Use of allele-specific qPCR and PCR-RFLP analysis for rapid detection of the SARS-CoV-2 variants in Tunisia: A cheap flexible approach adapted for developing countries

Wajdi Ayadi; Fahmi Smaoui; Saba Gargouri; Sana Ferjani; Zaineb Hamzaoui; Awatef Taktak; Amel Chtourou; Houda Skouri-Gargouri; Azza Hadj Sassi; Mouna Ben Sassi; Sameh Trabelsi; Ali Gargouri; Ilhem Boutiba-Ben Boubaker; Héla Karray-Hakim; Raja Mokdad-Gargouri; Lamia Feki-Berrajah

doi:10.1371/journal.pone.0321581

Peer Review History

Original SubmissionOctober 23, 2024
23 Oct 2024 Author Response https://doi.org/10.1371/journal.pone.0321581.r001
2 Jan 2025 Decision Letter - Huseyin Tombuloglu, Editor PONE-D-24-46700Use of allele-specific qPCR and PCR-RFLP analysis for rapid detection of the SARS-CoV-2 variants in Tunisia: a cheap flexible approach adapted for developing countriesPLOS ONE Dear Dr. Ayadi, 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 Feb 16 2025 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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Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: No ****** 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: Yes Reviewer #2: 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: ''Use of allele-specific qPCR and PCR-RFLP analysis for rapid detection of the SARS-CoV-2 variants in Tunisia: a cheap flexible approach adapted for developing countries'' has mentioned and explained the detection of allele and sublineage specific screening of SARS-CoV-2 Omicron variants. Restriction in sample collection was disadvantage of the study. Additionally: - Font type of Table 1 is different, - In Figure 3 'October' has been written as 'Octobre' mistakenly. - In lane 263, text looks selected and highlighted. - Even the title of article involves name of Tunisia, possible ethnical or local factors relevant to Tunisia and comparison with others haven't been mentioned. Discussion part would be getting stronger depending on this detail. Reviewer #2: The manuscript by Ayadi et al reports alternative methods for monitoring the SARS-CoV-2 variants. These methods seemed to be more suitable in the developing countries. I have several concerns: 1. When applying the as-PCR and PCR-RFLP for virus variant screening, it should be more cautious on determination the indies that could affect the results, including sample size, age and sex distribution of the enrolled samples etc. Although the authors declared that their variant monitoring results are in concordance with the epidemiological surveillance data, this is more like a concordance. 2. The authors should provide more details on the as-PCR and PCR-RFLP methods applied in the present study, how about their limit of detection, specificity, reproducibility etc.? 3. The structure of the manuscript is a little strange and recommended to be re-organized to make the manuscript more easy to follow. ****** 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: Yes: Jianguo Li ******** [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. 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Revision 1
19 Feb 2025 Author Response Responses to Reviewer 1's Comments: 'Use of allele-specific qPCR and PCR-RFLP analysis for rapid detection of the SARS-CoV-2 variants in Tunisia: a cheap flexible approach adapted for developing countries'' has mentioned and explained the detection of allele and sublineage specific screening of SARS-CoV-2 Omicron variants. Restriction in sample collection was disadvantage of the study. We greatly appreciate the reviewer’s insightful summary of our findings. The sample collection in our study encompasses nearly all SARS-CoV-2 positive samples detected at the microbiology laboratory of Habib Bourguiba University Hospital, Sfax, Tunisia, from June 2022 to April 2023. Samples with Cq values greater than 30 during routine RT-PCR diagnosis were excluded. Notably, we observed a decrease in the number of nasopharyngeal samples received during the study period compared to the earlier years of the pandemic. Additionally: - Font type of Table 1 is different The font type in Table 1 has been corrected to match the rest of the manuscript for consistency. -In Figure 3 'October' has been written as 'Octobre' mistakenly. Thank you for pointing this out. The typographical error has been corrected from 'October' to 'October' in Figure 3. Following the requested reorganization of the manuscript, the figure number has been changed to Figure 2 in this new version. -In lane 263, text looks selected and highlighted. The unintentionally highlighted text in the previous version has been corrected in the revised manuscript. -Even the title of article involves name of Tunisia, possible ethnical