Evaluation of rapid diagnostic test kits for detection of Treponema pallidum antibody

Sirinart Chomean; Palakorn Puttaruk; Phakawat Khamsophar; Waraphorn Fukpo; Chollanot Kaset

doi:10.1371/journal.pone.0303477

Peer Review History

Original SubmissionDecember 29, 2023
1 Apr 2024 Decision Letter - Anoop Kumar, Editor PONE-D-23-43206Evaluation of rapid diagnostic test kits for detection of Treponema pallidum antibodyPLOS ONE Dear Dr. Kaset, 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 May 16 2024 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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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 Reviewer #2: Partly ******** 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 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: The manuscript entitled “Evaluation of rapid diagnostic test kits for detection of Treponema pallidum antibody” is well written, structured and presents a comprehensive evaluation of two prototypes of the Onsite Syphilis Ab Combo Rapid Test (Fd and Ff), comparing their performance with the established chemiluminescent microparticle immunoassay (CMIA) method. The research addresses a significant gap in the field by assessing the efficacy and reliability of rapid syphilis testing, particularly in settings with limited resources where prompt and accurate diagnostics are essential. The reported sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and accuracies of the rapid test prototypes are well-documented and demonstrate their effectiveness as diagnostic tools. One notable strength of the study is the inclusion of diverse patient samples, including syphilis patients, healthy individuals, and cases with potential cross-reactions. This approach enhances the generalizability of the findings and highlights the utility of the rapid tests in real-world clinical scenarios. Overall, this article provides valuable insights into the performance of the Onsite Syphilis Ab Combo Rapid Test prototypes and their potential integration into current diagnostic algorithms. Reviewer #2: Dear Authors, Thank you for your study aiming to assess the accuracy and reliability of two new versions of the The Onsite Syphilis Ab Combo Rapid Test that demonstrated high sensitivity and specificity comparable to CMIA methods. Please, consider the suggestions below to improve on this good study. Materials and Methods Page 5, line 108. “Population Study”. I think Study population or Study participants would be a much-preferred Subheading here. Page 6, line 113 -115 “A total of 450 serum samples were collected from Thammasat University Hospital (TUH). These were classified into three groups: 200 reactive samples for T. pallidum antibodies obtained from syphilis patients…” Please, how and with what were these samples classified? How was the diagnosis of ‘syphilis’ made in your study participants before samples were collected from them? Please, give more details on your data collection procedures and sample collection and processing. Methods Page 6, line 124-125 “Each test utilized 40 µL of serum followed by 40 µL of the provided buffer.” If after 15-minute incubation at room temperature, results were visually interpreted, it then shows that that the buffer contains the recombinant antigen of the TP bacterium. If this is the case, please indicate that the buffer contains the antigen, or that both buffer and the recombinant antigen were added also. Correlation analysis of The Onsite Syphilis Ab combo Rapid Test with CMIA, RPR and TPPA test Page 8, line 170-171 “Onsite Syphilis Ab Combo Rapid Test were grouped into W+, 2+, and 4+”. Please explain what W+ stands for and rationale behind the grouping into +W, 2+, and 4+. Results Page 9, line 196 “…as illustrated in Table 1”. Please, where is the Table 1 mentioned here? Also, consider preparing a three-column table displaying overall sensitivity, specificity, predictive values, and likelihood ratios of The Onsite Syphilis Ab combo Rapid Test using CMIA as the standard. Measures of Diagnostic accuracy (Or Diagnostic Parameters) Estimated values (%) 95% Confidence intervals Sensitivity Specificity PPV NPV PLR NLR Accuracy Page 12, line 244 “…illustrated that its performs effectively across various…” Please, correct to “…illustrated that it performs effectively across various…” Page 15, line 272-276 “In conclusion, the Onsite Syphilis Ab combo Rapid Test (prototype Ff) demonstrates a high degree of efficacy in detecting syphilis infections…” I do not understand why your conclusion tend to project the performance of Prototype Ff over Fd while in your analysis, Prototype Fd out-performed Prototype Ff. I would suggest you reverse this conclusion in favour of Prototype Fd. Prototype Fd consistently gave strong positive reactions (100%) when compared with Ff (87.5%) against different TPPA tires. Please, what do you think are the major limitations of this study? Please, add this before your conclusion. Reference Page 24, line 465, reference no 15 “…Wilson C, Peterman TA. 2019. Frequency and characteristics of biological false-positive test results…” Please remove 2019 if it’s not part of the journal article title. ****** 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: Dr. Anthony A. Iwuafor ******** [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: Reviewer Comments for authors attention.docx https://doi.org/10.1371/journal.pone.0303477.r001
Revision 1
5 Apr 2024 Author Response Response to Reviewers Dear Editor and Reviewers, We are writing in response to the comments and suggestions provided on our manuscript entitled "Evaluation of Rapid Diagnostic Test Kits for Detection of Treponema pallidum Antibody," submitted to Plos One with manuscript number PONE-D-23-43206. We deeply appreciate the time and effort the reviewers have invested in evaluating our work, and we are grateful for the insightful feedback that has significantly contributed to the enhancement of our manuscript. We have carefully reviewed and considered all the comments and have made corresponding revisions to our manuscript. Below is a summary of the major changes and our responses to the specific comments: Materials and Methods Page 5, line 108. “Population Study”. I think Study population or Study participants would be a much-preferred Subheading here. Response to Reviewer: Thank you for the suggestion. We have revised the text accordingly, as per Page 5, line 108. Page 6, line 113 -115 “A total of 450 serum samples were collected from Thammasat University Hospital (TUH). These were classified into three groups: 200 reactive samples for T. pallidum antibodies obtained from syphilis patients…” Please, how and with what were these samples classified? How was the diagnosis of ‘syphilis’ made in your study participants before samples were collected from them? Please, give more details on your data collection procedures and sample collection and processing. Response to Reviewer: Syphilis diagnosis is based on the patient’s history, physical examination, laboratory testing, and sometimes radiology. However, many people with syphilis do not have any symptoms or have only minor symptoms and do not realize that anything is wrong. Identifying asymptomatic infection, especially among pregnant women, through screening using laboratory tests and treatment of positive cases will prevent further transmission and adverse pregnancy outcomes and congenital syphilis. The available laboratory tests for syphilis include direct detection methods (i.e., dark-field microscopy, direct fluorescent antibody test, and nucleic acid amplification test), serology (treponemal and non-treponemal tests), and examination of cerebrospinal fluids. Regarding our study, the 450 serum samples collected from Thammasat University Hospital were classified into three groups based on reactivity for T. pallidum antibodies. Specifically, 200 samples were obtained from the positive group using the CMIA method, following standard clinical diagnostic procedures that included clinical assessment and serological tests such as the rapid plasma reagin (RPR) test and Treponema pallidum particle agglutination assay (TPPA). We have revised the text accordingly, as per Page 6, line 118-121. Methods Page 6, line 124-125 “Each test utilized 40 µL of serum followed by 40 µL of the provided buffer.” If after 15-minute incubation at room temperature, results were visually interpreted, it then shows that that the buffer contains the recombinant antigen of the TP bacterium. If this is the case, please indicate that the buffer contains the antigen, or that both buffer and the recombinant antigen were added also. Response to reviewer: Thank you for your insightful comment regarding the composition of the buffer used in our study with the Onsite Syphilis Ab Combo Rapid Test, prototypes Fd and Ff. We appreciate the opportunity to clarify this aspect of our methodology. We would like to clarify that the buffer provided with the rapid test kits does not contain the recombinant antigen of the Treponema pallidum (Tp) bacterium. Instead, the buffer's role is to facilitate the migration of the sample across the test strip by capillary action. The buffer composition typically includes 1% Bovine Serum Albumin (BSA), 0.5% sucrose, 0.05% Tween 20, and is diluted in Phosphate-Buffered Saline (PBS). These components serve as a running buffer, ensuring optimal conditions for the detection reaction without interfering with the specificity or sensitivity of the test. The principle of the test, as detailed in our manuscript (PAGE 6, LINE 124-135 AND PAGE 7, LINE 136 -143), is based on the interaction between the antibodies present in the serum sample and the recombinant Tp antigens conjugated with colloidal gold, which are already pre-applied on the colored conjugate pad of the test cassette. This interaction occurs independently of the buffer composition, with the antigen-antibody complex then migrating towards the test and control lines to provide a visual result. We hope this explanation adequately addresses your concern and clarifies the role of the buffer in the rapid test kits used in our study. We have amended our manuscript to ensure this explanation is explicitly stated, to prevent any misunderstanding regarding the test components and their respective functions. Thank you once again for your valuable feedback, which has helped enhance the clarity and accuracy of our study's methodology. Correlation analysis of The Onsite Syphilis Ab combo Rapid Test with CMIA, RPR and TPPA test Page 8, line 170-171 “Onsite Syphilis Ab Combo Rapid Test were grouped into W+, 2+, and 4+”. Please explain what W+ stands for and rationale behind the grouping into +W, 2+, and 4+. Response to reviewer: Thank you for your inquiry regarding the grading system used for the Onsite Syphilis Ab Combo Rapid Test. In our study, the grading of positive results from the Onsite Syphilis Ab Combo Rapid Test was categorized into W+, 2+, and 4+. This grading system represents the strength of the positive band observed during the test, compared to the control band. Specifically: W+ indicates a weak positive result 2+ indicates a moderate positive result 4+ indicates the strongest positive result The purpose of this grading system is to provide a qualitative assessment of the intensity of the reaction observed on the test band. By categorizing results into different grades, we can compare the performance of the test with other testing methods. This helps us determine whether the two testing kits, which interpret reactions visually, exhibit concordance in reaction strength. Results Page 9, line 196 “…as illustrated in Table 1”. Please, where is the Table 1 mentioned here? Also, consider preparing a three-column table displaying overall sensitivity, specificity, predictive values, and likelihood ratios of The Onsite Syphilis Ab combo Rapid Test using CMIA as the standard. Measures of Diagnostic accuracy (Or Diagnostic Parameters) Estimated values (%) 95% Confidence intervals Sensitivity Specificity PPV NPV PLR NLR Accuracy Response to Reviewer: We apologize for the oversight. Table 1 was inadvertently omitted from the main manuscript. We have now inserted Table 1 into the manuscript as per your suggestion, and it can be found on Page 10, Lines 230-232. Thank you for bringing this to our attention, and we appreciate your thorough review. Table 1 Evaluation Summary of Rapid Diagnostic Test Kits for Treponema pallidum Antibody Detection Diagnosis Performance The Onsite Syphilis Ab combo rapid test Fd Ff Sensitivity % (95% Cl) 100.00 (98.17-100.00) 100 (98.17-100.00) Specificity % (95% Cl) 98.80 (96.53-99.75) 99.60 (97.79-99.99) PPV % (95% Cl) 1.64 (0.54-4.88) 4.76 (0.70-26.13) NPV % (95% Cl) 100.00 (98.52-100.00) 100 (98.53-100.00) PLR (95% Cl) 83.33 (27.06-256.63) 250.00 (35.35-1767.68) NLR (95% Cl) 0 0 Accuracy % (95% Cl) 98.00 (97.30-99.59) 99.60 (98.47-99.96) Page 12, line 244 “…illustrated that its performs effectively across various…” Please, correct to “…illustrated that it performs effectively across various…” Response to Reviewer: Thank you for your keen observation. We have promptly corrected the error in the manuscript following your comment on Page 13, Line 275. The phrase now accurately reads, "...illustrated that it performs effectively across various..." We appreciate your attention to detail and apologize for any confusion caused by the oversight. Page 15, line 272-276 “In conclusion, the Onsite Syphilis Ab combo Rapid Test (prototype Ff) demonstrates a high degree of efficacy in detecting syphilis infections…” I do not understand why your conclusion tend to project the performance of Prototype Ff over Fd while in your analysis, Prototype Fd out-performed Prototype Ff. I would suggest you reverse this conclusion in favour of Prototype Fd. Prototype Fd consistently gave strong positive reactions (100%) when compared with Ff (87.5%) against different TPPA tires. Response to Reviewer: We sincerely apologize for any confusion caused by the previous conclusion. We appreciate your insightful feedback and have revised the conclusion accordingly