Effects of vitamin B12 supplementation on neurodevelopment and growth in Nepalese Infants: A randomized controlled trial.

BACKGROUND: Vitamin B12 deficiency is common and affects cell division and differentiation, erythropoiesis, and the central nervous system. Several observational studies have demonstrated associations between biomarkers of vitamin B12 status with growth, neurodevelopment, and anemia. The objective of this study was to measure the effects of daily supplementation of vitamin B12 for 1 year on neurodevelopment, growth, and hemoglobin concentration in infants at risk of deficiency. METHODS AND
FINDINGS: This is a community-based, individually randomized, double-blind placebo-controlled trial conducted in low- to middle-income neighborhoods in Bhaktapur, Nepal. We enrolled 600 marginally stunted, 6- to 11-month-old infants between April 2015 and February 2017. Children were randomized in a 1:1 ratio to 2 μg of vitamin B12, corresponding to approximately 2 to 3 recommended daily allowances (RDAs) or a placebo daily for 12 months. Both groups were also given 15 other vitamins and minerals at around 1 RDA. The primary outcomes were neurodevelopment measured by the Bayley Scales of Infant and Toddler Development 3rd ed. (Bayley-III), attained growth, and hemoglobin concentration. Secondary outcomes included the metabolic response measured by plasma total homocysteine (tHcy) and methylmalonic acid (MMA). A total of 16 children (2.7%) in the vitamin B12 group and 10 children (1.7%) in the placebo group were lost to follow-up. Of note, 94% of the scheduled daily doses of vitamin B12 or placebo were reported to have been consumed (in part or completely). In this study, we observed that there were no effects of the intervention on the Bayley-III scores, growth, or hemoglobin concentration. Children in both groups grew on an average 12.5 cm (SD: 1.8), and the mean difference was 0.20 cm (95% confidence interval (CI): -0.23 to 0.63, P = 0.354). Furthermore, at the end of the study, the mean difference in hemoglobin concentration was 0.02 g/dL (95% CI: -1.33 to 1.37, P = 0.978), and the difference in the cognitive scaled scores was 0.16 (95% CI: -0.54 to 0.87, P = 0.648). The tHcy and MMA concentrations were 23% (95% CI: 17 to 30, P < 0.001) and 30% (95% CI: 15 to 46, P < 0.001) higher in the placebo group than in the vitamin B12 group, respectively. We observed 43 adverse events in 36 children, and these events were not associated with the intervention. In addition, 20 in the vitamin B12 group and 16 in the placebo group were hospitalized during the supplementation period. Important limitations of the study are that the strict inclusion criteria could limit the external validity and that the period of vitamin B12 supplementation might not have covered a critical window for infant growth or brain development.
CONCLUSIONS: In this study, we observed that vitamin B12 supplementation in young children at risk of vitamin B12 deficiency resulted in an improved metabolic response but did not affect neurodevelopment, growth, or hemoglobin concentration. Our results do not support widespread vitamin B12 supplementation in marginalized infants from low-income countries. TRIAL REGISTRATION: ClinicalTrials.gov NCT02272842 Universal Trial Number: U1111-1161-5187 (September 8, 2014) Trial Protocol: Original trial protocol: PMID: 28431557 (reference [18]; study protocols and plan of analysis included as Supporting information).

RCT Entities:   Population Interventions Outcomes

Introduction

Vitamin B12 (cobalamin) deficiency is common and affects all ages worldwide [1-3]. Animal-based foods are the primary sources, and poor status is prevalent in South Asia as well as in other low- and middle-income regions with low consumption of meat, animal milk, and fish [3,4].

Vitamin B12 is required for cell division and differentiation, utilization of energy, and other critical metabolic processes [5-7]. Failure to thrive, delayed development, and macrocytic anemia are typical manifestations in children with severe deficiency [8]. Several observational studies have demonstrated associations between biomarkers of vitamin B12 status with growth, neurodevelopment, and anemia [1,9-14]. The results from these studies suggest that the negative consequences of poor B12 nutrition are also seen in suboptimal B12 status, and not only in those with clinical deficiency. These observations, however, may be due to factors such as unmeasured confounding or limitations in the biomarkers used to assess vitamin B12 status.

Three randomized controlled trials (RCTs) have measured the effect of vitamin B12 supplementation on neurodevelopment in children with suboptimal B12 status. Of these, 2 facility-based RCTs in infants born at low birth weight or who had developmental delay showed that a high dose of vitamin B12 (400-μg hydroxycobalamin intramuscularly) substantially improved motor development [15,16]. In the third study, a population-based RCT in young North Indian children, there was a small borderline significant beneficial effect of daily oral supplementation with 1.8 μg of vitamin B12 for 6 months on neurodevelopment [17]. The evidence from these RCTs support the notion that poor vitamin B12 status may be a relevant public health concern; however, the evidence is currently not strong enough to alter widespread feeding recommendations. We therefore designed the current population-based RCT to measure the effects of daily supplementation of vitamin B12 for 1 year. In this study, we ensured that the baseline B12 status and metabolic response to the supplementation was well characterized, and we targeted marginally stunted infants due to their associated risk of vitamin B12 deficiency, delayed development, and stunted growth.

Participants and methods

Study design and participants

This study is reported according to the CONsolidated Standards of Reporting Trials (CONSORT) guidelines (S1 Checklist). The study was a community-based, double-blind placebo-controlled trial in Nepalese infants. We hypothesized that daily administration of 2 μg vitamin B12 for 12 months would improve neurodevelopment, growth, and hemoglobin concentration. We conducted the study in Bhaktapur municipality and surrounding peri-urban communities near the capital Kathmandu. For the last 20 years, we have shown that poor vitamin B12 status is common in both women and children in this population [4,18,19]. Bhaktapur is among the most densely populated municipalities in Nepal. The majority of births (96%) occur at health centers, and 1 in every second family is living in joint households and has a separate kitchen [20]. The primary outcomes were neurodevelopment measured by the Bayley Scales of Infant and Toddler Development 3rd ed. (Bayley-III), attained growth (cm and kg), and hemoglobin concentration. The secondary outcome was the metabolic response measured by plasma concentrations of cobalamin, total homocysteine (tHcy), and methylmalonic acid (MMA).

Ethics statement

The study received ethical clearance from the Nepal Health Research Council (NHRC; #233/2014) and from the Regional Committee for Medical and Health Research Ethics (REC; #2014/1528) in Norway. After detailed information was provided to parents, we obtained written informed consent or a thumbprint from those who were illiterate (in the presence of an impartial witness).

Enrollment, randomization, and blinding

We enrolled 600 children between April 17, 2015 and February 15, 2017. Inclusion criteria were age 6 to 11 months, length for age z-score <−1, intent to reside in the municipality and surrounding areas for the next 12 months, and availability of informed consent from parents. Children were excluded if they were taking (or planned to take) supplements that contained vitamin B12, had a severe systemic illness requiring hospitalization, if they were severely malnourished (weight for length z-score <−3), were severely anemic (hemoglobin concentration <7 g/dL), or had ongoing infections that required medical treatment. In cases of severe malnutrition, anemia, or infections, children received treatment and were screened again for eligibility after recovery.

Field workers identified eligible children from immunization clinics or through home visits. Infants were enrolled by a study supervisor or by a physician at the field office. We randomized the infants in a 1:1 ratio in blocks of 8 using a computer-generated randomization list. Randomization was concealed, and the study double-blinded as the participants were only linked to the intervention through the identification (id) number printed on the supplement labels. The list that linked this id number to the randomization code was kept with the producers of the supplements and the scientist who generated it. None of the investigators had access to this list until the data collection and cleaning for the primary outcomes were completed. The vitamin B12 supplements and placebo were produced specifically for the trial and were identical in taste and appearance. At enrollment and end of study, we measured neurodevelopment, weight, length, and hemoglobin concentration.

Intervention and co-interventions

All children received 2 μg of vitamin B12 (cyanocobalamin), corresponding to approximately 2 to 3 recommended daily allowances (RDAs) or placebo via a daily oral supplement for 12 months. The intervention was implemented using sachets containing 20 grams of a lipid-based paste produced by GC Rieber Compact (Gurgaon, Haryana, India; http://www.gcrieber-compact.com/). Each sachet provided the daily dose of supplements. To ensure that the effect of vitamin B12 was not limited by inadequate intake of other essential nutrients, both the placebo and vitamin B12 paste contained a base multi-micronutrient mixture with several other vitamins and minerals at approximately 1 RDA. All caregivers were given dietary recommendations according to national guidelines. Children who developed diarrhea during the intervention period received zinc and oral rehydration solution. Those with mild to moderate anemia (hemoglobin 7 to 10 g/dL) were treated with per oral iron for at least 30 days. Children with pneumonia, dysentery, or other illnesses were treated according to the most recent Integrated Management of Childhood Illness (IMCI) guidelines [21].

During weekly visits to the homes, field workers asked the mothers about intake of the paste during the past 7 days and recorded in detail the amount of paste given to the children (i.e., half, one-third, three-fourths, or less). All episodes of vomiting or regurgitation after supplementation of the paste were also recorded, and the total number of empty paste sachets were counted at the weekly visits to verify the reported compliance.

Outcomes

Neurodevelopment

The Bayley-III is a comprehensive assessment tool of neurodevelopment in infants and toddlers aged 1 to 42 months [22]. The Bayley-III is often regarded as the gold standard for assessing neurodevelopment in this age-group and is used in research worldwide. We administered the Bayley-III directly with the child at enrollment and end of the study at the study research office. The Bayley-III consists of a cognitive, language (receptive and expressive), motor (fine and gross motor), and socio-emotional scale. Three psychologists, of whom 1 had extensive experience with the Bayley-III, were trained to perform the assessments for the study. To ensure high-quality measurements, we performed standardization exercises in 20 children ahead of the enrollment where the Bayley assessments were scored by 2 raters. The psychologists were required to reach an intra-class correlation coefficient (ICC) >0.90 with the expert rater, who served as the gold standard. Of note, 7% of the sessions during the main study were double scored by the expert rater to ensure appropriate interobserver agreement throughout the study. The ICCs from the quality controls ranged from 0.95 to 0.99 [23]. All the Bayley-III assessments were video-recorded for quality purposes. The Bayley-III raw scores were converted into scaled and composite scores based on U.S. citizen normative data [22]. For the analyses, we used raw, scaled, and composite scores.

Anthropometry

Weight was measured with a portable electronic scale (model 877, Seca, California, United States of America) that measures to the nearest 0.01 kg, and length was measured with portable length board (model 417, Seca, California, USA). We measured length and weight at the clinic or at home during the monthly follow-up visits. All anthropometric measurements were performed twice. The mean values were used in the analyses.

Laboratory procedures

Blood samples were collected from the cubital veins into polypropylene tubes containing EDTA (Sarstedt, Germany), which were protected from direct sunlight exposure. Up to 4-mL blood was collected at enrollment and end of study. The hemoglobin concentration was analyzed immediately following blood sampling with HemoCue (HemoCue 201, Ångelholm, Sweden), which was calibrated as per the guidelines defined by the manufacturer. The blood was centrifuged at room temperature for 10 min at 2,000 to 2,500 g within 10 min after venipuncture (Model R-304, Remi, Mumbai, India). Plasma and blood cells were separated, transferred into polypropylene vials (Eppendorf, Germany), and immediately stored at <−80°C until analysis at Bevital Laboratory (Bergen, Norway; www.bevital.no). The samples were shipped to Norway on dry ice by World Courier. The plasma concentrations of cobalamin and folate were determined using microbiological assays [24,25] using a colistinsulfate-resistant strain of Lactobacillus leichmannii or chloramphenicol-resistant strain of Lactobacillus casei, respectively. The functional biomarkers plasma total tHcy and MMA are considered to be sensitive markers of B12 deficiency [26]. Plasma tHcy and MMA were analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS) based on methylchloroformate derivatization [27]. The within-day coefficient of variation was 4% for both cobalamin and folate and ranged from 1% to 5% for tHcy and MMA. The between-day coefficient of variation was 5% for both cobalamin and folate and ranged from 1% to 8% for MMA and tHcy. We also calculated a combined indicator of cobalamin status (3cB12) based on the 3 biomarkers cobalamin, tHcy, and MMA as suggested by Fedosov and colleagues [28]. In short, this index is the log of the cobalamin concentration divided by the product of the log of the tHcy and MMA concentrations.

Sample size

The study had 80% power to detect a standardized effect size of 0.22 and had 90% power to detect an effect size of 0.28. In these calculations, we assumed a loss to follow-up of 10%. Details on the samples size calculations are also presented in the previously published protocol paper [18].

Statistical analyses

The analyses in this study were carried out according to the predefined protocols and analysis plans (S1–S4 Texts). Weight for age, weight for length, and length for age z-scores were calculated using the most recent WHO growth charts [29]. We defined underweight, stunting, and wasting as z-scores below −2 [29]. We depicted the relationship between changes in vitamin B12 status (i.e., change in the 3cB12 values from baseline to end of study) according to vitamin B12 status (3cB12) at baseline. To do so, we performed a kernel-weighted local polynomial regression of delta 3cB12 on baseline 3cB12 values by treatment group and depicted these dose-response graphs of the predicted values with 95% confidence intervals (CIs). We also compared the concentrations of cobalamin, tHcy, and MMA between the study groups in all infants and according to 3cB12 categories (“possible deficient,” “low,” and “adequate”) at baseline. As these variables were left-skewed, we present the geometric means and the geometric standard deviation factors (GSDFs). We used the log-transformed values of the end-study biomarker concentrations when comparing the differences between the study groups. The exponentials of these mean differences are presented as the geometric mean ratio (GMR) between the study groups.

We compared the mean end-study Bayley-III scaled and composite scores between the intervention groups. For the other outcome variables (Bayley-III raw scores, growth, and hemoglobin concentrations), the effects of the intervention were estimated by comparing the changes from baseline to end study between the study groups. The precision of the effect estimates and corresponding P values were calculated using the Student t test assuming equal variances. We also analyzed the data by several predefined subgroups. The subgrouping variables were stunting (defined as length for age z-scores <−2), underweight (weight for age z-scores <−2), low vitamin B12 status (3cB12 <−0.5), low birth weight (birth weight <2,500 g), anemia (hemoglobin concentration <11.0 g/dL), and exclusive breastfeeding. For these analyses, we used univariate and multiple generalized linear models with the Gaussian distribution family and identity link function adjusting for a set of predefined potential confounders. The following variables were adjusted for in all subgroup analyses: length for age z-scores, maternal and paternal education, and age of the child at baseline. All analyses were performed using Stata version 16 (StataCorp, College Station, Texas, USA).

Results

From April 2015 to February 2017, we screened 733 infants and randomized 600 into the study (Fig 1). A total of 26 infants dropped out due to refusal or migration. Two children were unable to complete the end-study activities, leaving 572 infants with complete neurodevelopmental assessments. We were able to collect and analyze blood samples for biomarker assessments at end study from 567 children.

Fig 1

Trial flowchart of a study measuring the effect of daily vitamin B12 supplementation in Nepalese infants.

Baseline features by intervention and placebo groups are presented in Table 1. One in every five infants was born at low birth weight, and one-third were stunted (<−2 z-score length for age) at enrollment. The baseline features were evenly distributed between the intervention groups. The baseline status of vitamin B12 and its plasma biomarkers were also comparable between the intervention groups (S1 Table).

Table 1

Baseline characteristics in a study investigating the effect of daily vitamin B12 supplementation on neurodevelopment and growth in 600 Nepalese infants.

	Vitamin B12 group (n = 300)		Placebo group (n = 300)
	n	%	n	%
Infant characteristics
Mean age of child (months), mean ± SD	8.1 ± 1.7		8.0 ± 1.8
Male child	158	53	151	50
Have older siblings	155	52	153	51
Low birth weight (<2,500 gm)1	56	19	59	20
Hospitalization within first month of age	28	9.3	26	8.7
Demographic features
Mother’s age, mean ± SD	27.1 ± 4.7		27.5 ± 4.6
Father’s age2, mean ± SD	30.0 ± 7.1		30.6 ± 5.1
Mothers who completed secondary school or above	197	65.7	180	60
Fathers who completed secondary school or above	199	66.3	189	63
Mothers who work	117	39.0	110	36.7
Fathers who work	286	95.3	280	93.3
Socioeconomic status
Family staying in joint family	143	47.7	149	49.7
Family residing in rented house	152	50.7	139	46.3
Number of rooms in use by the household (<−2)	163	54.3	174	58
Kitchen and bedroom in the same room	148	49.3	150	50
Family having own land	138	46	144	48
Receiving remittance from abroad	30	10	27	9
Breastfeeding status
No breastfeeding at time of interview	8	2.7	6	2
Exclusive breastfeeding for 3 months or more	143	47.7	137	45.6
Nutritional status of infants
Underweight (weight for age z-score <−2)	62	20.7	50	16.6
Stunting (length for age z-score <−2)	96	32.1	98	32.7
Wasting (weight for length z-score <−2)	12	4.0	7	2.3
Hemoglobin, g/dL, mean ± SD	10.6 ± 0.96		10.6 ± 0.91
Anemia (hemoglobin <11 g/dL)	183	61	202	67.3
Nutritional status of mother
BMI of mother, mean ± SD	23.7 ± 3.5		23.7 ± 3.6
<18.5 kg/m2 BMI of mother	15	5	19	6.3

1Among 579 infants whose birth weights were recorded.

2Among 487 fathers who were available.

n, number.

Compliance

More than 94% of the prescribed doses were reportedly taken (94.3% in the vitamin B12 group and 94.0% in the placebo group; S2 Table). Of these, 86.3% and 84.7% of the vitamin B12 and placebo group, respectively, consumed the entire prescribed doses.

End points

Vitamin B12 supplementation had no effect on any of the neurodevelopmental outcomes (Table 2). For example, the cognitive composite scores were 0.73 points (95% CI: −0.55 to 2.02, P = 0.261) lower in the vitamin B12 group compared to the placebo group. There was also no effect on growth or hemoglobin concentration (Table 3). Children in both groups grew on an average 12.5 cm (SD: 1.8), and the mean difference was 0.20 cm (95% CI: −0.23 to 0.63, P = 0.354). The mean difference in hemoglobin concentration between the groups was 0.02 g/dL (95% CI: −1.33 to 1.37, P = 0.978). The adjusted effects of the interventions on growth and neurodevelopment by various subgroups are shown in S1 and S2 Figs. These analyses did not reveal any variable that modified the effect on any of the outcomes. Unadjusted subgroup analyses yielded the same results. We found no adverse effects of vitamin B12 supplementation (S3 Table).

Table 2

Effect of daily vitamin B12 supplementation for 1 year starting in infancy on the Bayley Scales of Infant Development scores among infants in Bhaktapur, Nepal.

Bayley-III subscales	Vitamin B12 group (n = 283)	Placebo group (n = 289)	Mean differences
	Mean ± SD	Mean ± SD	(95% CI) (P value)
Change in raw scores from baseline until end study
Cognitive	20.9 ± 4.3	20.7 ± 4.2	0.16 (−0.54 to 0.87) (0.648)
Language
Expressive	14.4 ± 4.7	14.8 ± 5.0	0.35 (−0.44 to 1.14) (0.387)
Receptive	10.9 ± 4.0	11.2 ± 3.6	−0.31 (−0.94 to 0.32) (0.334)
Motor
Fine motor	13.8 ± 3.3	13.7 ± 2.9	0.10 (−0.41 to 0.60) (0.712)
Gross motor	21.4 ± 5.1	21.7 ± 4.7	−0.28 (−1.08 to 0.52) (0.491)
Socio-emotional	29.4 ± 14.3	31.0 ± 12.0	−1.57 (−3.96 to 0.82) (0.197)
End-study composite and scaled score
Cognitive composite score	90.5 ± 8.2	91.2 ± 7.3	0.73 (−0.55 to 2.02) (0.261)
Language composite score	93.0 ± 12.8	92.6 ± 12.6	−0.42 (−2.51 to 1.66) (0.692)
Expressive scaled score	8.6 ± 2.6	8.5 ± 2.4	−0.01 (−0.43 to 0.41) (0.965)
Receptive scaled score	9.0 ± 2.3	8.9 ± 2.5	−0.13 (−0.53 to 0.27) (0.517)
Motor composite score	99.9 ± 8.7	100.2 ± 8.3	0.32 (−1.08 to 1.73) (0.652)
Fine motor scaled score	10.7 ± 1.6	10.9 ± 1.8	0.22 (−0.06 to 0.50) (0.131)
Gross motor scaled score	9.2 ± 2.0	9.1 ± 1.7	−0.12 (−0.42 to 0.19) (0.457)
Socio-emotional composite score	104.3 ± 16.7	103.4 ± 17.1	−0.92 (−3.70 to 1.86) (0.518)

The mean differences, the corresponding 95% CI, and P values were calculated using Student t test assuming equal variances.

Bayley-III, Bayley Scales of Infant and Toddler Development 3rd ed.; CI, 95% confidence interval; n, number.

Table 3

Effect of vitamin B12 supplementation on growth and hemoglobin concentrations among infants in Bhaktapur, Nepal.

	Vitamin B12 group (n = 283)	Placebo group (n = 290)	Mean differences
	Mean ± SD	Mean ± SD	(95% CI) (P value)
Change from baseline
Length (cm)	12.5 ± 1.8	12.5 ± 1.8	0.09 (−0.21 to 0.39) (0.574)
Weight (kg)	2.1 ± 0.5	2.1 ± 0.6	−0.01 (−0.10 to 0.09) (0.919)
Hemoglobin (g/dL)	1.0 ± 1.1	1.0 ± 1.2	−0.02 (−0.20 to 0.17) (0.876)
End study
Length (cm)	78.2 ± 2.6	78.4 ± 2.6	0.20 (−0.23 to 0.63) (0.354)
Weight (kg)	9.4 ± 0.9	9.4 ± 1.0	−0.02 (−0.18 to 0.13) (0.780)
Length for age z-score	−1.8 ± 0.7	−1.7 ± 0.7	0.05 (−0.06 to 0.16) (0.411)
Weight for height z-score	−0.7 ± 0.8	−0.8 ± 0.8	−0.08 (−0.22 to 0.05) (0.238)
Weight for length z-score	−1.4 ± 0.7	−1.4 ± 0.8	−0.04 (−0.16 to 0.08) (0.529)
Hemoglobin (g/dL)	11.6 ± 0.8	11.6 ± 1.0	0.02 (−1.33 to 1.37) (0.978)

The mean differences, the corresponding 95% CI, and P values were calculated using Student t test assuming equal variances.

CI, 95% confidence interval

The effect of vitamin B12 supplementation on B12 status expressed by the 3cB12 according to baseline status is depicted in Fig 2. The distance between the solid and dotted lines represents the metabolic effect of the intervention, which decreased as the baseline vitamin B12 status improved. A similar trend is displayed in S1 Table where the effects on the different biomarkers are shown. For the functional biomarkers, tHcy and MMA, poorer vitamin B12 status at baseline was associated with a larger effect of vitamin B12 supplementation. For example, when restricting the analyses to infants who were classified as possibly deficient, the placebo group had 60% higher MMA concentrations (indicating poorer vitamin B12 status) at end study compared to those in the vitamin B12 group (GMR 1.60, 95% CI: 1.20 to 2.14, P < 0.001). In the children who had adequate status at baseline, there was no effect of vitamin B12 supplementation on the MMA concentration (S1 Table).

Fig 2

The association between vitamin B12 status at baseline and change in vitamin B12 status from baseline to end study by randomized group.

The y-axis is the change in 3cB12 from baseline to end study, and the x-axis is the baseline 3cB12 value. The regression lines were generated by a kernel-weighted local polynomial regression with the change in 3cB12 as the dependent variable and 3cB12 at baseline as the independent variable. The shaded areas represent the 95% CI of the regression lines. The profiles were generated separately for the 2 intervention groups, and the distance between the 2 regression lines represents the effect of the vitamin B12 supplementation. The 3cB12 is a function of the plasma concentrations of cobalamin, tHcy, and MMA. CI, 95% confidence interval; MMA, methylmalonic acid; tHcy, total Homocysteine.

Discussion

In this year-long, double-blind, placebo-controlled RCT, daily intake of a supplement containing vitamin B12 improved vitamin B12 status in marginally stunted Nepalese infants. Those with poor status at the onset of the study benefitted more than those with adequate status. The intervention, however, did not result in any improvements in neurodevelopment, nor was there any effects on growth or hemoglobin concentration. Restricting the analyses to those with poor status at baseline, i.e., those who also had the best metabolic response, did not alter these results.

Our findings are in contrast to results from the observational studies where vitamin B12 status was positively associated with neurodevelopment, growth, and hemoglobin concentration [10,14,30]. The findings are also at odds with results from the 3 small (group sizes of 32 to 104) RCTs in infants and young children where vitamin B12 supplementation resulted in improved developmental scores [15-17].

Several elements of our study design and conduct support the veracity of our findings. The trial enrolled 600 children, of whom >95% could be included in the analyses. As a result, we have precise effect estimates of our primary outcomes, and thus, sufficient power to detect clinically meaningful differences between the treatment groups. The randomization was successful as indicated by no differences in baseline characteristics, and few participants were lost to follow-up. Finally, the compliance with supplement use was high, as it was given on nearly 95% of the scheduled days and resulted in an excellent metabolic response (S2 Table).

The study team has extensive experience with the Bayley-III, and the inter-rater reliability was excellent during both the initial standardization exercises as well as for the quality controls throughout the study. The Bayley-III scores were associated with established risk factors for poor neurodevelopment [23], which provided support for the validity of the test in this study setting. In addition, the study staff were trained and standardized for 2 decades in measuring infant growth, ensuring precise measurements for these outcomes. Lastly, we had an effective cold chain and used state-of-the-art biochemical methods to estimate the biomarker concentrations ensuring optimal description of the vitamin status.

The participants were moderately stunted infants and accordingly at risk of poor neurodevelopment and vitamin B12 deficiency. Thus, they constituted a group of children where we could expect an effect of vitamin B12 supplementation if subclinical deficiency affected any of our outcomes. For practical and ethical reasons, we could not target children with defined or overt B12 deficiency. We did not have the necessary diagnostic recourses to measure vitamin B12 status before randomization, and giving a placebo to infants with diagnosed vitamin B12 deficiency for a year would violate the principle of clinical equipoise. Thus, the study included some children with adequate vitamin B12 status at baseline. It is plausible that inclusion of vitamin B12 replete children could attenuate a potential effect of the intervention. However, this does not explain our null findings as the effect estimates did not change when we restricted the analyses to those with evidence of deficiency.

A higher dose or a different, more effective, mode of administration (i.e., injection) could have led to different results. The metabolic response, however, indicates a substantial biological effect. Vitamin B12 is involved in 2 biochemical reactions in humans [31]. In 1 of these, vitamin B12 is required for the transfer of methyl groups, which includes remethylation of homocysteine to methionine. Disruption of this pathway increases homocysteine and affects gene regulation and DNA synthesis [31]. Vitamin B12 also acts as an enzymatic cofactor for methylmalonyl-CoA mutase, an enzyme involved in the catabolism of fats and amino acids. Disruption of the latter pathway explains the increased MMA observed in vitamin B12 deficiency. Our study provides causal evidence that both of these metabolic pathways are affected by mild vitamin B12 deficiency in children, and their functioning improves with supplementation. Thus, despite no effects on the clinical outcomes, children in this population could benefit from increasing the vitamin B12 intake as indicated by their improved metabolic profile.

The period of supplementation in the present study might not have covered a critical window where adequate vitamin B12 status is crucial for optimal growth and neurodevelopment. Thus, initiating supplementation sooner, such as before or during pregnancy or earlier in infancy, could have yielded different results.

Restricting the participants to mildly stunted children increased the internal validity and statistical power of our study at the expense of the external validity. In other words, our recruitment strategy reduced the generalizability of our results, which is a limitation. It should be noted that enrolling all infants from the community would result in a larger proportion with adequate vitamin B12 status and, consequently, more children who would not respond to the intervention. A design ensuring higher external validity could accordingly attenuate a potential effect and, at the same time, increase the variability, both contributing to reduced statistical power. Reduced statistical power increases the risk of false-negative results (type II errors), which particularly question null findings such as here. In addition, our study was designed to measure the effects of vitamin B12 supplementation on several outcomes across several subgroups, which reduced the probability of overlooking relevant short-term clinical outcomes.

An important assumption of our study was that the inclusion criteria ensured we targeted those who would benefit from supplementation. It is possible, however, that these stunted children would suffer from deficiencies of other growth-limiting nutrients. We believe this potential bias was accounted for by providing other vitamins and minerals, by treating common infections, and by giving zinc for diarrhea.

In summary, our results show that in Nepalese infants, daily vitamin B12 supplementation improves vitamin B12 status and the metabolic profile expressed by the composite 3cB12 indicator. The metabolic response indicates that this population could benefit from vitamin B12 supplementation, but the clinical consequences of subclinical deficiency in early childhood remain uncertain.

The effect of vitamin B12 supplementation on the Bayley Scales of Infant Development subscale scores in different subgroups.

A point estimate to the left of the vertical line indicates a beneficial effect of vitamin B12. None of the subgroup specific effects were statistically significant. The effect estimates were calculated with multiple general linear models with the Gaussian distribution family and identity link function adjusting for length for age z-scores, maternal and paternal education, and age of the child at baseline. Stunting and underweight were defined as being <−2 length for age z-scores and weight for age z-scores, respectively. 3cB12: combined vitamin B12 status indicator as suggested by Fedosov and colleagues [28], low 3cB12 is <−0.5, low birth weight: birth weight <2,500 g, anemia: hemoglobin concentration <11 g/dL.

(TIF)

Click here for additional data file.

The effect of vitamin B12 supplementation on growth and hemoglobin concentration in different subgroups.

A point estimate to the left of the vertical line indicates a beneficial effect of vitamin B12. None of the subgroup specific estimates were statistically significant. The effect estimates were calculated with multiple general linear models with the Gaussian distribution family and identity link function adjusting for length for age z-scores, maternal and paternal education, and age of the child at baseline. Stunting and underweight were defined as being <−2 length for age z-scores and weight for age z-scores, respectively. 3cB12: combined vitamin B12 status indicator as suggested by Fedosov and colleagues [28], low 3cB12 is <−0.5, low birth weight: birth weight <2,500 g, anemia: hemoglobin concentration <11 g/dL.

(TIF)

Click here for additional data file.

Effects of daily vitamin B12 supplementation for 1 year starting in infancy on markers of vitamin B12 status.

(DOCX)

Click here for additional data file.

Compliance of vitamin B12 supplementation among Nepalese infants participating in clinical trial on the effect of vitamin B12 supplementation on growth, development, and hemoglobin concentration.

(DOCX)

Click here for additional data file.

Adverse effects of vitamin B12 supplementation among Nepalese infants participating in clinical trial on the effect of vitamin B12 supplementation on growth, development, and hemoglobin concentration.

(DOCX)

Click here for additional data file.

Main protocol version 1.0.

August 1, 2014.

(DOCX)

Click here for additional data file.

Main protocol version 2.1.

October 16, 2016.

(DOCX)

Click here for additional data file.

Plan of analysis version 1.

March 2018.

(DOCX)

Click here for additional data file.

Plan of analysis version 2.1.

October 2019.

(DOCX)

Click here for additional data file.

CONSORT Checklist.

(DOCX)

Click here for additional data file.

11 Feb 2020

Dear Dr Strand,

Thank you for submitting your manuscript entitled "A randomized controlled trial of vitamin B12 supplementation, neurodevelopment, and growth in Nepalese infants" 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.

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Kind regards,

Helen Howard, for Clare Stone PhD

Acting Editor-in-Chief

PLOS Medicine

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27 Aug 2020

Dear Dr. Strand,

Thank you very much for submitting your manuscript "A randomized controlled trial of vitamin B12 supplementation, neurodevelopment, and growth in Nepalese infants" (PMEDICINE-D-20-00074R1) 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.

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***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.***

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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,

Emma Veitch, PhD

PLOS Medicine

On behalf of Clare Stone, PhD, Acting Chief Editor,

PLOS Medicine

plosmedicine.org

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Requests from the editors:

*If possible, please present the title per the PLOS Medicine style, this should present the study objective/question in the first part of the title and then the study design (eg, "A randomized controlled trial," "A retrospective study," "A modelling study," etc.) in the subtitle (ie, after a colon).

*In the last sentence of the Abstract Methods and Findings section, please include a brief note about any key limitation(s) of the study's methodology.

*Please complete the CONSORT checklist (http://www.consort-statement.org/), designed to aid reporting of randomized trials, and ensure that all components of CONSORT are present in the manuscript. We'd suggest noting in the methods section that the trial is reported in line with CONSORT; please upload the completed CONSORT checklist as a supplementary file with your resubmission.

*Per the CONSORT guidance, we'd recommend that the abstract and Results section should present the estimates of effect size (with 95% CI's) on the main outcomes for the trial so that readers can get a sense of what the range of effects are that are plausible with the data - rather than just the narrative summary that there is no effect.

*It would be helpful if the authors could say something about possible harms (adverse effects) with the supplementation in the trial - the trial registration suggests that adverse effects would be monitored but the results section does not seem to present any data for this. Obviously in trials both benefits and risks are important.

*It would also be good to include some discussion about possible external validity (generalisability) of the trial findings given that, as noted by reviewers, the children in the study are marginally stunted.

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Comments from the reviewers:

Reviewer #1: This is a statistical review of manuscript PMEDICINE-D-20-00074_R1. The manuscript is well written and easy to follow. Some clarifications are needed in the statistical section.

Major comments:

* Statistical analyses section: "Differences in proportions were calculated using generalized linear models (GLM) with the binomial distribution family and identity link function". Please could you clarify with you did not use one of the most common link functions for binary data (i.e. logit, cloglog, etc). The identity link is rarely used.

* Statistical analyses section: "For these analyses we used crude and multiple GLMs with the gaussian distribution family and identity link function adjusting for a set of predefined potential confounders". Are you referring to continuous endpoints or binary endpoints? It is not clear currently, as this sentence follows the previous sentence that cover difference in proportions.

Minor comment:

* Abstract: please specify what was percentage of loss to follow up (26/600=4.3%). Also, 2 children could not be included in the analysis. I think it might be worth stating the size of your complete data set and stating how it compares to the original 600 children as percentage.

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Reviewer #2: This is a clearly written paper describing a blinded RCT of Vitamin B12 supplementation in Nepalese infants. The primary outcomes were neurodevelopment, growth and haemoglobin concentration. The authors have succinctly presented the rationale for the study in the introduction. The methods are clearly described but would benefit from the inclusion of the CONSORT checklist, in the supplementary section. It would be useful to include information about trial registration. The authors are to be commended in the presentation of the results which are presented primarily as mean differences with 95%CI, eliminating the need to include p-values. The intervention had no effect on the outcomes, even after sub-group analysis was undertaken. The authors complete the paper with a concise but well thought-out discussion. The inclusion of supplemental tables and figures are useful for those who are interested in more detail. A minor point is the use of tense; sometimes present and sometimes past. In conclusion, this paper provides an exemplary model for how clinical researchers can present data from a RCT.

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Reviewer #3: The manuscript under review by Strand et al. reports on a randomized controlled trial of B12 supplementation to Nepalese infants and potential effects on metabolic and developmental outcomes.

The manuscript is nicely written and generally fits the scope of PLoS Medicine, however, the results as presented may not be sufficient to be considered a substantial advance over existing knowledge. While they are clear and support the conclusion of the authors, the results may not be applicable to the general population as the study focus concentrated on marginally stunned children with a specific treatment dose of B12. While there is no question about the validity of this approach, it may have not resulted in improvements for neurodevelopment and growth but the dose was sufficient to improve selective B12 biomarkers in the deficient infant subgroup indicating that there could be a benefit of B12 supplementation. The authors rightfully discussed this topic in the manuscript, thus I would assume that they also consider the possibility that B12 supplementation can be beneficial for developmental markers, possible after dose adjustments. Thus, the current results may not be as exciting for the Bayley-III outcomes, but they are indicative of a selective metabolic response based on B12 status at baseline, which are not unexpected to be noticeable before notable changes in growth and development, and therefore important results for future studies in the field. I encourage to investigate the Bayley-III results between the B12-sufficient infants at baseline and the B12 deficient children to examine possible significant differences in the development tests (and not just exclude B12 sufficient children from the analysis), other than stunting. If the Bayley-III results do not differ by B12 status, then a non-response to the intervention is not surprising, since there wasn't a difference to begin with.

Moreover, the study included infants with sufficient B12 status, over 100 per group, which changes the sample size, and possibly effect size. We don't know if this may have affected the outcome, but it could be by reducing the sample numbers for certain statistical analyses the effect size just changed enough to not be significant. However, given the limited amount of studies on this topic, this manuscript does add to the knowledge base, but conclusions should be revisited. Additional comments and concerns should be addressed before this manuscript may be recommended for publication:

� Line numbers are missing. Please add as per journal guidelines, which is also beneficial for the reviewer.

� Please add a space before reference directly attached to the word, e.g. but not limited to P9 "charts[29]".

Abstract:

� RDA is an abbreviation and should be explained at the first use.

� As mentioned above, I do not recommend to generalize the results obtained to a population basis as some your results differ depending on the B12 status of the children.

Author Summary:

� The meaning of the findings should be revised based on my discussion above.

Introduction:

� P4 last paragraph: A daily oral supplementation with 1.8µg?

� P4 last paragraph: I am not quite clear how the results from the 3 RCTs support that the public health concern is not sufficient to alter feeding recommendations, given that all trials showed benefits of the B12 interventions. A little bit more about your thought process here would be beneficial to the reader.

Subjects and Methods:

� P6 Intervention and co-intervention: each sachet provided the daily dose of supplements.

� P7 1st line: home visits were conducted by whom?

� P8 1st paragraph: please add model and manufacturer with location for the used scales and any equipment used for height measurements.

� P8 first paragraph: please add reference(s) for the mentioned standard guidelines.

� P8 1st paragraph: mean values of how many replicate measurements?

� P8 Laboratory procedures: HemoCue model?

� P8 Laboratory procedures: Temperature during centrifugation? Which model, manufacturer of the centrifuge?

� I assume samples were shipped on dry ice to Norway for analysis?

� P8 Laboratory procedures: The B12 analysis is not based on the strain of bacteria but the strains were used for this.

� P8 Laboratory procedures: "The functional markers plasma homocysteine…". This sentence does not belong in the Methods section, but could be added in the Introduction and/or Discussion.

� P9 statistics: Generally, looks good to me but I am not an expert in statistics.

� P9 statistics: You defined z-scores -2 based on the WHO growth charts? Please specify or add references.

� P10: Results, 1st paragraph: 572 completed the Bayley-III, but did you also get blood from 572 children as well?

Results:

� P11, 2nd paragraph: "We did not find adverse effects…"

Discussion:

� P11, Discussion: the 2nd sentence is unnecessary if you have to explain it in the third sentence. Please omit.

� Please add a Discussion about my points in the opening paragraph regarding your results and their interpretation.

Tables and Figures:

� Where applicable, please add to all Tables footnotes and Figure legends the statistical test used to examine significant differences.

� Supplemental Table 3: 1st row = n (children), 2nd row = n (events). OK, but then the next 3 rows, which fall under n(events) appear to be n(children) undergoing the different numbers of events. The specific diagnosis, however, belongs with n(events). This needs to be clarified to the reader.

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

[LINK]

20 Sep 2020

Submitted filename: ResponsesPloSMedicine_R1.pdf

Click here for additional data file.

1 Oct 2020

Dear Dr. Strand,

Thank you very much for re-submitting your manuscript "The effect of vitamin B12 supplementation on neurodevelopment and growth in Nepalese Infants: A randomized controlled trial" (PMEDICINE-D-20-00074R2) 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]

Our publications team (plosmedicine@plos.org) will be in touch shortly about the production requirements for your paper, and the link and deadline for resubmission. DO NOT RESUBMIT BEFORE YOU'VE RECEIVED THE PRODUCTION REQUIREMENTS.

***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.

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 Oct 08 2020 11:59PM.

Sincerely,

Artur Arikainen

Associate Editor

PLOS Medicine

plosmedicine.org

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Requests from Editors:

1. Title: Please update to: “Effects of vitamin B12 supplementation on neurodevelopment and growth in Nepalese Infants: A randomized controlled trial”

2. Financial Disclosure: Please list the funders using full sentences, eg. “This study was funded by…”

3. Data Availability Statement: If the data are not freely available, please describe briefly the ethical, legal, or contractual restriction that prevents you from sharing it. Please also include an appropriate contact (web or email address) for inquiries (this cannot be the study authors).

4. Competing Interests: Please mention the paid or unpaid employment by one or more authors at “Bevital AS, Bergen, Norway”.

5. Abstract:

a. Around lines 9-11, please give the study recruitment dates.

b. Line 13: Please rephrase as: “The primary outcomes were ...; secondary outcomes included ...".

c. Line 15: Please give the numbers lost to follow-up by assigned group. Delete “Only”.

d. Line 16: Please correct to "94%" to match the main Results. Also, clarify that: "…reported to have been consumed..."

e. Line 17: State the primary outcome results first, even if negative.

f. Line 18: Delete “substantially”.

g. Please give point effect estimates, 95% CI and p values for all results mentioned, even if not significantly different, including: “The two indirect functional biomarkers of vitamin B12, plasma total homocysteine and methylmalonic acid, were significantly and substantially reduced in the vitamin B12 group compared to the placebo group. There were no effects on the Bayley-III scores, growth, or hemoglobin.”

h. Please mention whether any adverse events were recorded, and whether they were linked to treatment.

i. Line 22: Please mention another limitation.

j. Line 24: Please start with: “In this study, we observed that…”. Please also state the primary outcome first; followed by the other findings in the style "…appeared to result in an improved…”

k. Line 26: Please add a brief sentence on the interpretation or overall relevance of these results.

6. Author Summary:

a. Please use bullet points to break up the paragraphs.

b. Lines 41 and 46: Please perhaps clarify the link between anaemia and hemoglobin concentration for lay readers.

c. Line 46: Please define “subclinical” or remove the term, for clarity to a lay reader.

d. Line 48: Please define “marginally” in his context or remove the term, for clarity to a lay reader.

e. Lines 49-50 and 54: Please define “metabolic profile”, for clarity to a lay reader.

7. Please remove spaces from within citation callouts, eg “…and fish [3,4].”

8. Please replace the word “subjects” with “patients” throughout.

9. Line 114: Please give full recruitment dates, including day.

10. Line 245: Please state the primary outcomes first.

11. Results and Tables: Please include p values alongside 95% CIs, including where not significant.

12. Fig 2: Please describe in the legend what the shaded areas represent.

13. Please ensure all abbreviations are defined in Table footnotes.

14. PLOS does not permit "data not shown.” Please remove this claim, or do one of the following:

a) If you are the owner of the data relevant to this claim, please provide the data in accordance with the PLOS data policy, and update your Data Availability Statement as needed.

b) If the data not shown refer to a study from another group that has not been published, please cite personal communication in your manuscript text (it should not be included in the reference section). Please provide the name of the individual, the affiliation, and date of communication. The individual must provide PLOS Medicine written permission to be named for this purpose.

c) For any other circumstance, please contact me ASAP.

Please rename your CONSORT checklist file “S1 Checklist”.

15. When completing the CONSORT checklist, please use section and paragraph numbers, rather than page numbers.

16. Please mention in your Methods section: “This study is reported according to the CONSORT guidelines (S1 Checklist).”

17. Please rename your analysis plan and protocol documents S1 Text, S2 Text etc. Please mention in your Methods section: “This analyses in this study were carried out according to the predefined protocols and analysis plans (S1-S[n] Texts).” Please also describe any changes to the protocol that took place after the start of the study, and why they were made.

18. Lines 352-375: Please remove the Author Contribution, Conflict of Interest, Funding statements – these should be completed on the online submission form.

19. Line 376: Please rename “Additional Contributions” to “Acknowledgements”.

20. Please provide a URL or DOI for reference 21.

---

Comments from Reviewers:

Reviewer #1: I thank the authors for replying to my comments. I would only make a minor suggestion: instead of writing "crude and multiple generalized linear models" I would suggest "univariate and multivariate generalized linear models".

Reviewer #3: The authors have carefully addressed my comments, concerns, and questions. One last minor point would be that the calculation and use of 3cB12 was nicely explained in the Methods, there is no actual mentioning of "3cB12" in the Results and Discussion. Figure 2 is the main illustration where cB12 was used, which is the main Figure for End Point results (page 11). For the less familiar reader it would be beneficial to add the "3cB12' to the text to make the conception to the Methods section. Same applies to the Discussion section. Otherwise the manuscript can be considered for publication from my side.

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

[LINK]

23 Oct 2020

Dear Prof. Strand,

On behalf of my colleagues and the academic editor, Dr. Lars Åke Persson, I am delighted to inform you that your manuscript entitled "Effects of vitamin B12 supplementation on neurodevelopment and growth in Nepalese Infants: A randomized controlled trial" (PMEDICINE-D-20-00074R3) has been accepted for publication in PLOS Medicine.

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Thank you again for submitting the manuscript to PLOS Medicine. We look forward to publishing it.

Best wishes,

Emma Veitch,

Senior Editor

PLOS Medicine

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