or local factors relevant to Tunisia and comparison with others haven't been mentioned. Discussion part would be getting stronger depending on this detail. Thank you for this insightful suggestion. Based on our data and other previous findings, we conclude that the variant epidemiology in Tunisia has been largely influenced by the country's strong travel connections with Europe and other North African nations. We have expanded the following paragraph in the Discussion section (lines 406-411): “Consistent with previous findings, Tunisia exhibited variant dynamics similar to those observed in neighboring North African countries and parts of Europe [26,41], reflecting regional transmission patterns shaped by travel and epidemiological factors. Furthermore, our screening approach successfully identified variants that were more prevalent in other regions, such as BA.2.75, which was widespread in parts of Asia [42]. This underscores the potential of our method for detecting variants beyond the Tunisian context”. Responses to Reviewer 2's Comments: 1/ When applying the as-PCR and PCR-RFLP for virus variant screening, it should be more cautious on determination the indies that could affect the results, including sample size, age and sex distribution of the enrolled samples etc. Although the authors declared that their variant monitoring results are in concordance with the epidemiological surveillance data, this is more like a concordance. Thank you for raising this point. We have provided more details regarding these factors in lines 103-105. Overall, the samples were obtained from a population with a nearly balanced gender distribution (56% female, 44% male), covering all age groups from newborns to 92 years (mean: 47 ± 22 years). We believe that these demographic factors are unlikely to influence the results of variant screening, as current evidence indicates that SARS-CoV-2 lineage distribution is primarily shaped by viral evolution and epidemiological dynamics rather than host demographic characteristics. 2/ The authors should provide more details on the as-PCR and PCR-RFLP methods applied in the present study, how about their limit of detection, specificity, reproducibility etc? We fully agree with the reviewer. The analytical sensitivity, specificity, and reproducibility of our screening tests have been demonstrated using recombinant plasmids containing the main target sequences of this study. We have included the necessary details in the Materials and Methods section (lines 148-165, 183-188 and 208-210), in results section (lines 269-291) and discussion section (lines 356-360). 3/ The structure of the manuscript is a little strange and recommended to be re-organized to make the manuscript more easy to follow. The structure of the manuscript has been reorganized to enhance clarity and make it easier to follow. We adopted the following order: first, Validation of the selected genetic markers using publicly available global sequence data; second, Analytical performance of screening assays; followed by Omicron BA.4 and BA.5 screening, Screening of BA.2 and BA.5 descendant sublineages, and finally, Concordance between screening and sequencing results The repositioned paragraphs are highlighted in blue within the text for your convenience. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0321581.r003
10 Mar 2025 Decision Letter - Huseyin Tombuloglu, Editor Use of allele-specific qPCR and PCR-RFLP analysis for rapid detection of the SARS-CoV-2 variants in Tunisia: a cheap flexible approach adapted for developing countries PONE-D-24-46700R1 Dear Dr. Ayadi, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Huseyin Tombuloglu Academic Editor PLOS ONE Additional Editor Comments (optional): Please double-check punctuation and some terminologies (SARS-CoV-2) and be sure they are written correctly. 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 #2: 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 #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: 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 #2: 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 #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 #2: All of my comments have been addressed, I have no more question. I recommend to accept the manuscript. ****** 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 #2: Yes: Jianguo Li ******** https://doi.org/10.1371/journal.pone.0321581.r004
Formally Accepted
Acceptance Letter - Huseyin Tombuloglu, Editor PONE-D-24-46700R1 PLOS ONE Dear Dr. Ayadi, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Huseyin Tombuloglu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0321581.r005

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