PONE-D-23-34199Detection of Hepatitis Viruses in Suspected Cases of Viral Haemorrhagic Fevers in NigeriaPLOS ONE

Dear Dr. Salu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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ACADEMIC EDITOR: Please, improve the text of the manuscript as it is presently clumsy. Also, provide a description of the outcome of molecular analyses. It would be better if authors carried out sequencing of amplified products. 

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Please submit your revised manuscript by Apr 04 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Daniel Oladimeji Oluwayelu, D.V.M., M.Sc., Ph.D.

Academic Editor

PLOS ONE

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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: No

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

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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: Yes

Reviewer #3: Yes

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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

Reviewer #3: 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: In this manuscript, the authors described the outcome of a serological survey and molecular analysis of 350 Nigerian patients initially suspected of being affected from a viral hemorrhagic fever. All samples turned out to be negative for Lassa, Yellow Fever, and Dengue. Samples of these patients coming from all Nigerian provinces were screened by ELISA for the presence of serological scars signaling an infection with hepatitis A, B, C, or E viruses. Samples positive in ELISA were then analyzed by nested PCR.

A subset of 36% of patients presented a reactive serology. The most frequent was anti-Hepatitis E virus (20.3%). The authors suggest that in a certain number of cases, it is not a Haemorrhagic Fever virus (HFV) that is involved in the suspected disease but rather a more common hepatitis virus. They suggest to systematically extend screening of negative HFV to hepatitis.

The paper is interesting but asks many additional questions left unanswered and present some shortcomings as well.

The identification of the different causes of febrile illness is the topic of many publications in Africa and is not at all a trivial issue (see for example M.J. Maze et alii, The epidemiology of febrile illness in sub-Saharan Africa: implications for diagnosis and management. Clinical Microbiology and Infection 24, 2018, 808-814)

What is the proportion of samples routinely considered as negative for LASV, YFV or DENV in the laboratory of the authors?

The choice of the serological tests is puzzling. The authors targeted acute hepatitis A by using IgM anti-HAV kit but they looked in the meantime for markers used to detect chronic diseases for HCV and HBV or for past disease for HEV. They are thus mixing interpretations for acute and chronic hepatitis. It is probable that all anti-HCV or HBsAg positive patients were chronically infected for a while and that the fever that motivated the haemorrhagic fever diagnosis was not due to HBV or HCV. The 5% and 10% of smaples positive for HCV and HBV correspond roughly to what can be expected in the general Nigerian population while these figures should have been higher if they have been associated with the current disease. In addition, looking for anti-HEV IgG is also looking for past infection. Why not using anti-HBc IgM or anti-HEV IgM to detect an acute infection.

Why not looking for other HFV such as Chikungunya or West Nile Virus involved at high frequency in a former Nigerian survey (see Baba M et alii. Evidence of arbovirus co-infection in suspected febrile malaria and typhoid patients in Nigeria. J Infect Dev Ctries 2013;7:51e9) ?

“The cold chain of the samples was maintained, and randomly selected plasma samples”: what was the system of selection? It can significantly impact the final outcome of the study for example if a large proportion of sample coming from the North-East region has been “selected”.

Please provide a table comparing the samples positive and negative for hepatitis viruses with the outcome of statistical tests.

Please provide a table describing each type of patients: HBsAg(+), anti-HCV(+), anti-HEV(+).

There is a lengthy paragraph about the co-infections that represent only 5% of the whole series. They are only 15, and it is not sure that relevant conclusions can be draw from such a small subset.

Were some of the samples part of outbreaks or were they only sporadic cases of suspected VHF?

“In endemic areas, the knowledge of these hepatitis and other viruses that might be cocirculating in suspected cases of VHFs »: the provided evidence do not support the co-circulation of hepatitis and VHF in Nigeria. Regarding the co-circulation of hepatitis viruses it is well documented by the publication of papers dedicated to co-infections.

Likewise “The study also shows that there might be possible co-morbidity between viral hemorrhagic fevers and hepatitis viruses” is not a realistic statement. The study is not showing that and it was not meant to do so.

“This finding supports the documented evidence that globally, over 350 and 150 million people are chronically infected with HBV and HCV respectively”: I do not think that the findings of the present manuscript support these figures.

We do not know anything about the percentage of positivity by PCR for any viruses.

Why analyzing HAV by PCR while it was all negative by serology?

We do not know whether the patients with bleeding or those who died were infected or not with hepatitis virus. We do not know their demographical status as well.

Discussion

The long paragraph about the North East region should be shorten and substantiated by more references.

“this leads to corrupt and immoral practices »: I do not think that we can deem a subset of the Nigerian youth as corrupted and immoral. For sure, some young people are susceptible to adopt practices dangerous for their own life.

Minor:

Abstract: Please indicate clearly that the 350 samples analyzed were negative for LASV, YFV, and DENV.

“126 were positive for at least one hepatitis virus”

Background: “These hepatitis agents has been documented to be associated with high disease burden, morbidity, mortality and has the potential to cause outbreaks and epidemics of about 50% of hepatitis cases worldwide”. What are the cause of the other 50% of hepatitis outbreak and epidemics? It is weird.

“selected sample aliquots were extracted » : What was the volume of plasma used for extraction?

Results: « 212 (60.6%) males and 138 (39.4%) females with a ratio of 1:1.5” Please, precise the order of sexes to calculate the ratio M:F or F:M ?

Smoking and drinking cannot be linked directly to viral hepatitis.

“The findings in these age groups agrees with other studies of HEV infections in Nigeria…”: What does it mean?

Table 1: Please provide the reference of the primers used.

Table 2: “geo-political”, “geographical” instead?

Reviewer #2: Detection of Hepatitis Viruses in Suspected Cases of Viral Haemorrhagic Fevers in Nigeria

The significance of this investigation cannot be overstated, particularly given the ongoing global climate changes and the prevalence of arboviruses and other co-morbidities. The Sub-Saharan Africa (SSA) region, grappling with numerous health challenges and inadequate healthcare infrastructure, stands to benefit greatly from the findings of this scientific study.

That being said, I would like to share a few observations and comments.

Comments:

Background: The literature review should encompass an examination of different diagnostic procedures, their respective limitations, as well as the challenges faced by low- and middle-income countries (LMICs) in terms of availability and adequacy of these diagnostic tools for identifying viral hemorrhagic fevers (VHF) and other coinfection and co-morbidities.

Method.

Comments:

A comprehensive table outlining the clinical data pertaining to the signs, symptoms, and specific regions of the participants should be incorporated within the methods section.

The population delineation by the author was insufficient. It is imperative for the authors to provide a comprehensive definition of this population, taking into account factors such as age group, pregnant and non-pregnant individuals, with particular attention to the implications on HEV.

Does the author thinks the coinfections, co-circulation, low or high prevalence could potentially be attributed to antibody dependence enhancement (ADE)? In the context of arboviruses, is it plausible that the vector (namely mosquitoes) might have been capable of carrying both viruses, or perhaps acquiring and transmitting them simultaneously?

Reviewer #3: The manuscript was generally well written. Because the manuscript was line numbered, it was difficult writing separate report so the corrections were made in the document. I think the authors should follow the format of the journal to make review process easier.

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Reviewer #1: No

Reviewer #2: Yes: Peter Mac Asaga {MD, PhD)

Reviewer #3: No

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Attachments

Attachment

Submitted filename: PONE-D-23-34199.pdf

PONE-D-23-34199R1

Detection of Hepatitis Viruses in Suspected Cases of Viral Haemorrhagic Fevers in Nigeria PLOS ONE

Dear Dr. Salu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 11 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Daniel Oladimeji Oluwayelu, D.V.M., M.Sc., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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 #4: (No Response)

Reviewer #5: 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 #4: Yes

Reviewer #5: Yes

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

Reviewer #4: N/A

Reviewer #5: Yes

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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 #4: Yes

Reviewer #5: Yes

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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 #4: Yes

Reviewer #5: 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 #4: The manuscript is well research and well written. However, the statistical methods used for the analysis was not stated, and there some grammatical errors that will need to be corrected; for example:

1. Page 6-Ethical considerations, line 4, "accessed protected" should be edited to "access-protected".

2. Page 7-Data/specimen retrieval and storage, line 3, "randomly by......" should be edited to "randomly selected by.....".

Reviewer #5: I have read the manuscript with keen interest. It is an article of interest in the field of virology.

1. The abstract informative is informative and reflect the body of the paper.

2. The introduction provided sufficient background information for readers in the immediate field to understand the problem/hypotheses and it ended with the objectives of the study.

3. The text was well arranged and the logic is clear.

4. There are no grammatical errors in this article.

5. The related concepts were clearly introduced and the readability is sufficient.

6. The proposed simulation /experiment /scheme is quite novel.

7. The theoretical analysis in this article is strong.

8. The reference section is informative and accurate.

I therefore, recommend the manuscript for your acceptance

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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 #4: Yes: Oche Ochai Agbaji

Reviewer #5: Yes: Iheanyi Omezuruike Okonko PhD

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Detection of Hepatitis Viruses in Suspected Cases of Viral Haemorrhagic Fevers in Nigeria

PONE-D-23-34199R2

Dear Dr. Salu,

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,

Daniel Oladimeji Oluwayelu, D.V.M., M.Sc., Ph.D.

Academic Editor

PLOS ONE

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