Reviewer ’s report  
 
Title: Evaluation of breath, plasma, and urinary markers of lactose malabsorption to diagnose  
lactase non -persistence following lactose or milk ingestion  
 
Version: 0 Date: 07 Feb 2020  
 
Reviewer: Guy Vergères  
 
Reviewer's report:  
 
Review of article BMGE -D-19-01087 by Shrestha et al.  
 
General comments:  
In the present manuscript Shrestha et al. have evaluated the performance of three biologic 
markers in diagnosing lactose maldigestion and  thus, lactase non -persistence. The diagnostic 
tests have been evaluated using three different lactose sources (lactose solution, conventional 
milk and A2 milk). The article addressed relevant questions, i.e. the effect of the lactose matrix 
and the milks casein content on the results of 3 diagnostic tests. The article shows proper use of 
statistical tools used to evaluate diagnostic tests (i.e. Kohen's Kappa index, ROC AUC and 
Youden index). The authors show that recommended thresholds from literature do n ot performed 
systematically well when applied to their dataset; and further propose two different H2 cut -offs 
after the intake of either conventional or A2 milk.  
However, the article would benefit from a modification of its general structure to present the  
main findings in a clearer manner, and to better respond to the aims as presented in the title, 
abstract and introduction. Additional statistical analysis could have been added to complete 
results (predicted thresholds after lactose intake, show ROC curve s and kinetics) or mentioned if 
already published (comparison of H2 breath test results from CON and A2 milks).  
Also, the authors might reconsider their choice of a H2 &gt; 25 ppm cut -off (as a H2 &gt; 20 
ppm threshold is generally used in literature), or provide better rational for the use of 25 ppm.  
Some mistakes and inconsistencies have been found in the results, requiring either correction or 
further discussion.  
Finally, the article has some sections that read well, but some other present less clear phrasing 
(see "various remarks" below). Overall, the article would benefit from a proofreading.  
The above -mentioned concerns are detailed below.  
 
General structure of the article:  
As the aim of the article is to evaluate the three diagnostic methods, I suggest to focus the article 
on the results of the tests (Supp. Table 1 and Figure 1) and their diagnostic performance without 
applying literature thresholds (Table 4 and  calculated thresholds); and then evaluate the validity 
of the cut -offs proposed by the literature (Table 3). As it is now, it seems that the article gives 
too much weight on the evaluation of the literature thresholds.  
Suggestion of paragraphs' order in t he Results section:  
* Participant characteristics (including the Lactase genotyping section)  
* Malabsorption marker difference between LP and LNP individuals with lactose or milk (refer 
to Supplemental Table 1 and Figure 1)  
* Diagnostic accuracy of malabs orption marker using ROC curve analyses assessed against LCT 
genotypes (refer to Table 4)  
* Calculated cut -off values after milk and lactose challenge (include calculated cut -offs for 
lactose and milks intake in Table 4)  
* Validity of malabsorption markers  using literature cut -offs assessed against LCT genotype 
(refer to Table 3).  
 
Additional results that could have been added:  
* Provide, as Supplemental figures, the ROC curves (with Youden indexes when applicable).  
* Show, as Supplemental figures, the kin etics comparing LP vs LNP subjects, for breath H2, 
blood glucose or urine galactose/creatinine, after lactose or milk intake. Alternately, if the data 
has already been shown with the same subject grouping in Figure 4 of Milan et al 2019 (ref 20), 
make refe rence to this figure in the manuscript.  However, it appears that the subject grouping 
between both reports is different justifying the proposed supplementary figure.  
* Indicate the calculated cut -offs for the glucose and the galactose tests after lactose intake (since 
their accuracies were 'acceptable'). These calculated thresholds can later be compared and 
discussed with the literature -based thresholds.  
* Do the ROC curves AUCs differ statistically between the tests (e.g. H2 breath test using 
Lactose vs C ONmilk vs A2Mmilk)?  
 
Choice of the 25 ppm cut -off for the H2 breath test:  
The authors justify their choice of 25 ppm cut -off by citing 3 articles (ref. 19 -21). The article 
from Casellas et al. (ref. 20 and 21 also cite Casellas et al.) does not give ration al regarding the 
use of a 25 ppm cut -off. 
However, general consensus is to use a cut -off of 20 ppm (Rezaie et al., Am J Gastroenterol. 
2017;112(5):775 -84 or Gasbarrini et al., Aliment Pharmacol Ther. 2009; 29: 1 -49. 
doi:10.1111/j.1365 -2036.2009.03951.x).  
Author should give more rational for the use of 25 ppm cut -off, or provide results using the 20 
ppm cut -off. 
 
Results inconsistencies:  
* Breath H2 levels for LNP subjects are lower after CON milk intake compared to A2 milk 
intake (Supplem. Table 1, 65.22 pp m vs 89.86 ppm), is this difference significant? On the other 
hand, Figure 1, as well as the results from the same study from Milan et al. Fig. 4A (ref. 20), 
show the contrary. Please clarify and/or discuss.  
 
 
Various remarks:  
 
Table 2:  
* Please, present P -values in and the statistical test used.  
 
Table 3:  
* Cut -offs for urinary galactose are incorrect, please correct for 0.10  
* Not necessary to merge the results for CON and A2 milk as it does not improve the results  
 
Table 4:  
* Please i ndicate to what test the P -values refer to (should also be mentioned in the Methods 
section)  
* Add calculated thresholds  
 
Supp Table 1:  
* Please add units  
* Which statistical test was used?  
 
Figure 1:  
* Explanation of dotted/solid lines on graph is confu sing.  
* Please indicate that the subjects were classified as LP or LNP based on the genotyping test.  
* Use same Y -scale on the 3 plots?  
 
Introduction:  
* Line 27.  Rephrase: "Presence of C/T13910 and G/A22018 polymorphisms, determined by 
restriction fragmen t length polymorphism, was used as the diagnostic reference for LNP."  
* Line 29.  The Results section of the introduction should give less weight to the results obtained 
with literature cut -offs. 
* Line 33.  "urinary galactose/creatinine could not be compa red to…" explain why they could 
not be compared or rephrase "whereas values obtained for urine gal/creatinine…".  
* Line 38.  Please use the values obtained with the adjusted lactose doses, so it can be compared 
with lactose intake (92.9 % instead of 85.7 % , 78.6 % instead of 71.4 %).  
 
Background:  
* Line 55.  Remove 'the' before lactase.  
* Line 59.  "however, intolerance to other foods can…."  
* Line 60.  Specify what kind of digestive symptoms are observed for LI, and give an example or 
reference of a food causing the same types of digestive symptoms as dairy. LI also needs to be 
added to the list of abbreviations at the end.  
* Lines 68 -81.  Explain which homozygosity/heterozygosity of the genotypes translates to 
LP/LNP.  
* Line 94.  The phrase 'rarely been established' is a bit misleading, since some cut -offs have been 
proposed by other groups. Perhaps change to 'have not been validated'.  
* Lines 96 -99.  Ch eck grammar and phrasing of this sentence.  
* Line 99.  'Milk' cannot be argued. Consider changing to 'it has been argued that milk is a 
more…'.  
* Line 100.  The word 'medium' is used here seemingly to describe both milk and lactose, but 
lactose is not a me dium itself.  
* Lines 100 -103.  Check grammar and phrasing of this sentence.  
* Lines 105 -106.  Same comment as above regarding line 94.  
* Lines 111 -112.  Contrary to breath H2 and blood glucose, urinary galactose is not a commonly 
used LM measure: specify " breath H2, plasma glucose, and urinary galactose/creatinine" in the 
sentence.  
 
 
Methods:  
* Line 121.  Should be 'following ingestion of lactose and milk'. Check throughout manuscript.  
* Line 122. 'Digestive' symptoms?  
* Line 140.  Please give lactose source  and purity.  
* Line 155.  Centrifugation of whole blood?  
* Line 170.  Add company details for Lonza.  
* Line 179.  Spelling mistake. Should be 'manufacturer's'.  
* Line 203.  Spelling mistake. Should be 'assessed'.  
* Lines 206 -210.  Please give a short descr iption for "sensitivity", "specificity", PPV etc. or 
provide a reference, especially since these metrics are heavily used and described in the results.  
* Line 215.  0.70 &lt;AUC &lt;0.80 instead of AUC &gt;0.70&lt;0.80? (same at line 216).  
* Line 221.  Spelling mistake for 'malabsorption' in heading of Table 1.  
* Line 232.  P &lt;0.05?  
 
Results:  
* Lines 235 -236.  Check grammar. Consider changing to 'as Caucasian…with a minority of…'.  
* Line 245.  G/A2210 ?  
* Line 247.  AA??  
* Lines 249 -250.  'with lactose  or milk' consumption?  
* Line 263.  Why lower than previous reports? Elaborate in discussion.  
* Line 268.  Add P -value for k=0.319  
* Line 283.  "The sensitivity was unchanged (78.6 %)". The table shows a decrease to 71.4 %.  
* Line 303.  Specify: plasma glucose and urinary galactose/creatinine ratio.  
 
Discussion:  
* Line 318.  For discussion, put cut -off values obtained in this study in context with previous 
studies. Including for cut -off values calculated after lactose intake.  
* Line 326.  β-casein variation in milk was a further determinant of the breath H2 test 
performance.  
* Line 366.  Spelling mistake: 'one serving'.  
* Lines 339/340.  Or affected by the urine adjustment method (using creatinine or specific 
gravity).  
* Line 366.  Please precise that lactose dose adjustment avoided one false negative resulting in 
better sensitivity of the test.  
* Lines 374 -375.  Should rephrase: "these milk types resulted in different breath H2 levels 
(check if significant), different sensitivity for the b reath H2 test to detect LNP and different…"  
* Lines 381 -382.  "… when milk is used as a substrate to diagnose LNP using H2 breath test".  
* Lines 422 -425.  Unclear sentence, consider rephrasing.  
 
References:  
* Please revise references 29 &amp;  45 
 
 
 
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the  necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
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 am able  to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in t he 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.  
 
No competiting 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 webs ite 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 nam ed 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: Evaluation of breath, plasma, and urinary markers of lactose malabsorption to diagnose  
lactase non -persistence following lactose or milk ingestion  
 
Version: 0 Date: 07 Feb 2020  
 
Reviewer: Guy Vergères  
 
Reviewer's report:  
 
Review of article BMGE -D-19-01087 by Shrestha et al.  
 
General comments:  
In the present manuscript Shrestha et al. have evaluated the performance of three biologic 
markers in diagnosing lactose maldigestion and  thus, lactase non -persistence. The diagnostic 
tests have been evaluated using three different lactose sources (lactose solution, conventional 
milk and A2 milk). The article addressed relevant questions, i.e. the effect of the lactose matrix 
and the milks casein content on the results of 3 diagnostic tests. The article shows proper use of 
statistical tools used to evaluate diagnostic tests (i.e. Kohen's Kappa index, ROC AUC and 
Youden index). The authors show that recommended thresholds from literature do n ot performed 
systematically well when applied to their dataset; and further propose two different H2 cut -offs 
after the intake of either conventional or A2 milk.  
However, the article would benefit from a modification of its general structure to present the  
main findings in a clearer manner, and to better respond to the aims as presented in the title, 
abstract and introduction. Additional statistical analysis could have been added to complete 
results (predicted thresholds after lactose intake, show ROC curve s and kinetics) or mentioned if 
already published (comparison of H2 breath test results from CON and A2 milks).  
Also, the authors might reconsider their choice of a H2 &gt; 25 ppm cut -off (as a H2 &gt; 20 
ppm threshold is generally used in literature), or provide better rational for the use of 25 ppm.  
Some mistakes and inconsistencies have been found in the results, requiring either correction or 
further discussion.  
Finally, the article has some sections that read well, but some other present less clear phrasing 
(see "various remarks" below). Overall, the article would benefit from a proofreading.  
The above -mentioned concerns are detailed below.  
 
General structure of the article:  
As the aim of the article is to evaluate the three diagnostic methods, I suggest to focus the article 
on the results of the tests (Supp. Table 1 and Figure 1) and their diagnostic performance without 
applying literature thresholds (Table 4 and  calculated thresholds); and then evaluate the validity 
of the cut -offs proposed by the literature (Table 3). As it is now, it seems that the article gives 
too much weight on the evaluation of the literature thresholds.  
Suggestion of paragraphs' order in t he Results section:  
* Participant characteristics (including the Lactase genotyping section)  
* Malabsorption marker difference between LP and LNP individuals with lactose or milk (refer 
to Supplemental Table 1 and Figure 1)  
* Diagnostic accuracy of malabs orption marker using ROC curve analyses assessed against LCT 
genotypes (refer to Table 4)  
* Calculated cut -off values after milk and lactose challenge (include calculated cut -offs for 
lactose and milks intake in Table 4)  
* Validity of malabsorption markers  using literature cut -offs assessed against LCT genotype 
(refer to Table 3).  
 
Additional results that could have been added:  
* Provide, as Supplemental figures, the ROC curves (with Youden indexes when applicable).  
* Show, as Supplemental figures, the kin etics comparing LP vs LNP subjects, for breath H2, 
blood glucose or urine galactose/creatinine, after lactose or milk intake. Alternately, if the data 
has already been shown with the same subject grouping in Figure 4 of Milan et al 2019 (ref 20), 
make refe rence to this figure in the manuscript.  However, it appears that the subject grouping 
between both reports is different justifying the proposed supplementary figure.  
* Indicate the calculated cut -offs for the glucose and the galactose tests after lactose intake (since 
their accuracies were 'acceptable'). These calculated thresholds can later be compared and 
discussed with the literature -based thresholds.  
* Do the ROC curves AUCs differ statistically between the tests (e.g. H2 breath test using 
Lactose vs C ONmilk vs A2Mmilk)?  
 
Choice of the 25 ppm cut -off for the H2 breath test:  
The authors justify their choice of 25 ppm cut -off by citing 3 articles (ref. 19 -21). The article 
from Casellas et al. (ref. 20 and 21 also cite Casellas et al.) does not give ration al regarding the 
use of a 25 ppm cut -off. 
However, general consensus is to use a cut -off of 20 ppm (Rezaie et al., Am J Gastroenterol. 
2017;112(5):775 -84 or Gasbarrini et al., Aliment Pharmacol Ther. 2009; 29: 1 -49. 
doi:10.1111/j.1365 -2036.2009.03951.x).  
Author should give more rational for the use of 25 ppm cut -off, or provide results using the 20 
ppm cut -off. 
 
Results inconsistencies:  
* Breath H2 levels for LNP subjects are lower after CON milk intake compared to A2 milk 
intake (Supplem. Table 1, 65.22 pp m vs 89.86 ppm), is this difference significant? On the other 
hand, Figure 1, as well as the results from the same study from Milan et al. Fig. 4A (ref. 20), 
show the contrary. Please clarify and/or discuss.  
 
 
Various remarks:  
 
Table 2:  
* Please, present P -values in and the statistical test used.  
 
Table 3:  
* Cut -offs for urinary galactose are incorrect, please correct for 0.10  
* Not necessary to merge the results for CON and A2 milk as it does not improve the results  
 
Table 4:  
* Please i ndicate to what test the P -values refer to (should also be mentioned in the Methods 
section)  
* Add calculated thresholds  
 
Supp Table 1:  
* Please add units  
* Which statistical test was used?  
 
Figure 1:  
* Explanation of dotted/solid lines on graph is confu sing.  
* Please indicate that the subjects were classified as LP or LNP based on the genotyping test.  
* Use same Y -scale on the 3 plots?  
 
Introduction:  
* Line 27.  Rephrase: "Presence of C/T13910 and G/A22018 polymorphisms, determined by 
restriction fragmen t length polymorphism, was used as the diagnostic reference for LNP."  
* Line 29.  The Results section of the introduction should give less weight to the results obtained 
with literature cut -offs. 
* Line 33.  "urinary galactose/creatinine could not be compa red to…" explain why they could 
not be compared or rephrase "whereas values obtained for urine gal/creatinine…".  
* Line 38.  Please use the values obtained with the adjusted lactose doses, so it can be compared 
with lactose intake (92.9 % instead of 85.7 % , 78.6 % instead of 71.4 %).  
 
Background:  
* Line 55.  Remove 'the' before lactase.  
* Line 59.  "however, intolerance to other foods can…."  
* Line 60.  Specify what kind of digestive symptoms are observed for LI, and give an example or 
reference of a food causing the same types of digestive symptoms as dairy. LI also needs to be 
added to the list of abbreviations at the end.  
* Lines 68 -81.  Explain which homozygosity/heterozygosity of the genotypes translates to 
LP/LNP.  
* Line 94.  The phrase 'rarely been established' is a bit misleading, since some cut -offs have been 
proposed by other groups. Perhaps change to 'have not been validated'.  
* Lines 96 -99.  Ch eck grammar and phrasing of this sentence.  
* Line 99.  'Milk' cannot be argued. Consider changing to 'it has been argued that milk is a 
more…'.  
* Line 100.  The word 'medium' is used here seemingly to describe both milk and lactose, but 
lactose is not a me dium itself.  
* Lines 100 -103.  Check grammar and phrasing of this sentence.  
* Lines 105 -106.  Same comment as above regarding line 94.  
* Lines 111 -112.  Contrary to breath H2 and blood glucose, urinary galactose is not a commonly 
used LM measure: specify " breath H2, plasma glucose, and urinary galactose/creatinine" in the 
sentence.  
 
 
Methods:  
* Line 121.  Should be 'following ingestion of lactose and milk'. Check throughout manuscript.  
* Line 122. 'Digestive' symptoms?  
* Line 140.  Please give lactose source  and purity.  
* Line 155.  Centrifugation of whole blood?  
* Line 170.  Add company details for Lonza.  
* Line 179.  Spelling mistake. Should be 'manufacturer's'.  
* Line 203.  Spelling mistake. Should be 'assessed'.  
* Lines 206 -210.  Please give a short descr iption for "sensitivity", "specificity", PPV etc. or 
provide a reference, especially since these metrics are heavily used and described in the results.  
* Line 215.  0.70 &lt;AUC &lt;0.80 instead of AUC &gt;0.70&lt;0.80? (same at line 216).  
* Line 221.  Spelling mistake for 'malabsorption' in heading of Table 1.  
* Line 232.  P &lt;0.05?  
 
Results:  
* Lines 235 -236.  Check grammar. Consider changing to 'as Caucasian…with a minority of…'.  
* Line 245.  G/A2210 ?  
* Line 247.  AA??  
* Lines 249 -250.  'with lactose  or milk' consumption?  
* Line 263.  Why lower than previous reports? Elaborate in discussion.  
* Line 268.  Add P -value for k=0.319  
* Line 283.  "The sensitivity was unchanged (78.6 %)". The table shows a decrease to 71.4 %.  
* Line 303.  Specify: plasma glucose and urinary galactose/creatinine ratio.  
 
Discussion:  
* Line 318.  For discussion, put cut -off values obtained in this study in context with previous 
studies. Including for cut -off values calculated after lactose intake.  
* Line 326.  β-casein variation in milk was a further determinant of the breath H2 test 
performance.  
* Line 366.  Spelling mistake: 'one serving'.  
* Lines 339/340.  Or affected by the urine adjustment method (using creatinine or specific 
gravity).  
* Line 366.  Please precise that lactose dose adjustment avoided one false negative resulting in 
better sensitivity of the test.  
* Lines 374 -375.  Should rephrase: "these milk types resulted in different breath H2 levels 
(check if significant), different sensitivity for the b reath H2 test to detect LNP and different…"  
* Lines 381 -382.  "… when milk is used as a substrate to diagnose LNP using H2 breath test".  
* Lines 422 -425.  Unclear sentence, consider rephrasing.  
 
References:  
* Please revise references 29 &amp;  45 
 
 
 
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the  necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
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 am able  to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in t he 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.  
 
No competiting 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 webs ite 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 nam ed 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: Evaluation of breath, plasma, and urinary markers of lactose malabsorption to diagnose  
lactase non -persistence following lactose or milk ingestion  
 
Version: 0 Date: 07 Feb 2020  
 
Reviewer: Guy Vergères  
 
Reviewer's report:  
 
Review of article BMGE -D-19-01087 by Shrestha et al.  
 
General comments:  
In the present manuscript Shrestha et al. have evaluated the performance of three biologic 
markers in diagnosing lactose maldigestion and  thus, lactase non -persistence. The diagnostic 
tests have been evaluated using three different lactose sources (lactose solution, conventional 
milk and A2 milk). The article addressed relevant questions, i.e. the effect of the lactose matrix 
and the milks casein content on the results of 3 diagnostic tests. The article shows proper use of 
statistical tools used to evaluate diagnostic tests (i.e. Kohen's Kappa index, ROC AUC and 
Youden index). The authors show that recommended thresholds from literature do n ot performed 
systematically well when applied to their dataset; and further propose two different H2 cut -offs 
after the intake of either conventional or A2 milk.  
However, the article would benefit from a modification of its general structure to present the  
main findings in a clearer manner, and to better respond to the aims as presented in the title, 
abstract and introduction. Additional statistical analysis could have been added to complete 
results (predicted thresholds after lactose intake, show ROC curve s and kinetics) or mentioned if 
already published (comparison of H2 breath test results from CON and A2 milks).  
Also, the authors might reconsider their choice of a H2 &gt; 25 ppm cut -off (as a H2 &gt; 20 
ppm threshold is generally used in literature), or provide better rational for the use of 25 ppm.  
Some mistakes and inconsistencies have been found in the results, requiring either correction or 
further discussion.  
Finally, the article has some sections that read well, but some other present less clear phrasing 
(see "various remarks" below). Overall, the article would benefit from a proofreading.  
The above -mentioned concerns are detailed below.  
 
General structure of the article:  
As the aim of the article is to evaluate the three diagnostic methods, I suggest to focus the article 
on the results of the tests (Supp. Table 1 and Figure 1) and their diagnostic performance without 
applying literature thresholds (Table 4 and  calculated thresholds); and then evaluate the validity 
of the cut -offs proposed by the literature (Table 3). As it is now, it seems that the article gives 
too much weight on the evaluation of the literature thresholds.  
Suggestion of paragraphs' order in t he Results section:  
* Participant characteristics (including the Lactase genotyping section)  
* Malabsorption marker difference between LP and LNP individuals with lactose or milk (refer 
to Supplemental Table 1 and Figure 1)  
* Diagnostic accuracy of malabs orption marker using ROC curve analyses assessed against LCT 
genotypes (refer to Table 4)  
* Calculated cut -off values after milk and lactose challenge (include calculated cut -offs for 
lactose and milks intake in Table 4)  
* Validity of malabsorption markers  using literature cut -offs assessed against LCT genotype 
(refer to Table 3).  
 
Additional results that could have been added:  
* Provide, as Supplemental figures, the ROC curves (with Youden indexes when applicable).  
* Show, as Supplemental figures, the kin etics comparing LP vs LNP subjects, for breath H2, 
blood glucose or urine galactose/creatinine, after lactose or milk intake. Alternately, if the data 
has already been shown with the same subject grouping in Figure 4 of Milan et al 2019 (ref 20), 
make refe rence to this figure in the manuscript.  However, it appears that the subject grouping 
between both reports is different justifying the proposed supplementary figure.  
* Indicate the calculated cut -offs for the glucose and the galactose tests after lactose intake (since 
their accuracies were 'acceptable'). These calculated thresholds can later be compared and 
discussed with the literature -based thresholds.  
* Do the ROC curves AUCs differ statistically between the tests (e.g. H2 breath test using 
Lactose vs C ONmilk vs A2Mmilk)?  
 
Choice of the 25 ppm cut -off for the H2 breath test:  
The authors justify their choice of 25 ppm cut -off by citing 3 articles (ref. 19 -21). The article 
from Casellas et al. (ref. 20 and 21 also cite Casellas et al.) does not give ration al regarding the 
use of a 25 ppm cut -off. 
However, general consensus is to use a cut -off of 20 ppm (Rezaie et al., Am J Gastroenterol. 
2017;112(5):775 -84 or Gasbarrini et al., Aliment Pharmacol Ther. 2009; 29: 1 -49. 
doi:10.1111/j.1365 -2036.2009.03951.x).  
Author should give more rational for the use of 25 ppm cut -off, or provide results using the 20 
ppm cut -off. 
 
Results inconsistencies:  
* Breath H2 levels for LNP subjects are lower after CON milk intake compared to A2 milk 
intake (Supplem. Table 1, 65.22 pp m vs 89.86 ppm), is this difference significant? On the other 
hand, Figure 1, as well as the results from the same study from Milan et al. Fig. 4A (ref. 20), 
show the contrary. Please clarify and/or discuss.  
 
 
Various remarks:  
 
Table 2:  
* Please, present P -values in and the statistical test used.  
 
Table 3:  
* Cut -offs for urinary galactose are incorrect, please correct for 0.10  
* Not necessary to merge the results for CON and A2 milk as it does not improve the results  
 
Table 4:  
* Please i ndicate to what test the P -values refer to (should also be mentioned in the Methods 
section)  
* Add calculated thresholds  
 
Supp Table 1:  
* Please add units  
* Which statistical test was used?  
 
Figure 1:  
* Explanation of dotted/solid lines on graph is confu sing.  
* Please indicate that the subjects were classified as LP or LNP based on the genotyping test.  
* Use same Y -scale on the 3 plots?  
 
Introduction:  
* Line 27.  Rephrase: "Presence of C/T13910 and G/A22018 polymorphisms, determined by 
restriction fragmen t length polymorphism, was used as the diagnostic reference for LNP."  
* Line 29.  The Results section of the introduction should give less weight to the results obtained 
with literature cut -offs. 
* Line 33.  "urinary galactose/creatinine could not be compa red to…" explain why they could 
not be compared or rephrase "whereas values obtained for urine gal/creatinine…".  
* Line 38.  Please use the values obtained with the adjusted lactose doses, so it can be compared 
with lactose intake (92.9 % instead of 85.7 % , 78.6 % instead of 71.4 %).  
 
Background:  
* Line 55.  Remove 'the' before lactase.  
* Line 59.  "however, intolerance to other foods can…."  
* Line 60.  Specify what kind of digestive symptoms are observed for LI, and give an example or 
reference of a food causing the same types of digestive symptoms as dairy. LI also needs to be 
added to the list of abbreviations at the end.  
* Lines 68 -81.  Explain which homozygosity/heterozygosity of the genotypes translates to 
LP/LNP.  
* Line 94.  The phrase 'rarely been established' is a bit misleading, since some cut -offs have been 
proposed by other groups. Perhaps change to 'have not been validated'.  
* Lines 96 -99.  Ch eck grammar and phrasing of this sentence.  
* Line 99.  'Milk' cannot be argued. Consider changing to 'it has been argued that milk is a 
more…'.  
* Line 100.  The word 'medium' is used here seemingly to describe both milk and lactose, but 
lactose is not a me dium itself.  
* Lines 100 -103.  Check grammar and phrasing of this sentence.  
* Lines 105 -106.  Same comment as above regarding line 94.  
* Lines 111 -112.  Contrary to breath H2 and blood glucose, urinary galactose is not a commonly 
used LM measure: specify " breath H2, plasma glucose, and urinary galactose/creatinine" in the 
sentence.  
 
 
Methods:  
* Line 121.  Should be 'following ingestion of lactose and milk'. Check throughout manuscript.  
* Line 122. 'Digestive' symptoms?  
* Line 140.  Please give lactose source  and purity.  
* Line 155.  Centrifugation of whole blood?  
* Line 170.  Add company details for Lonza.  
* Line 179.  Spelling mistake. Should be 'manufacturer's'.  
* Line 203.  Spelling mistake. Should be 'assessed'.  
* Lines 206 -210.  Please give a short descr iption for "sensitivity", "specificity", PPV etc. or 
provide a reference, especially since these metrics are heavily used and described in the results.  
* Line 215.  0.70 &lt;AUC &lt;0.80 instead of AUC &gt;0.70&lt;0.80? (same at line 216).  
* Line 221.  Spelling mistake for 'malabsorption' in heading of Table 1.  
* Line 232.  P &lt;0.05?  
 
Results:  
* Lines 235 -236.  Check grammar. Consider changing to 'as Caucasian…with a minority of…'.  
* Line 245.  G/A2210 ?  
* Line 247.  AA??  
* Lines 249 -250.  'with lactose  or milk' consumption?  
* Line 263.  Why lower than previous reports? Elaborate in discussion.  
* Line 268.  Add P -value for k=0.319  
* Line 283.  "The sensitivity was unchanged (78.6 %)". The table shows a decrease to 71.4 %.  
* Line 303.  Specify: plasma glucose and urinary galactose/creatinine ratio.  
 
Discussion:  
* Line 318.  For discussion, put cut -off values obtained in this study in context with previous 
studies. Including for cut -off values calculated after lactose intake.  
* Line 326.  β-casein variation in milk was a further determinant of the breath H2 test 
performance.  
* Line 366.  Spelling mistake: 'one serving'.  
* Lines 339/340.  Or affected by the urine adjustment method (using creatinine or specific 
gravity).  
* Line 366.  Please precise that lactose dose adjustment avoided one false negative resulting in 
better sensitivity of the test.  
* Lines 374 -375.  Should rephrase: "these milk types resulted in different breath H2 levels 
(check if significant), different sensitivity for the b reath H2 test to detect LNP and different…"  
* Lines 381 -382.  "… when milk is used as a substrate to diagnose LNP using H2 breath test".  
* Lines 422 -425.  Unclear sentence, consider rephrasing.  
 
References:  
* Please revise references 29 &amp;  45 
 
 
 
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the  necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
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 am able  to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in t he 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.  
 
No competiting 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 webs ite 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 nam ed 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