low
      leptin and interleukin 6 levels reflect the adequacy of fat stores. Depletion of white
      adipose stores is postulated to limit the ability of a child to sustain energy production
      during the course of the illness and thereby increase the child's risk of death.
      Alternatively, hypoleptinemia may reduce viability affecting glucose and energy homeostasis
      or immune competence.

      Leptin and interleukin 6 targeted analysis will therefore be used to create a metabolic
      profile of SAM patients at presentation and during nutritional rehabilitation, and may
      predict mortality prior to and during treatment.

      Dr. Michael Freemark and colleagues at DUMC are currently developing novel point-of-care
      micro-assays to characterize the hormonal status of leptin and interleukin 6 in SAM children
      from a single fingerstick that will be piloted.

      MICRONUTRIENT AND IMMUNE RESPONSE BIOMARKERS

      Deficiencies of vitamin A and iron are among the most common micronutrient deficiencies
      related to childhood undernutrition and are both linked to compromised immune function.
      Manifestations of isolated iron deficiency include anemia, fatigue, impaired cognitive
      development and reduced growth and physical strength. Vitamin A deficiency contributes to
      anemia by immobilizing iron in the reticuloendothelial system, reducing hemopoiesis and
      increasing susceptibility to infections; it is essential for the functioning of the immune
      system and its deficiency has been clearly shown to be associated with diarrhea and related
      mortality.

      Vitamin A deficiency has been evidenced as frequent in SAM children. Vitamin A status,
      measured by the surrogate marker RBP, has been shown to be low in SAM children and to rise
      during nutritional rehabilitation. Mean serum vitamin A has been shown to decrease with
      increasing stunting (HAZ), wasting (WHZ) and underweight (WAZ). Additionally, there are
      indications that the storage levels of iron in SAM children are increased not decreased, even
      in the presence of quite severe anemia. However, there is a major lack of evidence on this
      point; we know that these parameters also need to be adjusted for inflammation biomarker,
      anemia and malaria, which was not done in the studies mentioned by Golden.

      Immune response biomarkers like C-reactive protein (CRP) are elevated in SAM children with
      severe bacterial infections. CRP is therefore a potentially valuable clinical tool for
      identifying bacterial infections, and recent research has shown that a rapid CRP could be
      useful in field settings to identify children most at risk for dying, with a relatively good
      negative predictive value (81% sensitivity, 85% specificity).

      There is a need to evaluate the relationship between micronutrient status, immune response
      and anthropometric diagnosis of SAM children at and to examine the extent to which
      nutritional rehabilitation is effective in treating deficiencies of vitamin A and iron and to
      prevent deficiencies during catch up growth.

      An inexpensive and sensitive simple sandwich enzyme-linked immunosorbent assay (ELISA)
      technique was recently developed to measure indicators of vitamin A and iron deficiency. Due
      to the low cost, high throughput, and comparability to traditional tests, this procedure has
      several advantages for assessing vitamin A and iron status on the field. Moreover, it can
      easily be combined with the measurement of immune response biomarkers like CRP and α1-acid
      glycoprotein (AGP).

      CRP, AGP, as well as biomarkers of iron (serum ferritin and serum transferrin receptor) and
      vitamin A status (serum retinol binding protein) can be assessed in a few drops of capillary
      blood. CRP and AGP will be used to adjust for the effect of inflammation on the micronutrient
      status indicators. Inflammation is indeed known to elevate serum ferritin and depress retinol
      binding protein as part of the biological acute phase response to inflammation.

      URINE TESTS

      The presence of ketones in the urine, indicating lipid catabolism (fat tissue disintegration
      and rapid weight loss) was evidenced during fasting and SAM. Metabolic status for SAM
      children at the time of enrollment in CMAM has been characterized by ketonemia; yet,
      lipolysis decreases in response to nutritional rehabilitation suggested by total ketones.

