Review for 10.1186/1471-230X-11-22 (2011)

Reviewer's report
Title:Gastrointestinal Flora and Gastrointestinal Status in Children with Autism --
Comparisons to Neurotypical Children and Correlation with Autism Severity
Version: 1Date:7 November 2010
Reviewer: Manya T Angley
Reviewer's report:
Comments 2, 4 and 6 require major compulsory revisions.
1. Is the question posed by the authors well defined?
The literature review is thorough and the research question is well defined
2. Are the methods appropriate and well described?
The authors need to state which instrument was used to diagnose the children
with autism.
Where were the controls drawn from? How was autism excluded in the control
participants?
The ATEC is not a robust measurement of autism severity . It is mainly used by
those associated with the people who developed it i.e. Bernard Rimland and
colleagues at the Autism Research Institute for the purposes of assessing
treatment effects in children with autism by assessing autism related behaviours.
It was not developed to assess autism severity per se. Other instruments such as
the ADI are much more rigorous. Please justify why you used the ATEC.
The manuscript indicates 1) that stool samples were collected and sent by 2-day
express shipping to Doctors Data (where it is assumed analyses were
undertaken), 2) that empty sterile tubes were used for chemistry testing (which
includes SCFA analyses) and 3) stool was frozen prior to sending in an insulated
bag. Our experience, and the experience of our collaborators who have decades
of experience in the collection of faeces and subsequent SCFA and
microbiological analysis, is that faecal samples should be frozen immediately and
only thawed immediately prior to processing for analysis to prevent fermentation
continuing. Failure to do so will lead to fermentation profiles that are different
from those in the faeces at the time of collection due to continued fermentation
and, in the case of prolonged periods at warm temperatures, depletion of SCFA
in the faeces will occur. It is the view of the reviewer that the transport method
used (2 days on dry ice in a bag, even though insulated) is likely to have resulted
in significant thawing of faecal samples in transit and the integrity of the sample
for SCFA analyses is therefore doubtful. In this respect the SCFA and possibly
the other biochemical analyses of faeces are not suitable for publication unless
the authors can verify that the faecal samples received following transport were
still frozen. Also, there is no indication of how long stool samples remained
unfrozen (if not frozen immediately) from the time after stool was passed from
subjects to the time it was sent to Doctors Data. Presumably someone processed
the faecal samples before transport to enable the samples to be transported.
The manuscript does not indicate whether the faecal sample was a one-time
collection or a pool over say 24 h or 48 h.
Another point of concern relates to the method by which SCFA have been
measured. The method used should be explained in more detail, especially since
the method that appears to be used is not a common method. Indeed the
method, using the papers described by Collin et al., have scarcely been used in
the years since they were published in 1974! A search of papers (eg. ISI Web of
Science) will show very few citations. However, this is not to say that the method
does not produce accurate results. The authors should therefore provide a more
detailed explanation of their analysis and give reassurance of its accuracy in their
hands.
3. Are the data sound?
It is difficult to know given the lack of detail supplied in the methodology re
transport and measurement of SCFAs
4. Does the manuscript adhere to the relevant standards for reporting and data
deposition?
Somewhat - there is not detail regarding what type of organisation Doctor's data
is
5. Are the discussion and conclusions well balanced and adequately supported
by the data?
Yes - although there are significant methodological issues that require
clarification.
6. Are limitations of the work clearly stated?
There is no evidence to support the statement "Internal studies by Doctor’s Data
have shown good recovery of organisms up to 14 days after collection." and this
is a limitation that is not stated. The limitations of the ATEC need to be clearly
stated.
7. Do the authors clearly acknowledge any work upon which they are building,
both published and unpublished?
Yes
8. Do the title and abstract accurately convey what has been found?
Yes
9. Is the writing acceptable?
Yes
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
Non-financial competing interest
Our group have recently completed similar work which has been presented at 2
conferences
1. Wang LV, Christophersen CT, Gerber, CP, Sorich MJ, Angley MT, Conlon MA,
(2010) Gut bacterial and fermentation profiles are altered in children with autism,
Australian Gastroenterology Week (AGW), Gold coast, Australia, and
2. Wang LV, Christophersen CT, Sorich MJ, Angley MT, Conlon MA, (2010)
Propionic acid concentrations are elevated in the large bowel of children with
autism. 2nd Nutritional Genomics Symposium 'A Healthy Start to Life' , Adelaide,
Australia.
We will submit a manuscript in the next fortnight outlining our findings.