Reviewer ’s report  
 
Title: Relationship between mucosal healing by tacrolimus and r elapse of refractory ulcerative 
colitis: A retrospective study  
 
Version: 1 Date: 01 Dec 2019  
 
Revi ewer: Holger Schäffler  
 
Reviewer's report:  
 
The medical therapy of UC can be a challenge, although new treatment options, especially 
biologica ls and small molecules do exist. The role of tacrolimus in the treatment of UC has been 
studied before, however, if mucosal healing is a valid treatment option in UC patients under 
tacrolimus is still under debate.  
 
Although the study was designed in a re trospective manner, several interesting observations 
could be made by the authors. However, in the present form, the manuscript cannot be 
recommended for acceptance. If several major and the minor points are adequately addressed, 
reconsideration may be giv en. 
 
 
General points:  
 
1. Statistical analysis is insufficient, statistical consult is recommended.  
 
2. Data presentation needs scrutiny. While the authors nicely describe the design of the 
study with three time points, data presentation is somewhat confusing with tables for 
the first two time points and graphs for the third time point.  
 
3. Translation by a native speaker is recommended.  
 
 
Specific points:  
 
Page 3:  
 
Abstract/Methods: It should be stated that a retrospective study was performed, it should be 
stated that a case -control study was performed. Single center should be mentioned.  
 
Abstract/Results: Restructuring is required. Suggestion: Baseline, end of treatm ent, follow up. It 
should be stated what the difference was for the parameters that were "different". What do you 
mean with "There were also significant differences and the steroid -free rate"?  
 
Line 31: Patients with MH at the end TAC -&gt; end of TAC  
 
Page 4: 
 
Line 4: The first word of the introduction should not be an abbreviation. I would suggest to 
write: the prevalence of UC has increased instead of "the number of patients".  
 
Line 7: "Surgery is indicated", rephrase "is" to "may be", please indicate that surgery is the 
option for severe subtypes of UC and indicate the incidence of this treatment for UC  
 
Line 16: Surgery "is", rephrase to "may be"  
 
Please rephrase this first paragraph or have it redacted professionally.  
 
Line 22/23, please delete the number "5 2.6%", or put this number into perspective.  
 
Line 28: "which" is missing, rephrase "important issue"  
 
Line 47: Please specify the type of your department, tertiary care? Reference center?  
 
Line 50: follow up period should be expressed as median, not mean, pl ease indicate the interval, 
please specify the name of the prescribed drug and the supplier.  
 
Line 56: Please indicate the measuring method (fluid phase chromatography, LC -MS/MS ?) 
Please indicate the sampling method (plasma, serum, whole blood, was a chela tor used?)  
Page 5:  
 
Line 10: bpatients to patients  
 
Line 19 -22: Avoid: "so all patients had prednisolone -dependent or prednisolone -resistant 
refractory UC."  
 
Line 25: It should be clarified that endoscopy was done before TAC, it should be stated the 
median ti me from last endoscopy to TAC initiation  
 
Line 35: It should be stated if the Lichtiger score was determined at each visit or if it was 
calculated retrospectively. Please also reference the Lichtiger score as the Modified Truelove and 
Witts Severity Index.  
 
Line 50: "total dose of prednisolone up to remission", it is difficult to understand that remission 
was achieved in the hospital, please clarify. Was there any other treatment than prednisolone 
before TAC?  
 
Page 6:  
 
Line 1: what are the ranges for the specif ied time points?  
 
Line 16: It should be stated that surgery was not required if this was the case.  
Line 25: Consultation of a medical statistician is highly recommended. Please test your data for 
normality and please provide appropriate statistical analysi s and data presentation. Why is a 
nonparametric test used but a parametric measure provided in the table? How about multivariate 
testing? Which test was used to test for differences in relapse rates?  
 
Line 37: Please provide the approval number  
 
Page 7:  
 
Line 16ff: when was the last endoscopy before treatment? Time to target trough levels is not 
possible before therapy. Total dose of prednisolone is misleading at this point. Looking at the 
table I realize at this point that the patients were not outpatients bu t inpatients. Were all patients 
started on TAC in the hospital? This should be stated in the methods section.  
 
Line 38: MN group?  
 
Line 44: Please specify which number belongs to which group.  
 
Page 8:  
 
Line 25: It should be described in the methods section, how the occurrence of AEs was 
determined. Was there a systematic screening for these "adverse events"? Was it patient 
reported?  
 
Line 28: adverse instead of advanced, significantly instead of significa nt, different instead of 
difference, rephrase this sentence  
 
Line 32: adverse  
 
Line 41: "features" is not applicable  
 
Line 47: rephrasing recommended, source recommended  
 
Line 56: The authors should cite the work of Ogata et al 2016 IBD. This study was a place bo 
controlled study of Tacrolimus for refractory UC and Tacrolimus therapy was associated with a 
higher  44% MH rate. In the following, the authors should discuss their findings in perspective to 
this paper.  
 
Page 9:  
 
Line 22ff: This is highly speculative an d unlikely. This speculation should be weakened  
significantly and other reasons should be discussed - different biology of the underlying disease? 
If the authors want to keep this claim, they have to show that trough levels were lower in this 
group, which  would prove incompliance.  
 
Line 37ff: Did the NMH group receive more other drugs. Authors have to show data on this if 
they want to keep this claim. Were clinical symptoms improved in the NMH group over the MH 
group? The data do not suggest this.  
 
Page 10:  
 
Line 13: Authors do not report on stool measures such as Calprotectin. It should be discussed, 
why.  
 
Line 19ff: This is one major weakness of the study. It has to be explained, why the treatment 
duration was different. Is the time from TAC initiation to sco pe different? Whithout this data, the 
following speculation is pointless.  
 
Line 35: of instead of if; combined use of AZA should be explained. The authors describe their 
finding that "combined use of AZA" was not different between the two groups. From this,  they 
conclude that it "was unclear whether AZA contributed to promoting MH". This claim may be 
true, but it cannot be made from the data.  
 
Line 38: The authors could speculate a little more about the reasons for the lower steroid free 
rate in the NMH group . They should state that tapering was not possible in a large fraction of the 
NMH group, if this was the case.  
 
Page 11:  
 
Line 10ff: It would be interesting to see how many patients were steroid free at 100 days, e.g. 
was the disease activity masked by the s teroids? The numbers do not need to be repeated, they 
are described in the results section.  
 
Line 32: Previous reports on TAC in UC need to be acknowledged and the results discussed in 
this context, for example Baumgart et al. 2003 Aliment Pharmacol Ther, S chmidt et al. 2016 
JCC, Ogata et al 2016 IBD, Schmidt et al 2013 Aliment Pharmacol Ther.  
 
Line 38 -57, nicely written, but how does this relate to the data?  
 
Page 12:  
 
Line 13: The novel findings of the paper need to be emphasized more clearly. Has age been 
shown to be a predictor for response?  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Unable to assess  
 
Does the work include the nece ssary controls?  
If not, please specify which controls are required in your comments to the authors.  
No 
 
Are the conclusions drawn adequate ly supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any s tatistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
 
I recommend additional statisti cal review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the  following questions:  
 
1.  Have you in the past five years received reimbursements, f ees, funding, or salary 
from an organisation that may in any way gain or lose financial ly from the 
publication of this manuscript, either now or in the future?  
 
2.  Do you hold any stocks or shares in an organisation t hat may in any way gain or 
lose financially from the publication of this manuscript, either now or in the 
future?  
 
3.  Do you hold or are you currently applying for any patents relating to the content 
of the manuscript?  
 
4.  Have you received reimbursements, fees, funding, or salary from an organization 
that holds or has applied for patents relating to the content of the manuscript?  
 
5.  Do you have any other financial competing interests?  
 
6.  Do you have any  non-financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have  no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing i nterests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any attachments I upload will be posted on  the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be in cluded as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Relationship between mucosal healing by tacrolimus and r elapse of refractory ulcerative 
colitis: A retrospective study  
 
Version: 1 Date: 01 Dec 2019  
 
Revi ewer: Holger Schäffler  
 
Reviewer's report:  
 
The medical therapy of UC can be a challenge, although new treatment options, especially 
biologica ls and small molecules do exist. The role of tacrolimus in the treatment of UC has been 
studied before, however, if mucosal healing is a valid treatment option in UC patients under 
tacrolimus is still under debate.  
 
Although the study was designed in a re trospective manner, several interesting observations 
could be made by the authors. However, in the present form, the manuscript cannot be 
recommended for acceptance. If several major and the minor points are adequately addressed, 
reconsideration may be giv en. 
 
 
General points:  
 
1. Statistical analysis is insufficient, statistical consult is recommended.  
 
2. Data presentation needs scrutiny. While the authors nicely describe the design of the 
study with three time points, data presentation is somewhat confusing with tables for 
the first two time points and graphs for the third time point.  
 
3. Translation by a native speaker is recommended.  
 
 
Specific points:  
 
Page 3:  
 
Abstract/Methods: It should be stated that a retrospective study was performed, it should be 
stated that a case -control study was performed. Single center should be mentioned.  
 
Abstract/Results: Restructuring is required. Suggestion: Baseline, end of treatm ent, follow up. It 
should be stated what the difference was for the parameters that were "different". What do you 
mean with "There were also significant differences and the steroid -free rate"?  
 
Line 31: Patients with MH at the end TAC -&gt; end of TAC  
 
Page 4: 
 
Line 4: The first word of the introduction should not be an abbreviation. I would suggest to 
write: the prevalence of UC has increased instead of "the number of patients".  
 
Line 7: "Surgery is indicated", rephrase "is" to "may be", please indicate that surgery is the 
option for severe subtypes of UC and indicate the incidence of this treatment for UC  
 
Line 16: Surgery "is", rephrase to "may be"  
 
Please rephrase this first paragraph or have it redacted professionally.  
 
Line 22/23, please delete the number "5 2.6%", or put this number into perspective.  
 
Line 28: "which" is missing, rephrase "important issue"  
 
Line 47: Please specify the type of your department, tertiary care? Reference center?  
 
Line 50: follow up period should be expressed as median, not mean, pl ease indicate the interval, 
please specify the name of the prescribed drug and the supplier.  
 
Line 56: Please indicate the measuring method (fluid phase chromatography, LC -MS/MS ?) 
Please indicate the sampling method (plasma, serum, whole blood, was a chela tor used?)  
Page 5:  
 
Line 10: bpatients to patients  
 
Line 19 -22: Avoid: "so all patients had prednisolone -dependent or prednisolone -resistant 
refractory UC."  
 
Line 25: It should be clarified that endoscopy was done before TAC, it should be stated the 
median ti me from last endoscopy to TAC initiation  
 
Line 35: It should be stated if the Lichtiger score was determined at each visit or if it was 
calculated retrospectively. Please also reference the Lichtiger score as the Modified Truelove and 
Witts Severity Index.  
 
Line 50: "total dose of prednisolone up to remission", it is difficult to understand that remission 
was achieved in the hospital, please clarify. Was there any other treatment than prednisolone 
before TAC?  
 
Page 6:  
 
Line 1: what are the ranges for the specif ied time points?  
 
Line 16: It should be stated that surgery was not required if this was the case.  
Line 25: Consultation of a medical statistician is highly recommended. Please test your data for 
normality and please provide appropriate statistical analysi s and data presentation. Why is a 
nonparametric test used but a parametric measure provided in the table? How about multivariate 
testing? Which test was used to test for differences in relapse rates?  
 
Line 37: Please provide the approval number  
 
Page 7:  
 
Line 16ff: when was the last endoscopy before treatment? Time to target trough levels is not 
possible before therapy. Total dose of prednisolone is misleading at this point. Looking at the 
table I realize at this point that the patients were not outpatients bu t inpatients. Were all patients 
started on TAC in the hospital? This should be stated in the methods section.  
 
