Elastography—A Bona Fide Non-Invasive Method for Assessing Non-Alcoholic Fatty Liver Disease in Children

Round 1

Reviewer 1 Report

A review of the various factors related to NAFLD in children is presented, while the diagnostic value of elastography as a supporting tool is discussed. Some reference elastographic values related to pathologies, such as fibrosis, are also collected.

As the authors claim, there are few references on this subject, the value of this work lies in compiling this information adequately, however, given the “review” nature of the proposed document, an alarming number of issues have not been considered, starting with the aim of the document. Also, the document clearly focuses on the clinical part, sometimes  leaving aside elastography.

General comments

1. With this title, the reader is expected to find a review of elastographic techniques and the various reference values that are or could be used in real clinical situations. Also, an extensive discussion of the current problems/challenges of using these techniques and how they are currently being solved (or intended to be solved), as well as propose where efforts should be concentrated.
2. The abstract does not make clear what the objective of the document is. It describes a little about motivation and quickly concludes by saying that elastography has some utility. Authors should emphasize how they want to demonstrate this claim.
3. At the end of the introduction, a paragraph describing the aim of the document is necessary, why are the authors writing this? Justify your work.
4. It is hard to read a review without any summarizing tables, with the most relevant data and values interesting for readers.
5. Also, no images are provided, liver elastogaphy images showing confounding artifacts or typical challenges would significantly improve the work.
6. The elastography techniques are barely described. Consider extending this paragraphs with more technical details, highlighting differences between techniques and where should they be applied.
7. In section 2.2, no elastography challenges are discussed, it is more about inherent limitations of elastography instead of liver/obesity related issues.
8. In general, it is difficult to follow the document, there are back and forth discussions on the same topic. A reorganization is required.
9. The conclusions section should be one the key parts of the document, where authors state their vision on the subject and concisely summarizes their compiled information. As with the abstract, it resembles a general reflection, but the reader would expect to get a new idea. Consider rewriting entirely.
10. In general, an important English proofreading is needed. Avoid “ones” and mixing the past and present tenses constantly.

Specific comments

1. Line18: not clear “diagnosis is essential for further development”, rewrite
2. Line41: do not use “ones”
3. Lines83-85: not clear the mixing of concepts between stiffness and elasticity. Use a general reference on elastography.
4. Line87: specify elastography methods for liver assessment
5. Line92: what process?
6. Line 102: “produced”, consider generated
7. Line103: rewrite
8. Line106: “stiffness”, change for stiffer
9. Lines161-162: not clear why differentiating shear wave and ARFI, the authors included shear wave in ARFI before
10. Lines163-166: comparison of speeds and elasticity values (are the Young modulus or stiffness/shear modulus), maybe a discussion on how this values are related is worth
11. Line177: increase respect to what
12. Line195: new line before “a major”
13. Lines221-222: do not describe a paper and cite it twice, summarize these lines
14. Lines240-242: if the title claims that elastography is convenient why this technique is doubted here?

Author Response

March the 28^th, 2021

To Editor/Reviewers of Applied Sciences,

Dear Editor/Reviewers,

Please find attached a revised version of the manuscript entitled: "Elastography – a bona fide non-invasive method for assessing non-alcoholic fatty liver disease in children" written by Cristina Oana Mărginean, Lorena Elena Meliț, and Maria Oana Săsăran,  Manuscript ID: applsci-1147143.

Firstly, we thank very much the reviewers for their valuable comments and suggestions in order to improve our paper.

Following the reviewers’ concerns and observations, we made some modifications to the initial version of our manuscript, which we described in great detail, according to their recommendations, highlighting them in blue in the attached manuscript as it follows:

Answer to Reviewer 1

Comment 1

A review of the various factors related to NAFLD in children is presented, while the diagnostic value of elastography as a supporting tool is discussed. Some reference elastographic values related to pathologies, such as fibrosis, are also collected.

As the authors claim, there are few references on this subject, the value of this work lies in compiling this information adequately, however, given the “review” nature of the proposed document, an alarming number of issues have not been considered, starting with the aim of the document. Also, the document clearly focuses on the clinical part, sometimes  leaving aside elastography.

Answer 1

Firstly, we thank you very much for your positive comments and valuable suggestions. We sincerely hope that the revised form of our manuscript according to your recommendations fulfills all of your concerns.

Comment 2

With this title, the reader is expected to find a review of elastographic techniques and the various reference values that are or could be used in real clinical situations. Also, an extensive discussion of the current problems/challenges of using these techniques and how they are currently being solved (or intended to be solved), as well as propose where efforts should be concentrated.

Answer 2

Thank you for your suggestion. According to your recommendation, we consider more appropriate to change our title into: ‘Elastography – a bona fide non-invasive method for assessing non-alcoholic fatty liver disease in children’ since it reflects better the aim of our review. Also, we introduced a more detailed approach of elastography techniques and their use in the clinical setting of NAFLD since this was the aim of our review. Moreover, we discussed the problems/challenges that might emerge as a result of these techniques in this clinical situation and reorganized the section  ‘The opportunities and challenges of ultrasound-based elastography in children NAFLD’ introducing multiple new information, all of them being highlighted in blue.

Comment 3

The abstract does not make clear what the objective of the document is. It describes a little about motivation and quickly concludes by saying that elastography has some utility. Authors should emphasize how they want to demonstrate this claim.

Answer 3

According to your recommendation, we rephrased the abstract, line 15-34: ‘Pediatric obesity became a major public health problem worldwide resulting in a wide spectrum of systemic complications. Liver disease associated to obesity, also known as nonalcoholic fatty liver disease (NAFLD) is currently the most common chronic liver condition in children. Therefore, its timely and proper diagnosis is essential for preventing further development of cirrhosis. Multiple studies focused on identifying the most accurate non-invasive diagnostic method for liver fibrosis or cirrhosis. Although liver biopsy remains the gold-standard in terms of this hepatopathy, elastography methods emerged as a relatively reliable alternative to liver biopsy. Thus, recent studies revealed the great importance of these non-invasive methods not only in diagnosing pediatric NAFLD, but also in its staging. MRE is commonly considered to have a greater accuracy than ultrasound-based elastography methods, but with lower availability and higher costs. Ultrasound-based elastography methods (TE, p-SWE and 2D-SWE) were proved to have similar accuracy in NAFLD staging. Nevertheless, multiple confounding factors account for potential challenges when using elastography for liver stiffness measurement, such as age, obesity itself (i.e. BMI), transaminase levels, or portal flow. A potential solution for facing these challenges might be represented by a complex approach based on the combination between elastography, clinical and laboratory findings. Although the studies that assessed the role of elastography in pediatric NAFLD staging are scarce, the current knowledge underlines a crucial role of these techniques taking into account their ability to distinguish between fibrosis degrees, their non-invasive patterns, lower costs and side effects when compared to liver biopsy. Therefore, elastography might become a cornerstone in staging pediatric NAFLD.’

Comment 4

At the end of the introduction, a paragraph describing the aim of the document is necessary, why are the authors writing this? Justify your work.

Answer 4

We apologize for not clearly mention the aim of our review. Nevertheless, according to your recommendation, we introduced the following sentence stating our aim at the end of introduction, line 94-95: ‘The aim of this review was to assess the role of elastography in pediatric NAFLD staging, as well as identifying its potential limitations and challenges in this age group.’

Comment 5

It is hard to read a review without any summarizing tables, with the most relevant data and values interesting for readers.

Answer 5

Thank you for your suggestions. We summarized the most important data from the literature on pediatric patients into 4 tables comprising cut-off values, elastography values in children with obesity as well as studies comparing elastography findings with biopsy scores. Each table was also mentioned in the text where appropriate:

• At the end of the paragraph regarding MRE, lines 177-178, we introduced: ‘The most important findings regarding MRE in children were summarized in Table 1.’
• In the section Current elastography methods, at the end of the paragraph discussing ultrasound-based elastography methods, lines 216-217: ‘Cut-off values for ultrasound-based elastography in children identified in the literature were summarized in Table 2.’
• In the section 2.2., lines 359-360: ‘The most relevant studies that compared the results between liver elastography and biopsy findings in children NAFLD were summarized in Table 3.’
• In the section 2.2., lines 381-383: ‘The most relevant studies that assessed NAFLD in children using ultrasound-elastography based methods were described in Table 4.’

Comment 6

Also, no images are provided, liver elastogaphy images showing confounding artifacts or typical challenges would significantly improve the work.

Answer 6

Thank you for your suggestion, we provided 4 liver elastography images from our clinical practice as you recommended: Figure 1. Liver TE in normal weight healthy children, Figure 2. Liver 2D-SWE in normal weight healthy children, Figure 3. TE – liver invalid measurement (children), Figure 4. 2D-SWE – liver invalid measurement (children)

Comment 7

The elastography techniques are barely described. Consider extending this paragraphs with more technical details, highlighting differences between techniques and where should they be applied.

Answer 7

Thank you for your suggestion. We improved the section of current elastography techniques as you recommended,

• lines 131-147: “Clinical liver MRE set up usually consists of an active driver, a passive driver positioned in the inferior part of the right chest wall closer to the liver, and a 25-foot-long plastic tube connecting these drivers. The active driver is represented by an acoustic driving system that generates 60Hz shear waves in tissue from outside the scanner room. The passive driver is activated as a result of varying acoustic pressure triggered from the active driver via the plastic tube. Patients must rest in supine position during the exam. Tissue displacement caused by the propagation of shear waves is determined by a modified phase contrast imaging sequence, which uses different conventional MR sequences, such as gradient recalled echo, echoplanar imaging, spin echo, or balanced steady state free precision. MRE has the capacity to spatially map and quantify displacement patterns, which are useful for the calculation of certain mechanical characteristics related to wave propagation. Several MRE algorithms are available for the inversion of wave information into stiffness maps such as direct inversion of differential equations of motion, spatial frequency measurement, or iterative method based on the finite element model. These algorithms are used for measuring an important mechanical tissue characteristic, i.e. ‘the magnitude of the complex shear modulus’ accounting for both elasticity and viscosity, expressed in kilopascals[34–36].”;
• lines 153-168: “Nevertheless, a more recent study performed on 71 healthy pediatric volunteers revealed a mean liver stiffness value of 2.1 kPa pointing out that sex, age or BMI had no impact on liver stiffness measurement[34]. Conflicting results might be explained by technical or platform-related differences in terms of strength, driver frequency used for MRE, MR scanner vendor, or they might reflect the differences between the populations included in the study[34]. Liver thresholds for MRE defined for healthy children differ from those in healthy adults[39]. Thus, Etchell et al revealed that healthy children present lower liver stiffness values as compared to healthy adults[40]. These findings came in contradiction to those of Sawh et al, who recently found that normal liver stiffness values were higher in children than previously reported for adults[33]. In terms of liver fibrosis, different studies pointed out that the cut-off values range between 2.4-2.93 kPa[41–45]. A strong correlation has been identified between MRE liver stiffness measurements and the amount of fibrosis in the liver biopsy samples suggesting that MRE has the same accuracy as biopsy for liver fibrosis staging[46]. The accuracy of MRE for detecting liver fibrosis increases with the stage of fibrosis, but with an excellent overall performance[37].”
• lines 200-214: “In practice, an initial push pulse is applied to the tissue inducing shear waves perpendicular to the ultrasound beam. The speed of the shear waves propagating through the tissue are estimated using B-mode imaging tracking, and it is proportional to tissue stiffness. Using these methods, liver stiffness can be expressed in m/s displaying the speed of the shear wave through the tissue, or in kPa, based on the Young’s modulus. 2D-SWE uses a large field of view with pixel color coding of the stiffness values, while p-SWE is based on the assessment of a fixed region of interest (ROI) of approximately 1 ml. Regarding the actual technique, several specifics are important such as the transducer’s position parallel to the liver capsule, the ROI parallel to the transducer and perpendicular to the ARFI pulse, as well as the observer’s focus on avoiding artefactual liver stiffening by taking the measurements 1.5-2 cm under the liver capsule. In order to obtain a value as accurate as possible, ten measurements are recommended in case of p-SWE and 5 for 2D-SWE[60]. Nevertheless, the quality criteria depend on the recommendation of each vendor[61]. The variability of measurements performed with p-SWE and 2D SWE was found to increase with liver stiffness[47].”

Comment 8

In section 2.2, no elastography challenges are discussed, it is more about inherent limitations of elastography instead of liver/obesity related issues.

Answer 8

Thank you for your suggestion. It is true that we mentioned mostly elastography limitations, but these limitations account also for potential challenges in daily practice. We reorganized section 2.2 according to your recommendations underlining elastography challenges/limitations and how they can be solved.

Comment 9

In general, it is difficult to follow the document, there are back and forth discussions on the same topic. A reorganization is required.

Answer 9

We apologize for repeating certain information but our intention was only to underline their current extreme importance in this field. Nevertheless, we reorganized our manuscript following your recommendations.

Comment 10

The conclusions section should be one the key parts of the document, where authors state their vision on the subject and concisely summarizes their compiled information. As with the abstract, it resembles a general reflection, but the reader would expect to get a new idea. Consider rewriting entirely.

Answer 10

Thank you for your valuable suggestions. We rephrased the section of conclusions in line with your recommendations, lines 401-423: ‘NAFLD is most-likely a real challenge for pediatricians and its proper diagnosis and close follow-up are essential for preventing further life-threatening complications, such as cirrhosis, end-stage liver disease or hepatocarcinoma. Taking into account the more expressed need for non-invasive approach in pediatric patients as compared to adults, elastography methods represent promising diagnostic tools with a relatively good accuracy for NAFLD staging. Nevertheless, multiple confounding factors should be closely monitored when assessing NAFLD children on elastography, such as age, BMI, transaminase levels, or portal blood flow. A complex approach combining elastography parameters with clinical and laboratory findings might increase the accuracy of ultrasound-based elastography in terms of NAFLD staging. Albeit elastography is limited in differentiating between mild fibrosis and normal liver, it is extremely useful for diagnosing liver fibrosis in children with obesity. Moreover, elastography is crucial for NAFLD monitoring and for guiding liver biopsy in selected cases. Still, multiple controversies arose due to their relatively novel use in small ages and further studies are required in order to define especially their role in delineating the staged of liver fibrosis in children with obesity.’

Comment 11

In general, an important English proofreading is needed. Avoid “ones” and mixing the past and present tenses constantly.

Answer 11

We apologize for our language mistakes. Our work was revised by a native English speaker.

Comment 12

Line18: not clear “diagnosis is essential for further development”, rewrite

Answer 12

We apologize for our writing mistakes, we corrected as it follows, line 18: ‘…diagnosis is essential for preventing further development…’.

Comment 13

Line 41: do not use “ones”

Answer 13

We replaced the word ‘ones’ with ‘disorders’, line 48.

Comment 14

Lines 83-85: not clear the mixing of concepts between stiffness and elasticity. Use a general reference on elastography.

Answer 14

According to your recommendation, we stated the general reference on elastography, lines 102-103: ‘Thus, when referring to elastography, the term liver stiffness seems to be more appropriate.’

Comment 15

Line 87: specify elastography methods for liver assessment

Answer 15

Thank you for your suggestion, line 113.. We specified elastography methods for liver assessment

Comment 16

Line 92: what process?

Answer 16

We rephrased for a better understanding, line 119.: ‘Liver fibrosis is a dynamic process’.

Comment 17

Line 102: “produced”, consider generated

Answer 17

We replaced ‘produced’ with ‘generated’ according to your recommendations, line 126.

Comment 18

Line103: rewrite

Answer 18

We rephrased as it follows, lines 127-129.: ‘…the liver and their images are obtained using a modified phase contrast MRI sequence, further converted into tissue stiffness maps or elastograms as a result of an inversion algorithm’,

Comment 19

Line 106: “stiffness”, change for stiffer

Answer 19

We changed ‘stiffness’ with ‘stiffer.’ – line 130

Comment 20

Lines 161-162: not clear why differentiating shear wave and ARFI, the authors included shear wave in ARFI before

Answer 20

We apologize for the confounding statement, but we referred to another shear-wave based technique, i.e. supersonic shear imaging. Therefore, we rephrased our statement as it follows, line 323-324: ‘with a higher sensitivity and specificity using supersonic shear imaging in comparison with ARFI’. - lines

Comment 21

Lines163-166: comparison of speeds and elasticity values (are the Young modulus or stiffness/shear modulus), maybe a discussion on how this values are related is worth

Answer 21

According to the most recent study of Feraiolli et al (Ferraioli G, Barr RG, Dillman JR. Elastography for Pediatric Chronic Liver Disease: A Review and Expert Opinion, Ultrasound Med 2020; 9999:1–20) liver stiffness can be measured either using the speed of the share is expressed in m/s or the Young’s modulus expressed in kPa. Therefore, for all ultrasound-based elastography techniques the results can be provided either as m/s or Young’s modulus in kPa depending on the radiologist or the referring clinician’s preference. Thus, we rephrased and introduced a discussion regarding the relationship between these values as it follows, lines 325-334: ‘The authors pointed out that the reference values according to ARFI are 1.34 m/s in case of stage 1 fibrosis, 1.57 m/s in stage 2, 1.85 m/s for stage 3, and 2.13 m/s for stage 4. Contrariwise, in terms of supersonic shear imaging, the authors found a median cutoff value of 7.9 kPa in case of stage 1, 9.4 kPa in stage 2, 14.2 kPa for stage 3, and 23.94 kPa for stage 4[110]. Although for ARFI the results are provided in m/s and for supersonic shear imaging as Young’s modulus in kPa, both elastography methods reveal increasing values as liver fibrosis advances. Similar trends were provided by Mărginean et al, who reported the results of 2D-SWE as both m/s and Young’s modulus in kPa[19].  (Mărginean, C.O.; Meliţ, L.E.; Ghiga, D.V.; Săsăran, M.O. The Assessment of Liver Fibrosis in Children with Obesity on Two Methods: Transient and Two Dimensional Shear Wave Elastography. Sci Rep 2019, 9, 19800, doi:10.1038/s41598-019-56358-2). Based on these findings we might assume that the values liver stiffness assessed in m/s or in kPa are directly related since an increase in both in noticed in stiffer livers.’

Comment 22

Line 177: increase respect to what

Answer 22

We rephrased for clarification, line 252: ‘directly related to the individual’s age’.

Comment 23

Line 195: new line before “a major”

Answer 23

Thank you for your suggestion. We introduced a new line as you recommended, line 346.

Comment 24

Lines 221-222: do not describe a paper and cite it twice, summarize these lines

Answer 24

Thank you for your valuable suggestion. We rephrased the lines you mentioned as summarized the content avoiding to cite twice the paper, lines 386-391: ‘Therefore, a study that compared SWE and conventional ultrasound findings in children with obesity and normal weight ones pointed out that mean SWE velocity values were significantly higher in the obese group with abnormal liver echogenicity at conventional ultrasound that in those with apparently normal aspect of liver, but the authors failed in identifying a significant difference in normal weight children[68].’.

Comment 25

Lines 240-242: if the title claims that elastography is convenient why this technique is doubted here?

Answer 25

Elastography is indeed a convenient technique for the assessment of liver fibrosis in children, but it still presents several limitation especially due to the scarce reports in small ages. Nevertheless, we rephrased the sentence in order to avoid confusions, lines 421-423: ‘Still, multiple controversies arose due to their relatively novel use in small ages and further studies are required in order to define especially their role in delineating the stages of liver fibrosis in children with obesity’.

Thus, by this letter and by the attached revised manuscript of our original manuscript, we hope to have fulfilled all the observations and recommendations made by the Reviewers.

Thank you for your time and consideration.

On behalf of all authors of this work,

Yours sincerely,

Lecturer Lorena Elena Meliț, MD, PhD

Departament of Pediatrics, University of Medicine and Pharmacy Tirgu Mures, 38 Gh. Marinescu St., 540136, Tirgu Mures, Romania. Phone: +40-742-984744, e-mail: [email protected]

Reviewer 2 Report

In the part on pSWE I suggest discussing a recent article on the impact of skin-to-liver distance in fibrosis staging in patients with NAFLD - DOI: 10.3390/diagnostics10100795, in particular  if any study on it was evaluated  in children  

Author Response

March the 28^th, 2021

To Editor/Reviewers of Applied Sciences,

Dear Editor/Reviewers,

Please find attached a revised version of the manuscript entitled: "Elastography – a bona fide non-invasive method for assessing non-alcoholic fatty liver disease in children" written by Cristina Oana Mărginean, Lorena Elena Meliț, and Maria Oana Săsăran,  Manuscript ID: applsci-1147143.

Firstly, we thank very much the reviewers for their valuable comments and suggestions in order to improve our paper.

Following the reviewers’ concerns and observations, we made some modifications to the initial version of our manuscript, which we described in great detail, according to their recommendations, highlighting them in blue in the attached manuscript as it follows:

Answer to Reviewer 2

Comment 1

In the part on pSWE I suggest discussing a recent article on the impact of skin-to-liver distance in fibrosis staging in patients with NAFLD - DOI: 10.3390/diagnostics10100795, in particular  if any study on it was evaluated  in children  

Answer 1

Thank you for your suggestion. We discussed the article you mentioned above and searched also for other studies using elastography methods on this topic for a better assessment. Therefore, we introduced the following paragraph as a major limitation and a limitation of elastography methods in assessing NAFLD, lines 287-306: ‘This limitation is defined by the impact of skin-to-liver distance on the feasibility and accuracy of liver elastography. In terms of elastography, it was hypothesized that skin-to-liver distance <34 mm is optimal for discriminating between fibrosis stages[100]. This hypothesis is in line with other studies which concluded that in patients with a BMI ≥30 kg/m², elastography has a relatively low accuracy in differentiating between the first two fibrosis stages[101,102]. It was also underlined that the success rate in obese patients depends on the technique being proved that TE has a higher failure rate in this group when compared to p-SWE[101]. Moreover, a recent study performed on obese adults emphasized that p-SWE is able to discriminate between fibrosis stages independently of its severity if skin-to-liver distance is taken into account following two rules: a thicker abdominal wall results in a lesser accuracy for a proper histological staging and the impact of skin-to-liver distance on accurate fibrosis staging is machine-dependent[103]. In terms of ARFI, it was showed that interoperator concordance increases when skin-to-liver distance is <2.5 cm[104], being previously documented that a higher concordance is reflected on the correlation between fibrosis stages assessed on ARFI and histology[105]. Using TE, another recent study proved that a skin capsular distance ≥25 mm results in overestimation of fibrosis affecting the detection of severe fibrosis in patients with NAFLD[106]. Thus, this is an important challenge that should be addressed also in children with NAFLD since to date we found no study on this topic in pediatric patients with NAFLD.’

Thus, by this letter and by the attached revised manuscript of our original manuscript, we hope to have fulfilled all the observations and recommendations made by the Reviewers.

Thank you for your time and consideration.

On behalf of all authors of this work,

Yours sincerely,

Lecturer Lorena Elena Meliț, MD, PhD

Departament of Pediatrics, University of Medicine and Pharmacy Tirgu Mures, 38 Gh. Marinescu St., 540136, Tirgu Mures, Romania. Phone: +40-742-984744, e-mail: [email protected]

Reviewer 3 Report

Mărginean CO et al. reviewed elastography for assessing liver impairment in children with obesity. They concluded that elastography might become a cornerstone in the diagnosis of pediatric NAFLD. The most serious problem with this paper is that it is not clearly separated from Andersen's report.¹ The following is my comments to the authors.

Major comments:

There are already many reviews of elastography. Many of the papers cited in the authors' paper are outdated, and the following numbered papers should be replaced with newer papers. Reference numbers 8, 12, 13, 16, 18, 23, 24, 25, 35, 38, 43.

The authors should cite and redraft the following article to include the most recent data listed below.^2-8

Reference number 18 should be changed to the latest guideline.

Minor comment:

Abstract. “Hepatopathy” is not a common expression. Please use another word.

References

1. Andersen SB, Ewertsen C, Carlsen JF, Henriksen BM, Nielsen MB. Ultrasound Elastography Is Useful for Evaluation of Liver Fibrosis in Children-A Systematic Review. J Pediatr Gastroenterol Nutr. 2016;63(4):389-399.
2. Younossi ZM, Noureddin M, Bernstein D, et al. Role of Noninvasive Tests in Clinical Gastroenterology Practices to Identify Patients With Nonalcoholic Steatohepatitis at High Risk of Adverse Outcomes: Expert Panel Recommendations. Am J Gastroenterol. 2021;116(2):254-262.
3. Trout AT, Anupindi SA, Gee MS, et al. Normal Liver Stiffness Measured with MR Elastography in Children. Radiology. 2020;297(3):663-669.
4. Trout AT, Xanthakos SA, Bennett PS, Dillman JR. Liver Shear Wave Speed and Other Quantitative Ultrasound Measures of Liver Parenchyma: Prospective Evaluation in Healthy Children and Adults. AJR Am J Roentgenol. 2020;214(3):557-565.
5. Sawh MC, Newton KP, Goyal NP, et al. Normal range for MR elastography measured liver stiffness in children without liver disease. J Magn Reson Imaging. 2020;51(3):919-927.
6. Li DK, Khan MR, Wang Z, et al. Normal liver stiffness and influencing factors in healthy children: An individual participant data meta-analysis. Liver Int. 2020;40(11):2602-2611.
7. Ferraioli G, Barr RG, Dillman JR. Elastography for Pediatric Chronic Liver Disease: A Review and Expert Opinion. J Ultrasound Med. 2020.
8. Hwang JY, Yoon HM, Kim JR, et al. Diagnostic Performance of Transient Elastography for Liver Fibrosis in Children: A Systematic Review and Meta-Analysis. AJR Am J Roentgenol. 2018;211(5):W257-W266.

Author Response

Answer to Reviewer 3

Comment 1

Mărginean CO et al. reviewed elastography for assessing liver impairment in children with obesity. They concluded that elastography might become a cornerstone in the diagnosis of pediatric NAFLD. The most serious problem with this paper is that it is not clearly separated from Andersen's report.¹ The following is my comments to the authors.

There are already many reviews of elastography. Many of the papers cited in the authors' paper are outdated, and the following numbered papers should be replaced with newer papers. Reference numbers 8, 12, 13, 16, 18, 23, 24, 25, 35, 38, 43.

Answer 1

Thank you very much for your valuable time spent on assessing our manuscript. It is true that Andersen et al also performed a review on the role of elastography in children with liver fibrosis, but our aim was to focus and assess more in detail the role of these techniques in children with NAFLD since obesity has become a global public health problem. The review of Andersen discusses the general aspects of elastography methods in children with liver fibrosis indicating the especially the specific aspects in healthy children vs. children with liver fibrosis, the differences between measurements, without assessing in particular NAFLD as a cause of liver fibrosis.

Thank you for your suggestion. According to your recommendation we replaced the indicated references.

Comment 2

The authors should cite and redraft the following article to include the most recent data listed below.^2-8

Answer 2

We redrafted the manuscript following your recommendations as it follows

• We replaced the reference 8 and rephrased accordingly, lines 55-59 and lines 84-87: ‘NAFLD defined as accumulation of fat in the liver is generally a silent, potentially reversible condition, but it might also progress into a more severe form, i.e. non-alcoholic steatohepatitis (NASH) associating steatosis, inflammation and cellular injury with or without fibrosis[8], with an increased the risk for cirrhosis and end-stage liver disease[9,10]. (Younossi ZM. Et al. Role of Noninvasive Tests in Clinical Gastroenterology Practices to Identify Patients With Nonalcoholic Steatohepatitis at High Risk of Adverse Outcomes: Expert Panel Recommendations. Am J Gastroenterol 2021;116:254–262. https://doi.org/10.14309/ajg.0000000000001054), increasing the risk for cirrhosis and end-stage liver disease’ (Setiawan VW, Stram DO, Porcel J, et al. Prevalence of chronic liver diseaseand cirrhosis by underlying cause in understudied ethnic groups: The Multiethnic Cohort. Hepatology 2016;64:1969–77; Mohamad B, Shah V, Onyshchenko M, et al. Characterization of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD) patients without cirrhosis. Hepatol Int 2016;10:632–9)
• References 12, 13, 16 and 18 were replaced and we rephrased the statements regarding serum biomarkers using the references you recommended as it follows, lines 71-80: ‘The American Association for the Study of Liver Disease (AASLD) recommends the use of two algorithms, FIB-4 and NFS for the assessment of liver fibrosis based on routine clinical and laboratory parameters, which seem to be useful in predicting advanced hepatic cirrhosis or fibrosis[14]. (Chalasani N, Younossi Z, Lavine JE, et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology 2018;67:328–57). Thus, FIB-4 takes into account the age, platelets count, aspartate aminotransferase (AST) and alanine aminotransferase and it was proved to be useful for the stratification of NAFLD patients differentiating between those with advanced fibrosis versus none[15–17] (Sterling RK, Lissen E, Clumeck N, et al. Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection. Hepatology 2006;43:1317–25; Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepatol 2018;68:305–15; Anstee QM, Lawitz EJ, Alkhouri N, et al. Noninvasive tests accurately identify advanced fibrosis due to NASH: Baseline data from the STELLAR trials. Hepatology. 2019;70:1521–30). NFS is an even more complex algorithm including not only the age, the platelets and AST/ALT ration, but also BMI, albumin levels and the assessment of glucose tolerance/diabetes, representing a reliable alternative for liver biopsy[8] (Younossi ZM, Noureddin M, Bernstein D, et al. Role of Noninvasive Tests in Clinical Gastroenterology Practices to Identify Patients With Nonalcoholic Steatohepatitis at High Risk of Adverse Outcomes: Expert Panel Recommendations. Am J Gastroenterol 2021;116:254–262.)’ A clear bidirectional interrelation has been stated between NAFLD and metabolic syndrome, whose components (e.g. type 2 diabetes mellitus, obesity, hypertension, dyslipidemia) additionally increase the risk of developing NAFLD[20–23]. (Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64:73–84; Byrne CD, Targher G. NAFLD: a multisystem disease. J Hepatol. 2015;62:S47–64; Younossi ZM, Stepanova M, Afendy M, Fang Y, Younossi Y, Mir H, et al. Changes in the prevalence of the most common causes of chronic liver diseases in the United States from 1988 to 2008. Clin Gastroenterol Hepatol. 2011;9:524–530.e1; quiz e60; Ting YW, Wong SW, Anuar Zaini A, Mohamed R, Jalaludin MY. Metabolic syndrome is associated with advanced liver fibrosis among pediatric patients with non-alcoholic fatty liver disease. Front Pediatr. 2019;7:491. https://doi.org/10.3389/fped.2019.00491)’
• Reference 23 was replaced with ‘Hoodeshenas, S.; Yin, M.; Venkatesh, S.K. Magnetic Resonance Elastography of Liver: Current Update. Top Magn Reson Imaging 2018, 27, 319–333, doi:10.1097/RMR.0000000000000177.’
• References 24 were removed and we introduced more recent information according to your recommendation as it follows, lines 234-236: ‘Multiple confounding factors identified in pediatric populations were reported to influence liver stiffness on elastography, such as age, sedation, hepatic steatosis or inflammation[64,65]. (Li DK, Khan MR, Wang Z, et al. Normal liver stiffness and influencing factors in healthy children: An individual participant data meta-analysis Liver International. 2020;40:2602–2611, Raizner A, Shillingford N, Mitchell PD, et al. Hepatic inflammation may influence liver stiffness measurements by transient elastography in children and young adults. J Pediatr Gastroenterol Nutr. 2017;64(4):512-517. https://doi.org/10.1097/MPG.00000 00000 001376).’
• Reference 25 was replaced with ‘Sawh MC, Newton KP, Goyal NP, et al. Normal Range for Magnetic Resonance Elastography Measured Liver Stiffness in Children without Liver Disease. J Magn Reson Imaging. 2020 March; 51(3): 919–927. doi:10.1002/jmri.26905’
• Reference 35 was replaced and we rephrased as following, lines 220-223: ‘Liver fibrosis in pediatric patients is commonly seen in the setting of fatty liver disease, hepatitis, primary sclerosing cholangitis, autoimmune hepatitis, congestive hepatopathy, cystic fibrosis, biliary atresia, Alagille syndrome, Fontan-associated liver disease, a-1 antitrypsin deficiency, storage disorders and Wilson’s disease[36,60] (Serai, S.D.; Wallihan, D.B.; Venkatesh, S.K.; Ehman, R.L.; Campbell, K.M.; Sticka, J.; Marino, B.S.; Podberesky, D.J. Magnetic Resonance Elastography of the Liver in Patients Status-Post Fontan Procedure: Feasibility and Preliminary Results. Congenit Heart Dis 2014, 9, 7–14, doi:10.1111/chd.12144; Ferraioli G, Barr RG, Dillman JR. Elastography for Pediatric Chronic Liver Disease: A Review and Expert Opinion, Ultrasound Med 2020; 9999:1–20)’
• Reference 38 was replaced by: ‘Li DK, Khan MR, Wang Z, et al. Normal liver stiffness and influencing factors in healthy children: An individual participant data meta-analysis Liver International. 2020;40:2602–2611’
• Reference 43 was removed and more recent information was introduced according to your recommendations, lines 239-244: ‘Multiple studies revealed an increase in liver stiffness with age, but the results remain controversial since the association was found to be weak[64,67–70]. (Trout AT, Xanthakos SA, Bennet PS, Dilmann JR. Liver Shear Wave Speed and Other Quantitative Ultrasound Measures of Liver Parenchyma: Prospective Evaluation in Healthy Children and Adults AJR 2020; 214:1–9; Bailey SS, Youssfi M, Patel M, Hu HH, Shaibi GQ, Towbin RB. Shear-wave ultrasound elastography of the liver in normal-weight and obese children. Acta Radiol 2017; 58:1511–1518; Fontanilla T, Cañas T, Macia A, et al. Normal values of liver shear wave velocity in healthy children assessed by acoustic radiation force impulse imaging using a convex probe and a linear probe. Ultrasound Med Biol 2014; 40:470–477; Matos H, Trindade A, Noruegas MJ. Acoustic radiation force impulse imaging in paediatric patients: normal liver values. J Pediatr Gastroenterol Nutr 2014; 59:684–688; Li DK, Khan MR, Wang Z, et al. Normal liver stiffness and influencing factors in healthy children: An individual participant data meta-analysis Liver International. 2020;40:2602–2611). Thus, Li et al proved a strong association between increasing age and liver stiffness measurements in children ≥3 years[64]. (Li DK, Khan MR, Wang Z, et al. Normal liver stiffness and influencing factors in healthy children: An individual participant data meta-analysis Liver International. 2020;40:2602–2611).’. Moreover, we considered important to mention the potential explanation for the positive association between age and liver stiffness: ‘Most-likely, these findings might be related to microstructure, vascular and metabolic changes that occur during maturation as it was proved on animal models[71,72]. (Pauleau G, Sandoz B, Thollon L, Serre T, Brunet C. Anthropometric characterization of the child liver. Surg Radiol Anat. 2010;32(8):767- 775; Yarpuzlu B, Ayyildiz M, Tok OE, Aktas RG, Basdogan C. Correlation between the mechanical and histological properties of liver tissue. J Mech Behav Biomed Mater. 2014;29:403-416. https://doi.org/10.1016/j.jmbbm.2013.09.016. https://doi.org/10.1007/s0027 6-010-0675-8).’
• We also introduced the findings of Hwang et al. according to your recommendations, lines 378-381: ‘Another recent meta-analysis involving 723 patients underlined that TE represents is a highly accurate method for the diagnosis of liver fibrosis in children with a sensitivity of 95% and a specificity of 90% in the setting of significant liver fibrosis, above stage 2[118].’ (Hwang JY, Yoon HM, Kim JR, et al. Diagnostic Performance of Transient Elastography for Liver Fibrosis in Children: A Systematic Review and Meta-Analysis. AJR:211, November 2018)’
• As well as the findings of Trout in the field of MRE, lines 153-155: ‘Nevertheless, a more recent study performed on 71 healthy pediatric volunteers revealed a mean liver stiffness value of 2.1 kPa pointing out that sex, age or BMI had no impact on liver stiffness measurement[34].’ (Trout AT, Anupindi SA, Gee MS, et al. Normal Liver Stiffness Measured with MR Elastography in Children. Radiology 2020; 00:1–7 https://doi.org/10.1148/radiol.2020201513).

Comment 3

Reference number 18 should be changed to the latest guideline.

Answer 3

Thank you for your suggestion we used the reference in order to underline the risk of hepatic cirrhosis and hepatocarcinoma and therefore we did not consider mandatory to use the latest guideline since we did not use it to introduce recommendations. Nevertheless, we replaced the reference 18 with ‘Chalasani N. et al. The Diagnosis and Management of Nonalcoholic Fatty

Liver Disease: Practice Guidance from the American Association for the Study of Liver Diseases. Hepathology 2017’

Comment 4

Abstract. “Hepatopathy” is not a common expression. Please use another word.

Answer 4

Thank you for your suggestion. We replaced ‘hepatopathy’ with ‘liver condition’ as you recommended.

Reviewer 4 Report

The authors want to report current findings in liver elastography as a genuine method to diagnose NAFLD. Throught the paper, in my opinion, there is a great confusion between NAFLD diagnosis (that can be done by a mere ultrasound examination) and NAFLD staging, where liver fibrosis assessment is of utmost importance. The authors need to clarify this.

Major:

• the term liver impairment is not used correctly, is best to use liver disease or disease staging, the authors are not only talking about liver failure;
• Lines 61-65: the authors should cite and also refer to this recent paper that evaluated the performance of non-invasive markers and elastography.
• 2.1 Current elastography methods: the authors should create a short paragraph on the current state of the art of liver elastography
• Line 98-101: the authors should stress the fact that even blood flow can affect liver stiffness especially in portal hypertension. In terms of confounding factors these are some recent papers that the authors should cite: 1) transaminases - DOI: https://doi.org/10.3390/microorganisms8030348 // DOI: 10.1007/s40477-020-00456-9 ; 2)
  
• pSWE/TE/2D-SWE: the authors should actually compare the various elastography techniques as reliable in nafld fibrosis staging, also in terms of different histology scores that can be used (Brunt vs. Kleiner).
  

Minor: english should be revised by a native speaker

Author Response

Answer to Reviewer 4

Comment 1

The authors want to report current findings in liver elastography as a genuine method to diagnose NAFLD. Throught the paper, in my opinion, there is a great confusion between NAFLD diagnosis (that can be done by a mere ultrasound examination) and NAFLD staging, where liver fibrosis assessment is of utmost importance. The authors need to clarify this.

Answer 1

Thank you for your suggestion. Indeed, our intention was to discuss NAFLD staging using elastography, and we corrected for clarification as you recommended where appropriate, and we also introduced the following phrase in the revised form of our manuscript in order to clarify the role of ultrasound in diagnosing NAFLD, as well as elastography in NAFLD staging, lines 394-396: ‘Thus, conventional ultrasound might represent the first step in assessing children with obesity and diagnosing NAFLD[119] in order to guide them for elastography staging, which is definitely associated with greater costs.’ (Hamaguchi, M.; Kojima, T.; Itoh, Y.; Yuichi, H.; Kota, F.; Tomoaki, N.; Takahiro, K.; Noriyuki, T.; Junichi, O.; Kazunori, I.; et al. The Severity of Ultrasonographic Findings in Nonalcoholic Fatty Liver Disease Reflects the Metabolic Syndrome and Visceral Fat Accumulation. Am. J. Gastroenterol. 2007).

Comment 2

• the term liver impairment is not used correctly, is best to use liver disease or disease staging, the authors are not only talking about liver failure;

Answer 2

Thank you for your suggestion. We corrected the term liver impairment and replaced it with liver disease.

Comment 3

• Lines 61-65: the authors should cite and also refer to this recent paper that evaluated the performance of non-invasive markers and elastography.

Answer 3

Thank you for your suggestion. We rephrased the entire paragraph regarding the performance of non-invasive parameters and added important, novel information, lines 71-80: ‘The American Association for the Study of Liver Disease (AASLD) recommends the use of two algorithms, FIB-4 and NFS for the assessment of liver fibrosis based on routine clinical and laboratory parameters, which seem to be useful in predicting advanced hepatic cirrhosis or fibrosis[14] (Chalasani N, Younossi Z, Lavine JE, et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology 2018;67:328–57). Thus, FIB-4 takes into account the age, platelets count, aspartate aminotransferase (AST) and alanine aminotransferase and it was proved to be useful for the stratification of NAFLD patients differentiating between those with advanced fibrosis versus none[15–17] (Sterling RK, Lissen E, Clumeck N, et al. Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection. Hepatology 2006;43:1317–25; Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepatol 2018;68:305–15; Anstee QM, Lawitz EJ, Alkhouri N, et al. Noninvasive tests accurately identify advanced fibrosis due to NASH: Baseline data from the STELLAR trials. Hepatology. 2019;70:1521–30). NFS is an even more complex algorithm including not only the age, the platelets and AST/ALT ration, but also BMI, albumin levels and the assessment of glucose tolerance/diabetes, representing a reliable alternative for liver biopsy[8]. (Younossi ZM, Noureddin M, Bernstein D, et al. Role of Noninvasive Tests in Clinical Gastroenterology Practices to Identify Patients With Nonalcoholic Steatohepatitis at High Risk of Adverse Outcomes: Expert Panel Recommendations. Am J Gastroenterol 2021;116:254–262.)’

Comment 4

• 2.1 Current elastography methods: the authors should create a short paragraph on the current state of the art of liver elastography

Answer 4

Thank you for your suggestion. We introduced a paragraph regarding liver elastography, lines 104-112: ‘Liver elastography has gained a lot of interest in the context of NAFLD, a current global public health problem in all age groups. Thus, liver elastography aims to objectively characterize diffuse liver disease and it might reflect pathologic processes such as fibrosis, inflammation or congestion[27–30]. (DiPaola FW, Schumacher KR, Goldberg CS, Friedland-Little J, Parameswaran A, Dillman JR. Effect of Fontan operation on liver stiffness in children with single ventricle physiology. Eur Radiol 2017; 27:2434–2442; Barr RG, Ferraioli G, Palmeri ML, et al. Elastography assessment of liver fibrosis: society of radiologists in ultrasound consensus conference statement. Radiology 2015; 276:845–861; Hanquinet S, Rougemont AL, Courvoisier D, et al. Acoustic radiation force impulse (ARFI) elastography for the noninvasive diagnosis of liver fibrosis in children. Pediatr Radiol 2013; 43:545–551; Samir AE, Dhyani M, Vij A, et al. Shear-wave elastography for the estimation of liver fibrosis in chronic liver disease: determining accuracy and ideal site for measurement. Radiology 2015; 274:888–896). Taking into account the increased rates of both morbidity and mortality in patients with diffuse liver disease[31](Dhyani M, Anvari A, Samir AE. Ultrasound elastography: liver. Abdom Imaging 2015;40(4):698-708), a proper management is essential for the best outcome in these patients. Furthermore, the management depends mostly on the accurate staging of liver fibrosis. Liver elastography seems to be a promising diagnostic tool in the assessment of liver fibrosis irrespectively of the age and type of chronic liver disease. ’

Comment 5

• Line 98-101: the authors should stress the fact that even blood flow can affect liver stiffness especially in portal hypertension. In terms of confounding factors these are some recent papers that the authors should cite: 1) transaminases - DOI: https://doi.org/10.3390/microorganisms8030348 // DOI: 10.1007/s40477-020-00456-9 ; 2)

Answer 5

Thank you for your valuable suggestion. Following your recommendations, we introduced additional information regarding the confounding factors, lines 262-280: ‘The most recent confounding factor identified in a study performed on patients with Hepatitis C virus (HCV) is represented by serum transaminases hypothesized to increase liver stiffness measurements. Thus, Giuffrè et al proved as a result of a study on 110 patients with HCV that the probability of liver fibrosis overestimation of two or more grades equals 50% for AST of 99 IU/L and ALT of 90.5 IU/L; 80% for AST of 123.5 IU/L and ALT of 101.5 IU/L; and up to 100% for AST of 211 IU/L and ALT of 140 IU/L[93]. (Giuffrè M, Fouraki S, Comar M, et al. The Importance of Transaminases Flare in Liver Elastography: Characterization of the Probability of Liver Fibrosis Overestimation by Hepatitis C Virus-Induced Cytolysis. Microorganisms 2020, 8, 348; doi:10.3390/microorganisms8030348).

Neverhtless, on data are available to date in pediatric patients regarding the impact of serum transaminases on liver stiffnes measurements.’; ‘Nevertheless, no data is available to date in pediatric patients regarding the impact of serum transaminases on liver stiffness measurements. Blood flow is another potential confounding factor of liver stiffness since previous studies underlined that increased blood flow as a result of food intake leads to an increased liver stiffness[94].  (Popescu A, Bota S, Sporea I, et al. The influence of food intake on liver stiffness values assessed by acoustic radiation force impulse elastography-preliminary results. Ultrasound Med Biol 2013;39:579-84). Thus, a period of at least 4 hours of fasting is recommended before the examination in order to avoid this confounding factor[95].( Yin, M.; Talwalkar, J.A.; Glaser, K.J.; Venkatesh, S.K.; Chen, J.; Manduca, A.; Ehman, R.L. Dynamic Postprandial Hepatic Stiffness Augmentation Assessed with MR Elastography in Patients with Chronic Liver Disease. AJR Am J Roentgenol 2011, 197, 64–70, doi:10.2214/AJR.10.5989.)

‘It is well-documented that elastography is of major importance in assessing portal hypertension, but it was proved that severe portal hypertension impairs the correlations between liver stiffness and hepatic venous pressure gradient emphasizing that the assessment of spleen stiffness could be more reliable in these cases[96].’ (Roccarina D, Rosselli M, et al. Elastography methods for the non-invasive assessment of portal hypertension. Expert Review of Gastroenterology & Hepatology, DOI: 10.1080/17474124.2017.1374852). ‘In terms of spleen stiffness assessment, a recent study performed on HCV patients proved that spleen stiffness increased by 3.220 kPa for each mm of portal vein diameters and by 0.7 kPa for each cm/s of portal vein velocity underlining that it might be an accurate parameter for portal hypertension stratification[97].’(Giuffrè M, Fouraki S, Campigotto M, et al. Alanine aminotransferase and spleno‑portal dynamics affect spleen stiffness measured by point shear‑wave elastography in patients with chronic hepatitis C in the absence of significant liver fibrosis. Journal of Ultrasound https://doi.org/10.1007/s40477-020-00456-9).’

Comment 6

• pSWE/TE/2D-SWE: the authors should actually compare the various elastography techniques as reliable in nafld fibrosis staging, also in terms of different histology scores that can be used (Brunt vs. Kleiner).

Answer 6

Thank you for your valuable suggestion. According to your recommendation, we discussed the correlations between ultrasound-based elastography methods and different histology scores and introduced the following paragraphs in the revised form of our manuscript, lines 362-378: ‘Multiple studies aimed to identify if elastography is able to replace liver biopsy in order to diagnose this condition, an emerging concern worldwide. Two histology scoring systems were used for assessing liver fibrosis: Brunt classification (Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94(9):2467–2474) defined as grade 0 for no fibrosis, grade 1 for zone 3 perisinusoidal and/or pericellular fibrosis, grade 2 - the changes identified in grade 1 plus focal or extensive periportal fibrosis, grade 3 as in grade 2 plus focal or extensive bridging fibrosis and grade 4 defining cirrhosis; and Kleiner activity scoring system with 0 for no fibrosis, 1 for periportal or perisinusoidal fibrosis, 2 for perisinusoidal and portal or periportal fibrosis, 3 for bridging fibrosis, and 4 when cirrhosis occurs (Kleiner DE, Brunt EM, Van Natta M, et al; Nonalcoholic Steatohepatitis Clinical Research Network. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41(6):1313-1321). Garcovich et al performed a study on 68 pediatric patients with histologically proven NASH according to the Brunt scoring system, and found that SWE established correctly the fibrosis stage in 57 of 68 patients (84%), with higher accuracy in patients with advanced fibrosis. The authors encountered that 6.7 kPa might be a reliable cutoff value for delineating fibrosis stages ( Garcovich, M.; Veraldi, S.; Di Stasio, E.; Zocco, M.A.; Monti, L.; Tomà, P.; Pompili, M.; Gasbarrini, A.; Nobili, V. Liver Stiffness in Pediatric Patients with Fatty Liver Disease: Diagnostic Accuracy and Reproducibility of Shear-Wave Elastography. Radiology 2017, 283, 820–827, doi:10.1148/radiol.2016161002); ‘Similar findings were reported by Alkhouri et al on 67 children with histologically proven NAFLD according to the Kleiner’s system, who stated that TE is a useful non-invasive indicator of clinically significant liver fibrosis guiding the proper selection of patients that require liver biopsy (Alkhouri N, et al. Combined paediatric NAFLD fibrosis index and transient elastography to predict clinically significant fibrosis in children with fatty liver disease. Liver International 2012, DOI:10.1111/liv.12024).’

Comment 7

Minor: english should be revised by a native speaker

Answer 7

We greatly appreciate all your comments and suggestions. According to your recommendation, our manuscript was revised by a native English speaker.

Round 2

Reviewer 1 Report

The authors have addressed most of the reviewers's concerns and the document has improved significantly.

I did not see the images in the new submission, but I assumed they were appropriate.

Consider reducing the size of the tables, more compact. Also, be careful with stiffness and Young modulus, stiffness sometimes refers to shear modulus in elastography.

Reviewer 3 Report

This reviewer thinks it was a good piece of writing, reflecting the reviewer(s)' suggestions. I think this paper is suitable for publication in Journal of Applied Sciences.

Reviewer 4 Report

I have read with great interest the revised version of the manuscript, where the authors have implemented most of the changes suggested by each reviewer. 

I believe the manuscript can be accepted for publication in its current form.