Distribution of naturally -occurring NS5B resistance-associated substitutions in Egyptian patients with chronic Hepatitis C

Hala Rady Ahmed; Nancy G. F. M. Waly; Rehab Mahmoud Abd El-Baky; Ramadan Yahia; Helal F. Hetta; Amr M. Elsayed; Reham Ali Ibrahem

doi:10.1371/journal.pone.0249770

Peer Review History

Original SubmissionDecember 25, 2020
1 Feb 2021 Decision Letter - Philippe Gallay, Editor PONE-D-20-39695 Distribution of naturally occurring NS5B resistant associated substitutions among treatment naïve HCV Egyptian patients and non-responders PLOS ONE Dear Rehab Mahmoud Abd El-Baky, 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. Although the reviewers found your study relevant, some issues need to be addressed including the small number of analyzed samples, English editing, a too small NS5B region sequenced, to compare sequences to a set of HCV NS5B sequences, etc. Please submit your revised manuscript by Mar 14 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Philippe Gallay Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2.Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found some minor occurrences of overlapping text with the following previous publication(s), some of which you are an author, which needs to be addressed: - https://www.sciencedirect.com/science/article/abs/pii/S2210740119300439?via%3Dihub We would like to make you aware that copying extracts from previous publications word-for-word, especially outside the methods section, is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications. Please revise the manuscript to quote or rephrase the duplicated text and cite your sources for text outside the methods section. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 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 ****** 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: No Reviewer #2: No ****** 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: This is a cross-sectional study of NS5B resistance mutations in Egypt. Given the very high prevalence of HCV in this part of the world, such studies are relevant. However, there are a number of significant limitations to this study. Most importantly, the very small sample size is quite surprising, since there are clearly many more individuals that could have been included / studied. The manuscript requires editing by a native English speaker and/or professional editing service. How was HCV RNA quantified? What was the lower limit of detection for the assay? Only 18 of the 35 patient samples could be amplified by PCR suggesting very low sample quality. Samples storage conditions should be stated explicitly. Were the samples that were PCR negative then extracted again and the PCR tried a second time? The authors have amplified only a small portion (~400 bases) of the entire NS5B region, representing a significant study limitation. “normal” and “abnormal” levels for ALT and other clinical variables should be explicitly defined. Are the authors comparing their sequences to a single NS5B consensus to identify mutations? If so, this approach has major limitations. The consensus may change with more or fewer sequences to generate the consensus, and minor – but important – mutations may be missed altogether. It is better to compare the query sequence to a set of HCV references (rather than to one) and then define a mutation as greater than X percentage of samples. A phylogenetic tree must be included to show the relationship amongst the sequences isolates. Reviewer #2: In this manuscript authors analyzed the RASs occurrence in 17 treatment-naïve and 1 non-responder samples out of 35 total number of patients. Samples were collected from patients who failed to achieve an early viral response with combined sofosbuvir (400mg/day), daclatasvir (60mg/day) for 6 months therapy. Authors reported several mutations as possible RASs linked to SOF. Although these mutations may be of a great value to understand DAA resistance, however, the study lack important aspects that need to be addressed to be publishable. Examples for these shortcomings are the small number of the analyzed samples especially the non-responders, lack of comparison with other cohort of patients who achieved an early viral response, and the use of multiple DAAs but analyzing one target gene. Although daclatasvir targets NS5A, nothing was done on this gene as patients may be resistant to daclatasvir and this resistance was related to NS5A and not NS5B. The manuscript has many places that need rephrasing or improvement to read better. In addition, the references need to be updated as the authors did not include several important and recent papers about the topic. More detailed comments will be attached below. ****** 6. 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. 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 #1: No Reviewer #2: No [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. Attachments Attachment Submitted filename: Reponse_Plos.docx https://doi.org/10.1371/journal.pone.0249770.r001
Revision 1
27 Feb 2021 Author Response Reviewers’ comments Author’s replies The manuscript requires editing by a native English speaker and/or professional editing service. The authors made the required English editing to the manuscript. How was HCV RNA quantified? What was the lower limit of detection for the assay? The HCV-RNA was amplified on Stratagene Mx3000P system using Artus HCV QS-RGQ-PCR Kit reagents. Amplification followed by simultaneous detection was carried out using RT-PCR, with TaqMan assay for a particular region of the HCV genome. the lower limit of detection for the assay is 30 IU/ml. Only 18 of the 35 patient samples could be amplified by PCR suggesting very low sample quality. Samples storage conditions should be stated explicitly. Approximately, 3 ml of blood were collected from each patient, the samples were shipped cooled (4°C) within 6 hours. centrifuged at 1500 rpm, sera were separated then distributed in aliquots, stored at -20°C. Then after RNA extraction the eluted RNA was stored at -80°C until further use. The sensitivity of the assay can be reduced as the samples stored for a longer period of time. The assay may be subjected to many issues such as difficulty to amplify NS5B fragments, low viral load and the need to extremely trained professionals. These issues were discussed in the following references: • Abdel-Hamid M, El-Daly M, Molnegren V, El-Kafrawy S, Abdel-Latif S, Esmat G, et al. Genetic diversity in hepatitis C virus in Egypt and possible association with hepatocellular carcinoma. Journal of general virology. 2007;88(5):1526-31. • Li Z, Zhang Y, Liu Y, Shao X, Luo Q, Cai Q, et al. Naturally occurring drug resistance associated variants to hepatitis C virus direct-acting antiviral agents in treatment-naive HCV genotype 1b-infected patients in China. Medicine. 2017;96(19). • Warkad SD, Nimse SB, Song K-S, Kim T. HCV detection, discrimination, and genotyping technologies. Sensors. 2018;18(10):3423. Were the samples that were PCR negative then extracted again and the PCR tried a second time? Yes, the samples that were PCR negative extracted again using The MagMAX Viral RNA Isolation Kit For viral nucleic acid isolation which used to deliver yields of high quality RNA and DNA with little sample-to-sample variation as 35–75% of the input RNA should be recovered. The MagMAX™ magnetic bead technology useful for concentrating RNA from dilute samples. Then, the PCR tried a second time but gave no bands. The authors have amplified only a small portion (~400 bases) of the entire NS5B region, representing a significant study limitation. Because Sofosbuvir (SOF) is the predominant available NS5B polymerase inhibitor Nucleoside/nucleotide inhibitors (NI)] in Egypt, we mainly investigated the positions which are related to resistance associated variants (RAVs) to sofosbuvir and other positions such as 368, 411, 414, 448, 553, 554 and 556 which are related to resistance to another NS5B polymerase inhibitor, Dasabuvir [non-nucleoside inhibitor] NNI)], not fully covered in this study. As, NNIs delivered as a monotherapy came with a limited success, with a low to average antiviral activity, a low resistance barrier, and a minimal range of genotypic activity (active only against subtypes 1b and 1a). Fadl, N, Salem, TZ. Hepatitis C genotype 4: A report on resistance‐associated substitutions in NS3, NS5A, and NS5B genes. Rev Med Virol. 2020; 30:e2120 “normal” and “abnormal” levels for ALT and other clinical variables should be explicitly defined. Normal and abnormal levels for all clinical variables defined in the manuscript. Are the authors comparing their sequences to a single NS5B consensus to identify mutations? If so, this approach has major limitations. The consensus may change with more or fewer sequences to generate the consensus, and minor – but important – mutations may be missed altogether. It is better to compare the query sequence to a set of HCV references (rather than to one) and then define a mutation as greater than X percentage of samples. In order to undergo alignment step, HCV GT-4a reference strain with accession number DQ988074, HCV GT-4l reference strain FJ839870, HCV GT-4o reference strain FJ462440, HCV GT-3a reference strain D17763 and HCV GT-1g reference strain AM910652 from NCBI (National Center for Biotechnology Information) was chosen to be aligned with the sequenced samples. As a result, for the alignment, we noticed the presence of the mutations or the substitutions listed in our study in the tested samples and their absence in the reference strains. So, further investigations are needed to prove their relation to resistance. A phylogenetic tree must be included to show the relationship amongst the sequences isolates. The authors added the phylogenetic tree to the manuscript. The small number of the analyzed samples, especially the non-responders. . Most of HCV Egyptian patients received the treatment at private clinics and pharmacies outside the viral hepatitis management center so hardly to reach to be included in our study. Additionally, the number of non-responder cases are very low due to the high rate of virological success of SOF-based regimens. lack of comparison with other cohort of patients who achieved an early viral response. There is some difficulty to follow up the patients after they end their treatment. We already tried to follow up our cases responsiveness to the treatment but failed. In Egypt, the routine follow-up depends mainly on performing RT-PCR to detect the viral load every 6 months, Then every year. So, patients can perform this test in private laboratories not the center which is considered one of the limitations of this study. the use of multiple DAAs but analyzing one target gene. Although daclatasvir targets NS5A, nothing was done on this gene as patients may be resistant to daclatasvir and this resistance was related to NS5A and not NS5B. . Because sequencing step has high cost and this research did not receive any specific grant from funding agencies. The manuscript has many places that need rephrasing or improvement to read better The authors made the required rephrasing to the manuscript. In addition, the references need to be updated as the authors did not include several important and recent papers about the topic. The authors made the required updates to the references. In the paragraph started with “Ribavirin-associated resistance mutations at positions D244N, Q309R, and A333E of NS5B were analyzed.” I am not sure why the authors compare their mutations with non-DAAs drugs and ignore comparing them with other DAAs drugs. Either they include the most related drugs or remove this paragraph altogether. Ribavirin is included in HCV treatment protocol in Egypt. RBV is a nucleotide analogue that can be incorporated in the RNA-dependent polymerase, although the precise mechanism of action on viral replication remains elusive. Raj, V. S., Hundie, G. B., Schürch, A. C., Smits, S. L., Pas, S. D., Le Pogam, S., ... & Haagmans, B. L. (2017). Identification of HCV resistant variants against direct acting antivirals in plasma and liver of treatment naïve patients. Scientific reports, 7(1), 1-10. ‏ Petronella Gededzha, M., Mphahlele, M. J., Blackard, J. T., & Gloria Selabe, S. (2017). Prevalence of NS5B resistance mutations in hepatitis C virus (HCV) treatment naïve South Africans. Hepatitis Monthly, 17(6).‏ Also, in Table 1, non-responders showed low viral load in comparison to naïve patients. This has to be addressed and reasonable explanation has to be made. As the virus was under the stress of the antiviral therapy which results in the decrease in the replication of the virus and low viral load while naïve treatment patients are not subjected to the antiviral therapy stress. In addition, many patients under the antiviral therapy showed inaccurate low viral load or false negativity when testing plasma for HCV RNA due to the presence of HCV distributed in hepatic tissues and in extrahepatic tissues such as pancreas, kidney, brain, lymphoid tissues or elsewhere. So, Direct testing of these tissues in individuals who test negatively for HCV RNA in blood will be required to confirm or refute that these other tissue sites serve as potential reservoirs of persistent HCV infection and as a source of viral relapse. - Gurakar A, Fagiuoli S, Faruki H, De Maria N, Balkan M, Van Thiel DH, Friedlander L. Utility of hepatitis C virus RNA determinations in hepatic tissue as an end point for interferon treatment of chronic hepatitis C. Hepatology. 1995 Oct;22(4 Pt 1):1109-12. doi: 10.1016/0270-9139(95)90616-9. PMID: 7557858. Change all treatment naïve to “treatment-naïve” The authors change all treatment naïve to “treatment-naïve” in the manuscript Title - For “Distribution of naturally occurring NS5B resistant associated substitutions among treatment- naïve HCV Egyptian patients and non-responders” The title is not accurate as the authors should not consider the non-responders data as it came out from only one patient. We suggested this title Distribution of naturally NS5B resistant associated substitutions between Egyptian HCV in Minia, Egypt Abstract - Authors mentioned “Results: NS5B amino acid sequence analysis showed the presence of several novel NS5B mutations (more than 40 substitution mutations) that have not been previously reported to be associated with a resistant phenotype.” Without comparing with another cohort with EVR, these mutations may be not related to resistance. We compare our results with 5 standard strains sequences. we noticed the presence of the substitutions that were listed in our study in the tested samples and their absence in the reference strains and we did not find these substitutions in any of the research that were carried out in our area. So, further investigations are needed to prove their relation to resistance. - In Table 4, it is not clear why the authors arrange ID numbers or the mutations this way. Neither IDs, number of mutations, or location of the mutations are in order. I recommend to order the table according to the no. of substitutions as well as the locations of the mutations. Each column should include one location and if at that position different mutations exist, it should be market with asterisk. Example, first column should be only for position 231 with asterisk for A231G* and moving 244 to different column in order. All changes were done Note: the alignment for the substitutions were put in separate columns but the table became too large. So, we add it in the manuscript as an image. The actual excel form of the table will be attached as supplementary file. All typing errors written in the response file of the reviewer were corrected in the manuscript. Attachments Attachment Submitted filename: response (1) (1).docx https://doi.org/10.1371/journal.pone.0249770.r002
8 Mar 2021 Decision Letter - Philippe Gallay, Editor PONE-D-20-39695R1 Distribution of naturally occurring NS5B resistant associated substitutions among treatment naïve HCV Egyptian patients and non-responders PLOS ONE Dear Dr. Rehab Mahmoud Abd El-Baky, 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. ============================== The authors have addressed a majority of the previous issues. However, their explanation/comments need to be fully integrated in the text starting with the title "....treatment-naive". ============================== Please submit your revised manuscript by Apr 22 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Philippe Gallay 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. Additional Editor Comments (if provided): The authors addressed a majority of the issues and provided reasonable answers to address the comments of the reviewers. However, all their explanation need to be integrated in the revised manuscript starting with the title: "....treatment-naive.....". [Note: HTML markup is below. Please do not edit.] [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. https://doi.org/10.1371/journal.pone.0249770.r003
Revision 2
23 Mar 2021 Author Response Authors respond to all reviewers' comments and the file was attached Attachments Attachment Submitted filename: response to reviwers (1).docx https://doi.org/10.1371/journal.pone.0249770.r004
25 Mar 2021 Decision Letter - Philippe Gallay, Editor Distribution of Naturally -Occurring NS5B Resistance-Associated Substitutions In Egyptian Patients with Chronic Hepatitis C PONE-D-20-39695R2 Dear Dr. Rehab Mahmoud Abd El-Baky, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Philippe Gallay Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0249770.r005
Formally Accepted
6 Apr 2021 Acceptance Letter - Philippe Gallay, Editor PONE-D-20-39695R2 Distribution of Naturally -Occurring NS5B Resistance-Associated Substitutions In Egyptian Patients with Chronic Hepatitis C Dear Dr. Abd El-Baky: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Philippe Gallay Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0249770.r006

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