Rapid quantification assay of hepatitis B virus DNA in human serum and plasma by Fully Automated Genetic Analyzer μTASWako g1

Moto Watanabe; Hidenori Toyoda; Tomohisa Kawabata

doi:10.1371/journal.pone.0278143

Peer Review History

Original SubmissionNovember 9, 2022
13 Dec 2022 Decision Letter - Md. Golzar Hossain, Editor PONE-D-22-30604Rapid Quantification Assay of Hepatitis B Virus DNA in Human Serum and Plasma by Fully Automated Genetic Analyzer μTASWako g1PLOS ONE Dear Dr. Kawabata, 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 Jan 27 2023 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, Md. Golzar Hossain, Ph.D. 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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following in the Financial Disclosure section: "Specific grant numbers; None Initials of authors who received each award; M.W Full names of commercial companies that funded the study or authors; Fujifilm Wako Pure Chemical Corporation Initials of authors who received salary or other funding from commercial companies; M.W and T.K URLs to sponsors’ websites; https://www.fujifilm.com/ffwk/en The funders had role in decision to publish of the manuscript." We note that one or more of the authors have an affiliation to the commercial funders of this research study: Fujifilm Wako Pure Chemical Corporation (1) Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. (2) Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: ""This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 5. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. 6. 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. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 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 ****** 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 ****** 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: Comments to the authors In this study, Watanabe et al. assessed the analytical and clinical performance of HBV DNA assay on the μTASWako g1 platform in human serum and EDTA-plasma. The authors found that the HBV DNA assay on the μTASWako g1 is potentially applied for alternative method of the HBV viral load test, in particular with the advantage of the HBV DNA result availability within 2h, improving the HBV infection management. The results of this study were interesting. I have several comments. 1. In the methods section, authors described as follows: “Afterward, 33 PCR cycles with a longer extension (70 s at 73.5°C) was performed with one CE injection from the PCR chamber for each subsequent cycle to analyze the accumulated target amplicons and took 56 min to complete the entire qPCR-CE step.” How did you sample from the PCR chamber into the electrophoresis channel? 2. Normally in PCR reactions, it is thought that the amplification reaction efficiency changes with changes in the concentration of the multiplex partner when the reaction is multiplexed, but what is the reason why the IC reactivity did not change when the HBV virus concentration was changed? 3. How can this "quick results" measurement method be useful in real-world clinical practice? Please discuss this point. ****** 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 ******** [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.0278143.r001
Revision 1
10 Jan 2023 Author Response To Reviewer Question 1: In the methods section, authors described as follows: “Afterward, 33 PCR cycles with a longer extension (70 s at 73.5°C) was performed with one CE injection from the PCR chamber for each subsequent cycle to analyze the accumulated target amplicons and took 56 min to complete the entire qPCR-CE step.” How did you sample from the PCR chamber into the electrophoresis channel? Answer: PCR product (DNA) is moved from PCR chamber toward electrophoresis channel by applying electrical current (0.2 μA) to PCR chamber via two microchannels which filled with a reagent for electrophoresis. The reagent wells were physically separated from electrode contact wells to prevent instrument contamination from reagent and amplicons. Electrical connection between the two wells was made through printed carbon electrodes on the chip. The detail of microfluidic chip design and the movement of DNA during the CE were well illustrated in previous research article (reference#18). The article is also helpful to understand how it works. Question 2: Normally in PCR reactions, it is thought that the amplification reaction efficiency changes with changes in the concentration of the multiplex partner when the reaction is multiplexed, but what is the reason why the IC reactivity did not change when the HBV virus concentration was changed? Answer: There are three reasons why the IC reactivity did not change when the HBV virus concentration was changed. 1. In the μTASWAKO g1 HBV assay, IC DNA (approximately 1,200 copies/assay) was added to the sample at the beginning of the DNA purification step, therefore, the IC DNA concentration in PCR mixture is almost consistent every times. 2. IC DNA was designed so that IC target sequence can be amplified by HBV primers. Basically, the PCR amplification efficiency of IC were equivalent to that of HBV target due to the same primer performance. 3. The IC DNA concentration, the primer concentration, and IC detection threshold for qPCR-CE have been optimized to reproducibly detect IC Cq by testing the samples containing high concentration of HBV (~3.5×10^8 IU/mL). The balance of those three factors is most important to enable IC detection without interference by HBV-amplification even when 3.5×10^8 IU/mL of HBV sample tested. Primer consumption in a reaction-mixture results in reduction of amplification efficiency as a progressing of PCR cycles. In the case of co-amplification of HBV (3.5×10^8 IU/mL) and IC, the HBV primers were consumed, however, the remaining primers maintains IC-amplification efficiency until PCR cycle (at 26-27) when IC-amplicons exceed the detection threshold for qPCR-CE. We have confirmed that IC Cq does not change across dynamic range of HBV quantification (~3.5×10^8 IU/mL), while IC Cq may change when testing of the sample containing HBV more than 10^9 IU/mL. If the assay detected HBV DNA at over dynamic range, the sample will need to be diluted with HBV-negative sample for re-test. Question 3: How can this "quick results" measurement method be useful in real-world clinical practice? Please discuss this point. Answer: HBV is potentially reactivated in some patients who undergo chemotherapy or immunosuppressive therapies and HBV reactivation often causes life-threatening liver failure. Reactivation occurs not only in HBsAg-seropositive patients but also in those with resolved HBV infection who are seronegative for HBsAg but seropositive for antibody against hepatitis B core antigen (anti-HBc) and/or antibody against HBsAg (anti-HBs). To prevent HBV reactivation-related hepatitis, HBV DNA monitoring-guided preemptive antiviral therapy using anti-HBV nucleos(t)ide analogs (NAs) is recommended by several guidelines for patients with resolved HBV infection, and it is mandatory to detect the emergence of serum HBV DNA as early as possible to start anti-HBV therapy early and effectively. However, the duration required for HBV DNA measurements, including both qualitative and quantitative, hinders agile and effective preventive strategies in real clinical practice. HBV DNA measurement technology with rapidly available results will be a strong advantage for monitoring patients at risk of HBV reactivation since the “quick results” possibly contribute to minimal delay in clinical decision-making after blood collection from the patients. Best regards, Tomohisa Kawabata Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0278143.r002
23 Jan 2023 Decision Letter - Md. Golzar Hossain, Editor Rapid Quantification Assay of Hepatitis B Virus DNA in Human Serum and Plasma by Fully Automated Genetic Analyzer μTASWako g1 PONE-D-22-30604R1 Dear Dr. Kawabata, 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, Md. Golzar Hossain, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 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 ****** 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 ****** 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: Comments to the Authors I have carefully reviewed your replies. You have responded to all questions appropriately. I did not think any further revision was necessary. ****** 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. 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 ******** Attachments Attachment Submitted filename: PONE_D_22_30604R1_Comments.docx https://doi.org/10.1371/journal.pone.0278143.r003
Formally Accepted
31 Jan 2023 Acceptance Letter - Md. Golzar Hossain, Editor PONE-D-22-30604R1 Rapid Quantification Assay of Hepatitis B Virus DNA in Human Serum and Plasma by Fully Automated Genetic Analyzer μTASWako g1 Dear Dr. Kawabata: 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 Dr. Md. Golzar Hossain Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0278143.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .