Methodological establishment and diagnostic value of a multiplex fluorescent PCR assay for the detection of three fastidious respiratory pathogens

Jingchao Shi; Yijun Zhu; Shuyun Chen; Xiaoyun Shan; Lihong Bo; Kai Shen; Keqiang Chen

doi:10.1371/journal.pone.0328651

Peer Review History

Original SubmissionDecember 2, 2024
Decision Letter - Daniela Flavia Hozbor, Editor Dear Dr. Zhu, 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 all the points raised during the review process. Please submit your revised manuscript by Aug 02 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. 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'. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: Dear Authors, I commend you for your work. Here are some comments and suggestions to further strengthen your manuscript: Abstract To enhance the abstract, consider adding a brief background to provide context for the study. Mention that Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are common fastidious bacteria causing respiratory tract infections. Highlight the challenges in their detection using traditional methods. Introduction 1. To strengthen the introduction, consider expanding on the clinical significance of mixed infections and the challenges they pose for diagnosis and treatment 2. You might also want to include a brief discussion of the mechanisms by which these pathogens cause disease and the host immune response to these infections Methods 1. Consider providing more information on the quality control measures taken to ensure the accuracy and reliability of the DNA extraction and PCR amplification steps Overall, this is a well-conducted study with significant clinical implications. Addressing the comments and suggestions will further enhance the quality and impact of your manuscript. Reviewer #2: This paper outlines a novel multiplex PCR assay for the identification of fastidious bacteria related to paediatric inner ear infections. This multiplex PCR method is shown to be sensitive down to 20-100 copies/ml within a reasonable number of PCR cycles to avoid overamplifications. This assay is shown to be specific for the three bacteria of interest compared to both species of the same genus and other unrelated bacteria. Finally, the authors show, using 173 clinical samples, that this multiplex assay outperforms traditional culture methods in the detection of concurrent bacteria within a sample, confirmed through NGS methods when in doubt. Overall, this paper lays out a novel diagnostic approach which would aid in early identification of infection causing bacteria and inform on antibiotic usage if implemented in the clinic. Minor Revisions 1. Although the complete list of bacteria tested for specificity is given in the methodology, I suggest, as a minimum, highlighting in the results text that the bacterial strains tested included both other Streptococcus, Haemophilus and Moraxella strains as well as other unrelated infectious bacteria types. The exhaustive list is not required in the results. 2. From the text and methods it is not exactly clear what concentrations were used for the repeatability test. It would be good to have this test put in context of the sensitivity test. For example, for the concentrations used for the repeatability test, are these similar to 10 copies/ml or 500 copies/ml. This is important to understand the reproducibility of the assay at low bacterial numbers. 3. For figure 2: It is not clear, are these curves representative of 4 samples: 3 single positive and a negative? Or is it a single triple positive sample and a negative. Ideally the former would be shown alongside a triple positive for comparison, as individual graph panels. 4. From line 333: This paragraph is repetitive of the results. If leaving this section in, expand on why the ability to detect low copy numbers is important and possible discuss whether this detectability is overly sensitive. ****** 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 https://doi.org/10.1371/journal.pone.0328651.r001
Revision 1
25 Jun 2025 Author Response Journal: PLOS ONE Manuscript title: Methodological Establishment and Diagnostic Value of a Multiplex Fluorescent PCR Assay for the Detection of Three Fastidious Respiratory Pathogens Manuscript ID: PONE-D-24-54222 Authors: Jingchao Shi, Yijun Zhu, Shuyun Chen, Xiaoyun Shan, Lihong Bo, Kai Shen, Keqiang Chen Dear editor and reviewers We appreciate your assessments of our manuscript (Manuscript ID: PONE-D-24-54222), entitled “Methodological Establishment and Diagnostic Value of a Multiplex Fluorescent PCR Assay for the Detection of Three Fastidious Respiratory Pathogens”, and have found that the reviewers’ comments and suggestions are valuable in revising the manuscript. We have made the necessary revisions as recommended by the reviewers. In the revised manuscript, we have highlighted all the amendments using yellow text. Thank you once again, and please don't hesitate to contact us if you have any further comments or questions. We are looking forward to your final decision. Thank you very much for your great help! Sincerely, Jingchao Shi The following are our point-by-point responses, in order of the comments about the manuscript: Reviewer #1: 1. Abstract To enhance the abstract, consider adding a brief background to provide context for the study. Mention that Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are common fastidious bacteria causing respiratory tract infections. Highlight the challenges in their detection using traditional methods. Introduction Response�We thank the reviewer for this constructive suggestion. In response, we have revised the abstract to include a brief background that highlights the clinical importance of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis as fastidious pathogens commonly associated with respiratory tract infections, and the limitations of traditional culture-based diagnostic methods. (line15-19) 2. Introduction To strengthen the introduction, consider expanding on the clinical significance of mixed infections and the challenges they pose for diagnosis and treatment. You might also want to include a brief discussion of the mechanisms by which these pathogens cause disease and the host immune response to these infections Response�We appreciate the reviewer’s valuable comments. In response, we have revised the Introduction section to provide a more comprehensive background. We have now added discussion on (1) the clinical significance of mixed infections and their diagnostic and therapeutic challenges, and (2) the pathogenic mechanisms and host immune responses associated with S. pneumoniae, H. influenzae, and M. catarrhalis. These changes enhance the rationale for developing a rapid and accurate detection method for these pathogens. (line61-76) 3. Methods Consider providing more information on the quality control measures taken to ensure the accuracy and reliability of the DNA extraction and PCR amplification steps Overall, this is a well-conducted study with significant clinical implications. Addressing the comments and suggestions will further enhance the quality and impact of your manuscript. Response�We appreciate the reviewer’s insightful comment regarding the need to elaborate on quality control measures in the Materials and Methods section. To address this, we have added specific details on the quality assurance steps taken during DNA extraction, PCR setup, and amplification, including the use of internal controls, reagent handling protocols, and contamination prevention practices. These additions enhance the transparency and reproducibility of our methods and underscore the reliability of our assay. (line113-116, line133-134,line152-158, line 205-206) Reviewer #2: 1. Although the complete list of bacteria tested for specificity is given in the methodology, I suggest, as a minimum, highlighting in the results text that the bacterial strains tested included both other Streptococcus, Haemophilus and Moraxella strains as well as other unrelated infectious bacteria types. The exhaustive list is not required in the results. Response�We thank the reviewer for the suggestion. We have now added a summary in the results section indicating that the specificity panel included closely related species from the same genera as the targets (e.g., Streptococcus mitis, Haemophilus haemolyticus, Moraxella osloensis), as well as other common respiratory and unrelated bacterial pathogens. (line245-249) The full list of bacterial species used for specificity testing was provided in the Methods section to ensure methodological transparency and reproducibility. This allows other researchers to replicate the study or assess potential cross-reactivity with specific organisms of interest. 2. From the text and methods it is not exactly clear what concentrations were used for the repeatability test. It would be good to have this test put in context of the sensitivity test. For example, for the concentrations used for the repeatability test, are these similar to 10 copies/ml or 500 copies/ml. This is important to understand the reproducibility of the assay at low bacterial numbers. Response�Thank you for your insightful comment. We have clarified the relationship between the concentrations used in the repeatability test and those in the sensitivity analysis. In the repeatability test, three concentration levels were selected for each pathogen, representing high, moderate, and low template amounts. These corresponded approximately to Ct values of ~24, ~28–29, and ~31–35, respectively. Based on the standard curve generated during the sensitivity test, these Ct values are estimated to correspond to the following DNA concentrations: Ct ≈ 24: ~10⁵–10⁶ copies/mL Ct ≈ 28–29: ~10³–10⁴ copies/mL Ct ≈ 31–35: ~10–100 copies/mL Therefore, the repeatability test included low concentrations close to the detection limit (10–20 copies/mL), ensuring assessment of assay reproducibility across the entire analytical range. The detailed Ct values and corresponding coefficients of variation (CVs) are provided in Supplementary Table 3, showing low intra-assay variability (CVs from 0.03 to 0.21), even at low template levels. We have also added this explanation to the Methods section for clarity. (line198-206) 3. For figure 2: It is not clear, are these curves representative of 4 samples: 3 single positive and a negative? Or is it a single triple positive sample and a negative. Ideally the former would be shown alongside a triple positive for comparison, as individual graph panels. Response�We appreciate the reviewer’s helpful suggestion regarding the clarification of Figure 2. In the revised version of Figure 2, we have included individual amplification curves for five representative samples: Panel A: S. pneumoniae-only positive Panel B: H. influenzae-only positive Panel C: M. catarrhalis-only positive Panel D: Negative control Panel E: Triple-positive sample (simultaneous presence of all three pathogens) Each panel clearly shows the fluorescence amplification signal for the respective pathogen(s), and the inclusion of both single and mixed infections enhances the clarity and interpretability of the assay’s multiplex capability. We believe this revision more effectively illustrates the specificity and multiplex detection capacity of our assay and fully addresses the reviewer’s concern. 4. From line 333: This paragraph is repetitive of the results. If leaving this section in, expand on why the ability to detect low copy numbers is important and possible discuss whether this detectability is overly sensitive. Response�We appreciate the reviewer’s insightful comment. We agree that the original paragraph in line 333 was somewhat repetitive and did not adequately explain the clinical relevance of detecting low bacterial copy numbers. In response, we have revised this section of the manuscript to highlight why this feature is important in clinical practice. Specifically, we emphasize that the ability to detect low-copy-number pathogens can aid in the diagnosis of early-stage infections, cases with low bacterial loads due to prior antibiotic treatment, or polymicrobial infections where certain pathogens are present in minor proportions. We have also addressed the concern of potential over-sensitivity. The inclusion of appropriate negative controls and the use of t-NGS confirmation in discordant cases help minimize the risk of false positives due to contamination or nonspecific amplification. Therefore, we believe that the assay maintains a clinically relevant balance between high sensitivity and specificity. (line371-379) Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0328651.r002
Decision Letter - Daniela Flavia Hozbor, Editor Methodological Establishment and Diagnostic Value of a Multiplex Fluorescent PCR Assay for the Detection of Three Fastidious Respiratory Pathogens PONE-D-24-54222R1 Dear Dr. Yijun Zhu, 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, Daniela Flavia Hozbor Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: N/A Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: Thank you for addressing the comments, I think your paper is now suitable for publication. Congratulations. Reviewer #2: (No Response) ****** 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 ******** https://doi.org/10.1371/journal.pone.0328651.r003
Formally Accepted
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