Reviewer's report
Title: Impact of micronutrient fortification of yoghurt on micronutrients
statusmarkers and growth - a randomized double blind controlled trial among
school children in Bangladesh
Version: 1 Date: 16 July 2012
Reviewer: ParminderSuchdev
Reviewer's report:
This trial addressed an important area of research, evaluating the effects of foodbased fortification on the micronutrient status and growth of school-aged
children. There are several methodological and presentation concerns with
manuscript, including numerous grammatical errors, which made the manuscript
difficult to read. In addition, the ~45% drop-out rate between baseline and followup is very high, which makes the findings difficult to interpret. Also, the
conclusions of the authors do not appear to be supported by the data presented.
More detailed comments follow.
Major Compulsory Revisions
1.1. There is insufficient rationale provided by authors for conducting the study
in the first place (given that other food-based fortification interventions, including
micronutrient powders, have already been shown to be efficacious). Why was
yogurt chosen as a vehicle for food fortification (especially when calcium content
may interfere with absorption of iron and other micronutrients)?
Response: The introduction has been modified as per the given suggestions to
clarify the rationale of the study. In Bangladesh, yoghurt is a readily available and
consumed food. Based on this premise Grameen in Bangladesh in collaboration
with social responsibility division of Dannon have established non-profit social
sector factory to produce yoghurt at low cost for local consumption in Bogra
Bangladesh, Global Alliance for Infant Nutrition (GAIN), used this opportunity to
use that as a vehicle for delivery of 4 important micronutrients and what emerged
was a product with micronutrient fortified yoghurt (33% RDA of iron, zinc, vitamin
A and iodine). The study was undertaken to evaluate potential impact of this
intervention in an efficacy study to establish proof of principle. The second
question regarding calcium (which we do not totally agree with) actually
underlines a need to evaluate a vehicle even when other vehicles may have
been shown to have an impact or no impact. Again we have clarified that there is
a need for multiple vehicles for deliver in public health setting given varied food
preferences and consumptions.
1. 2) The micronutrient composition for the fortified yogurt needs to be
described (list of micronutrients and dose). It would also be useful to know how
this differs from naturally occurring micronutrient in the non-fortified yogurt.
Response: The composition table in the paper has been modified to include
comparison of fortified and control yoghurt. The two were identical except for the
4 micronutrients.
1. 3) The sample size calculations are not clear, since as described it appears
that these were post hoc power calculations rather than the sample size needed
in each treatment group to detect a pre-determined difference (effect size) in the
outcome variable of interest.
Response: The sample size was estimated prior to the start of the study and
the table providing data on this is included as part of supplementary table 1
about which editors can make a decision regarding inclusion in supplementary
tables. The description in the paper has been modified to reflect this more
clearly.
1. 4) Several concerns are present with the analysis. First, additional
description is needed on how the authors accounted for the effects of
inflammation on biomarkers of iron and vitamin A status. Was this using an
internally derived correction factor as propped by Thurman, et al? Why were
RBP values not adjusted for inflammation? Second, the analyses looking at
mean changes in biomarkers rather than prevalence of deficiency may not be
clinically relevant and affect the authors’ conclusions. The authors go back and
forth between the use of % and mean values, which is confusing to follow.
Response: The reviewer is right for accounting for impact of inflammation we
did Thurman et al method, but the etiological fraction of the inflammation is so
low that it did not change anything therefore we rather than using estimated
numbers which may or may not be valid used original and described that results
did not change upon applying correction. We agree also that sometimes looking
at prevalence of deficiency may be helpful especially if there is a tail effect on
the other hand if there is not a tail effect but shift in distribution tail effect may
underestimate the effect. We are providing the cutoff data table as part of the
supplementary tables (Supplementary table 2) again for review and editorial
decision regarding its inclusion in supplementary table.
1. 5) The conclusions stated in the abstract are not consistent with those
listed in the paper. The conclusion in the paper as stated (that fortification of
yoghurt is efficacious) does not appear to be supported by the data, given the
lack of impact on iron, vitamin A, or iodine biomarkers. In fact vitamin A and
iodine status were worse at end line in the fortified yogurt group.
Response: In a controlled trial the change in intervention is comparison to the
control group. In event of a decline in both groups a substantially lesser decline
in the intervention group is also an indicator of efficacy of the intervention.
However, in follow up to the advice of the reviewer the conclusion has been
moderated more consistency established between abstract and paper.
Minor Essential Revisions
1) Abstracta. a. 2nd and 3rd sentences need to be combined (grammatical error).
Response: Sentences modified as per the suggestion.
a. b. Would be useful to include sample size in methods as well as
composition of fortified yogurt.
Response: Sample size as well as composition of fortified yogurt has been
included in the abstract.
a. c. Methods state that only blood samples were collected when in fact
urine samples were also evaluated for urinary iodine.
Response: Necessary change has been done as per suggestion.
a. d. Results, 1st sentence: unclear use of the term “status markers”
Response: Sentence has been modified as suggested.
a. e. Results: confusing to present some findings as % and others as mean
values. Data should be presented as % above or below a cut-off, since this is
more useful to the reader.
Response: As explained in earlier response the distribution shift in this
case is important and we are already providing cutoff data in
supplementary table, the abstract has been made consistent and mainly
reflects difference in means.
a.f.
Results: it is unnecessary to include acronyms for WAZ and BMIZ,
since these are not used again in the abstract.
Response: Acronyms are deleted as suggested.
a. g. The conclusion is confusing as written. It is unclear why the impact
of the intervention on iron status cannot be evaluated.
Response: The conclusion has been reworded. The impact of
intervention on iron status is not expected and hence cannot be evaluated
for example in comparison to a population with higher deficiency level.
2) Backgrounda. a.
b. b.
Line 1- “remains” should be changed to “remain”
Response: The background has been changed.
a. c. 1st paragraph, sentence “Such deficiencies can have far-reaching….”
Needs to be re-written.
Response: The background has been changed.
a. d.
Sentence “Previous research suggests…” needs a reference.
Response: The background has been changed.
a. e. Statement that “new vehicles need to be tested for their better
effectiveness” seems to be out of place given citation of systematic review of
fortified foods in previous sentence.
Response: The back ground has been changed.
a. f. Last paragraph- list of micronutrients and doses in fortified yogurt
should be listed
Response: The list of micronutrients with its dosage in the fortified yogurt
has been mentioned in a table 1.
3) Methodsa. a. Unclear why 2 schools were assigned as “pure control.” What does this
mean?
Response: The main reason for assigning 2 schools as “pure control” was to
enable evaluating combined benefit of consumption of fortified yoghurt in
comparison to non-consumption of yoghurt. There was interest from sponsors
that in a RCT though we would evaluate the effect of micronutrient fortification
but we may miss the impact of yoghurt and nutritional benefit which was one of
focuses of the Grameen Danone project to begin with.
a. b. Study population: Would be helpful to list anemia prevalence and
prevalence of malnutrition in general in target population. Why did the authors
choose to do the study in this area?
Response: The data for anemia and malnutrition in this population was not
available, which was one of the reasons we did not know low prevalence of iron
deficiency in the study population. The population was selected as the factory
was established in Bogra and that was supposed to be the primary population
where the yoghurt would be distributed and so there was interest in undertaking
study in that population rather than selecting a known deficient population in
some lower socio-economic slum. It was primarily as sponsor decision but made
sense in terms of external validity of immediate results.
c. c. Sample size- as stated earlier, this appears to be post-hoc power
calculation, rather than sample size estimate prior to analysis. Were these
calculations done to estimate changes in the intervention group vs. control
group?
Response: Responded earlier and table is provided.
c. d. Consent- How was “severe malnutrition” defined for exclusion?
Response: “Severe malnutrition” was defined as a clinical diagnosis made by
a physician that child needed nutritional rehabilitation or required
hospitalization or special treatment. We made it little broader than grade 3
malnutrition as other clinical factors may be important at times. However no
such child was encountered.
c. e.
Yoghurt distribution- what % of children received yoghurt at home vs.
school? Were results compared by this factor?
Response: Both the groups received yogurt in school during regular days,
only on holidays and if the child was absent from school yoghurt was
distributed at home. Thus percentage of receiving at home is not percentage of
children but the percentage of total days of follow up when children received
yoghurt at home. A segregated analysis of this is not possible and
methodological does make any merit. The distribution was similar in both the
groups (84 % of days in FY and 83 % of days in the NFY group received
yogurt in school and in14% of days vs 13% of days received at home).
f. f.
Height measurement- use of tape vs. height boards is unlikely to be valid
measurement. Please comment. How were height measurements validated (e.g.,
it would be helpful to list SD of these measurements for assessment of data
quality and compare to WHO standards). See: World Health Organization.
Physical Status: The Use and Interpretation of Anthropometry. Report of a WHO
Expert Committee. Technical Report Series No. 854. Geneva: WHO, 1995
Response: Microtoise tape for school age children is a valid tool for measuring
height (Ahmed et al, 1990; Voss et al, 1990) and has been extensively used in
number of studies conducted among school age children (Ghattu et al, 2011;
Banjong et al, 2003). We have used WHO standards for assessing growth
parameters (WHO, 2006), reference of the same has already been provided in
the manuscript. Below are some of the references from this area. We are fairly
confident about the measurements which were undertaken with appropriate
reliability established in training phase and two measurements taken each time.
Voss LD, Bailey BJR, Cumming K, Wilkin TJ, Betts PR. The reliability of height
measurement (The Wessex Growth Study). Archives ofDisease in Childhood
1990; 65: 1340-1344.
Ahmed ML, Yudkin PL, Macfarlane JA, McPherson K, Dunger DB. Are
measurements of height made by health visitors sufficiently accurate for routine
screening of growth? Archives of Diseases of Children 1990;65(12):1345-1348.
Ghattu V Krishnaveni, Sargoor R Veena, Nicola R Winder, Jacqueline C Hill,
Kate Noonan, Barbara J Boucher, Samuel C Karat, and Caroline HD Fall.
Maternal vitamin D status during pregnancy and body composition and
cardiovascular risk markers in Indian children: the Mysore Parthenon study. Am J
ClinNutr. 2011; 93(3): 628–635.
Banjong O, Menefee A, Sranacharoenpong K, Chittchang U, Eg-kantrong P,
Boonpraderm A, and X Tamachotipong S. Dietary assessment of refugees living
in camps: A case study of Mae La Camp, Thailand. Food and Nutrition Bulletin,
vol. 24, no. 4 © 2003, The United Nations University.
WHO Multicentre Growth Reference study Group. WHO Child Growth
Standards:Length/height-for-age, weight-for-age, weight-for-length, weight-forheight and body mass index-for-age:Methods and development. Geneva. Who:
2006
f. g. Definitions: There appears to be a missing “-“ in the formula for iron stores
Response: Correction has been made in the manuscript.
f. h. There is known association between inflammation and RBP, and
typically a similar approach as taken for ferritin (exclusion or adjustment) is
taken for RBP. However, this does not appear to be the case here. Why?
Response: As already stated we did evaluate this and as the etiological
fraction was so low that exclusion or correction was making no difference and
so we decided to present actual data which is more clean and less biased. But
the results or conclusion did not change.
f. i.
Definition of inflammation: Authors state that depleted iron stores were
defined as low ferritin where CRP and AGP were normal. Later, they state
concentration of elevated CRP and AGP were considered an indication of an
acute response. Do they mean “or”?
Response: Yes it is ‘or’, correction has been made to this Typo.
4) Results
a. a. Figure 1: Would be helpful to list number of children excluded due to
malnutrition, severe illness, or other conditions as stated in the methods. The
numbers of children in the last 2 boxes for each study arm are repeated, so one
of these could be deleted.
Response: As stated earlier there was no child found with exclusion criteria
and hence only exclusions were refusal to consent that is why there is only one
box. Though the numbers in the last two boxes are identical the last box has
been included to give the reader understanding that no exclusions were made
for non compliance or for any other reason in line with CONSORT guideline.
a. b. Tables 2 and 4 could be merged
Response: The tables have been merged and now labeled as Table 3.
a. c. Baseline data: Sentence “…this was not affected by even if the children
with infection were removed…” needs further explanation. How adjustment for
inflammation performed? Would be helpful to show these results.
Response: As already explained and as is evident from the data the
etiological fraction is so low that it is immaterial and therefore we do not feel
elaborating on this would contribute. It is a potential confounder, which does
not exist in this data, it merits explanation where it exists and we estimate
magnitude of change. In fact in our experience from multiple community-based
studies it is rather overrated as confounder. However language has been
changed to make it clear.
a. d. Table 1: unclear what the categories for Type of House mean; Age is
listed 2 decimal places, while all other figures are to 1 decimal place
Response: The type of house refers to the material used in the construction of
the house and whether it is a permanent structure or a temporary structure. Use
of baked bricks; concrete or sheeted house vs the house built with unbaked
bricks/mud and thatched, or a combination of both. This is used as a standard
variable or component for socio-economic status more reliable than stated
income in this part of world. Age also has been now reported to 1 decimal place
to be consistent in table 2 (earlier Table 1).
a. e. Table 2: Rather than presenting means, it would be more helpful to
present % above or below cutoffs for defining abnormal values. Also, it appears
that the children presented in this table are only those who were followed to endline, rather than all of those enrolled at baseline. This is in contrast to the
description in the Results section text. CRP and AGP should use the word
“elevated”, since these are % values, not means. The use of “infection” should be
replaced with “inflammation”
Response: This has already been addressed and a table with cutoffs has been
provided as supplementary table 2. The impact table is a paired analysis of the
difference of difference and for that reason can only include the paired data.
Other suggested changes have been made.
a.
f.
Compliance: would be helpful to state actual compliance
Response: The overall compliance was ~97.1 % and this has been stated in the
compliance section of the manuscript also.
a. g. Effect on micronutrient status: Application of correction for inflammation
needs to be described as stated earlier. Unclear what is meant by “etiologic
fraction of infection/inflammation.” This was not assessed by the study.
Response: This has been stated and explained earlier we appreciate that
reviewer may be getting confused with etiological fraction – it does not need to
be measured separately, in simple terms it means that actually the number of
children who have infection/inflammation is low and then out of those the
association with elevated levels of, let us say ferritin is low that net effect
becomes negligible. Definition-wise it means what part of elevated status market
is attributable or potentially caused by infection/inflammation being present.
a. h.
Table 3: Unclear how Mean Diff was calculated. Table needs a legend.
a.
i.
Effect of growth: Unclear how the authors corrected for
baseline measurements. This needs to be described in the methods.
There is no description of any multivariate analysis.
Response: The results analyzed are the difference of paired
differences, which are corrected for baseline differences. Changes
have been made in the methods to make it clearer.
5) Discussion:
a. a. 1st paragraph- Decline in excretion of urinary iodine is presented as a
positive finding, which is confusing and should be re-written
Response: The sentence has been rephrased to be clearer.
a. b. In addition to the low intake of micronutrients (which are not detailed in
the report), another hypothesis to explain the lack of impact could be calcium
and other inhibitors of iron absorption present in the yogurt.
Response: It has been reported that calcium intake can inhibit the
absorption of iron but the amount of calcium provided in this study was well
below the reported dosages of 300 mg that may affect iron absorption, hence
this possibility seems unlikely. Added one needs to keep in view that status
at baseline, there seems to be some more reports from the area and seems
water has high iron and that is causing the profile. We have undertaken and
reported milk fortification study in India (published in BMJ) and shown fairly
robust impact on iron status so are very confident calcium is not the reason.
a. c. 3rd paragraph- would be careful with using terminology “incidence” of
inflammation, since this was only measured at specific time points, and we do not
know the inflammation status of the children over the entire study period.
Furthermore, “chronic inflammation cannot be ruled out”- this is unlikely to be
true, given the measure of AGP in the study.
Response: The sentence has been rephrased. However with AGP and CRP one
rules out recent or ongoing infections and they are important for elevation of
acute phase proteins like ferritin. Presence of chronic infection would only cause
more anemia and anemia profile in population is not clinically consistent with that
hypothesis.
a. d. Hypothesis that Hb impact may be due to increased mobilization of
iron secondary to fortification with vitamin A and zinc, does not appear to
be supported, since mean levels of these micronutrients did not improve in
the fortified group.
Response: Zinc and Vitamin A are co-enzymes and the impact does not
have to be necessarily via improved status and improved availability may
not be reflected by status markers. Yet there was a status difference for
Vitamin A and marginal for Zinc so we believe that possibility can not be
excluded and may be the most plausible one which results in better
utilization of iron. However we agree there are only hypothesis and that is
why we have kept the wording toned down and given as a “possible”
explanation.
a. e. There is no discussion of study limitations in this Section.
Response: A Short paragraph on the limitation of the study has been
added. The major limitation of the study was that the study population was
not severely deficient of the micronutrients that was added in the yoghurt.
a.
f.
Conclusions- 1st paragraph- seems to be out of place and
should be moved/deleted. Final paragraph does not seem to be supported
by data and should be carefully reworded.
Response: The first paragraph and final paragraph have been reworded.
1.
6)
Author contributions: would be helpful to state who wrote the first
draft of the paper
Response: Author contributions have been revised as suggested.
Discretionary Revisions
None
Level of interest: An article whose findings are important to those with
closelyrelated research interests
Quality of written English: Not suitable for publication unless extensively edited
Statistical review: Yes, but I do not feel adequately qualified to assess
thestatistics.
Declaration of competing interests:
I declare that I have no competing interests.