to better reflect the comparative performance of Prototype Fd and Prototype Ff. New Conclusion: Prototype Fd showed a marginally higher consistency in eliciting strong positive reactions across all TPPA titer levels, suggesting its slightly superior sensitivity in detecting syphilis antibodies. Prototype Ff, while also highly effective, presented minor variability at certain titer levels, which could allow for nuanced interpretation in specific clinical scenarios. Both prototypes prove valuable for syphilis screening, with Fd offering a slight advantage in uniform sensitivity, and Ff providing flexibility in response interpretation. This comparative analysis underscores the importance of selecting an appropriate diagnostic tool based on the clinical context and the need for confirmatory testing to ensure diagnostic accuracy. As Page 16, Line 340-311 We believe this revised conclusion better captures the nuanced performance of both prototypes and provides a balanced assessment of their strengths and limitations. Thank you for guiding us towards this improvement. Please, what do you think are the major limitations of this study? Please, add this before your conclusion. Response to Reviewer: In addressing the major limitations highlighted in the reviewer's comment, we've added a comprehensive section before the conclusion to discuss these aspects. This addition aims to transparently acknowledge the constraints of our study, enhancing its contextual understanding. Firstly, we acknowledge the limitation regarding the diversity and size of our sample group. While our sample included a range of participants, including syphilis patients, healthy individuals, and those with conditions potentially leading to cross-reactivity, it may not fully capture the broader demographic affected by syphilis. This limitation might impact the generalizability of our findings to all populations. Secondly, our investigation into cross-reactivity was specifically focused on HIV, HBV, and HCV, without encompassing other infectious or non-infectious diseases that might influence the rapid tests' outcomes. This selective approach could potentially limit the comprehensiveness of our assessment regarding test specificity and sensitivity. Moreover, our reliance on the CMIA method as the sole standard for comparison might not provide a fully rounded evaluation of the rapid tests’ performance across diverse diagnostic contexts. This singular comparison basis may restrict our understanding of the tests' efficacy in different settings. Additionally, the cross-sectional nature of our study limits our ability to observe syphilis progression over time or assess test performance across the disease's various stages. This aspect could be crucial for understanding the dynamics of syphilis infection and the tests' diagnostic utility throughout the disease course. Lastly, the manual interpretation of the rapid tests introduces a subjective element to our study. This subjectivity underscores the necessity for objective, automated reading solutions to ensure consistent and reliable result interpretation. We hope this response addresses the concerns raised and provides a clearer picture of the steps taken to acknowledge and discuss the study's limitations within the manuscript. Revised conclusion has been added as indicated on Page 21, Lines 430-435 and Page 22, 436-444 of our manuscript. Reference Page 24, line 465, reference no 15 “…Wilson C, Peterman TA. 2019. Frequency and characteristics of biological false-positive test results…” Please remove 2019 if it’s not part of the journal article title. Response to Reviewer: Thank you for bringing this to our attention. We have removed the year "2019" from Reference No. 15 in accordance with your suggestion. We appreciate your careful review of our manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0303477.r002
25 Apr 2024 Decision Letter - Anoop Kumar, Editor Evaluation of rapid diagnostic test kits for detection of Treponema pallidum antibody PONE-D-23-43206R1 Dear Dr. Kaset, 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, Anoop Kumar, 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 #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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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: Dr. Anthony A. Iwuafor ******** https://doi.org/10.1371/journal.pone.0303477.r003
Formally Accepted
15 May 2024 Acceptance Letter - Anoop Kumar, Editor PONE-D-23-43206R1 PLOS ONE Dear Dr. Kaset, 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. Anoop Kumar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0303477.r004

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