Line 38: MN group?  
 
Line 44: Please specify which number belongs to which group.  
 
Page 8:  
 
Line 25: It should be described in the methods section, how the occurrence of AEs was 
determined. Was there a systematic screening for these "adverse events"? Was it patient 
reported?  
 
Line 28: adverse instead of advanced, significantly instead of significa nt, different instead of 
difference, rephrase this sentence  
 
Line 32: adverse  
 
Line 41: "features" is not applicable  
 
Line 47: rephrasing recommended, source recommended  
 
Line 56: The authors should cite the work of Ogata et al 2016 IBD. This study was a place bo 
controlled study of Tacrolimus for refractory UC and Tacrolimus therapy was associated with a 
higher  44% MH rate. In the following, the authors should discuss their findings in perspective to 
this paper.  
 
Page 9:  
 
Line 22ff: This is highly speculative an d unlikely. This speculation should be weakened  
significantly and other reasons should be discussed - different biology of the underlying disease? 
If the authors want to keep this claim, they have to show that trough levels were lower in this 
group, which  would prove incompliance.  
 
Line 37ff: Did the NMH group receive more other drugs. Authors have to show data on this if 
they want to keep this claim. Were clinical symptoms improved in the NMH group over the MH 
group? The data do not suggest this.  
 
Page 10:  
 
Line 13: Authors do not report on stool measures such as Calprotectin. It should be discussed, 
why.  
 
Line 19ff: This is one major weakness of the study. It has to be explained, why the treatment 
duration was different. Is the time from TAC initiation to sco pe different? Whithout this data, the 
following speculation is pointless.  
 
Line 35: of instead of if; combined use of AZA should be explained. The authors describe their 
finding that "combined use of AZA" was not different between the two groups. From this,  they 
conclude that it "was unclear whether AZA contributed to promoting MH". This claim may be 
true, but it cannot be made from the data.  
 
Line 38: The authors could speculate a little more about the reasons for the lower steroid free 
rate in the NMH group . They should state that tapering was not possible in a large fraction of the 
NMH group, if this was the case.  
 
Page 11:  
 
Line 10ff: It would be interesting to see how many patients were steroid free at 100 days, e.g. 
was the disease activity masked by the s teroids? The numbers do not need to be repeated, they 
are described in the results section.  
 
Line 32: Previous reports on TAC in UC need to be acknowledged and the results discussed in 
this context, for example Baumgart et al. 2003 Aliment Pharmacol Ther, S chmidt et al. 2016 
JCC, Ogata et al 2016 IBD, Schmidt et al 2013 Aliment Pharmacol Ther.  
 
Line 38 -57, nicely written, but how does this relate to the data?  
 
Page 12:  
 
Line 13: The novel findings of the paper need to be emphasized more clearly. Has age been 
shown to be a predictor for response?  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Unable to assess  
 
Does the work include the nece ssary controls?  
If not, please specify which controls are required in your comments to the authors.  
No 
 
Are the conclusions drawn adequate ly supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any s tatistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
 
I recommend additional statisti cal review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the  following questions:  
 
1.  Have you in the past five years received reimbursements, f ees, funding, or salary 
from an organisation that may in any way gain or lose financial ly from the 
publication of this manuscript, either now or in the future?  
 
2.  Do you hold any stocks or shares in an organisation t hat may in any way gain or 
lose financially from the publication of this manuscript, either now or in the 
future?  
 
3.  Do you hold or are you currently applying for any patents relating to the content 
of the manuscript?  
 
4.  Have you received reimbursements, fees, funding, or salary from an organization 
that holds or has applied for patents relating to the content of the manuscript?  
 
5.  Do you have any other financial competing interests?  
 
6.  Do you have any  non-financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have  no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing i nterests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any attachments I upload will be posted on  the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be in cluded as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Relationship between mucosal healing by tacrolimus and r elapse of refractory ulcerative 
colitis: A retrospective study  
 
Version: 1 Date: 01 Dec 2019  
 
Revi ewer: Holger Schäffler  
 
Reviewer's report:  
 
The medical therapy of UC can be a challenge, although new treatment options, especially 
biologica ls and small molecules do exist. The role of tacrolimus in the treatment of UC has been 
studied before, however, if mucosal healing is a valid treatment option in UC patients under 
tacrolimus is still under debate.  
 
Although the study was designed in a re trospective manner, several interesting observations 
could be made by the authors. However, in the present form, the manuscript cannot be 
recommended for acceptance. If several major and the minor points are adequately addressed, 
reconsideration may be giv en. 
 
 
General points:  
 
1. Statistical analysis is insufficient, statistical consult is recommended.  
 
2. Data presentation needs scrutiny. While the authors nicely describe the design of the 
study with three time points, data presentation is somewhat confusing with tables for 
the first two time points and graphs for the third time point.  
 
3. Translation by a native speaker is recommended.  
 
 
Specific points:  
 
Page 3:  
 
Abstract/Methods: It should be stated that a retrospective study was performed, it should be 
stated that a case -control study was performed. Single center should be mentioned.  
 
Abstract/Results: Restructuring is required. Suggestion: Baseline, end of treatm ent, follow up. It 
should be stated what the difference was for the parameters that were "different". What do you 
mean with "There were also significant differences and the steroid -free rate"?  
 
Line 31: Patients with MH at the end TAC -&gt; end of TAC  
 
Page 4: 
 
Line 4: The first word of the introduction should not be an abbreviation. I would suggest to 
write: the prevalence of UC has increased instead of "the number of patients".  
 
Line 7: "Surgery is indicated", rephrase "is" to "may be", please indicate that surgery is the 
option for severe subtypes of UC and indicate the incidence of this treatment for UC  
 
Line 16: Surgery "is", rephrase to "may be"  
 
Please rephrase this first paragraph or have it redacted professionally.  
 
Line 22/23, please delete the number "5 2.6%", or put this number into perspective.  
 
Line 28: "which" is missing, rephrase "important issue"  
 
Line 47: Please specify the type of your department, tertiary care? Reference center?  
 
Line 50: follow up period should be expressed as median, not mean, pl ease indicate the interval, 
please specify the name of the prescribed drug and the supplier.  
 
Line 56: Please indicate the measuring method (fluid phase chromatography, LC -MS/MS ?) 
Please indicate the sampling method (plasma, serum, whole blood, was a chela tor used?)  
Page 5:  
 
Line 10: bpatients to patients  
 
Line 19 -22: Avoid: "so all patients had prednisolone -dependent or prednisolone -resistant 
refractory UC."  
 
Line 25: It should be clarified that endoscopy was done before TAC, it should be stated the 
median ti me from last endoscopy to TAC initiation  
 
Line 35: It should be stated if the Lichtiger score was determined at each visit or if it was 
calculated retrospectively. Please also reference the Lichtiger score as the Modified Truelove and 
Witts Severity Index.  
 
Line 50: "total dose of prednisolone up to remission", it is difficult to understand that remission 
was achieved in the hospital, please clarify. Was there any other treatment than prednisolone 
before TAC?  
 
Page 6:  
 
Line 1: what are the ranges for the specif ied time points?  
 
Line 16: It should be stated that surgery was not required if this was the case.  
Line 25: Consultation of a medical statistician is highly recommended. Please test your data for 
normality and please provide appropriate statistical analysi s and data presentation. Why is a 
nonparametric test used but a parametric measure provided in the table? How about multivariate 
testing? Which test was used to test for differences in relapse rates?  
 
Line 37: Please provide the approval number  
 
Page 7:  
 
Line 16ff: when was the last endoscopy before treatment? Time to target trough levels is not 
possible before therapy. Total dose of prednisolone is misleading at this point. Looking at the 
table I realize at this point that the patients were not outpatients bu t inpatients. Were all patients 
started on TAC in the hospital? This should be stated in the methods section.  
 
