Serum cobalamin in children with moderate acute malnutrition in Burkina Faso: Secondary analysis of a randomized trial.

BACKGROUND: Among children with moderate acute malnutrition (MAM) the level of serum cobalamin (SC) and effect of food supplements are unknown. We aimed to assess prevalence and correlates of low SC in children with MAM, associations with hemoglobin and development, and effects of food supplements on SC. METHODS AND
FINDINGS: A randomized 2 × 2 × 3 factorial trial was conducted in Burkina Faso. Children aged 6 to 23 months with MAM received 500 kcal/d as lipid-based nutrient supplement (LNS) or corn-soy blend (CSB), containing dehulled soy (DS) or soy isolate (SI) and 0%, 20%, or 50% of total protein from milk for 3 months. Randomization resulted in baseline equivalence between intervention groups. Data on hemoglobin and development were available at baseline. SC was available at baseline and after 3 and 6 months. SC was available from 1,192 (74.1%) of 1,609 children at baseline. The mean (±SD) age was 12.6 (±5.0) months, and 54% were females. Low mid-upper arm circumference (MUAC; <125 mm) was found in 80.4% (958) of the children and low weight-for-length z-score (WLZ; <-2) in 70.6% (841). Stunting was seen in 38.2% (456). Only 5.9% were not breastfed. Median (IQR) SC was 188 (137; 259) pmol/L. Two-thirds had SC ≤222 pmol/L, which was associated with lower hemoglobin. After age and sex adjustments, very low SC (<112 pmol/L) was associated with 0.21 (95% CI: 0.01; 0.41, p = 0.04) and 0.24 (95% CI: 0.06; 0.42, p = 0.01) z-score lower fine and gross motor development, respectively. SC data were available from 1,330 (85.9%) of 1,548 children followed up after 3 months and 398 (26.5%) of the 1,503 children after 6 months. Based on tobit regression, accounting for left censored data, and adjustments for correlates of missing data, the mean (95% CI) increments in SC from baseline to the 3- and 6-month follow-up were 72 (65; 79, p < 0.001) and 26 (16; 37, p < 0.001) pmol/L, respectively. The changes were similar among the 310 children with SC data at all 3 time points. Yet, the increase was 39 (20; 57, p < 0.001) pmol/L larger in children given LNS compared to CSB if based on SI (interaction, p < 0.001). No effect of milk was found. Four children died, and no child developed an allergic reaction to supplements. The main limitation of this study was that only SC was available as a marker of status and was missing from a quarter of the children.
CONCLUSIONS: Low SC is prevalent among children with MAM and may contribute to impaired erythropoiesis and child development. The SC increase during supplementation was inadequate. The bioavailability and adequacy of cobalamin in food supplements should be reconsidered. TRIAL REGISTRATION: ISRCTN Registry ISRCTN42569496.

Introduction

In the first few years of life, growth velocity is high, and the brain reaches close to adult size. Hence, the nutritional requirements are high, and inadequate dietary intake may have long-lasting effects on growth and development. The diet of children in low-income settings is based on a starch-rich staple, with limited amounts of fruits, vegetables, and pulses, and little animal source foods [1]. Lack of animal source foods contribute to a range of coexisting deficiencies [1].

Exposure to pathogens and mycotoxins may impair intestinal absorption and nutritional status [2]. On a background of food insecurity, these factors may contribute to the widespread micronutrient deficiencies. Research and programs have focused on iron, zinc, and vitamin A deficiency. Cobalamin (B-12) deficiency has received less attention. Since cobalamin is important for DNA synthesis, developing deficiencies will first impair rapidly dividing cell lines such as enterocytes and erythrocytes [3]. Cobalamin is also involved in sprouting and myelination of neurons and affects brain development [3]. Cobalamin is solely found in animal source or fortified foods, and emerging data confirm that low or marginal cobalamin status is widespread, especially in Asia and Africa [4].

We conducted a randomized food supplementation trial among young children with moderate acute malnutrition (MAM) in Burkina Faso and used a factorial design to assess the effects of food type, soy quality, and milk content on the primary outcome fat-free mass index [5] and on the key secondary outcomes: iron status [6] and child development [7]. We have previously reported that food supplements were beneficial for child development, which motivated us to assess the role of cobalamin.

The aim of this paper is 3-fold. First, to assess the level and correlates of serum cobalamin (SC) at baseline, i.e., before initiation of supplementation. Second, to assess the associations of low SC with hemoglobin and child development at baseline. Third, during 3 months food supplementation, to assess the effects of matrix, soy quality, and milk content on SC.

Methods

Study design, area, and participants

The study was based on data from Treatfood (ISRCTN42569496), a randomized 2 × 2 × 3 factorial trial assessing the effects of food supplements among children with MAM [5] in Province du Passoré, Northern Region, Burkina Faso, in 2013 to 2014. Children in the area were screened by community health workers based on mid-upper arm circumference (MUAC) or by screening teams based on both MUAC and weight-for-length z-score (WLZ). Children could also be taken to the site by the caregiver or be referred from a health center. The final assessment for eligibility was carried out by study staff at the sites. Children between 6 and 24 months who were residents in the catchment area were recruited if a diagnosis of MAM was confirmed, i.e., MUAC 115 to <125 mm and/or WLZ −3 to <−2 in the absence of edema. Children were excluded if treated for severe acute malnutrition (SAM; i.e., WLZ <−3 or MUAC <115 mm or edema) or hospitalized within the past 2 months, if already in a nutritional program, or requiring hospitalization, e.g., hemoglobin <50 g/l. Children with a severe disability, limiting the feasibility of investigations, or with suspected allergy to milk, peanuts, corn–soy blend (CSB), or lipid-based nutrient supplement (LNS) were also excluded. In order to reduce mixing or sharing of the supplements, we only included the first child with MAM identified from a household, but provided the same supplement to siblings with MAM, as well as to discordant twins. During the intervention period, children visited the health center every 2 weeks. Serious adverse events were defined as anaphylaxis and death.

Study intervention

The children were randomized to receive a daily supplement for 3 months, with either a LNS or a CSB, and with either dehulled soy (DS) or soy isolate (SI), and either 0%, 20%, or 50% of protein from dried skimmed milk. LNS was provided in sachets of 92 g per daily ration and CSB in 1.7-kg bags per 14-day ration. All supplements consisted of 500 kcal/daily serving (120 g CSB or 92 g LNS). LNS was ready to use, whereas CSB needed to be mixed with water and made into porridge. Supplements were manufactured by GC Rieber Compact A/S (Søfteland, Norway). The nutrient composition of the products complied with the World Health Organization (WHO)’s technical note for management of MAM [8]. The content of water-soluble vitamins was higher in CSB to account for degradation during cooking. The cobalamin content per daily ration from premix was 4.1 μg in CSB and 3.2 μg in LNS. The dried skimmed milk contained additional cobalamin, so that the cobalamin content in products with 20% and 50% of protein from milk was 9.5% and 23.8% higher than products without milk.

As described previously [5,7], we individually randomized children to 1 of 12 combinations of the 3 factors, with variable block size and stratified by site, using www.randomization.com by a person not otherwise involved in the trial. The supplements were designated by a 1-letter code by the manufacturer, which was placed in a 12-letter sequence labeled on each supplement. The code key was kept in a sealed envelope in a safe until study completion. Only one person, otherwise not involved in the study, was aware of the random sequence and code system and relabeled supplements with individual study identifications. Study participants, outcome assessors, and other study staff were blinded with respect to soy quality, milk content, and matrix, while it was not possible to blind study participants with respect to matrix.

Data collection

At baseline, study nurses collected information about sociodemographics, 2-week morbidity, and carried out clinical examination. Children who were not up to date with vaccinations were referred to a health center. Children received albendazole (200 mg if <8 kg body weight; 400 mg if >8 kg body weight) and vitamin A (100,000 IU if 4 to 8 kg body weight; 200,000 IU if >8 kg body weight) if they had not received a supplement in the previous 6 months. Weight was measured to the nearest 100 g using an electronic scale with double weighing function (Seca model 881, Hamburg, Germany). Length was measured to the nearest 1 mm with a wooden length board. WLZ was determined at sites using WHO simplified field tables [9] and later recalculated using the zscore06 package [10] in Stata 14 (Stata, College Station, Texas, US). MUAC was measured on the left arm to the nearest 1 mm using a standard measuring tape. Anthropometric measurements were taken in duplicate by trained staff. Child development was assessed using an adapted version of the Malawi Development Assessment Tool (MDAT) [11]. Adaptation, validation, and pilot testing of the MDAT tool has been reported previously [12]. In brief, we assessed the gross motor, fine motor, and language domains of the MDAT. Items were rated as passed (1 point) or failed (0 points) until the child had failed 6 consecutive items. The assessor would then mark the remaining items as failed and move to the next domain. We calculated MDAT z-scores from a Malawian population of healthy nonmalnourished children [11].

Dietary data were collected using qualitative 24-hour recall. A list of 25 food groups was based on internationally available questionnaires from WHO [13], Food and Agriculture Organization (FAO) [14], as well as research about diets in Ouagadougou carried out by the Institut de Recherche pour le Développement [15] and adapted to the local context. The 25 food groups were then aggregated into 7 food groups as suggested by WHO [13].

Venous blood was collected from the arm at baseline and after 3 and 6 months. One drop was used for diagnosis of malaria (Plasmodium falciparum) using a rapid diagnostic test (SD Bioline Malaria Ag P.f), and 1 drop was used to measure hemoglobin on site using a HemoCue device (Hb 301, Ängelholm, Sweden). Remaining blood was put into a sample tube with clot activator (BD reference #368,492) and transported to the trial laboratory at 2 to 8°C. Serum was isolated following centrifugation at 700 g for 5 minutes (EBA 20S Hettich) and stored at −20°C until shipment and then at −80°C until analysis. Serum C-reactive protein (CRP), α1-acid glycoprotein (AGP), and ferritin were determined at VitMin Lab in Willstaedt, Germany using a combined sandwich enzyme-linked immunosorbent assay [16]. All samples were measured in duplicate and the intra- and interassay coefficients of variation were <10%. Serum ferritin was adjusted for inflammation using regression models (15). The cutoffs used was 12 and 24 μg/L for serum ferritin, 2 and 10 mg/L for serum CRP, and 0.8 and 1.2 g/L for serum AGP.

SC was determined at Rigshospitalet, Denmark, using standard hospital assays on a Cobas 8000, e801 module fulfilling accreditation from the Danish Accreditation Fund. The quantification method was competitive electrochemiluminescence immunoassay, and the measuring range was 112 to 1,480 pmol/L. Coefficient of variation was 6% at both 220 pmol/L (control H1. Immunoassay Plus Control Liquicheck Bio-Rad Kat. 361, Human serum) and 550 pmol/L (control H3. Immunoassay Plus Control Liquicheck Bio-Rad Kat. 363, Human serum). SC was considered low if ≤148 pmol/L and marginal if between 149 and 222 pmol/L, to allow comparison with data from surveys published by Allen and colleagues [4]. The detection limit of 112 pmol/L defined very low SC.

Statistical analyses

As previously reported, the sample size was calculated for the primary outcome of the Treatfood trial [5]. With a sample size of 1,608 (i.e., 134 in each of the 12 combinations resulting from the 2 × 2 × 3 factorial design), we had 80% power to detect a 0.6 SD difference in any pairwise comparison with 5% significance level, while allowing for 20% loss to follow-up. If no interaction, the sample size would allow detection of smaller effects.

Data were double entered into Epidata 3.1 software (Epidata Association, Odense, Denmark) and double entry checks were carried out on a daily basis. All statistical analyses were carried out using Stata 14. SC was not a preplanned outcome of the trial; however, we specified which covariates to assess as potential correlates and confounders before we received the cobalamin data and performed the statistical analyses.

Baseline

Tobit regression was used to assess for potential correlates of baseline SC, while adjusting for age and sex and taking the detection limit of 112 pmol/L into account as left censoring [17]. Linear regression was used to assess the associations between baseline SC and baseline hemoglobin and MDAT z-scores, while adjusting for age, sex, serum ferritin (hemoglobin only), and elevated serum CRP and AGP.

Follow-up

Mean SC at 0, 3, and 6 months and changes from 0 to 3 and 6 months were estimated using mixed-effects tobit regression with normally distributed random effects to describe the variation between children. Adjustments were made for age, sex, month of admission, site, and baseline MUAC, WLZ, length-for-age z-score (LAZ), as well as other correlates of missing cobalamin data. Mixed-effects tobit regression was also used to evaluate the effect of matrix, soy quality, and amount of milk on SC. First, 3-way interactions between the factors were tested for using likelihood ratio tests and, where possible, reduced to 2-way interactions or main effects. Analyses were adjusted for baseline SC as a covariate, and age, sex, MUAC, WLZ, LAZ, site, and elevated serum CRP and morbidity. Model checking was based on residual and normal probability plots. The change in SC during and after supplementation were presented as estimated means with 95% CIs. p-Values <0.05 were considered significant.

Ethical considerations

All children in need received treatment free of charge according to an adapted version of the Integrated Management of Childhood Illnesses guidelines [18,19] and the national protocol. Children who developed SAM during the intervention period were treated with ready-to-use therapeutic food (RUTF; Plumpy’Nut, Nutriset, Malaunay, France). Children who did not recover from MAM subsequently received RUTF or were referred for medical investigation. The study was carried out in accordance with the Declaration of Helsinki. Consent was obtained from caregivers, prior to inclusion, verbally and in writing (signature or fingerprints). Data were kept confidential and in a locked facility. The Ethics Committee for Health Research of the Government of Burkina Faso (2012-8-059) approved the study, and the Danish National Committee on Biomedical Research Ethics (1,208,204) gave consultative approval. The trial was registered as ISRCTN42569496.

Results

Data on SC at baseline were available from 1,192 (74.1%) of 1,609 children in the Treatfood trial conducted from September 2013 to August 2014. SC data were also available from 1,330 (85.9%) of the 1,548 children followed up after 3 months and 398 (26.5%) of the 1,503 children followed up after 6 months (). As reported previously, 4 children died, and no child developed an allergic reaction to supplements [5].

Trial profile.

Of 1,609 children enrolled, cobalamin data were available on 1,192 at baseline. After 12 and 24 weeks, 1,548 and 1,503 children were followed up, and cobalamin data were available on 1,330 and 398, respectively. CSB, corn–soy blend; DS, dehulled soy; LNS, lipid-based nutrient supplement; SI, soy isolate.

Among the 1,192 with baseline SC data, mean (±SD) age was 12.6 (±5.0) months, and 54% were females. Low MUAC (<125 mm) was found in 80.4% (958) of the children and low WLZ (<−2) in 70.6% (841). Stunting was seen in 38.2% (456). Only 5.9% were not breastfed. The 417 children without baseline SC were younger (11.7 versus 12.6 months, p = 0.001), had higher serum CRP (2.8 versus 2.2 mg/L, p = 0.03) and AGP (1.5 versus 1.3 g/L, p < 0.001), and were more likely to be breastfed (96.6 versus 94.0%, p = 0.04), but there were no differences with respect to sex distribution, anthropometry, morbidity, hemoglobin, and measures of child development (). There were no differences between children with and without SC data after 3 months. After 6 months, children with no data on SC were older and had higher serum concentrations of inflammatory markers and hemoglobin (p < 0.05, ). As previously reported, randomization resulted in baseline equivalence between intervention groups [7]. Missing SC was not associated with any of the 3 interventions neither at baseline, 3 months nor 6 months (all p > 0.18, ).

Level and correlates of baseline serum cobalamin

The median (IQR) SC at baseline was 188 (137; 259) pmol/L and ≤222 pmol/L in 63.4%. SC was lower in the youngest children (). After adjustment for age and sex, there were no differences by category of WLZ or MUAC. Children with LAZ <−3, i.e., severe stunting, had 23 (95% CI: −1; 46) pmol/L lower levels, although not significant (p = 0.06). Positive malaria test was associated with 21 (95% CI: 6; 35, p = 0.004) pmol/L lower levels, whereas elevated markers of inflammation were not.

Not currently breastfed was associated with 89 (95% CI: 60; 119, p < 0.001) pmol/L higher SC (). Only 3.8% had dairy, and 6.3% had meat the previous day. Yet, intake of dairy was associated with 38 (95% CI: 3; 74, p = 0.03) pmol/L higher SC, whereas intake of meat or any other food groups was not (p > 0.05). If assessed in the same model, dairy intake was associated with a 30 (95% CI: −5; 66, p = 0.09) pmol/L higher and not currently breastfed with 87 (95% CI: 57; 116, p < 0.001) pmol/L higher SC.

Serum cobalamin and hemoglobin

SC <112 pmol/L was associated with 5.7 (95% CI: 3.0; 8.3, p < 0.001) g/L lower hemoglobin (), and levels 112–148 and 149–222 pmol/L with 1.8 (95% CI: -0.6; 4.1, p = 0.15) and 2.3 (95% CI: 0.3; 4.3, p = 0.02) g/L lower hemoglobin after adjustment for other known correlates.

Serum cobalamin and child development

SC <112 pmol/L was associated with 0.21 (95% CI: 0.01; 0.41, p = 0.04) lower fine motor and 0.24 (95% CI: 0.06; 0.42, p = 0.01) lower gross motor z-score (). Further adjustment for elevated levels of markers of inflammation did not change the associations, although it became not significant for fine motor z-score (−0.19, 95% CI: −0.39; 0.003, p = 0.054). SC between either 112 to 148 or 149 to 222 pmol/L was not associated with fine or gross motor z-scores. SC was not associated with language z-score.

Effects of interventions

During food supplementation, the median (IQR) SC increased from 188 (137; 259) to 255 (188; 344) pmol/L, at which time 38.0% still had values ≤222 pmol/L. After an additional 3 months without supplementation, median SC dropped to 206 (154; 271) pmol/L. The medians were similar among 310 children with SC data at all 3 time points, i.e., 187 (137; 258), 250 (178; 336), and 215 (160; 275) pmol/L at baseline, 3 and 6 months, respectively. This was consistent with findings from a tobit regression model with adjustment for age, sex, site, month of admission, and baseline MUAC, WLZ, LAZ, as well as inflammation, morbidity, and hemoglobin, found to be correlates of missing cobalamin data at 6 months (). Based on this model, the mean (95% CI) SC at baseline, 3 and 6 months were 205 (198; 212), 277 (270; 283), and 231 (220; 241) pmol/L, respectively. The mean (95% CI) increments in SC from baseline to the 3- and 6-month follow-up were 72 (65; 79, p < 0.001) and 26 (16; 37, p < 0.001) pmol/L, respectively (). The means were similar among 310 children with SC determined at all 3 time points (S1 Fig).

SC during and after supplementation.

Mean (95% CI) SC at baseline (n = 1,192), after 3 months supplementation (n = 1,330), and after an additional 3 months without supplementation (n = 398). Based on mixed-effects tobit regression and adjusted for age, sex, months of admission, site and baseline values of MUAC, WLZ, and LAZ, serum CRP, inflammation and morbidity, and baseline SC. Horizontal line at 222 pmol/L indicates cutoff to define normal SC. CRP, C-reactive protein; LAZ, length-for-age z-score; MUAC, mid-upper arm circumference; SC, serum cobalamin; WLZ, weight-for-length z-score.

The effects of food matrix, soy quality, and milk content on SC after 3 months of supplementation are shown in . There was a 3-way interaction between the experimental factors (p = 0.02) explained by an interaction between matrix and soy quality (p < 0.001), due to a larger increase in SC in those given LNS (versus CSB) if SI was used. The main effect of LNS (versus CSB) of 16 (95% CI: 3; 29, p = 0.02) pmol/L was larger with SI (39, 95% CI: 20; 57, p < 0.001) than with DS (−8, 95% CI: −26; 11, p = 0.42). There were no effects of milk content.

Discussion

SC was low in children with MAM in Burkina Faso, as two-thirds had marginal or low levels. This is expected in low-income populations with a low intake of animal source and fortified foods and a high burden of gastrointestinal infections. Children will be born with low body stores of cobalamin, receive breastmilk with low content, and subsequently be given complementary foods with little if any animal source or cobalamin-fortified foods. A recent review summarized prevalence data from nationally representative studies and large nonrepresentative studies [4]. Of 6 studies among children in Asia and Africa, the prevalence of low and marginal cobalamin combined, i.e., below 222 pmol/L, ranged from to 33% to 80%.

Our study was conducted among children with MAM, diagnosed by low MUAC and/or low WLZ. Within these ranges of MUAC and WLZ, there were no association with SC but severe stunting was associated with lower SC. Although we are unable to make causal inferences based on cross-sectional data, this association could be due to a diet with little animal source foods being a cause of both cobalamin deficiency as well as impaired linear growth. Animal source foods are the only sources of natural cobalamin, but also an excellent source of growth nutrients, such as zinc, phosphorus, and amino acids, which are essential to synthesis of lean body mass and to linear growth [1]. Although cobalamin is essential to purine and pyrimidine metabolism and hence to DNA synthesis, it is not considered a growth nutrient [20]. However, a trial among young children in India found that cobalamin supplementation resulted in a 0.07 increase in weight-for-age z-score [21]. While this was due to an increase in ponderal, but not linear growth, cobalamin supplementation increased linear growth in subgroups of children who were either stunted or wasted. It is also possible that the association between impaired linear growth and cobalamin in our study could be explained by environmental enteric dysfunction [2]. This multifactorial condition is characterized by increased permeability of the gut which leads to systemic inflammation and down-regulation of insulin-like growth factor 1 (IGF-1), an important growth factor. It is also accompanied by impaired absorptive capacity, which would lead to deficiencies of growth nutrients, hence contributing to impaired growth. It would also lead to cobalamin deficiency, which may impair regeneration of enterocytes and thereby further contribute to impaired absorptive capacity.

In contrast to other markers of micronutrient status, SC was not associated with elevated serum levels of the acute phase reactants CRP and AGP. While this is in accordance with findings from the US National Health and Nutrition Examination Survey (NHANES) study among adults [22], as well as nationally representative surveys among women and children [23], prospective studies are needed to confirm if indeed the validity of SC is unaffected by inflammation. Yet, a positive malaria antigen test was associated with reduced SC. If this reflects that malaria infection impairs cobalamin status through a mechanism independent of the inflammatory response, then it might be through uptake of cobalamin from the blood by the plasmodium parasite, which also has cobalamin-dependent methionine synthase [24]. Interestingly, it has been reported that the fish tapeworm (Diphyllobothrium latum) may cause megaloblastic anemia by digesting cobalamin in the human small intestine [25,26].

Only around 5% of the children had been fed dairy or meat the previous day, which means that the majority of children get dairy or meat less then weekly, if ever. While intake of animal source foods is likely to be low among the general population of children in the area, it is most probably lower in these children with MAM and may have contributed to its development. Intake of dairy, but not meat, was associated with higher cobalamin. We neither had data on type of dairy, i.e., local or commercial cow’s milk, cheese, etc., nor on amount or frequency of intake. A study among 2-year-old Norwegian children assessed the correlation between dietary intake and SC [27]. Despite better dietary data, based on 7-day food records, and much larger intakes of animal source foods, only intake of dairy was correlated with SC.

In our study, children who were not breastfed had higher SC compared to those being breastfed, even after adjustment for age and sex. From a large study among 6 to 30 months old north Indian children, a similar difference in SC between breastfed and nonbreastfed children (183 versus 334 pmol/L) was reported. Dietary data were not available, but intake of animal source or fortified foods was considered low [28]. In our study, further adjustment for intake of animal source foods did not change the association. This may well be due to residual confounding, since we only had crude data on intake from food groups.

Despite limitations of a cross-sectional study, the considerably lower hemoglobin in children with marginal or low SC probably reflects that cobalamin deficiency impairs erythropoiesis. Cobalamin and folate play important roles in pyrimidine and purine metabolism and are therefore essential to DNA synthesis and cell division [3]. Since the turnover of blood cells is rapid, these cells are affected early in the development of cobalamin deficiency. In contrast to folate, which is found in a large range of foods, including green leafy vegetables and legumes, cobalamin is only available from animal source or fortified foods. Since the intake of animal source foods is low in most low-income settings, it is likely that cobalamin deficiency is among the most widespread deficiencies and may contribute considerably to anemia.

Suboptimal SC was associated with lower fine and gross motor domain z-scores, but not with language domain z-scores. Deficits were only seen with very low SC (<112 pmol/L). This is in contrast to hemoglobin, for which lower levels were seen even in children with marginal SC. A prospective study among 12- to 18-month-old children in India showed that plasma cobalamin was not associated with mental development index score at baseline, but positively associated with mental development index score after 4 months [29]. A later factorial trial found that cobalamin supplements increased gross motor development, whereas cobalamin and folic acid combined also increased problem-solving [30].

The lack of effects of milk, the only animal source ingredient in the foods, might be due to its modest contribution to cobalamin intake compared to the premix. Addition of dried skimmed milk to achieve 20% and 50% of protein from milk only resulted in 9.5% and 23.8% more cobalamin compared to the 4.1-μg cobalamin in CSB products and 3.2 μg in the LNS products without milk, which only contained cobalamin from premix. Interestingly, although the daily ration of CSB contained more cobalamin, those given LNS (versus CSB) had a greater increase in SC, but only if the LNS contained SI. A higher increase in those given LNS could be explained by a greater degradation during preparation of porridge from CSB than accounted for by increasing the content. It could also be due to a relatively greater consumption of the LNS compared to CSB rations [31,32]. Finally, it could be due to a greater bioavailability of cobalamin from the LNS matrix. The latter explanation is more likely given the interaction with soy quality. We have previously reported the same interaction between matrix and soy quality in relation to weight gain from this trial, i.e., children receiving LNS had a greater weight gain than those receiving CSB, but only if the products contained SI [5].

The overall increase in SC during supplementation was insufficient as more than one-third of the children were left with marginal or low SC. Furthermore, during the subsequent 3 months, there was a considerable decline reflecting low intake and continued diversion of cobalamin to target tissues. Our data show that the best supplement with respect to repletion of cobalamin status is LNS with SI. However, the content of cobalamin in LNS should be reconsidered, and cobalamin bioavailability in children with MAM should be studied. Cobalamin is primarily absorbed through a specific, saturable system involving binding to an intrinsic factor (IF) released in the stomach and uptake through IF receptors in the ileum [3]. An additional 1% to 2% of cobalamin in the diet can be absorbed through the unspecific system, and this is particularly important if IF-mediated absorption is impaired or saturated [3]. Both specific and unspecific absorption is likely to be affected, but does not seem to have been studied. Low cobalamin status and inadequate repletion during treatment are likely bigger problems in children with SAM, known to have reduced gastric acidity accompanied by bacterial overgrowth [33] and reduced enterocyte mass [34].

Strengths and limitations

The main strength is the large sample size, giving precision and power, and data on dietary intake, hemoglobin and development, as well as the randomized trial allowing us to assess effects of the experimental factors on SC. The findings are likely to be generalizable to children with MAM in low-income settings in sub-Saharan Africa, with limited access to animal source and fortified foods.

The main limitation is missing data on SC, mainly due to lack of serum. For the longitudinal data, missing data were associated with age, morbidity, inflammation, and hemoglobin, which may indicate that data were missing at random (i.e., missing data might be predicted by observed data). Consequently, analyses using mixed-effects models will still provide unbiased results. The observation is further corroborated by results from analyses that included adjustment for correlates of missingness and, also, complete case analyses only including children with data at all 3 time points; all these analyses provided very similar findings. For the cross-sectional analyses, missing values were also associated with age and inflammation, and, therefore, only results from analyses adjusted for correlates of missing were reported.

Another limitation is the cross-sectional design used to assess associations between SC and hemoglobin and child development. However, it is well established that adequate cobalamin intake and status is critically important for hemopoiesis and child development. The aim of assessing these associations was therefore merely to corroborate SC cutoffs, based on these known cobalamin-dependent outcomes, and to assess to what extent low cobalamin in the population is associated with functional deficits. While the associations we find between low SC and both low hemoglobin and low child development scores are likely to reflect the well-known cause–effect relationship, it cannot be inferred from the cross-sectional design.

The lack of data on serum methylmalonic acid, the only specific marker of cobalamin deficiency, is not necessarily a limitation with respect to the analysis on hemoglobin, since the aim was to assess associations between categories of SC and known cobalamin-dependent outcomes. It is likely that the role of cobalamin for these outcomes is through the essentiality of methylcobalamin for de novo synthesis of purine and pyrimidine, rather than through the essentiality of deoxyadenosylcobalamin as a cofactor for methylmalonyl-CoA mutase. If, furthermore, erythropoiesis and early child development rank higher in the hierarchy of biological functions requiring cobalamin than the functions dependent on methylmalonyl-CoA mutase, then impaired erythropoiesis and child development may develop before any increase in serum methylmalonic acid.

Conclusions

Children with MAM have low SC, which may contribute to impaired erythropoiesis and child development. Food products used for treatment fail to replete cobalamin status. The bioavailability and adequacy of cobalamin in food supplements for children with acute malnutrition should be reconsidered.

Mean (95% CI) SC at baseline, after 3 months supplementation, and after an additional 3 months without supplementation among 398 children with SC data at all time points. Based on tobit regression and adjusted for age, sex, months of admission, site and MUAC, WLZ and LAZ, inflammation and morbidity. Horizontal line at 222 pmol/L indicates cutoff to define normal SC. LAZ, length-for-age z-score; MUAC, mid-upper arm circumference; SC, serum cobalamin; WLZ, weight-for-length z-score.

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STROBE checklist.

STROBE, Strengthening the Reporting of Observational Studies in Epidemiology.

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CONSORT checklist.

CONSORT, Consolidated Standards of Reporting Trials.

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9 Jul 2021

Dear Dr Friis,

Thank you for submitting your manuscript entitled "Serum cobalamin in children with moderate acute malnutrition: findings from a randomized trial in Burkina Faso" for consideration by PLOS Medicine.

Your manuscript has now been evaluated by the PLOS Medicine editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

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Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Feel free to email us at plosmedicine@plos.org if you have any queries relating to your submission.

Kind regards,

Beryne Odeny

Associate Editor

PLOS Medicine

8 Oct 2021

Dear Dr. Friis,

Thank you very much for submitting your manuscript "Serum cobalamin in children with moderate acute malnutrition: findings from a randomized trial in Burkina Faso" (PMEDICINE-D-21-02962R1) for consideration at PLOS Medicine.

Your paper was evaluated by a senior editor and discussed among all the editors here. It was also discussed with an academic editor with relevant expertise, and sent to independent reviewers, including a statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

In light of these reviews, I am afraid that we will not be able to accept the manuscript for publication in the journal in its current form, but we would like to consider a revised version that addresses the reviewers' and editors' comments. Obviously we cannot make any decision about publication until we have seen the revised manuscript and your response, and we plan to seek re-review by one or more of the reviewers.

In revising the manuscript for further consideration, your revisions should address the specific points made by each reviewer and the editors. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

In addition, we request that you upload any figures associated with your paper as individual TIF or EPS files with 300dpi resolution at resubmission; please read our figure guidelines for more information on our requirements: http://journals.plos.org/plosmedicine/s/figures. While revising your submission, please upload your figure files to the PACE digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at PLOSMedicine@plos.org.

We expect to receive your revised manuscript by Oct 29 2021 11:59PM. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

We ask every co-author listed on the manuscript to fill in a contributing author statement, making sure to declare all competing interests. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. If new competing interests are declared later in the revision process, this may also hold up the submission. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT. You can see our competing interests policy here: http://journals.plos.org/plosmedicine/s/competing-interests.

Please use the following link to submit the revised manuscript:

https://www.editorialmanager.com/pmedicine/

Your article can be found in the "Submissions Needing Revision" folder.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

We look forward to receiving your revised manuscript.

Sincerely,

Beryne Odeny,

PLOS Medicine

plosmedicine.org

-----------------------------------------------------------

Requests from the editors:

1) Please revise your title according to PLOS Medicine's style. Your title must be nondeclarative and not a question. It should begin with main concept if possible. For example, please place the study design in the subtitle (i.e., after a colon). For example, “Serum cobalamin in children with moderate acute malnutrition in Burkina Faso: A cross-sectional study of findings from a randomized trial”

2) Please delete the “RESEARCH IN CONTEXT” section – this is not required by PLOS Medicine

3) At this stage, we ask that you write a non-technical Author Summary. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract. The summary should be accessible to a wide audience that includes both scientists and non-scientists. Please see our author guidelines for more information: https://journals.plos.org/plosmedicine/s/revising-your-manuscript#loc-author-summary.

4) Abstract:

a) Please report your abstract according to CONSORT for abstracts: http://www.consort-statement.org/extensions?ContentWidgetId=562 . Structure your abstract using the PLOS Medicine headings (Background, Methods and Findings, Conclusions).

b) Please combine the Methods and Findings sections into one section, “Methods and findings”. Replace “Interpretation” with “Conclusions”

c) Please ensure that all numbers presented in the abstract are present and identical to numbers presented in the main manuscript text.

d) Please quantify all results (with p values in addition to 95% CI).

e) Please include a summary of adverse events if these were assessed in the study.

f) In the last sentence of the Abstract Methods and Findings section, please describe the main limitation(s) of the study's methodology.

5) Did your study have a prospective protocol or analysis plan? Please state this (either way) early in the Methods section:

a) If a prospective analysis plan (from your funding proposal, IRB or other ethics committee submission, study protocol, or other planning document written before analyzing the data) was used in designing the study, please include the relevant prospectively written document with your revised manuscript as a Supporting Information file to be published alongside your study, and cite it in the Methods section. A legend for this file should be included at the end of your manuscript.

b) If no such document exists, please make sure that the Methods section transparently describes when analyses were planned, and when/why any data-driven changes to analyses took place.

c) In either case, changes in the analysis-- including those made in response to peer review comments-- should be identified as such in the Methods section of the paper, with rationale.

6) Please ensure that the study is reported according to the STROBE guideline for observational studies, and include the completed STROBE checklist as Supporting Information. Please add the following statement, or similar, to the Methods: "This study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline (S1 Checklist)." The STROBE guideline can be found here: http://www.equator-network.org/reporting-guidelines/strobe/

7) Your study is observational and therefore causality cannot be inferred. Please remove language that implies causality throughout the manuscript, such as “effect/s.” Refer to associations instead.

8) In the Methods and Results section, please consistently provide 95% CIs and p values for estimates in the main text and tables.

9) In table 5 and 6, please provide the meaning of the bolded numbers in the footnotes.

10) Please provide the meaning of bars and whiskers in your figures.

11) Please replace the terms “girls” with “females,” and “boys” with “males”

12) Please replace the term “marginally significant” with “not significant”

13) Please remove the ‘Funding’, from the end of the main text. In the event of publication, this information will be published as metadata based on your responses to the submission form.

14) Please use the "PLOS Medicine" citation style for reference formatting and see our website for other reference guidelines https://journals.plos.org/plosmedicine/s/submission-guidelines#loc-references.

a) Please ensure that only six names precede et al.

b) Remove the “Public Library of Science” from ref #7

Comments from the reviewers:

Reviewer #1: This is an important report of secondary data analysis of an RCT with results published in 2017 in this journal. There are other analyses from this same data (well cited in this manuscript) however the authors do not tell us relevant findings from those in the background. Here the authors look at the i) level and the determinants/correlates of cobalamin serum levels (and haemoglobin and child development) at the baseline of trial, and ii) the effects of food supplementation (matrix, soy quality and milk content) on serum cobalamin during the follow-up. The analysis is not conducted as in an RCT. Interaction coefficients were decided upon a p-value calculation. It is a well-written report however there are a few issues to be addressed before publication.

Major issue:

- I have to commend the authors to conduct a Tobit regression for the multivariable analysis of the serum cobalamin to address the fact that measurement is left censored. However, some of the analysis was conducted with the replacement of these censored values with 112/√2. This is an arbitrary choice. Did you conduct sensitivity analysis with another value? Why not use Tobit when comparing this serum cobalamin in all analyses rather than the t-tests or ANOVA?

Minor issues:

- Line 145 better to write weeks rather than wk

- Line 151 please cite the zscore06 package and tell us the version of these WHO reference tables

- Line 151 cite properly Stata 12; and line 189 Stata 14.

- Line 188 cite properly the Epidata 3.1 software

- Line 158 please add a justification for using Malawian MDAT z-scores for Burkina Faso children

- Line 199 please write "linear mixed-effects models" rather than "xtmixed models". Xtmixed is a Stata command therefore only a few Stata users would recognise it.

- Line 233 I think it would be better if it is written "level and correlates of baseline serum cobalamin"

- There is an important amount of missing data on cobalamin. For the longitudinal data analysis, the linear mixed-effects analysis implicitly deals with missing data under Missing at Random assumption (I think that is fine). At the baseline, the randomization may have offered the MCAR (missing completely at - random) so it is correct to ignore the missed measurements (although table 1 offers some concerning p-values at baseline). I think it would be good to discuss the data missing in strengths/limitations.

Reviewer #2:

Thank you very much for asking me to look at this work. In general, this is a very well written manuscript and follows on several papers that have been written from the clinical trial

There are a number of minor issues worthy of comment here:

1. The paper combines two study designs: a cross sectional study and a randomized controlled trial. Clearly a cross sectional study has its own limitations since the exposure and outcome are being are studied at the same time.

2. The second issue is that of the sample size for the study. This section is missing in this manuscript. The basis for the sample size including the effect size, power and precision is missing.

3. While the objectives were clearly stated, the RCT should have had a clear hypothesis regarding the intervention and cobalamin etc.

4. The process of randomization was not adequately described in the manuscript. This includes the process itself, allocation concealment etc. It is possible that the process was described in more detail elsewhere in the papers preceding this one, so the authors could describe it to a certain extent and refer to the detailed description in the previous papers from the same work.

5. Why were cobalamin results available for only 1192/1609 children? Despite this comment, the authors have clearly compared those with the data and those without. This is commendable.

6. The issue of other infections other than bacteria were not considered in detail. Is it possible that other infections such as Ascaris lumbricoides, hookworm and HIV affected the baseline levels? We note that the children were give albendazole at the beginning of the study. Be that as it may, it would appear that this is an area this is an area needing further study.

7. There is need for study profile

8. The authors have published part of this work [1] but without looking at cobalamin. In that paper the authors assessed gross motor, fine motor, language and cognitive skills. It is not clear why the current paper has not looked at cognitive function and cobalamin deficiency.

References

1. Olsen MF, Iuel-Brockdorff AS, Yaméogo CW, Cichon B, Fabiansen C, et al. (2020) Impact of food supplements on early child development in children with moderate acute malnutrition: A randomised 2 x 2 x 3 factorial trial in Burkina Faso. PLOS Medicine 17(12): e1003442. https://doi.org/10.1371/journal.pmed.1003442

Reviewer #3: General

The objective of this study was to describe the prevalence of low vitamin B12 status (serum cobalamin, SC) among Burkinabé children 6-23 mo of age with moderate acute malnutrition (MAM) participating in a food supplementation trial, and examine the impact of various food supplements on SC. As noted by the authors, there is a general lack of information on B12 status among children in Africa, and particularly among children with MAM. This paper thus adds new information on vitamin B12 deficiency and the effects of food supplements on micronutrient status in this population. In general the paper is well written and methods are appropriate.

There was a relatively high proportion of missing baseline SC values (26%). The authors' analysis of characteristics of children with available vs missing samples provides insight on the possible direction of bias. However, I do not see a comparison of whether/how missing samples differed by intervention group. This would be helpful to report as differential missing values by group could potentially influence the conclusions on the intervention effects.

The abstract concludes that "The SC increase during supplementation was inadequate. The bioavailability and adequacy of cobalamin in food supplements should be reconsidered." And in Lines 361-364: "The overall increase in serum cobalamin during supplementation was insufficient as more than one-third of the children were left with marginal or low serum cobalamin. Furthermore, during the subsequent 3 months there was a considerable decline. This suggests that the bioavailability of cobalamin is compromised in children with MAM."

The observation that a large proportion still had low SC after 3 months of treatment is important and understanding the role of bioavailability would be useful. I wonder if another explanation (just speculation) might be that B12 is diverted quickly to functional sites (including bone marrow) during repletion so that less remains in circulation compared to a person with adequate B12 status.

However, I disagree that the interpretation of the decrease after 3 months of no supplementation signals a bioavailability problem. This decrease is not surprising as SC concentrations are responsive to recent intake and would be expected to decline if children discontinue supplemental food are returning to a diet that is very low in animal-sourced foods or other sources of B12.

Minor

Lines 126-128: Any relevant exclusion criteria?

Lines 140-141: "The cobalamin content from premix was 4.1 μg in CSB and 3.2 μg in LNS." Is this the content per recommended serving?

The detection limit of 112 pmol/L seems somewhat high compared with the cutoff for deficiency <149 pmol/L.

Methods: What year(s) was the study carried out vs what year was SC measured? That is, how long were samples stored, and under what conditions, prior to analysis? This would be especially useful since the outcome is not specifically listed in the trial registration.

Lines 194-196 and 199-200 and 206-207: How was multicollinearity assessed in the models (especially if multiple anthropometric measures were used in the same model?)

Minor note: Line 200 refers to WHZ while line 206 refers to WLZ.

Lines 303-305: For associations between B12 and inflammation, please see this recent paper by the BRINDA group (Young et al, AJCN 2020). This study also found no association in multiple cross-sectional surveys.

Lines 353-355: What information is available on the stability of B12 in LNS or CSB? Are there data to suggest that stability is lower in CSB?

Reviewer #4: See attachment

Michael Dewey

Any attachments provided with reviews can be seen via the following link:

[LINK]

Submitted filename: friis.pdf

Click here for additional data file.

22 Dec 2021

Submitted filename: Friis-Cobalamin-Response-letter-22Dec21-final.docx

Click here for additional data file.

26 Jan 2022

Dear Dr. Friis,

Thank you very much for re-submitting your manuscript "Serum cobalamin in children with moderate acute malnutrition: findings from a randomized trial in Burkina Faso" (PMEDICINE-D-21-02962R2) for review by PLOS Medicine.

I have discussed the paper with my colleagues and the academic editor and it was also seen again by two reviewers. I am pleased to say that provided the remaining editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.

We expect to receive your revised manuscript within 1 week. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

We ask every co-author listed on the manuscript to fill in a contributing author statement. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

Please note, when your manuscript is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you've already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosmedicine@plos.org.

If you have any questions in the meantime, please contact me or the journal staff on plosmedicine@plos.org.

We look forward to receiving the revised manuscript by Feb 02 2022 11:59PM.

Sincerely,

Beryne Odeny,

PLOS Medicine

plosmedicine.org

------------------------------------------------------------

Requests from Editors:

1. Please ensure that the updated title is submitted in the next version

2. Abstract: minor edit at line #77 “The limitation … was”

3. Author summary: please restructure this section such that there are 2 to 4 bullet points per subheading. Each bullet point should not exceed 4 sentences

4. Please remove the “Role of funding source” from the methods section as this will be published based on your responses in the submission form

5. Conclusion: minor edit at line #494, should read “… products used for treatment fail …”

6. References – please provide weblinks and access dates for ref #8, 13, 14, 18, & 19

7. Please attach your STROBE checklists and any related flow diagrams.

Comments from Reviewers:

Reviewer #2: I reviewed verison R1 of this work. I have now looked at the revised manuscript and like to confirm that the comments have been, largely, addressed. In particular they have addressed the following issues: the study design, sample size calculation, randomization, missing cobalamin results, other infections, study profile, etc.

Therefore I am be happy to recommend publication

Reviewer #4: The authors have addressed all my points.

Michael Dewey

Any attachments provided with reviews can be seen via the following link:

[LINK]

27 Jan 2022

Submitted filename: Friis-Cobalamin-Response-letter-22Dec21-final.docx

Click here for additional data file.

3 Feb 2022

Dear Dr. Friis,

Thank you very much for re-submitting your manuscript "Serum cobalamin in children with moderate acute malnutrition in Burkina Faso: secondary analysis of a randomized trial" (PMEDICINE-D-21-02962R3) for review by PLOS Medicine.

I am pleased to say that provided the remaining editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.

We expect to receive your revised manuscript within 1 week. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

We ask every co-author listed on the manuscript to fill in a contributing author statement. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

Please note, when your manuscript is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you've already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosmedicine@plos.org.

If you have any questions in the meantime, please contact me or the journal staff on plosmedicine@plos.org.

We look forward to receiving the revised manuscript by Feb 10 2022 11:59PM.

Sincerely,

Beryne Odeny,

PLOS Medicine

plosmedicine.org

------------------------------------------------------------

Requests from Editors:

1) Thank you for providing your STROBE checklist. Please replace the page numbers with paragraph numbers per section (e.g. "Methods, paragraph 1"), since the page numbers of the final published paper may be different from the page numbers in the current manuscript.

2) The manuscript you have uploaded is not current as there are no observable edits. Please double check the version you uploaded and ensure you address prior editorial requests (no.2-6) as follows:

a. Main abstract: minor grammatical edit at line #77. It should read “The main limitation of this study was…”

b. Author summary: please restructure this section such that there are 2 to 4 bullet points per subheading.

c. Please remove the “Role of funding source” from the methods section as this will be published based on your responses in the submission form

d. Conclusion: minor grammatical edit at line #494. It should read “… products used for treatment fail …”

e. References – please provide weblinks and access dates for ref #8, 13, 14, 18, & 19

Any attachments provided with reviews can be seen via the following link:

[LINK]

4 Feb 2022

Submitted filename: Friis-Cobalamin-Response-letter-22Dec21-final.docx

Click here for additional data file.

4 Feb 2022

Dear Dr. Friis,

Thank you very much for re-submitting your manuscript "Serum cobalamin in children with moderate acute malnutrition in Burkina Faso: secondary analysis of a randomized trial" (PMEDICINE-D-21-02962R4) for review by PLOS Medicine.

There is one more editorial remaining issue that needs to be addressed and it is listed at the end of this email. Please take this into account before resubmitting your manuscript:

[LINK]

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.

We expect to receive your revised manuscript within 1 week. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

We ask every co-author listed on the manuscript to fill in a contributing author statement. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

Please note, when your manuscript is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you've already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosmedicine@plos.org.

If you have any questions in the meantime, please contact me or the journal staff on plosmedicine@plos.org.

We look forward to receiving the revised manuscript by Feb 11 2022 11:59PM.

Sincerely,

Beryne Odeny,

PLOS Medicine

plosmedicine.org

------------------------------------------------------------

Requests from Editors:

1. Both STROBE and CONSORT checklists need revision. Please replace the page numbers with paragraph numbers per section (e.g. "Methods, paragraph 1"), since the page numbers of the final published paper may be different from the page numbers in the current manuscript

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8 Feb 2022

Submitted filename: Friis-Cobalamin-Response-letter-22Dec21-final.docx

Click here for additional data file.

11 Feb 2022

Dear Dr Friis,

On behalf of my colleagues and the Academic Editor, Prof. James K Tumwine, I am pleased to inform you that we have agreed to publish your manuscript "Serum cobalamin in children with moderate acute malnutrition in Burkina Faso: secondary analysis of a randomized trial" (PMEDICINE-D-21-02962R5) in PLOS Medicine.

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Table 1

Baseline characteristics of 1,609 children with MAM with or without cobalamin data at baseline and 3- and 6-month follow-up.

	Baseline			3-month follow-up			6-month follow-up
	Data n = 1,192	No data n = 417	p-Value	Data n = 1,330	No data n = 279	p-Value	Data n = 398	No data n = 1,211	p-Value
Sociodemographic characteristics
Age, months	12.6 (±5.0)	11.7 (±4.4)	0.001	12.4 (±4.9)	12.0 (±4.6)	0.18	11.8 (±4.7)	12.5 (±4.9)	0.006
Sex, female	54.1% (645)	56.3% (234)	0.48	53.8 (716)	58.4 (163)	0.16	51.8% (206)	55.6% (673)	0.19
Breastfed	94.0% (1,119)	96.6% (402)	0.04	94.4% (1,254)	96.0% (267)	0.26	94.5% (376)	94.7% (1,145)	0.86
Anthropometry
MUAC, mm	122.7 (±4.0)	122.5 (±3.9)	0.34	122.6 (±3.9)	122.5 (±4.2)	0.54	123.0 (±4.1)	122.5 (±3.9)	0.051
WLZ	−2.2 (±0.5)	−2.2 (±0.5)	0.16	−2.2 (±0.5)	−2.3 (±0.5)	0.20	−2.2 (±0.5)	−2.2 (±0.5)	0.21
LAZ	−1.7 (±1.1)	−1.7 (±1.1)	0.88	−1.7 (±1.1)	−1.7 (±1.2)	0.77	−1.7 (±1.2)	−1.7 (±1.1)	0.42
Morbidity
Ill last 2 weeks	37.6% (445)	39.2% (163)	0.56	38.8% (513)	34.2% (95)	0.15	46.9% (186)	35.1% (422)	<0.001
Serum CRP, mg/L	2.2 (0.7; 9.2)	2.8 (1.0; 10.5)	0.03	2.4 (0.8; 9.8)	2.2 (0.7; 8.5)	0.41	3.3 (0.8; 11.5)	2.1 (0.7; 8.6)	0.01
Serum AGP, g/L	1.3 (±0.6)	1.5 (±0.7)	<0.001	1.4 (±0.7)	1.3 (±0.7)	0.66	1.4 (±0.7)	1.3 (±0.6)	0.003
Hemoglobin, g/L	100 (±16)	100 (±17)	0.69	100 (±16)	101 (±16)	0.13	97 (±15)	101 (±16)	<0.001

Data shown as mean (±SD), median [IQR], or n (%).

AGP, α1-acid glycoprotein; CRP, C-reactive protein; LAZ, length-for-age z-score; MAM, moderate acute malnutrition; MUAC, mid-upper arm circumference; WLZ, weight-for-length z-score.

Table 2

Growth, inflammation, and morbidity as correlates of baseline SC (pmol/L) in 1,192 children with MAM.

		Unadjusted		Age–sex adjusted
	n	B (95% CI)	p	B (95% CI)	p
Sex
Female	645	-
Male	547	−8 (−22; 6)	0.27	−11 (−25; 2)	0.10
Age (month)
6 to 11	629	-		-
12 to 18	339	26 (11; 42)	0.001	28 (12; 44)	0.001
18 to 24	224	52 (34; 70)	<0.001	53 (35; 71)	<0.001
Anthropometry
MUAC (mm)
>125	234	-
≤125	958	3 (−15; 20)	0.75	4 (−14; 22)	0.65
WLZ
>−2	351	-
≤−2	841	8 (−7; 23)	0.29	5 (−11; 20)	0.55
LAZ
>−2	736	-
>−3 and ≤−2	334	10 (−6; 25)	0.23	1 (−14; 17)	0.85
≤−3	122	−10 (−33; 13)	0.40	−23 (−46; 1)	0.06
Inflammation and morbidity
Serum CRP (mg/L)
≤2	569	-
>2 and ≤10	341	6 (−10; 22)	0.46	2 (−14; 18)	0.78
>10	282	−6 (−24; 11)	0.47	−9 (−26; 8)	0.31
Serum AGP (g/l)
≤0.8	241	-		-
>0.8 and ≤1.2	366	11 (−8; 31)	0.26	7 (−12; 27)	0.45
>1.2	585	15 (−4; 33)	0.11	7 (−11; 25)	0.46
Ill last 2 weeks
No	739	-		-
Yes	445	−6 (−20; 9)	0.43	−8 (−22; 6)	0.25
Malaria (Rapid test)
Negative	726	-		-
Positive	463	−16 (−30; −2)	0.03	−21 (−35; −6)	0.004

1Data shown as number (n), regression coefficient B (95% CI), and p-value. Numbers in categories may not add up, due to missing values.

AGP, α1-acid glycoprotein; CRP, C-reactive protein; LAZ, length-for-age z-score; MAM, moderate acute malnutrition; MUAC, mid-upper arm circumference; SC, serum cobalamin; WLZ, weight-for-length z-score.

Table 3

Breastfeeding and food groups as correlates of baseline SC (pmol/L) in 1,192 children with MAM.

		Unadjusted		Age–sex adjusted
	n	B (95% CI)	p	B (95% CI)	p
Breastfeeding and food intake 2
Breastfed currently
Yes	1,119	-		-
No	72	107 (79; 135)	<0.001	89 (60; 119)	<0.001
Grains, roots, and tubers
No	178	-		-
Yes	1,009	24 (4; 43)	0.02	9 (−11; 29)	0.39
Legumes and nuts
No	1,014	-		-
Yes	178	14 (−4; 34)	0.13	6 (−14; 25)	0.57
Vitamin A–rich fruits and vegetables
No	857	-		-
Yes	334	8 (−7; 24)	0.29	1 (−15; 16)	0.92
Other fruits and vegetables
No	529	-		-
Yes	663	13 (−1; 27)	0.06	1 (−14; 15)	0.94
Dairy
No	1,147	-		-
Yes	45	40 (3; 76)	0.03	38 (3; 74)	0.03
Meat
No	1,117	-		-
Yes	75	15 (−14; 43)	0.32	9 (−20; 37)	0.60

1Data shown as number (n), regression coefficient B (95% CI), and p-value. Numbers in categories may not add up, due to missing values.

2Dietary data based on 24-hour recall.

MAM, moderate acute malnutrition; SC, serum cobalamin.

Table 4

SC and other correlates of baseline hemoglobin (g/L) in 1,192 children with MAM.

		Age–sex adjusted		Multivariable model
	n	B (95% CI)	p	B (95% CI)	p
Sex
Female	645	-
Male	547	−3.9 (−5.7; −2.2)	<0.001	−3.4 (−5.0; −1.7)	<0.001
Age (month)
6 to 12	629	-
12 to 18	339	−3.8 (−5.8; −1.7)	<0.001	−2.6 (−4.6; −0.6)	0.01
18 to 24	224	−4.4 (−6.7; −2.0)	<0.001	−3.3 (−5.6; −1.1)	0.004
Inflammation
Serum CRP (mg/L)
≤2	569	-		-
>2 and ≤10	341	−6.6 (−8.6; −4.6)	<0.001	−4.6 (−6.7; −2.4)	<0.001
>10	282	−10.6 (−12.7; −8.6)	<0.001	−7.4 (−10.0; −4.9)	<0.001
Serum AGP (g/L)
≤0.8	241	-		-
>0.8 and ≤1.2	366	−4.0 (−6.4; −1.7)	0.001	−2.9 (−5.4; −0.5)	0.02
>1.2	585	−10.2 (−12.5; −8.0)	<0.001	−6.2 (−8.8; −3.5)	<0.001
Micronutrient status
Serum ferritin (adj, μg/L)
≤12	462	−3.7 (−5.8; −1.6)	<0.001	−4.3 (−6.3; −2.3)	<0.001
>12 and ≤24	341	−0.3 (−2.5; 2.0)	0.81	0.1 (−2.0; 2.2)	0.95
>24	384	-
SC (pmol/L)
<112	159	−5.5 (−8.3; −2.8)	<0.001	−5.7 (−8.3; −3.0)	<0.001
≥112 and ≤148	212	−1.7 (−4.2; 0.8)	0.18	−1.8 (−4.1; 0.6)	0.15
>148 and ≤222	387	−2.4 (−4.5; −0.3)	0.03	−2.3 (−4.3; −0.3)	0.02
>222	437	-		-

1Data shown as numbers (n), regression coefficients B (95% CI), and p-value. Numbers in categories may not add up, due to missing values. The multivariable model contains all variables shown.

AGP, α1-acid glycoprotein; CRP, C-reactive protein; MAM, moderate acute malnutrition; SC, serum cobalamin.

Table 5

SC as correlate of MDAT z-scores in 1,192 children with MAM.

		Language domain		Fine motor domain		Gross motor domain
		Age–sex	Multivariable	Age–sex	Multivariable	Age–sex	Multivariable
	n	B (95% CI)	B (95% CI)	B (95% CI)	B (95% CI)	B (95% CI)	B (95% CI)
SC (pmol/L)
<112	159	−0.07 (−0.26; 0.12) p = 0.47	−0.05 (−0.23; 0.13) p = 58	−0.21 (−0.41; −0.01) p = 0.04	−0.19 (−0.39; 0.003) p = 0.054	−0.24 (−0.42; −0.06) p = 0.01	−0.23 (−0.47; −0.05) p = 0.01
≥112 and ≤148	212	0.003 (−0.17; 0.17) p = 0.98	0.02 (−0.15; 0.19) p = 83	0.07 (−0.11; 0.25) p = 0.44	0.08 (−0.10; 0.26) p = 0.38	−0.06 (−0.22; 0.11) p = 0.52	−0.05 (−0.22; 0.12) p = 0.56
>148 and ≤222	387	0.01 (−0.13; 0.15) p = 0.90	0.02 (−0.12; 0.16) p = 0.81	0.10 (−0.05; 0.25) p = 0.21	0.11 (−0.04; 0.26) p = 0.17	−0.08 (−0.22; 0.06) p = 0.26	−0.08 (−0.22; 0.06) p = 0.24
>222	437	-	-	-	-	-	-

1Data shown as number (n), regression coefficient B (95% CI), and p-value. Numbers in categories may not sum up, due to missing values. In the multivariable model further adjustments are made for elevated serum CRP and AGP.

AGP, α1-acid glycoprotein; CRP, C-reactive protein; MDAT, Malawi Development Assessment Tool; MUAC, mid-upper arm circumference; SC, serum cobalamin.

Table 6

Effects of matrix, soy quality, and milk content in supplementary foods on SC (pmol/L) at end of the 3 months supplementation among 1,016 children with MAM.

	Main effects			Effect of LNS versus CSB by soy quality				Effects of SI versus DS by matrix
				SI		DS		LNS		CSB
Matrix
LNS	162	(3; 29)	0.02	39 (20; 57)	<0.001	−8 (−26; 11)	0.42
CSB	-	-		-		-
Soy quality
SI	−112	(−24; 2)	0.10					11 (−6; 30)	0.27	−35 (−53; −16)	<0.001
DS	-	-						-		-
Milk content (% of protein)
50%	9	(−7; 25)	0.30
20%	−2	(−18; 14)	0.84
0%	-	-

1Data shown as mean difference (95% CI) based on intention-to-treat population. Tobit regression adjusted for baseline measure of the outcome, WLZ, LAZ, MUAC, age, sex, month of admission, and site.

2Interaction between matrix and soy quality: p < 0.001.

CSB, corn–soy blend; DS, dehulled soy; LAZ, length-for-age z-score; LNS, lipid-based nutrient supplement; MAM, moderate acute malnutrition; MUAC, mid-upper arm circumference; SC, serum cobalamin; SI, soy isolate; WLZ, weight-for-length z-score.