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PONE-D-22-02044Multiplex real-time PCR using SYBR Green: unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluidPLOS ONE

Dear Dr. Campos,

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: 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

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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: No

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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: The authors present an adaptation of a previously proposed assay, but now based on melting curve analysis, for the identification of penicillin, macrolide and lincosamide resistant pneumococci in culture negative CSF specimens. The authors advance no justification for adapting the existing assay. The paper then presents a lengthy description of the statistical analyses performed and of the method development which can be greatly shortened and simplified. It is unclear what can be expected from 4/51 strains used to validate the assay since they do not seem to have been tested for their susceptibility to clindamycin or clindamycin and erythromycin. Sensitivity and specificity seem to be high (only 3 strains out of 47 had inconsistent results with phenotypic assay). A major undiscussed point is the portability of the assay to a different platform than the one used by the authors, which can limit its applicability. The authors then analyzed 86 samples received at their center between 2014-2020 (but only 61 were culture negative). The paper should be shortened to focus on the method and its appropriateness to determine pneumococcal susceptibility in CSF samples. Although overall the English is acceptable the paper would still benefit from a revision of English use.

Major points

1) The authors should clearly state the benefits of adapting the existing probe-based assay to a melting-curve based assay. If the motivation is economical, a paragraph stating the price differences should be presented and a convincing argument presented to support this.

2) The detailed description of the statistical tests employed should be shortened and only the relevant sections included. The main message of the paper is the method and its validation, and the paper should focus on this. The entire section on the choice of Tm prediction software (and the related sections in the discussion) should be removed. It is nonsensical to choose a prediction software and method based on its agreement with the empiric determined Tm, as is stated in lines 379-380.

3) The paper should clearly state what were the validation conditions used. If this was against the PCR on the DNA extracted from all the strains or, which would me much more interesting, against the DNA extracted from the CSF from which some of the strains were isolated. The discussion of results with different PCR conditions than the ones used (ex: lines 291-297) should be deleted.

4) The susceptibility of all isolates to all targeted antimicrobials must be known (table 2).

5) Given the variations in melting curves seen when DNA was prepared from different sources, what would be the robustness of the assay when run in different platforms? Although the Roche Lightcycler 480 II is quite a popular platform it is by no means the only one. A few sentences discussing the potential portability of the assay to other platforms would be important for a wider interest in the paper.

6) The purpose of the retrospective analysis and how it could impact on the clinical management of the patients or how the information could help in the surveillance of pneumococcal infections in this area must be clarified or this section removed. The fact that macrolides and lincosamides are not used in the treatment of pneumococcal meningitis raises the question of the usefulness of determining the potential susceptibility to these antimicrobials, a point that should be addressed.

7) The discussion should be shortened and focused on the main message of the paper without repetition of what was stated in the results section

Other points

8) Line 121. The authors changed the reverse primer amplifying the mef gene from the previously published assay. Why was this done? A sentence should be included to explain this option.

9) English use: line 116 “and thus it is sensitivity” should be “and thus its sensitivity”; line 164 “Comparison of technics” should be “Comparison of techniques”; line 181 “Characterization of applied samples” should be “Characterization of analyzed samples”; line 291 and elsewhere “acceptation criteria” should be “acceptance criteria”

10) Lines 222-224. The central limit theorem is not applicable to 39 data points which is what is available for the pbp2 gene (table 1). This sentence must be removed.

11) Table 1. The P values have comas instead of points separating the decimals. This table repeat the data presented in figure 1. I would suggest offering the figure as supplementary material.

Reviewer #2: The paper describes validation of a previously published Taqman multiplex real-time PCR assay for detecting 3 antimicrobial resistance genes, pbp2b, erm B and mef and instead using primers with SYBR green intercalating dye. The authors describe their approach for validation and then apply the assay to several culture-negative samples previously identified as lytA positive indicating presence of S. pneumoniae.

Overall, I found the paper quite difficult to read in its current format and could use some review for correct English sentence structure and grammar.

Specific comments

Line 21-22 and 59. Not necessary to have capital letters for Public Health?

Line 71. It is not clear what you mean by qPCR “provides more complete results than the standard techniques”?

Line 164. “technics” should be “techniques”

Line 206-208. I am curious why you would need to have 2 different primer concentrations when you use isolate vs CSF DNA? Can you explain and would you be concerned with performance of the assay with varying amounts of DNA in your sample? Did you do a serial dilution series to determine LOD?

Line 267-269 and Table 1. It Is not clear why mef has only 2 groups for comparison? Why was CSF silica and CSF heating combined? Was it due to sample size?

Line 292-297. Do you think that this approach using SYBR green without a probe is accurate enough given that you mention false positive and negative results with changes in temperature and primer concentrations. I did not see any data provided showing effects of sample concentration of DNA on the Tm and positive vs negative results. Can you explain in Fig 1 the large differences between Tm for CSF (silica) vs CSF (heating)? Is this related to DNA concentrations?

Line 387-388. You state “Thus, an interval must be adopted to be 388 considered positive according to the origin of the genetic material”. I have my doubts on how accurate this method would be if say for example you used a pleural fluid or blood sample? Would you be confident that this assay would accurately determine penicillin, erythromycin or clindamycin susceptibility in these other specimen types give the variability of the assay?

Lines 399-402. Again, here you indicate that differences such as changes in PCR instrumentation would affect results? I don’t see how this method could be useful to a larger community? Perhaps validated in your own lab for testing but seems problematic in that every lab would need to revalidate the assays in their own hands.

Line 444-445. Did you look for inducible clindamycin resistance by testing this strain with D-zone test?

Line 473-475. I would be very cautious using lytA as a SYBR green assay without the probe. I would be concerned about false positives from oral streptococci.

Line 490-491. In your concluding sentences you make reference to qPCR for 3 main bacteria? I don’t see any of this data in the paper and not really sure of the relevance to the data you are showing in this paper.

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Reviewer #1: No

Reviewer #2: No

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Multiplex real-time PCR using SYBR Green: unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid

PONE-D-22-02044R1

Dear Dr. Campos,

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,

Jose Melo-Cristino, M.D., 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

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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 #1: Yes

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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

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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 #1: Yes

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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: The authors have addressed most of the comments raised by the reviewers and provided necessary information in the text.

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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