Deficiency of mannose-binding lectin is a risk of Pneumocystis jirovecii pneumonia in a natural history cohort of people living with HIV/AIDS in Northern Thailand

Kunio Yanagisawa; Nuanjun Wichukchinda; Naho Tsuchiya; Michio Yasunami; Archawin Rojanawiwat; Hidenori Tanaka; Hiroh Saji; Yoshiyuki Ogawa; Hiroshi Handa; Panita Pathipvanich; Koya Ariyoshi; Pathom Sawanpanyalert

doi:10.1371/journal.pone.0242438

Peer Review History

Original SubmissionJuly 2, 2020
11 Aug 2020 Decision Letter - Aftab A. Ansari, Editor PONE-D-20-20525 Association between Mannose-binding Lectin Expression and Risk of Pneumocystis jirovecii Pneumonia in People Living with HIV/AIDS in Northern Thailand PLOS ONE Dear Dr. Yanagisawa, 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 pay close attention to the concerns of reviewer # 1 with regards to statistical analysis and the role of other confounders. There is also a minor issue raised by reviewer # 2 that you and your co-authors should be able to readily address. This is a nice and well performed study. Please submit your revised manuscript by Sep 25 2020 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. Please include the following items when submitting your revised manuscript: 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'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Aftab A. Ansari, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please provide the following details about the stored biological samples used in your study: (1) source of the samples (2) whether the samples were completely de-identified before researchers accessed the samples. We note that you state that "stored samples derived from the Lampang HIV cohort in “unlinked anonymous” pattern can be used in subsequent analyses without requiring additional ethical approval." Please clarify whether the samples were "unlinked anonymous" before you accessed them. Please add this information to the methods section and your ethics statement in your online submission form. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know 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 by Yanagisawa and colleagues describes the association between mannose-binding lectin and susceptibility to Pneumocystis jiroveccii pneumonia (PCP) in HIV+ ART-naive patients in Northern Thailand. PCP was diagnosed in 15% of patients and the authors report MBL expression to be associated with PCP onset in patients with CD4 counts > 50 /ul. The study is straightforward, and the data are well presented. However, the Kaplan Meir curves showing decreased likelihood of PCP over time is confusing. Also, the lower risk of PCP incidence in patients with CD4 counts < 50/ul is unclear. Were other important confounders not accounted for such as susceptibility to other OIs? Another caveat is that presence of coinfections impacting liver function could impact MBL levels and this confounder was not controlled for. Reviewer #2: The manuscript submitted by Yanagisawa and colleagues addresses the role of mannose binding lectin to susceptibility to Pneumocystis jirovecii infection. Specifically, they address the role of genotypes associated with MBL deficiency in the development of PCP in HIV infected subjects. The link between MBL deficiency and infection is an important subject and of significant interest to a broad array of biomedical and public health researchers. Susceptibility to PCP among HIV infected subjects is well-known and provides an opportunity to investigate this important question. The authors utilize a special cohort of HIV infected subjects from Northern Thailand for which samples were taken in a time period prior to the administration of ART. Such samples are rare and valuable. The authors carry out an MBL genotypic analysis of samples from the cohort, that allows them to identify subjects with high, intermediate and low MBL genotypes. This analysis is validated by measuring MBL levels in a subset of subjects by direct measurement of MBL. The association is strong. This analysis allows for identification of any correlations between PCP and MBL genotype. Such an analysis is however nontrivial. HIV infected subjects encompass a range of immunocompetence, which itself can have a profound impact on opportunistic infection. The authors correctly address this variable by evaluating the relationship between PCP, MBL genotype and blood CD4 counts. CD4 counts are a strong measure of immune dysfunction in HIV infected subject and is significantly associated with PCP. Indeed, this variable has the potential to mask any effect of MBL genotype on susceptibility. In fact, for subjects with a CD4 count <50 the association between MBL genotype and PCP infection was lost. However, for subjects with CD4 counts <200 they identified strong and convincing correlation between PCP and genotypes associated with MBL deficiency. I found this manuscript to be clearly presented. It was well organized and a pleasure to read. The subject is an interesting one. Numerous questions remain in relation to MBL deficiency and infection. I expect that these researchers will continue to contribute to this subject area. The only suggestion that I might offer involves the distinction between MBL low genotypes and MBL deficiency. Although the authors demonstrate a very strong correlation between genotype and deficiency, their analysis of susceptibility to PCP is with the genotype, not the phenotype. Perhaps they could make this distinction in their concluding remarks. ****** 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: 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. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: PLOSE ONE Review.docx https://doi.org/10.1371/journal.pone.0242438.r001
Revision 1
30 Oct 2020 Author Response To reviewer #1: “The manuscript by Yanagisawa and colleagues describes the association between mannose-binding lectin and susceptibility to Pneumocystis jiroveccii pneumonia (PCP) in HIV+ ART-naive patients in Northern Thailand. PCP was diagnosed in 15% of patients and the authors report MBL expression to be associated with PCP onset in patients with CD4 counts > 50 /ul. The study is straightforward, and the data are well presented. However, the Kaplan Meir curves showing decreased likelihood of PCP over time is confusing. “ Thank you for your suggestion. The Y-axis of Kaplan-Meier curves in Fig1 in the first manuscript may give misunderstanding that the incidence of the PCP looks to be decreased over time as you mentioned. In the revised manuscript, we modified Fig1. that Y-axis show the increase of PCP incidence. “Also, the lower risk of PCP incidence in patients with CD4 counts < 50/ul is unclear. ” Thank you for your suggestion. In this study, all patients having baseline CD4<200/µl were taken TMP/SMX for the PCP prophylaxis, so that the incidence of PCP in patients with CD4<50/µl group was modified by this medication. However, after the re-categorization of MBL expression type in the revised manuscript (deficient/low/intermediate/high), the cut-off level of CD4 counts below or over 50/µl were not a critical factor of PCP. Therefore, we added Fig 1. b (CD4<200) and c (≧200) in revised version to clarify the impact of PCP prophylaxis that they have been taken or not. As a result, we confirmed that deficient MBL expression type was an independent risk factor of PCP incidence whether CD4 counts were below nor over 200/µl through K-M analysis, univariate and multivariate Cox’s proportional hazard analysis (P10, line 204-210; P11, line 220-226). “Were other important confounders not accounted for such as susceptibility to other OIs? Another caveat is that presence of coinfections impacting liver function could impact MBL levels and this confounder was not controlled for.” Thanks for your suggestion. Because the data associated with liver function (i.e. liver transaminase, prothrombin time and choline-esterase activity) and all of plasma samples were not available, we could not conduct the analysis including these confounders. We also agree that MBL synthesis can be affected by many other confounders including liver functions, co-infections such as hepatitis B and C virus and other OIs. In consideration of these concerns, we retried cross-sectional, univariate and multivariate analysis including HBsAg and HCV-Ab as additional confounding variables. As a result, deficient expression type remained as a significant risk factor of PCP histories (P9, line 190-193, Table 2), and its incidence (P11, line 220-226, Table 3). We believe these data show the critical role of MBL expression type at the onset of PCP upon the co-infection of hepatitis B and C virus. To reviewer #2: The only suggestion that I might offer involves the distinction between MBL low genotypes and MBL deficiency. As you mentioned, patients classified “low” in our first manuscript includes complete deficiency to relatively low expression. Therefore, their plasma MBL concentrations distributed between undetectable to relatively low levels (shown in Fig 2). We re-analyzed our data according to your kind advice that we should distinguish the deficiency and low. As a result, we achieved more impressive results. Briefly, we confirmed that MBL deficiency associated with PCP histories (Table 2), cumulative provability (Fig 1) and incidence independently upon sex, age, CD4 and log10VL (Table 3). We appreciate your great advice and believe that our revised data improved to show the association between MBL expression type and PCP onset more clearly through the new manuscript. Although the authors demonstrate a very strong correlation between genotype and deficiency, their analysis of susceptibility to PCP is with the genotype, not the phenotype. Perhaps they could make this distinction in their concluding remarks. As you mentioned, we agree the limitation that we could not analyze the MBL phenotype and the PCP onset, because all of plasma samples were not available. Therefore, we added this discussion that further studies are needed to clarify the association of MBL phenotype and PCP onset (P15, line 314-318). Response to Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We apologize that first manuscript was not suitable for the PLOS ONE format. We have made the revised manuscript according to the instruction included in the URL you provided, then please confirm it again. 2. Please provide the following details about the stored biological samples used in your study: (1) source of the samples　(2) whether the samples were completely de-identified before researchers accessed the samples. We note that you state that "stored samples derived from the Lampang HIV cohort in “unlinked anonymous” pattern can be used in subsequent analyses without requiring additional ethical approval." Please clarify whether the samples were "unlinked anonymous" before you accessed them. Please add this information to the methods section and your ethics statement in your online submission form. Thanks for your suggestion. (1) We used the genomic DNA extracted by the standard protocol of phenol-chloroform method from the peripheral blood of registered patients. We clarified this preparation steps in the revised manuscript (P6, line 128-129). (2) This study was performed by the second use of anonymity samples and information provided in Lampang HIV cohort. Before the analysis and manuscript preparation, authors have not been provided any information that can identify the individuals registered in cohort. We added this point in ethical statement (P8, line 177-179), and input in the submission form. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ I (Kunio Yanagisawa) have already applied my information to ORCID form that you indicated. 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. We apologize our lack of understanding that the description of "data not shown " is not accepted. We deleted this description, then the data necessary to make discussion was included in supportive information at the end of revised manuscript. Attachments Attachment Submitted filename: Response to Reviewers_201030.docx https://doi.org/10.1371/journal.pone.0242438.r002
3 Nov 2020 Decision Letter - Aftab A. Ansari, Editor Deficiency of Mannose-binding Lectin is a Risk of Pneumocystis jirovecii Pneumonia in a Natural History Cohort of People Living with HIV/AIDS in Northern Thailand PONE-D-20-20525R1 Dear Dr. Yanagisawa, 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, Aftab A. Ansari, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0242438.r003
Formally Accepted
24 Nov 2020 Acceptance Letter - Aftab A. Ansari, Editor PONE-D-20-20525R1 Deficiency of Mannose-binding Lectin is a Risk of Pneumocystis jirovecii Pneumonia in a Natural History Cohort of People Living with HIV/AIDS in Northern Thailand Dear Dr. Yanagisawa: 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. Aftab A. Ansari Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0242438.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .