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: 26 July 2012
Reviewer: PrashanthThankachan
Reviewer's report:
Major comments
1. 1) The introduction should provide data on the current burden of
micronutrient deficiencies especially that of iron, zinc, vitamin A and Iodine
and establish the need for fortification with these nutrients. In the current
version, the reader has to assume that these deficiencies are high as no
prevalence estimates are provided, or read through the references provided
to get this information.
Response: The introduction has been modified as suggested.
1. 2) The rationale for doing this study as stated is “To test new vehicles for
better effectiveness”. The introduction until here does not speak anything
about the need for such a vehicle and as a result the introduction is not
coherent with the question being asked.
Response: The introduction has been modified so that the rationale and
need is clear.
1. 3) The introduction is very generic and does not bring forth the
background of the problem being addressed, it is more suited for a paper on
the benefits of fortification. It would be more appropriate to state the problem,
what has been done so far in Bangladesh with regard to this problem and
then state why the authors feel yogurt may be a vehicle that is worth
studying.
Response: The introduction has been modified to clearly state the question
being addressed and information on the findings so far from Bangladesh has
also been added. The rationale has been clarified that study was testing what
had already been established or in process of being established.
1. 4) The authors state 33% of the RDA was chosen as the level of
fortification. What was the basis of this? Did the authors do % inadequacy
calculation for the nutrients being fortified in the population?
Response: This has been clarified in background, the selection of product
and levels of fortification were pre-decided and study was only evaluating a
product, which was being introduced. The levels of 1/3rd RDA we were told
were based on multiple considerations, regulatory requirements, concerns
that in actual practice multiple fortified products may be consumed and
practical question was to evaluate the idea within efficacy framework.
1. 5) My concern about the report is the insufficient information about the
fortificants used. It would be useful to have the information in a table with
columns for a) nutrient description (that further specifies the chemical form
used, e.g., iron as ferrous sulphate), b) amounts of each nutrient by weight,
and c) amounts expressed as % of recommended intake, d) the same
nutrients contributed from the yogurt. In addition, it should specify other
ingredients, if any is added during the fortification process along with the
number of total kcal, protein and fat per serving.
Response: A table has been included in the manuscript detailing the nutrient
description and the amount of each nutrient present in the yoghurt cup that
has been provided by the study to the participants both in fortified group and
unfortified group the product as already described is a product that is being
marketed and information is available on web. Some of the details may be
proprietary and not available as the product is a Grameen-Danone
collaborative product but composition is available and has been presented in
the paper. No other nutrients were added during the process.
1. 6) The study was for a period of one year, however, the methods
sections indicate that the study was conducted between June 2008 and
March 2010.
Response: The intervention was provided for 12 months but all the
children in all the four schools were not enrolled at the same time. In
such a study to conduct baseline assessments and then enroll children
takes time and because of phased enrollment and then follow up of 12
months and similar staggering at end study the timeline of study is
reflected.
1. 7) The methods section specify that the study was conducted in 6
schools, however data has not been used in the present paper from 2
schools and therefore the significance of the 2 schools that was enrolled has
not been specified. If there was no intent to use the data from the children in
these two schools. From an ethical perspective what was the need to enroll
them in the first place as the fortified and unfortified yogurt was identical in
packaging, appearance, taste and smell. Was a concurrent control group
needed for this study?
Response: The study effective was a concurrent implementation of two
studies with two different questions. The blinded two group study was
evaluating the effect of micronutrient fortification as both groups got same
yoghurt (which is the question of this paper), a second question was to
evaluate a cumulative impact of yoghurt and micronutrients which need a
pure control without yoghurt. In those children for ethical reasons we did not
collect blood sample and so do not have data on status markers. This paper
addresses one question from this project and the sample relevant to that
question has been presented. Obviously there will be other analysis from the
project addressing different question, the sampling frame and data collection
needs to be understood in that context.
1.
8)
Ethical approval document numbers is missing and based on
information from Clinical Registry, it appears that data/results on several
other primary parameters that were measured have not been reported in this
manuscript. If this paper is pertaining to a part of that data/subset analysis,
reference to the main manuscript be provided and stated explicitly in the
methods section that this manuscript refers to only a part of a larger study so
that the reader has access to this information. Also inconsistencies are seen
between the information on the study as registered and as given in the this
manuscript, Eg: 30% RDA for fortificants in the registration document vs 33%
in the manuscript
Response: The trial has been registered with the Clinical Trials.gov. The
clinical trial registration number is NCT00980733. It is customary to provide
the registration number and not IRB document numbers in manuscript. This
trial, which as has been explained earlier essentially, was conduct of two
trials concurrently. The project document and registration is combined for
both and that is why reference has been made to pure control. However
there is no main study result and sub-study analysis or secondary analyses,
there are questions to be addressed and each question or set of questions is
reported in a manuscript. The growth data and blood data were only part of
the two groups presented here and all the date from the trial for those
outcomes is presented here. The inconsistencies relating to the %RDA of the
fortificants have been rectified and corrected to 30% RDA in the manuscript.
This is because of mention of 1/3 RDA, we have rechecked from the
company website and made all consistent to reflect 30% of the RDA.
1.
9)
The manuscript states that blood and urine samples were
collected from all children at baseline mid and at the end of the study. The
authors need to clarify if samples were taken from the 2 schools that served
as concurrent controls? Data is also not reported on the mid point
measurement for the FY and NFY groups, please add these.
Response: As already stated 2 schools that served, as pure controls were
not part of this question in these children blood/urine sample was not
collected and weight and height was not measured. The intent for data for
midline collection was to evaluate if fortification trials can be of a 6-month
duration. These data to not contribute to the present question and intent is to
at some point present it as a sub analysis to make a point you do not see
changes at 6 months that you see at 1 year. If we included that analysis in
this paper it will double the number of tables and increase the text and dilute
primary analyses and that is why has not been included.
1.
10)
If the separated plasma was stored in two aliquots, that was
used for the analysis of tfr, ferritin, RBP, Zinc, CRP and AGP, a total of 6
assays, one aliquot was probably sent to Germany and the other to
Chennai, India. How was the analysis carried out such that freeze thaw
stability issues were avoided from a single micro centrifuge tube?
Response: The zinc measurements were done in Annamalai university,
Chennai, India with one of the aliquots. The second aliquot was sent to
Germany where all the other assays were done using a sandwich ELISA
which simultaneously measures RBP, Ferritin, CRP, AGP, and Tfr (Erhardt
JG et al, 2004, J Nutr, 11:3127-32).
1.
11)
Sample size-The main outcome variables considered for
sample size computation are height and weight. But the computation is for
change over time and not for comparison between group which is a
deviation from the study design. The data for analysis has been chosen
based on availability of blood at endline. This does not correspond to the
variables considered for sample size. How much difference between group
was expected to arrive at sample size in each group
Response: The study sample size section has been rewritten to clarify
what was done and also a supplementary table (Supplementary Table 1) of
sample size calculation has been provided which can be included a
supplementary table. Sample size was not based on weight and height but
on a matrix of all parameters, the assumptions we had may not have
withheld but post facto power calculation is not recommended.
1.
12)
Statistics-This section states that ITT was employed but only
those children consenting for blood at endline were included for analysis and
this is nearly half of the entire sample. This is not ITT. Did all children
consenting to give blood give blood at endline? When was this consent
obtained? Why were 3 different software’s used for analysis when all of them
can perform all the analyses mentioned.
Response: Intent to treat analysis aims to include all data available
irrespective of compliance and that is what had been done, we did not impute
missing data as we feel that has a potential to introduce more bias.
Consent to participate was taken at baseline but as part of that consent
children could refuse to participate and withdraw at anytime and there were
withdrawals at 2nd and 3rd blood sampling. However there were no
differences in baseline parameters between those consenting and those
refusing. Use of three software’s was a preference issue of individuals
however we have streamlined that by repeating it in same software for reconformation.
1. 13)
Baseline observation corrected analysis not mentioned in
statistical methods
1. 14)
No report of paired analyses mentioned in the statistical
methods.
Response: For 13 and 14 comment, the suggested change has been
incorporated in the statistical method section to clarify a paired analysis was
performed.
1.
15)
Compliance data is missing from the text and tables. What
was the % of children that reported to the intervention in both the
groups?
Response: Compliance was a total of 97.16%. This information has been
added in the text.
16. 16)
In the result section, anemia prevalence in the present study
population has been stated as 53%.However, similar prevalence estimates
for iron deficiency in % be given instead of mean Hb, ferritin and tfr. How did
these prevalence estimates change in the FY and NFY groups at the end of
the study
Response: A separate analysis has been performed using cut off values and
we are providing this as part of the Supplementary table 2. It can be added
as a web appendix if you think it necessary.
16. 17)
The last sentence on the findings of biochemistry seems to be a
repetition.
Response: The sentence has been corrected.
16. 18)
Table 1-This table is for the entire sample. What about the
subset considered for the study? This should be presented as well. Is this
subset different from the rest of the sample?
