Role and effects of zinc supplementation in HIV-infected patients with immunovirological discordance: A randomized, double blind, case control study

Macarena Silva; Carmen G. Montes; Andrea Canals; Maria J. Mackenna; Marcelo Wolff

doi:10.1371/journal.pone.0244823

Peer Review History

Original SubmissionJune 24, 2020
16 Sep 2020 Decision Letter - Giuseppe Vittorio De Socio, Editor PONE-D-20-19537 Role and Effects of Zinc Supplementation in HIV-infected Patients with Immunovirological Discordance: A Randomized, Double Blind, Case Control Study PLOS ONE Dear Dr. Silva, 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 October 15 2020. 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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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: No ****** 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: In this paper Silva et al. investigate the role of zinc plasmatic levels and zinc supplementation in patients failing satisfactory CD4 recovery on virally-suppressive ART (IVD). The authors find higher proportion of hypozincemic patients in the IVD vs control group. However, they failed to find a positive effect of zinc supplementation. The paper is well-written and straight forward, addressing an unexplored issue. I find that a major bias in the patients population consists in the significant difference in the time on ART between IVD and controls, with IVD being on ART for significantly lower time. Lower ART time would mean lower possibility to achieve full immune recovery. The authors should either enroll a 3rd cohort of IVD with equal time on ART, or at least acknowledge this bias. In Table 1 the authors report data on hepatitis B co-infection? Can the authors provide also data on HCV co-infection, which has been described to associate to low immune recovery on cART. Reviewer #2: This study determined whether zinc supplementation in virologically suppressed patients who do not achieve complete immune recovery, prevents immune failure after 12 months of supplementation. The study included 80 patients, 40 with incomplete immune recovery (IVD) and 40 controls with CD4 cell count >350 cells/mm3. The mean plasma zinc levels were not different between the groups as baseline and were within the normal ranges. While the CD4 count in the incomplete recovery group increased more than in the control group, the difference was not significantly different. The study is well planned and described. The investigators, however, did not provide sample size calculation and as they note, there may not have been sufficient number of study participants to show significant difference between the groups. Moreover, the manuscript does not show the HIV viral load of the patients and it is not clear if the IVD patients were virally suppressed. This may play a role in the incomplete immune recovery. The mean plasma zinc levels were within normal range at baseline, with a few participants having hypozincemia. This contrasts with previous studies showing a positive effect of zinc supplementation on CD4 cell count of patients with low plasma zinc levels at baseline. While there were participants with very low zinc levels in this study, there were too few of them to make a judgement about the effectiveness of zinc supplementation in patients with low levels of zinc. Another problem is that it is not clear whether the data was analyzed with intent to treat analyses. It also does not appear that the analyses controlled for baseline CD4 cell count which was significantly different between the groups. There is no table showing differences between the treatment and the placebo groups at the end of the study among all participants that started the study, only those with incomplete immune recovery. The results for the 40 participants who did not have incomplete immune recovery are not shown after 12 months of supplementation, only their baseline characteristics. Thus, this manuscript is basically about 40 people with IVD who were either supplemented with zinc or received placebo. There are minor errors in English language than need to be corrected. ****** 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:** Giulia Marchetti 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. https://doi.org/10.1371/journal.pone.0244823.r001
Revision 1
4 Dec 2020 Author Response We thank the reviewers and editorial comments for their critical assessment of our manuscript. In the following we address their concerns point by point. Reviewer 1 Point 1.1: “I find that a major bias in the patients population consists in the significant difference in the time on ART between IVD and controls, with IVD being on ART for significantly lower time. Lower ART time would mean lower possibility to achieve full immune recovery. The authors should either enroll a 3rd cohort of IVD with equal time on ART, or at least acknowledge this bias.” R: We recognize de bias since the longer time of ART exposure, the greater immune recovery achieved, especially during the first 5 years. For this reason, we included a comparative analysis of immune recovery based on final CD4 cell count for both groups (IVD and controls). We found no differences on the median CD4 cell count at the end of the study (IVD: 39 cell/mm3, controls: 19 cell/mm3; p 0,1433). Furthermore, we do not know if hypozinchemia observed in the IVD group could be related to the time of ART exposure. This aspect was incorporated on the discussion. Point 1.2: “Can the authors provide also data on HCV co-infection, which has been described to associate to low immune recovery on ART”. R: We added the HCV frequency for both groups on table 1 (IVD: 2.9%; controls: 2.8%; p 0.739) Reviewer 2 Point 2.1: “The investigators, however, did not provide sample size calculation and as they note, there may not have been sufficient number of study participants to show significant difference between the groups” R: We calculated the sample size needed to assess whether the relative risk of a CD4 cell count increase above 30%. We were based on the percentages of CD4 cell increase greater than 30% from de study of Asdamongkol et al (46.1% in patients with zinc and 23.53% in patients with placebo), with a power of 80% and a confidence level of 95%. According to this calculation, a sample size of 28 patients per group was required. The calculation was made with nMaster 2.0 software. Power was calculated for the comparison of the increasing average CD4cell count between cases (zinc) and controls (placebo), obtaining a value of 100%. This result indicates that, although we did not reach the previously calculated sample size (28 patients per group of zinc and placebo), the sample size obtained was sufficient to assess whether the increase in CD4 cell levels is different between cases and controls. This item is added on Methods. Point 2.2 : “the manuscript does not show the HIV viral load of the patients and it is not clear if the IVD patients were virally suppressed. This may play a role in the incomplete immune recovery” R: All patients had to be virologically suppress to enter the study. This item is added on table 1 and reinforced on discussion. Point 2.3 “This contrasts with previous studies showing a positive effect of zinc supplementation on CD4 cell count of patients with low plasma zinc levels at baseline. While there were participants with very low zinc levels in this study, there were too few of them to make a judgement about the effectiveness of zinc supplementation in patients with low levels of zinc” R: The study of Asdamongkol is the only one that has demonstrated a benefit of zinc supplementation on hypozinchemic patients and also included a low number of patients (hypozinchemia:12 (5 supplemented), normozinchemia:18 (8 supplemented) Their findings were very similar to ours (hypozinchemia:12 (6 supplemented); normozinchemia: 25 (13 supplemented) , but our p value was not satistically significant. The table in the article is above: (table can be seen on file attached "Response to Reviewers") Asdamongkol N, Phanachet P, Sungkanoparph S. Low plasma zinc levels and immunological responses to zinc supplementation in HIV‐in- fected patients with immunological discordance after antiretroviral ther- apy. J Int AIDS Soc. 2012;15 Suppl 4:152‐3. Our findings: (the proper format of the table can be seen on file attached "Response to Reviewers" Table 4. Response of CD4 of supplemented patients accord to zinc level. (12 hypozinchemia: 6 supplemented; 25 normozinchemia 13 supplemented) CD4+ (cells/mm3) * Hypozinchemia(n=6) Normozinchemia(n=13) P- value Baseline 188.5 (179-224) 193 (163.5-213) 0.543 Final 235 (191-256) 222.5 (206.5-261) 0.752 � CD4+ 56 (11-59) 31.5 (10-93.5) 0.752 Futhermore, on a recent review neither Baum, Bobat, Green, Range or Reich found differences on increase of CD4 cell count after zinc supplementation at any zinc blood level. (AIDS Rev. 2020;22:1-10) This aspect is added on the discussion and reference at bibliography. Point 2.4: “another problem is that it is not clear whether the data was analyzed with intent to treat analyses” R: the data was analyzed with intent to treat. This item is reinforced on the Methods. Point 2.5” It also does not appear that the analyses controlled for baseline CD4 cell count which was significantly different between the groups. There is no table showing differences between the treatment and the placebo groups at the end of the study among all participants that started the study, only those with incomplete immune recovery” R: we added on table 1 the immune recovery at the end of the study of IVD and control patients showing no differences. This aspect was reinforced on the discussion. Thank you for your consideration of this manuscript. Kind regards, Macarena Silva, MD Infectious Diseases Hospital San Borja Arriarán University of Chile School of Medicine Santiago, Chile Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0244823.r002
17 Dec 2020 Decision Letter - Giuseppe Vittorio De Socio, Editor Role and Effects of Zinc Supplementation in HIV-infected Patients with Immunovirological Discordance: A Randomized, Double Blind, Case Control Study PONE-D-20-19537R1 Dear Dr. Silva, 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, Giuseppe Vittorio De Socio, MD, PhD 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 ******** 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 ****** 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 ****** 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 ****** 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: (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 #1: Yes:** Giulia Marchetti https://doi.org/10.1371/journal.pone.0244823.r003
Formally Accepted
11 Jan 2021 Acceptance Letter - Giuseppe Vittorio De Socio, Editor PONE-D-20-19537R1 Role and effects of zinc supplementation in HIV-infected patients with immunovirological discordance: A randomized, double blind, case control study. Dear Dr. Silva: 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. Giuseppe Vittorio De Socio Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244823.r004

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