Aptamer based proteomic pilot study reveals a urine signature indicative of pediatric urinary tract infections

Liang Dong; Joshua Watson; Sha Cao; Samuel Arregui; Vijay Saxena; John Ketz; Abduselam K. Awol; Daniel M. Cohen; Jeffrey M. Caterino; David S. Hains; Andrew L. Schwaderer

doi:10.1371/journal.pone.0235328

Peer Review History

Original SubmissionOctober 23, 2019
13 Jan 2020 Decision Letter - Mehreen Arshad, Editor PONE-D-19-29587 Aptamer based proteomic pilot study reveals a urine signature indicative of pediatric urinary tract infections. PLOS ONE Dear Dr Schwaderer, 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. We would appreciate receiving your revised manuscript by Feb 27 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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 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 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 authors have provided evidence to suggest that there are compositional differences in the urinary proteome of individuals with UTI and pyuria and those with pyuria but no evidence of UTI. Several of the makers appear to be unique in this context. While many markers appear to provide high sensitivity their specificity is quite often low. This last point is truly difficult to assess because of the low numbers of patients examined. Since the use of dipstix and esterase monitoring was so heavily criticized and it is the current standard it would have been useful to include data regarding the results from the assay to compare performance. The manuscript is comparing a very expensive assay to a relatively inexpensive one. Clearly the intent would be to hone the panel down to a much smaller set of analytes but the comparison might have further strengthened the authors case. I question the value/point of providing the three sets of machine learning results as they are quite disparate and there is no significant discussion of the relative merits or limitations of these approaches. Thus it is unclear what this data adds to the current document. I found the reporting of fluorescence intensity rather confusing as I am familiar with significantly higher signals with somascan than were listed. This may arise from the normalization to creatinine levels. It would be beneficial to determine if the creatinine levels between each group were significantly different. The data to do this is available in the supplementary tables. I failed to see the value of the inclusion of supplementary figures 6 and 7 as this could just as easily been stated in the discussion. The data derives from protein atlas and has no relationship to the current study other than saying there is evidence to suggest the presence of the candidate proteins in renal and bladder tissues. Similarly I do not see the need/utility of the PCR data on unrelated and incompletely rationalized samples from different tissue sources. Somascan is very robust and sensitive with a very board dynamic range. It is essential to have some form of validation using an antibody based approach to demonstrate that ELISA is actually feasible for these analytes at the concentrations present in urine. Reviewer #2: This paper by Dong et al. presents an interesting work and potentially holds very promising impact on POC diagnostics for UTI, and I found this work interesting. However, there are few queries and suggestions that need to be addressed to improve the quality of manuscript. Comment 1: Language at times is bit difficult to understand, particularly in the paragraph 2-5 of the discussion, the linkage between the paragraphs are also missing. Comment 2: Similarly, in the introduction many lines are lengthy which can be difficult to comprehend for readers, should be broken into smaller sentences. For instance, consider this line “Infectious conditions including renal tuberculosis, herpes simplex virus and chlamydia along with noninfectious inflammatory conditions including Kawasaki disease, appendicitis, foreign bodies and interstitial nephritis can cause what has been historically described as sterile pyuria” Comment 3: Proper referencing is missing at times for instance, consider “Several challenges have limited protein-based biomarker discovery including the difficulty to develop high throughput assays such as enzyme-linked immunosorbent assay (ELISA) for identifying biomarker candidates.” Comment 4: The problems with existing technologies, like ELISA should be briefly discussed in the introduction and how aptamer-based technologies can alleviate them should also be discussed in brief with proper references. Comment 5: It would be good to include some information on the current potential and market of aptamers in Introduction. Some the recent publications on this aspect is as follows. 1- Aptamer-based point-of-care diagnostic platforms 2- Aptamers in the Therapeutics and Diagnostics Pipelines 3- The point behind translation of aptamers for point of care diagnostics 4- Defining Target Product Profiles (TPPs) for Aptamer-Based Diagnostics ****** 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: Yes: John A Wilkins 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0235328.r001
Revision 1
13 May 2020 Author Response We have revised the manuscript based on the reviewers comments resulting in a more concise and focused study. We have also performed additional studies when recommended. Please see below for an itemized response. Since the use of dipstix and esterase monitoring was so heavily criticized and it is the current standard it would have been useful to include data regarding the results from the assay to compare performance. The manuscript is comparing a very expensive assay to a relatively inexpensive one. Clearly the intent would be to hone the panel down to a much smaller set of analytes but the comparison might have further strengthened the authors case. Response: For this study we purposely chose 16 patients with a positive culture and positive leukocyte esterase (LE) on dipstick, 8 with a negative culture and positive LE and 8 with negative LE and culture. Therefore LE was predetermined to have a sensitivity of 50% and specificity of 33%. In our previously reported larger cohort of 199 patients that we selected our samples from LE >/= small had a sensitivity of 83% and specificity of 85% (PMID: 26885759). We have added this language and the reference to our discussion. I question the value/point of providing the three sets of machine learning results as they are quite disparate and there is no significant discussion of the relative merits or limitations of these approaches. Thus it is unclear what this data adds to the current document. Response: We have removed the former figure 3 with three machine learning data sets and focused the results on the Random Forest algorithm. I found the reporting of fluorescence intensity rather confusing as I am familiar with significantly higher signals with somascan than were listed. This may arise from the normalization to creatinine levels. It would be beneficial to determine if the creatinine levels between each group were significantly different. The data to do this is available in the supplementary tables. The urine creatinine levels were not different between groups, P = 0.523 using the Kruskal-Wallis test because the data was not parametric. Additionally we, in the initial submission, presented the ratio of urine biomarker to creatinine with the Somalogic results as relative fluorescent units (RFU) per ml and the creatinine as mg/dl for a dimensionally complex ratio of biomaker(RFU/ml)/ creatine (mg/dl). The higher creatinine volume (dl as opposed to ml) results in lower ratio values. We have revised Figure 2 and Table 2 with the creatinine results as mg/ml so mls cancel out and the results are the more traditional and simple biomarker (RFU)/creatinine (mg) units. Response: I failed to see the value of the inclusion of supplementary figures 6 and 7 as this could just as easily been stated in the discussion. The data derives from protein atlas and has no relationship to the current study other than saying there is evidence to suggest the presence of the candidate proteins in renal and bladder tissues. Similarly I do not see the need/utility of the PCR data on unrelated and incompletely rationalized samples from different tissue sources. Response: We have removed supplementary figures 6 and 7 and instead added the presence of candidate biomarkers in renal and bladder tissue to the discussion as recommended. Somascan is very robust and sensitive with a very board dynamic range. It is essential to have some form of validation using an antibody based approach to demonstrate that ELISA is actually feasible for these analytes at the concentrations present in urine. Response: We have validated the Somalogic results for GM-CSF with an antibody based approach in a separate patient cohort (an adult patient cohort) which resulted in a similar area under the curve value. The results are presented in a new Figure 3. We have also updated the methods, results and discussion based on this finding. Comment 1: Language at times is bit difficult to understand, particularly in the paragraph 2-5 of the discussion, the linkage between the paragraphs are also missing. Response: We have revised the wording and added linkage for paragraphs 2-5 Comment 2: Similarly, in the introduction many lines are lengthy which can be difficult to comprehend for readers, should be broken into smaller sentences. For instance, consider this line: “Infectious conditions including renal tuberculosis, herpes simplex virus and chlamydia along with noninfectious inflammatory conditions including Kawasaki disease, appendicitis, foreign bodies and interstitial nephritis can cause what has been historically described as sterile pyuria” Response: We have shortened sentences and simplified the introduction as recommended. Comment 3: Proper referencing is missing at times for instance, consider “Several challenges have limited protein-based biomarker discovery including the difficulty to develop high throughput assays such as enzyme-linked immunosorbent assay (ELISA) for identifying biomarker candidates.” Response: We have add references for the example cited and in other places as well. Comment 4: The problems with existing technologies, like ELISA should be briefly discussed in the introduction and how aptamer-based technologies can alleviate them should also be discussed in brief with proper references. Response: We have provided an extended explanation for ELISA limitation in the introduction: “Several challenges have limited protein-based biomarker discovery with traditional antibody based ELISAs. Specifically, ELISAs are time consuming to perform and the required antibodies have inherent costs, instability, batch to batch variation, storage requirements limited dynamic ranges and are difficult to multiplex [10-12].” Comment 5: It would be good to include some information on the current potential and market of aptamers in Introduction. Some the recent publications on this aspect is as follows. 1- Aptamer-based point-of-care diagnostic platforms 2- Aptamers in the Therapeutics and Diagnostics Pipelines 3- The point behind translation of aptamers for point of care diagnostics 4- Defining Target Product Profiles (TPPs) for Aptamer-Based Diagnostics Response: We have added more information about the current and market potential of aptamers in the introduction: Aptamers are being explored as affordable, sensitive, specific, user friendly point of care tests on a variety of platforms [15]. Aptamers have the ability to perform in formats where antibodies often perform poorly such as homogenous multiplex assays, do not degrade when stored at room temperature as a dry lyophilized reagent at room temperature and have minimal to no batch to batch variation [12]. Thus there is speculation that aptamers may replace antibodies in future diagnostics [16]” Attachments Attachment Submitted filename: PLOSone response to reviewers-v1[1].docx https://doi.org/10.1371/journal.pone.0235328.r002
15 Jun 2020 Decision Letter - Mehreen Arshad, Editor Aptamer based proteomic pilot study reveals a urine signature indicative of pediatric urinary tract infections. PONE-D-19-29587R1 Dear Dr. Schwaderer, 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, Mehreen Arshad, M.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0235328.r003
Formally Accepted
23 Jun 2020 Acceptance Letter - Mehreen Arshad, Editor PONE-D-19-29587R1 Aptamer based proteomic pilot study reveals a urine signature indicative of pediatric urinary tract infections. Dear Dr. Schwaderer: 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. Mehreen Arshad Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0235328.r004

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