      Moreover, biomarkers of urinary infections like urinary nitrites and urinary leucocyte
      esterase (LE) have also been shown to be associated with an increased mortality risk in SAM
      children. Positive dipstick urinalysis administered as a bedside screening test for either
      nitrates or LE is associated with a higher case fatality and was shown to be a strong
      predictor of mortality in children admitted with SAM.

      Non-sterile urine sample will be also carried out when possible, and these biological
      parameters will be measured using through the urinary multiple indicator strips (e.g. Roche
      laboratory, or Combi Screen of Analyticon).

      BIOELECTRICAL IMPEDANCE (BI)

      It has been suggested that different anthropometric diagnoses identify children with
      different body composition, and associated nutritional needs. Restoration of body composition
      indicates successful management of SAM. Bioelectral impedance (BI) is a safe, rapid and easy
      technique often used to assess body composition, predicting total body water (TBW) in
      non-edematous children. It has demonstrated utility for indexing acute changes in hydration
      in children with SAM during in-patient treatment. This technique could also potentially
      distinguish tissue versus hydration relates weight catch-up during or post treatment. Lastly,
      BI analysis may predict survival outcomes for children hospitalized for SAM.

      BI parameters will therefore be used to describe body composition at admission and the
      restoration of body composition throughout nutritional recovery.

      METHODS

      STUDY DESIGN

      This study consists of three prospective follow-up studies (Bangladesh, Burkina Faso and
      Liberia) including cohorts of SAM children between 6 and 59 months of age.

      Children will be recruited according to the current WHO recommended anthropometric criteria
      for SAM diagnosis, WHZ and MUAC. Clinical examination, interviews with caregivers and blood
      and hair samples will be collected at admission and follow-up.

      Prospective follow-up cohort studies will be nested into currently operational
      Community-based Management of Acute Malnutrition (CMAM) programs run with the technical
      support of ACF-France, in hospitals and primary health-care centers involved in SAM
      management. All participants will be treated according to the standard of care outlined in
      the national protocol for SAM management of the country; this included a medical examination
      and standard treatment for infections as well as hospital referral for any complications
      requiring medical attention. The follow-up duration for enrolled SAM cases will be three
      months at minimum. Each individual cohort study will last approximately one year.

      This study design is multi-centric, and will be conducted in Bangladesh, Burkina Faso and
      Indonesia to account for potential contextual variation in the link between anthropometry and
      nutritional status.

      The various biomarkers assessed in this study, alongside anthropometry and clinical
      characteristics, at admission and follow-up times, can be grouped into the following three
      main groups:

        1. Biomarkers of micronutrient deficiencies: (1) iron status biomarkers like serum ferritin
           and serum transferrin receptor; (2) vitamin A status biomarkers like retinol binding
           protein; and (3) vitamin C in the urine.

        2. Biomarkers and indicators of body composition and energy metabolism: (1) urinary
           ketones; (2) natural enrichment of nitrogen and carbon stable isotopes in hair; and (3)
           circulating leptin and IL-6.

        3. Biomarkers of non-specific immune response or urinary infections: (1) c-reactive protein
           level; (2) urinary nitrites; and (3) urinary leucocytes esterase.

      QUESTIONNAIRES

      Data collection sheets, hereafter referred to as case report forms (CRF) will be linked to
      the patient's information by his or her unique study ID number. All data will be collected by
      trained ACF research staff.

      A baseline questionnaire will be administered upon admission by means of a structured
      interview with the caregiver. The interview content includes socioeconomic indicators, family
      size, income, expenditure as well as the medical history of the child (including changes in
      the child's weight, quantity and quality of food consumed and global health status). This
      questionnaire will include changes in the child's weight, quantity and quality of food
      consumed and overall health status.

      At each weekly visit, caregivers will answer a morbidity questionnaire (regarding fever,
      diarrhea, respiratory infection, and appetite) over the past week. Additionally, the
      caregiver will be asked to score the child's health status using a visual analog scale (VAS),
      table 4 the VAS is a psychometric response scale used in questionnaires to measure subjective
      characteristics or attitudes that cannot otherwise be directly measured ("Visual Analog
      Scale," 2015). The use of a VAS in the assessment of severity of illness has been shown to be
      a strong predictor of mortality.

      These data will be compared to the patient's nutritional progress to assess the relationship
      between maternal health perception and nutritional indicators of recovery. Additional
      questions will probe adherence to treatment ready-to-use therapeutic food (RUTF) on a weekly
      basis.

      ANTHROPOMETRY

      Weight, height, MUAC, edema, will be measured weekly in all children.

      Weight will be measured to the nearest 0.1 kg with an electronic SECA scale, which will allow
      for simultaneous weighing of caregiver and patient. A standard weight of 5-10 kg will be used
      for daily calibration of the scale, and it will be stabilized on wooden plank to ensure the
      scale stays in a horizontal position.

      Length and height will be measured to the nearest 0.1 cm with a UNICEF model wooden height
      board with graduated index strips in millimeters on each side. A standardized length stick
      will be used to check the accuracy of the equipment. Children less than 2 years of age will
      be measured lying down and older children will be measured standing up. In case the age
      cannot be verified, children less than 87cm will be measured lying down. Children above 2
      years of age, or above 87 cm who are not able to stand, will be measured lying, and 0.7 cm
      will be subtracted the recumbent length during data analysis.

      MUAC will be measured with a non-stretchable MUAC tape on the left arm to the nearest
      millimeter.

      Anthropometry will be measured and recorded twice. Measurements will be repeated by the same
      person and directly after each other to minimize discomfort for the child. The measurer will
      read out loud his measurement, which will then be repeated by the assistant who will record
      the results. In case of large differences between the measurements, the procedure will be
      repeated. All measuring tools will be calibrated and checked daily for accuracy and replaced
      if needed. To quantify the inter-measurer error, as part of training and refresher training
      programs, anthropometry measurements will be repeated by a second person. This procedure will
      be done on a small sample of children and will take place at the beginning, mid-way and end
      of the trial.

      CLINICAL ASSESSMENT AND EDEMA

      Presence of bilateral pitting edema of nutritional origin will be assessed by applying normal
      thumb pressure on the tops of both feet for three seconds. In the presence of edema (a
      remnant impression remains for some time, where the fluid has been temporarily pressed out of
      the tissue) the same procedure will be repeated on the lower legs, hands. Generalized, severe
      edema can be observed sacral pad and face (forehead, eyelids). The degree of edema
      generalization will be recorded according to WHO categorization of edema severity outlined in
      the guidelines for the management of severe acute malnutrition in children 6 to 59 months of
      age with edema, presented in table 5.

      The nurse will conduct a weekly clinical assessment of the child (i.e. temperature,
      respiratory rate, pulse rate, diarrhea, vomiting, and malaria). Symptoms, diagnosis and
      treatments prescribed will be recorded. The clinical assessment will also serve to monitor
      the development of medical complications requiring inpatient care. Any serious adverse
      effects or development of medical complications will be immediately reported to the study
      supervisor and the child will be referred for inpatient treatment if necessary.

      BLOOD SAMPLES AND ANALYSES

      Since no laboratory in the study area performs all the desired analyses required, the samples
      must be exported for analysis.

      A serum sample of 0.5 mL of serum from baseline and at two weeks and two months after
      nutritional rehabilitation will be sent to Dr. Juergen Ehardt at VitMin Laboratory in
      Wilstaett, Germany for the following analyses:

        -  C-reactive protein (CRP),

        -  a1-acid glycoprotein (AGP),

        -  Serum ferritin

        -  Retinol Binding Protein (RBP)

        -  Soluble Transferrin Receptor (sTfR).

      A duplicate serum sample will be kept in the event that the first sample is lost or destroyed
      during transport.

      Serum RBP will be used to assess vitamin A status. Serum ferritin and sTfR will be used to
      assess iron status. Additionally, the two acute phase reactants, CRP and AGP, will be
      measured and used to adjust for the effect of inflammation on the micronutrient status
      indicators. These five proteins will be measured using a specialized ELISA kit which analyses
      all five components simultaneously.

      HAIR SAMPLES AND ANALYSES

      A single lock of 20 - 25 hair follicles will be shaved at the back of the skull at admission
      and throughout treatment to both retrospectively characterize the nature and magnitude of
      metabolic deficits at admission; and also the efficacy of treatment in correcting them. Hair
      samples will be sent to Dr. Jean-Francois Huneau and Dr. Helene Fouillet at the Human Biology
      and Nutrition Laboratory at AgroParisTech in Paris, France. Hair samples will be
      sub-sectioned into 2.5 mm samples and analyzed for d13C and d15N through EA-IRM analysis.

      URINE SAMPLES AND ANALYSES

      Freshly voided, clean catch urine samples will be tested using reagent test strips (e.g.
      Multistix) at admission and at the end of treatment to assess biomarkers of urinary
      infections, urinary nitrites and urinary LE.

      BIOELECTRICAL IMPEDANCE

      BI parameters will be measures using a NutriGuard-S (DataInput, Germany) using protocols
      described elsewhere. Self-adhesive disposable electrodes will be attached to the right hand
      and foot. Measurements will be taken in triplicate, each spaced 5 minutes apart, while
      children are supine with limbs abducted from the body.

      DATA MANAGEMENT AND ANALYSIS

      DATA MANAGEMENT

      All questionnaire data and measurements will be measured on paper print-outs. The field
      supervisor will digitize, back-up and share data with the project coordinator at least
      weekly. Appropriate consistency checks and completeness rules will be applied to all data
      templates.

      Anthropometric measurements will be standardized using the ENA SMART software standardization
      tools; additionally, regular supervision and refresher training workshops will be organized
      to maintain the highest quality data.

      In addition to a digital database, paper forms including the date, the patient's and
      caregiver's name, study ID, age, length and height at admission will be produced at
      enrollment. These forms will be kept at each health center and new anthropometric values will
      be added at each visit to track the child's health progress.

      Field tables for anthropometric conversion to z-scores will be available at all health
      centers.

      To maximize the follow-up rate, all participants will be registered in a digital logbook (for
      example using Epidata) where each visit will be registered with the date of attendance.
      Weekly lists of expected participants will be produced and printed for reminder calls.
      Participants who do not show up at the health center will be contacted per telephone, and if
      not successful, a team of community health workers will try to trace the family at the home
      address and encourage them to go to the health center for continuation of the treatment.

      DATA ANALYSIS

      All data will be analyzed using STATA Data Analysis and Statistical Software version 13
      (StataCorp, College Station; Lakeway, Texas, U.S.A.).

      ETHICAL CONSIDERATIONS

      This research protocol will be submitted for ethical clearance to:

        1. The Institutional Review Board (IRB) of the Institute of Tropical Medicine Antwerp,
           Belgium; and

        2. The Committee of Medical Ethics (CME) at Antwerp University Hospital, Universitair
           Ziekenhuis Antwerpen (UZA) and the University of Antwerp, Universiteit Antwerpen
           (UAntwerp);

           Context adapted versions of this protocol will be submitted for ethical clearance to:

        3. The Comite National d'Ethique pour la Recherche en Sante (CNERS), Burkina Faso

        4. The IRB of the University of Liberia - Pacific Institute of Research and Evaluation
           (UL-PIRE;

        5. The National Research Ethics Committee (NERC) at the Bangladesh Medical Research Council
           (BMRC).

      CONFLICTS OF INTEREST

      None of the project affiliates have declared any conflicts of interest.
    

Intervention name(s): Severe Acute Malnutrition
Intervention type(s): Other
Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        In the effort to describe and compare nutritional needs and risks associated with the
        different types of anthropometric diagnoses as they are present in the community, inclusion
        criteria for this study are designed to create a cohort to match the population of children
        who will be detected and referred to treatment in the catchment areas of community-based
        acute malnutrition management programs.
      
Keywords: Severe Acute Malnutrition