Line 38: MN group?  
 
Line 44: Please specify which number belongs to which group.  
 
Page 8:  
 
Line 25: It should be described in the methods section, how the occurrence of AEs was 
determined. Was there a systematic screening for these "adverse events"? Was it patient 
reported?  
 
Line 28: adverse instead of advanced, significantly instead of significa nt, different instead of 
difference, rephrase this sentence  
 
Line 32: adverse  
 
Line 41: "features" is not applicable  
 
Line 47: rephrasing recommended, source recommended  
 
Line 56: The authors should cite the work of Ogata et al 2016 IBD. This study was a place bo 
controlled study of Tacrolimus for refractory UC and Tacrolimus therapy was associated with a 
higher  44% MH rate. In the following, the authors should discuss their findings in perspective to 
this paper.  
 
Page 9:  
 
Line 22ff: This is highly speculative an d unlikely. This speculation should be weakened  
significantly and other reasons should be discussed - different biology of the underlying disease? 
If the authors want to keep this claim, they have to show that trough levels were lower in this 
group, which  would prove incompliance.  
 
Line 37ff: Did the NMH group receive more other drugs. Authors have to show data on this if 
they want to keep this claim. Were clinical symptoms improved in the NMH group over the MH 
group? The data do not suggest this.  
 
Page 10:  
 
Line 13: Authors do not report on stool measures such as Calprotectin. It should be discussed, 
why.  
 
Line 19ff: This is one major weakness of the study. It has to be explained, why the treatment 
duration was different. Is the time from TAC initiation to sco pe different? Whithout this data, the 
following speculation is pointless.  
 
Line 35: of instead of if; combined use of AZA should be explained. The authors describe their 
finding that "combined use of AZA" was not different between the two groups. From this,  they 
conclude that it "was unclear whether AZA contributed to promoting MH". This claim may be 
true, but it cannot be made from the data.  
 
Line 38: The authors could speculate a little more about the reasons for the lower steroid free 
rate in the NMH group . They should state that tapering was not possible in a large fraction of the 
NMH group, if this was the case.  
 
Page 11:  
 
Line 10ff: It would be interesting to see how many patients were steroid free at 100 days, e.g. 
was the disease activity masked by the s teroids? The numbers do not need to be repeated, they 
are described in the results section.  
 
Line 32: Previous reports on TAC in UC need to be acknowledged and the results discussed in 
this context, for example Baumgart et al. 2003 Aliment Pharmacol Ther, S chmidt et al. 2016 
JCC, Ogata et al 2016 IBD, Schmidt et al 2013 Aliment Pharmacol Ther.  
 
Line 38 -57, nicely written, but how does this relate to the data?  
 
Page 12:  
 
Line 13: The novel findings of the paper need to be emphasized more clearly. Has age been 
shown to be a predictor for response?  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Unable to assess  
 
Does the work include the nece ssary controls?  
If not, please specify which controls are required in your comments to the authors.  
No 
 
Are the conclusions drawn adequate ly supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any s tatistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
 
I recommend additional statisti cal review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the  following questions:  
 
1.  Have you in the past five years received reimbursements, f ees, funding, or salary 
from an organisation that may in any way gain or lose financial ly from the 
publication of this manuscript, either now or in the future?  
 
2.  Do you hold any stocks or shares in an organisation t hat may in any way gain or 
lose financially from the publication of this manuscript, either now or in the 
future?  
 
3.  Do you hold or are you currently applying for any patents relating to the content 
of the manuscript?  
 
4.  Have you received reimbursements, fees, funding, or salary from an organization 
that holds or has applied for patents relating to the content of the manuscript?  
 
5.  Do you have any other financial competing interests?  
 
6.  Do you have any  non-financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have  no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing i nterests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any attachments I upload will be posted on  the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be in cluded as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant