PONE-D-22-04328Lymphatic Filarial Serum Proteome Profiling for Identification and Characterization of Diagnostic BiomarkersPLOS ONE

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PLOS ONE

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Additional Editor Comments:

Authors need to revise the manuscript. Alongside the comments of the reviewers, please modify the discussion and include important findings (e.g. https://doi.org/10.1093/infdis/jix067; 10.1038/s42003-019-0392-8) to highlight the importance of the present study.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I have read with interest the manuscript “Lymphatic Filarial Serum Proteome Profiling for Identification and Characterization of Diagnostic Biomarkers” having MS no: PONE-D-22-04328

In the current study, the authors collected the serum of persons living in endemic areas of LF and analyzed the presence and abundance of C-reactive protein, α-1-antitrypsin, heterogeneous nuclear ribonucleoprotein D like apolipoproteins A-I and A-IV. The authors projected them as biomarkers of LF and proposed them as diagnostic measures. This work sounds interesting and does have fair medical importance, however, I have some concerns over the manuscript that should be addressed properly.

The major problems with this manuscript are:

1. Correctly denote the full name of GPELF where used, it has been denoted differently in places, authors should have been more careful.

2. Authors mentioned that “The proteomics analysis results showed that various proteins were differentially expressed (p<0.05), including C-reactive protein, α-1-antitrypsin, heterogeneous nuclear ribonucleoprotein D like, apolipoproteins A-I and A-IV which have not been reported in Lymphatic Filariasis previously”; However, according to available literature the claim appeared far more enthusiastic and not based on facts as there are reports. Some of them are

CRP:

i. J Clin Immunol. 1991 Jan;11(1):46-53. doi: 10.1007/BF00918794.

ii. https://doi.org/10.1371/journal.ppat.1002749

α-1-antitrypsin:

i. Indian J Med Res. 2011 Jul; 134(1): 79–82.

ii. Human Parasitic Pulmonary Infections; Gary W. Procop, Ronald C. Neafie, in Pulmonary Pathology (Second Edition), 2018; Tropical Pulmonary Eosinophilia and Aberrant Filaria

Apolipoproteins:

i. https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(06)69100-9.pdf

ii. doi: 10.1002/14651858.CD003753.pub4

The claim should be justified properly and I suggest a thorough revision of the literature of the manuscript literature and citations they had made.

3. The authors claimed that “To our knowledge, this is the first report of comparative human serum profiling in different categories of LF patients.” This is partly true as there are reports on IgG4 antibodies on IgE-activated granulocytes in patients. (doi: 10.1371/journal.pntd.0005777).

I do request the authors a further detailed survey of existing literature before confirming any claim. Though the merit of a research article may not decrease the unnecessary and false claim can lead the readers to a fix and discourage them from a further study on the particular aspect.

4. Regarding the supporting information, I suggest that the authors should upload a single pdf or Docx supplementary file to ease the reading of the reviewer and readers.

5. Serum profiling is alright. However, what they have claimed that is a maiden report is not there are reports of the serum profiling and precisely for some of the proteins they emphasize on. How these serum or precise combinations of them can be of diagnostic purpose should be elaborated and given more emphasis. Additionally why anyone would opt for these complex and costly methods against the available methods is not properly elaborated. Are they are thinking of any on-field diagnosis? Then this should also be elaborated.

6. In the introduction, the epidemiological status should be cited properly.

7. In the introduction, section Authors should elaborate in a few sentences what are the available immunoassay tests and their diagnostic criteria, restrictions, limitations, and scope of further and other immunoassay diagnostics. There are a few test kits already available and authors choose to completely avoid them.

8. In the Materials and Method section and to the heading Clinical characterization of LF cases: "Lymphatic Filariasis.......secondary infections.” this should go to the introduction. There is no page or line number, please provide the page and line number to your manuscript.

9. What are the selection criteria of the participants? Are they were previously screened for LF? What is their age, sex? if they are selected randomly based on their consent (it should be properly mentioned) then the percentage of population positive for LF (77%) is very hard to believe. and this should be properly verified and revised.

10. Separation of immune complexes: By the method of whom?

11. In the Result section: Authors denoted that "serum protein concentration altered

Significantly in response to LF infections", however, the applied method of significance is not mentioned and if there is any significant change between groups of infected peoples, i.e. asymptomatic, acute and chronic is not properly mentioned. Authors should employ extensive statistical means of exploring serum parameters between these groups and with the non-infected ones when advocating these parameters for diagnostic means.

12. As the authors just predicted the functional interaction the heading should be written like that. For a proper evaluation of functional interaction, a far more detailed study is needed with inhibitors and siRNA and other detailed molecular biology methods.

Reviewer #2: The present study evaluates the proteome profile of serum from individuals infected with Lymphatic Filariasis as a possible source for identification and characterization of diagnostic biomarkers that may be useful in detecting LF infections in asymptomatic cases and can also serve as indicators for differentiating among different clinical stages of the disease. Based on the results obtained, the authors attest to the potential of human serum profiling for detection of LF and claim that their study is the first report of comparative human serum profiling in different categories of LF patients.

The study addresses an important area of identifying LF biomarkers in active infections undergoing IDA so as to assess the outcome of GPELF, however, substantial concerns remain that needs to be addressed before the work is accepted for publication.

Reviewer’s comments:

The study dwells on an important area of human health that is of major concern in many developing countries. However, the study even though concise, suffers from major drawbacks that limits the publication of this piece of work in the present form. The authors need to address the following concerns.

Major concerns:

1. LF patients were infected with W. bancrofti. But the authors have not segregated them on the basis of age and gender. This should have been done and data presented accordingly. Also, it would have been interesting if some B. malayi infected cases were also part of the study as it would have demonstrated the robustness of the methodologies used. Nevertheless, the data presented should be segregated and discussed keeping in mind the differences between gender and age.

2. The authors claim that serum protein concentration was significantly altered in response to LF infections (Results section-1st Paragraph). However, the values of protein concentration (Table-01) in normal serum (69 ± 8.0 mg/ml), asymptomatic (78 ± 12.0 mg/ml), acute cases (81 ± 14 mg/ml) and chronic cases (93 ± 19 mg/ml) do NOT look drastically different, especially when one looks at the SD value carefully between groups. The authors have indicated p values at three different places in table 1, but it is very very difficult to comprehend how come with such high SD value between groups, the authors could still generate p value < 0.05. The authors must work out the statistical data again or clearly mention which statistical tool was used to calculate the p value. Same concern holds true for IFF and immune complex data as well.

3. Fold change analysis of FT-IR data shows significantly altered peaks at 3300, 2950, 1645, 1540 and 1448 cm-1 between asymptomatic and acute cases. What about the altered peaks in chronic cases? The authors need to discuss this point.

4. The authors claim that a combination of different techniques (FTIR, MMP zymography, SDS-PAGE, 2DGE, MALDI-TOF/MS etc.) was used to identify LF biomarkers from serum samples of different stages of LF patients. How do the authors conclude that the biomarkers thus detected were due to LF infection only and not due to any other previous ailment or other diseased conditions? Merely writing, “LF infected cases were examined by a clinician and were categorized based on the above mentioned manifestations and presence/absence of microfilariae in the bloodstream” does not hold much value as patients exhibiting Tropical Pulmonary Eosinophilia do not show presence of Mf in the peripheral blood. What was the criteria used for exclusion by the clinician, needs to be mentioned in detail.

5. Can the adopted approach be used to distinguish LF infection caused by Wuchereria bancrofti, Brugia malayi and Brugia timori? This needs to be discussed by the authors.

6. What was the duration of the study? Since acute infection cases would progress to chronic cases, won’t their serum profile differ over a period of time and match with the chronic cases? What were the time points of blood withdrawal? Single time point withdrawal is not sufficient to provide the complete picture as gravid females would continuously produce Mf which may alter serum profile? Also, age and gender would make a difference.

7. Zymography and MMP data is not very convincing, as very small differences exist between groups. When one takes SD into account, there would be hardly any differences.

8. Fig 2 , Marker well has different contrast (top and bottom) how come?

Minor concerns:

1. The authors need to factually correct the starting line of abstract and introduction. As per latest WHO figures, 863 million people are affected by LF, the number citing 120 million infections is a very old data (https://www.who.int/news-room/fact-sheets/detail/lymphatic-filariasis).

2. Under “Separation of immune complexes”, either the authors must mention the name of author/research group or write “as described previously”. Just writing “LF cases were isolated by the method of [15] with minor modification” is not correct.

3. Length of IPG strips and pH gradient used in 2DGE should be included in M & M, rather than just in the results.

4. Fig 3b, y axis should be “change”

5. Clearly state statistical comparisons have been made between which groups in fig 1 a and 1b?

6. In Abbreviation and abstract, GPELF should read as Global Programme to Eliminate Lymphatic Filariasis.

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Reviewer #1: Yes: Niladri Mukherjee

Reviewer #2: No

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Lymphatic Filarial Serum Proteome Profiling for Identification and Characterization of Diagnostic Biomarkers

PONE-D-22-04328R1

Dear Dr. Singh,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Suprabhat Mukherjee, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Authors have addressed all the comments/concerns and the revised version is acceptable for publication.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Most of the comments have been addressed. However, I still see that minor concern 4 has not been addressed, and minor concern 5 has not been responded properly.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Niladri Mukherjee

Reviewer #2: No

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