Response: The baseline table for the subset contributing to study was not
any different and a table has been provided in supplementary tables as
Supplementary Table 3.
16. 19)
In Table 2 infection column is empty, also provide footnote to
describe the different forms of AGP data. The units are also confusing for
CRP/AGP (mg/L) (n,%).
Response: The table has been rectified. Infection for the present analysis
has been defined as CRP >5g/L or AGP >1g/L. CRP >5 g/L is defined as
incubation while AGP >1g/L is defined as convalescence. We have
change the table layout to make it more understandable. The mg/L refers
to the CRP or AGP values whereas the n % refers to the number of
children with the values given in brackets.
16. 20)
It would be better to provide the mean± SD data at
endline measures alongside the variables of table 2
Response: Formatting wise as the second reviewer has asked to
combine the tables we may not be to show the mean difference and
CI in the combined format. But for your reference, a table for end
line measures is added as Supplementary Table 4.
16. 21)
From Table 3, it seems that both ferritin and total body iron
stores improved in the non fortified group, was this baseline to endline
change significant in the non fortified group.
Response: The baseline to end line change was not significant in the non
fortified group. The p value for paired difference between baseline and endstudy body iron store in the NFY arm was 0.35. The p value for paired
difference between baseline and end-study ferritin in the NFY arm was 0.35
16. 22)
Table 3: Can be clubbed with table 2. RBP expansion given in
table 2. SF and Iodine seem to be not normally distributed. TTest may not be
appropriate. Examine normality for the difference from baseline as these data
are used in ttest to compare between groups. P value cannot be 0.00. If very
low it has to be reported as p<0.001.
Response: Change in ferritin and iodine were normally distributed. The
necessary correction has been made.
16. 23)
Table 5: weight CI wrong. Can be clubbed with table 4
Response: Now Table 6, the CI value has been corrected.
16. 24)
The discussion need to bring forth mechanistic approaches with
regard to yogurt and the fortificants used in this population, for Eg: the use of
ferric pyrophosphate as the fortificant in yogurt needs to be discussed with
regard to its relative bioavailability and the fact that milk and milk products
are inhibitory to iron absorption, may have resulted in no impact on iron
status markers in this study.
Response: Discussion has been modified in the light of the above
suggestion. This has already been stated in response to the comments of the
1st reviewer.
16. 25)
Milk and yogurt have also shown to increase zinc absorption
by as much as 78%, this needs to be discussed (J.Nutr, 2005, Vol 135,
No3, 465-468)
Response: Discussion has been modified in the light of the above
suggestion.
16. 26)
In the discussion it is stated that the iron and zinc ratios are
1:1, are these molar ratios? And at 33% of RDA are the iron and zinc
molar ratio 1:1?
Response: Yes the molar ratios of iron and zinc are 1:1, Yes, 30% RDA
(33% RDA corrected to 30% RDA) the ratio is 1:1.
16. 27)
Based on the data, the study population is not iron deficient
and only 1% vitamin A deficient and only some children deficient in Iodine,
discuss if there is a need to fortify with iron, Vitamin A and Iodine in such
populations.
Response: An explanation why the study was conducted in this population
has been added to paper and responses, the results would suggest that it
has some benefit, the benefits observed are conservative estimate of those
that would be observed else where. Usually the criticism is reverse that
researchers find a highly deficient population and demonstrate an impact and
advocate use of intervention. In this study we had a simple paradigm, there is
a factory established in Bogra and first area of distribution will have to be that
area; what is the impact in that area? Then the secondary question was what
is its implication for rest of country and world. By that logic there is some
benefit and that benefit is going to be more in places with more deficiency.
16.
28)
The discussion states that some children were iodine deficient
at baseline, the same can be provided in the results section along with other
prevalence estimates. Also discuss the use of iodine as a fortificant in this
study in the background to existing universal salt iodization in Bangladesh.
Response: Necessary modification has been incorporated
29) The conclusion should be specific to this study and explicitly stated with
regard to the significant biochemical/anthropometric changes seen both in
the abstract and discussion.
Response: The conclusion has been changed as per suggestion.
Minor comments
1. 1) Space to be given after the subscript in the affiliation of authors
Response: Corrections have been made
1. 2) Page 12 line 2 and 3 space after after “was”
Response: Corrections have been made
1. 3) Page 13, line 8, space to be added after ;
Response: Corrections have been made
1. 4) Page 16, line 16, space after ref 40.
Response: Corrections have been made
1. 5) Tables space before the bracket ()
Response: Corrections have been made
Level of interest: An article of importance in its field
Quality of written English: Needs some language corrections before
beingpublished
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests'