Reviewer’s report
Title: Fecal carriage of multidrug-resistant Escherichia coli by community children in Southern Taiwan
Version: 0 Date: 05 Dec 2016
Reviewer: Oscar Gómez
Reviewer's report:
General comments
This manuscript assesses the prevalence of MDR E. coli in children less than 18 years of age in
Southern Taiwan. The study collects stools from subjects hospitalized subjects and evaluate them with
conventional microbiology and molecular biology techniques for antibiotics susceptibilities, presence
of ESBLs, MLST sequence types, and ESBL gene detection.
Overall, the manuscript provides relevant information on the carriage rate of MDR E. coli in stools
from children in Taiwan and the identification of the MLST 131 clones in these samples. The main
drawbacks of this study are the insufficient information provided in materials and methods and results
sections.  Insufficient  information  is  provided  on  study  design,  inclusion  criteria,  type  of  stools
collection and processing before plating, geographic origin of subjects enrolled, subject diagnosis at the
type of enrollment, number of subjects with gastrointestinal disease, infection diseases, among other
diagnostic information. 
One major drawback of this manuscript is the evaluation of MDR E. coli data from different countries
with respect to GDP. Data collected from different countries is not comparable because it was collected
decades apart, using subjects with different ages, and using data from rural vs urban data. We strongly
believe that this type of comparison significantly decreases the quality of this study. 
Specific comments
Abstract
It is recommended that this manuscript be proof-read by a native English speaker. Phrases throughout
the manuscript need revision. 
Page2, lines 35 to 37. Recommend the authors to clearly state the objective or objectives of the study
rather than describing what they did. 
Page 3, lines 56 to 57. The phrase: "The prevalence of the fecal carriage of resistant E. coli in children
was high among these E. coli, 37% were MDR, 18.5% were not susceptible to ciprofloxacin", needs to
be revised for English. Recommend consistency with terminology: antibiotic resistance vs antibiotic
non-susceptibility.
Background
Background should state clearly the objectives of the study, and briefly state the study strategy.
Page 4, line 65 to 69. Phrase needs revision: "Escherichia coli is a finely tuned, ubiquitous pathogen
that commonly causes urinary tract infections (UTIs) and bacteremia in humans of all ages". E. coli
being fine-tuned is unclear. E. coli include multiple strains that include commensal, intestinal and
extraintestinal pathogens. Only some of these strains cause UTI or bacteremia 
Page 5, lines 76 to 78. Phrase needs revision: "Current strategies to monitor antibiotic resistance in
bacteria mainly rely upon examining resistance in pathogenic organisms." 
Materials and Methods
Page 5, line 88 to 90. Authors are advised to describe the study design, the type of study, the inclusion
criteria as well as the exclusion criteria. Authors indicate that subjects enrolled were admitted to the
hospital. Author should clearly describe whether subjects were sick. If they were sick it is important to
mention  if  inclusion  criteria  allowed  patient  with  diarrhea,  infectious  diarrhea,  other  infections,
abdominal non-infectious conditions. Authors should mention what type of survey was applied to
subjects  in  addition  to  demographics.  Was  information  collected  on  travel,  antibiotics  use,
hospitalization, direct or indirect contact with individuals in nosocomial environments, contact with
international travelers, contact with farm animals, wild animals or pets. 
Authors should include information on ethics committee evaluation and approval in materials and
methods section. 
Page 6, lines 96 to 97. Authors need to define CHROMagar, and describe the reason why they are using
this. The authors make the assumption that blue colonies are all E. coli. Authors should provide
evidence that all or some of the blue colonies detected by CHROMagar were subject to conventional
biochemical identification testing to confirm that blue colonies were all E. coli. 
Page 8, lines 142 to 145. Authors need to provide a rational for evaluating GDP with respect to ESBL
E. coli carriage. This reviewer questions the relevance of comparing countries GDP and ESBL E. coli
carriage  state.  Other  epidemiological  risk  factors  however  were  not  evaluated:  travel,  pets,  prior
antibiotics  use,  prior  hospitalization,  parents  contact  with  hospital  environments.  Authors  should
indicate whether epidemiological risk factors were collected for this study.
Results
Page 9, line 167, E. coli here and throughout manuscript should be written with italics.
Page 10, lines 184 to 188. Authors are not analyzing comparable data. The reported studies were
conducted decades apart and the subjects age was also wide apart. The data was not uniform with
respect to location, some studies were from rural areas and some from urban areas. Any conclusion
drawn from this type of comparison is probably inaccurate. The authors should indicate why data
obtained  from  Taiwan  was  to  analyzed  with  respect  to  socioeconomic  status,  educational  level,
housing, health insurance, water sources, pet exposure, among other epidemiological factors.
Discussion
Page 11, lines 193 to 195. This phrase needs to be revised for English: "Antimicrobial resistance in
commensal flora is a serious threat because a very highly populated ecosystem, such as the gut, may
become a source of additional intestinal infections at a later stage; these infections may then spread to
other hosts or transfer genetic resistance elements to other members."
Page 11, lines200 to 204. This statement is not accurate based on data presented: "A significant inverse
relationship between the GDP per capita and prevalence of ESBL E. coli was observed." None of the
studies were representative of each individual country, they were obtained decades apart, subject ages
were not uniform, and geographic regions within each country vary from urban to rural. Data obtained
for comparison is not reliable. 
Page 11, lines 212- 214.  This phrase needs revision: "By contrast, the rate of nonsusceptibility to rarely
used antibiotics such as imipenem, amikacin, tigecycline, and colistin was 0%." In addition to English
revision,  no information is provided to infer that these antibiotics are rarely used in Taiwan. 
Page 12, lines 225 to 227. Phrase needs revision: " most patients with bacteremia or UTIs were
previously healthy and did not exhibit any apparent risk factors." Author should clearly state to which
risk factors they are referring to. 
Page 13, lines 235 to 237. This phrase is incorrect: "By contrast, in the present study, ST131 (7/13) was
the most common ESBL-producing E. coli clone in the stools of children. These findings suggest that
community-acquired pathogenic ESBL-producing E. coli might be transferred from fecal material
rather than river." This study is not about transmission but rather about carriage state. Even if the
statement is correct it is meaningless because in this study the reservoir of E. coli ESBL ST131 positive
has not been found in any other source but stools.
Page 14, lines 254 to 256. This statement is inaccurate: "Third, because the diseases of the enrolled
patients were not further classified, we could not determine whether the different diseases affected the
fecal carriage and antimicrobial resistance rate of E. coli." Authors should provide information on
subjects diagnosis at the time admission to the hospital. Based on diagnosis it may or may not be
possible to determine if the results from this study represent MDR E. coli carriage or rather new
acquisition due to infection. 
Conclusion
Page 14, lines 262 to 264: children. This statement is vague: "These concerning results highlight the
importance of using hygiene procedures and appropriate antibiotics to control the emergence and
spread of MDR organisms." What the authors may want to say is that the results should highlight the
importance  in  establishing  an  antibiotic  stewardship  and  infection  control  Programs  to  decrease
inappropriate antibiotic use and to decrease transmission of MDR E. coli. 
Figures 1 and 2 should be better explained in a single table. Figure 3 should be removed.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
No
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
Unable to assess
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
No
Are you able to assess any statistics in the manuscript or would you recommend an additional 
statistical review?
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.
I recommend additional statistical 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, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that 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 interests
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 for 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 included 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: Fecal carriage of multidrug-resistant Escherichia coli by community children in Southern Taiwan
Version: 0 Date: 05 Dec 2016
Reviewer: Oscar Gómez
Reviewer's report:
General comments
This manuscript assesses the prevalence of MDR E. coli in children less than 18 years of age in
Southern Taiwan. The study collects stools from subjects hospitalized subjects and evaluate them with
conventional microbiology and molecular biology techniques for antibiotics susceptibilities, presence
of ESBLs, MLST sequence types, and ESBL gene detection.
Overall, the manuscript provides relevant information on the carriage rate of MDR E. coli in stools
from children in Taiwan and the identification of the MLST 131 clones in these samples. The main
drawbacks of this study are the insufficient information provided in materials and methods and results
sections.  Insufficient  information  is  provided  on  study  design,  inclusion  criteria,  type  of  stools
collection and processing before plating, geographic origin of subjects enrolled, subject diagnosis at the
type of enrollment, number of subjects with gastrointestinal disease, infection diseases, among other
diagnostic information. 
One major drawback of this manuscript is the evaluation of MDR E. coli data from different countries
with respect to GDP. Data collected from different countries is not comparable because it was collected
decades apart, using subjects with different ages, and using data from rural vs urban data. We strongly
believe that this type of comparison significantly decreases the quality of this study. 
Specific comments
Abstract
It is recommended that this manuscript be proof-read by a native English speaker. Phrases throughout
the manuscript need revision. 
Page2, lines 35 to 37. Recommend the authors to clearly state the objective or objectives of the study
rather than describing what they did. 
Page 3, lines 56 to 57. The phrase: "The prevalence of the fecal carriage of resistant E. coli in children
was high among these E. coli, 37% were MDR, 18.5% were not susceptible to ciprofloxacin", needs to
be revised for English. Recommend consistency with terminology: antibiotic resistance vs antibiotic
non-susceptibility.
Background
Background should state clearly the objectives of the study, and briefly state the study strategy.
Page 4, line 65 to 69. Phrase needs revision: "Escherichia coli is a finely tuned, ubiquitous pathogen
that commonly causes urinary tract infections (UTIs) and bacteremia in humans of all ages". E. coli
being fine-tuned is unclear. E. coli include multiple strains that include commensal, intestinal and
extraintestinal pathogens. Only some of these strains cause UTI or bacteremia 
Page 5, lines 76 to 78. Phrase needs revision: "Current strategies to monitor antibiotic resistance in
bacteria mainly rely upon examining resistance in pathogenic organisms." 
Materials and Methods
Page 5, line 88 to 90. Authors are advised to describe the study design, the type of study, the inclusion
criteria as well as the exclusion criteria. Authors indicate that subjects enrolled were admitted to the
hospital. Author should clearly describe whether subjects were sick. If they were sick it is important to
mention  if  inclusion  criteria  allowed  patient  with  diarrhea,  infectious  diarrhea,  other  infections,
abdominal non-infectious conditions. Authors should mention what type of survey was applied to
subjects  in  addition  to  demographics.  Was  information  collected  on  travel,  antibiotics  use,
hospitalization, direct or indirect contact with individuals in nosocomial environments, contact with
international travelers, contact with farm animals, wild animals or pets. 
Authors should include information on ethics committee evaluation and approval in materials and
methods section. 
Page 6, lines 96 to 97. Authors need to define CHROMagar, and describe the reason why they are using
this. The authors make the assumption that blue colonies are all E. coli. Authors should provide
evidence that all or some of the blue colonies detected by CHROMagar were subject to conventional
biochemical identification testing to confirm that blue colonies were all E. coli. 
Page 8, lines 142 to 145. Authors need to provide a rational for evaluating GDP with respect to ESBL
E. coli carriage. This reviewer questions the relevance of comparing countries GDP and ESBL E. coli
carriage  state.  Other  epidemiological  risk  factors  however  were  not  evaluated:  travel,  pets,  prior
antibiotics  use,  prior  hospitalization,  parents  contact  with  hospital  environments.  Authors  should
indicate whether epidemiological risk factors were collected for this study.
Results
Page 9, line 167, E. coli here and throughout manuscript should be written with italics.
Page 10, lines 184 to 188. Authors are not analyzing comparable data. The reported studies were
conducted decades apart and the subjects age was also wide apart. The data was not uniform with
respect to location, some studies were from rural areas and some from urban areas. Any conclusion
drawn from this type of comparison is probably inaccurate. The authors should indicate why data
obtained  from  Taiwan  was  to  analyzed  with  respect  to  socioeconomic  status,  educational  level,
housing, health insurance, water sources, pet exposure, among other epidemiological factors.
Discussion
Page 11, lines 193 to 195. This phrase needs to be revised for English: "Antimicrobial resistance in
commensal flora is a serious threat because a very highly populated ecosystem, such as the gut, may
become a source of additional intestinal infections at a later stage; these infections may then spread to
other hosts or transfer genetic resistance elements to other members."
Page 11, lines200 to 204. This statement is not accurate based on data presented: "A significant inverse
relationship between the GDP per capita and prevalence of ESBL E. coli was observed." None of the
studies were representative of each individual country, they were obtained decades apart, subject ages
were not uniform, and geographic regions within each country vary from urban to rural. Data obtained
for comparison is not reliable. 
Page 11, lines 212- 214.  This phrase needs revision: "By contrast, the rate of nonsusceptibility to rarely
used antibiotics such as imipenem, amikacin, tigecycline, and colistin was 0%." In addition to English
revision,  no information is provided to infer that these antibiotics are rarely used in Taiwan. 
Page 12, lines 225 to 227. Phrase needs revision: " most patients with bacteremia or UTIs were
previously healthy and did not exhibit any apparent risk factors." Author should clearly state to which
risk factors they are referring to. 
Page 13, lines 235 to 237. This phrase is incorrect: "By contrast, in the present study, ST131 (7/13) was
the most common ESBL-producing E. coli clone in the stools of children. These findings suggest that
community-acquired pathogenic ESBL-producing E. coli might be transferred from fecal material
rather than river." This study is not about transmission but rather about carriage state. Even if the
statement is correct it is meaningless because in this study the reservoir of E. coli ESBL ST131 positive
has not been found in any other source but stools.
Page 14, lines 254 to 256. This statement is inaccurate: "Third, because the diseases of the enrolled
patients were not further classified, we could not determine whether the different diseases affected the
fecal carriage and antimicrobial resistance rate of E. coli." Authors should provide information on
subjects diagnosis at the time admission to the hospital. Based on diagnosis it may or may not be
possible to determine if the results from this study represent MDR E. coli carriage or rather new
acquisition due to infection. 
Conclusion
Page 14, lines 262 to 264: children. This statement is vague: "These concerning results highlight the
importance of using hygiene procedures and appropriate antibiotics to control the emergence and
spread of MDR organisms." What the authors may want to say is that the results should highlight the
importance  in  establishing  an  antibiotic  stewardship  and  infection  control  Programs  to  decrease
inappropriate antibiotic use and to decrease transmission of MDR E. coli. 
Figures 1 and 2 should be better explained in a single table. Figure 3 should be removed.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
No
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
Unable to assess
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
No
Are you able to assess any statistics in the manuscript or would you recommend an additional 
statistical review?
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.
I recommend additional statistical 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, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that 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 interests
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 for 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 included 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: Fecal carriage of multidrug-resistant Escherichia coli by community children in Southern Taiwan
Version: 0 Date: 05 Dec 2016
Reviewer: Oscar Gómez
Reviewer's report:
General comments
This manuscript assesses the prevalence of MDR E. coli in children less than 18 years of age in
Southern Taiwan. The study collects stools from subjects hospitalized subjects and evaluate them with
conventional microbiology and molecular biology techniques for antibiotics susceptibilities, presence
of ESBLs, MLST sequence types, and ESBL gene detection.
Overall, the manuscript provides relevant information on the carriage rate of MDR E. coli in stools
from children in Taiwan and the identification of the MLST 131 clones in these samples. The main
drawbacks of this study are the insufficient information provided in materials and methods and results
sections.  Insufficient  information  is  provided  on  study  design,  inclusion  criteria,  type  of  stools
collection and processing before plating, geographic origin of subjects enrolled, subject diagnosis at the
type of enrollment, number of subjects with gastrointestinal disease, infection diseases, among other
diagnostic information. 
One major drawback of this manuscript is the evaluation of MDR E. coli data from different countries
with respect to GDP. Data collected from different countries is not comparable because it was collected
decades apart, using subjects with different ages, and using data from rural vs urban data. We strongly
believe that this type of comparison significantly decreases the quality of this study. 
Specific comments
Abstract
It is recommended that this manuscript be proof-read by a native English speaker. Phrases throughout
the manuscript need revision. 
Page2, lines 35 to 37. Recommend the authors to clearly state the objective or objectives of the study
rather than describing what they did. 
Page 3, lines 56 to 57. The phrase: "The prevalence of the fecal carriage of resistant E. coli in children
was high among these E. coli, 37% were MDR, 18.5% were not susceptible to ciprofloxacin", needs to
be revised for English. Recommend consistency with terminology: antibiotic resistance vs antibiotic
non-susceptibility.
Background
Background should state clearly the objectives of the study, and briefly state the study strategy.
Page 4, line 65 to 69. Phrase needs revision: "Escherichia coli is a finely tuned, ubiquitous pathogen
that commonly causes urinary tract infections (UTIs) and bacteremia in humans of all ages". E. coli
being fine-tuned is unclear. E. coli include multiple strains that include commensal, intestinal and
extraintestinal pathogens. Only some of these strains cause UTI or bacteremia 
Page 5, lines 76 to 78. Phrase needs revision: "Current strategies to monitor antibiotic resistance in
bacteria mainly rely upon examining resistance in pathogenic organisms." 
Materials and Methods
Page 5, line 88 to 90. Authors are advised to describe the study design, the type of study, the inclusion
criteria as well as the exclusion criteria. Authors indicate that subjects enrolled were admitted to the
hospital. Author should clearly describe whether subjects were sick. If they were sick it is important to
mention  if  inclusion  criteria  allowed  patient  with  diarrhea,  infectious  diarrhea,  other  infections,
abdominal non-infectious conditions. Authors should mention what type of survey was applied to
subjects  in  addition  to  demographics.  Was  information  collected  on  travel,  antibiotics  use,
hospitalization, direct or indirect contact with individuals in nosocomial environments, contact with
international travelers, contact with farm animals, wild animals or pets. 
Authors should include information on ethics committee evaluation and approval in materials and
methods section. 
Page 6, lines 96 to 97. Authors need to define CHROMagar, and describe the reason why they are using
this. The authors make the assumption that blue colonies are all E. coli. Authors should provide
evidence that all or some of the blue colonies detected by CHROMagar were subject to conventional
biochemical identification testing to confirm that blue colonies were all E. coli. 
Page 8, lines 142 to 145. Authors need to provide a rational for evaluating GDP with respect to ESBL
E. coli carriage. This reviewer questions the relevance of comparing countries GDP and ESBL E. coli
carriage  state.  Other  epidemiological  risk  factors  however  were  not  evaluated:  travel,  pets,  prior
antibiotics  use,  prior  hospitalization,  parents  contact  with  hospital  environments.  Authors  should
indicate whether epidemiological risk factors were collected for this study.
Results
Page 9, line 167, E. coli here and throughout manuscript should be written with italics.
Page 10, lines 184 to 188. Authors are not analyzing comparable data. The reported studies were
conducted decades apart and the subjects age was also wide apart. The data was not uniform with
respect to location, some studies were from rural areas and some from urban areas. Any conclusion
drawn from this type of comparison is probably inaccurate. The authors should indicate why data
obtained  from  Taiwan  was  to  analyzed  with  respect  to  socioeconomic  status,  educational  level,
housing, health insurance, water sources, pet exposure, among other epidemiological factors.
Discussion
Page 11, lines 193 to 195. This phrase needs to be revised for English: "Antimicrobial resistance in
commensal flora is a serious threat because a very highly populated ecosystem, such as the gut, may
become a source of additional intestinal infections at a later stage; these infections may then spread to
other hosts or transfer genetic resistance elements to other members."
Page 11, lines200 to 204. This statement is not accurate based on data presented: "A significant inverse
relationship between the GDP per capita and prevalence of ESBL E. coli was observed." None of the
studies were representative of each individual country, they were obtained decades apart, subject ages
were not uniform, and geographic regions within each country vary from urban to rural. Data obtained
for comparison is not reliable. 
Page 11, lines 212- 214.  This phrase needs revision: "By contrast, the rate of nonsusceptibility to rarely
used antibiotics such as imipenem, amikacin, tigecycline, and colistin was 0%." In addition to English
revision,  no information is provided to infer that these antibiotics are rarely used in Taiwan. 
Page 12, lines 225 to 227. Phrase needs revision: " most patients with bacteremia or UTIs were
previously healthy and did not exhibit any apparent risk factors." Author should clearly state to which
risk factors they are referring to. 
Page 13, lines 235 to 237. This phrase is incorrect: "By contrast, in the present study, ST131 (7/13) was
the most common ESBL-producing E. coli clone in the stools of children. These findings suggest that
community-acquired pathogenic ESBL-producing E. coli might be transferred from fecal material
rather than river." This study is not about transmission but rather about carriage state. Even if the
statement is correct it is meaningless because in this study the reservoir of E. coli ESBL ST131 positive
has not been found in any other source but stools.
Page 14, lines 254 to 256. This statement is inaccurate: "Third, because the diseases of the enrolled
patients were not further classified, we could not determine whether the different diseases affected the
fecal carriage and antimicrobial resistance rate of E. coli." Authors should provide information on
subjects diagnosis at the time admission to the hospital. Based on diagnosis it may or may not be
possible to determine if the results from this study represent MDR E. coli carriage or rather new
acquisition due to infection. 
Conclusion
Page 14, lines 262 to 264: children. This statement is vague: "These concerning results highlight the
importance of using hygiene procedures and appropriate antibiotics to control the emergence and
spread of MDR organisms." What the authors may want to say is that the results should highlight the
importance  in  establishing  an  antibiotic  stewardship  and  infection  control  Programs  to  decrease
inappropriate antibiotic use and to decrease transmission of MDR E. coli. 
Figures 1 and 2 should be better explained in a single table. Figure 3 should be removed.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
No
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
Unable to assess
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
No
Are you able to assess any statistics in the manuscript or would you recommend an additional 
statistical review?
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.
I recommend additional statistical 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, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that 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 interests
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 for 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 included as confidential comments to the editors, which will not
be published.
I agree to the open peer review policy of the journal
assistant