The use of holding regimens for people living with HIV (PLWH) without effective antiretroviral options can have effects on outcomes and future treatment options. We aimed to investigate the use of holding regimens for PLWH in Asian countries. Data from adults enrolled in routine HIV care in IeDEA Asia-Pacific cohorts were included. Individuals were considered to be on holding regimen if they had been on combination antiretroviral therapy for at least 6 months, had two confirmed viral loads (VL) ≥1000 copies/mL, and had remained on the same medications for at least 6 months. Survival time was analyzed using Fine and Gray’s competing risk regression. Factors associated with CD4 changes and VL <1000 copies/mL were analyzed using linear regression and logistic regression, respectively. A total of 425 PLWH  met criteria for being on a holding regimen. From high-income countries, 63.0% were on protease inhibitors (PIs); from low-to-middle-income countries, 58.4% were on non-nucleoside reverse transcriptase inhibitors (NNRTIs); overall, 4.5% were on integrase inhibitors. The combination of lamivudine, zidovudine, and efavirenz was the most commonly used single regimen , followed by lamivudine, zidovudine, and nevirapine . Forty-one PLWH (9.7%) died during follow-up . Age >50 years compared to age 31–40 years , and VL ≥1000 copies/ml compared to VL <1000 copies/mL  were associated with increased mortality, while higher CD4 counts were protective. In our Asia regional cohort, there was a diversity of holding regimens, and the patterns of PI vs. NNRTI use differed by country income levels. Considering the high mortality rate of PLWH with holding regimen, efforts to extend accessibility to additional antiretroviral options are needed in our region. Combination antiretroviral therapy (cART) suppresses HIV replication, prevents opportunistic infections, and allows people living with HIV (PLWH) to live longer . BecauseFor PLWH with no available effective treatment options, guidelines consider continuing treatment to avoid clinical deterioration , 8. PrevWhen no fully suppressive cART options are available, providers may choose to maintain patients on “holding regimens” in anticipation of future treatments . The useThis study included data from the TREAT Asia HIV Observational Database (TAHOD) and TAHOD-Low Intensity TransfEr (TAHOD-LITE). TAHOD is a prospective observational cohort study involving 21 participating clinical sites in 12 countries in the Asia and Pacific region, which is a contributing cohort to the International Epidemiology Databases to Evaluate AIDS (IeDEA) global cohort consortium. TAHOD-LITE collects a simplified dataset from all PLWH who have received care at participating 10 sites in Cambodia, Hong Kong SAR, India, Indonesia, Singapore, South Korea, Taiwan, and Vietnam, contributing data from over 49,000 PLWH. A detailed description of the TAHOD and TAHOD-LITE databases and methods has been previously published –17. EthiTAHOD subjects were included if they were enrolled up to the September 2018 data transfer, whilst TAHOD-LITE subjects were included from the 2017 data transfer. For TAHOD, enrolment was the date the patient was recruited into the cohort which can be after ART initiation. For TAHOD-LITE, enrolment was the date the patient was first seen at the site. The latest follow-up date for TAHOD-LITE was July 2017. We defined PLWH meeting all of the following criteria as taking holding regimens: 1) have initiated cART and been on it for at least 6 months; 2) had virologic failure defined as viral load (VL) ≥1000 copies/mL on two consecutive tests performed within 6 months while on the same regimen  33 to 46), and 72.9% of subjects were male. Heterosexual contact was the primary route of HIV exposure (69.4%). Median baseline VL was 12,300 copies/mL , and median baseline CD4 counts were 240 cells/μL (IQR 137 to 389). Majority of subjects were from HICs (45.2%), followed by LMICs (37.9%). There were 211 (49.7%) patients who had no previous ART regimen changes, i.e. currently failing first-line ART. Overall, the combination of nucleoside reverse transcriptase inhibitor (NRTI) + NNRTI and the combination of NRTI + PI were used almost equally as holding regimens  .The combination of 3TC, zidovudine (AZT), and efavirenz (EFV) was the most commonly used single regimen , followed by 3TC + AZT + nevirapine (NVP) , 3TC + tenofovir disoproxil fumarate (TDF) + atazanavir/ritonavir (ATV/r) , and 3TC + TDF + EFV . Combinations other than NRTI + NNRTI and NRTI + PI accounted for 11.3% of the total, with 19 PLWH (4.5%) using INSTIs , of which a regimen consisted of 3TC + TDF + ATV/r was the most popular regimen . In LMICs and UMICs, NNRTI-based regimens were mainly used (58.4%). The combination of 3TC + AZT + NVP was the most popular regimen in LMICs , followed by 3TC + AZT + EFV . In UMICs, 3TC + AZT + EFV was the most commonly used , followed by didanosine + D4T + EFV  .In cases where PLWH were left on failing first regimens, 3TC + AZT + EFV was the most commonly used , followed by 3TC + AZT + NVP . Among those who had previously changed their regimens , 3TC + TDF + ATV/r was the most commonly used regimen in both patients who changed once  and who changed more than once before  deaths , and 80 (18.8%) were LTFU. The median follow-up time from baseline was 3.1 years (IQR 1.4–6.8). Out of 41 deaths, 21 were AIDS-related, 14 were non-AIDS-related, and 6 were deaths by unknown causes. Univariate analysis showed that baseline age (p = 0.026), VL (p = 0.001), and CD4 count (p <0.001) were associated with mortality. In multivariate analysis, factors associated with increased mortality, whilst adjusting for country-income, were ages >50 years compared to ages 31 to 40 years , and VL ≥1000 copies/ml compared to VL <1000 copies/mL . On the other hand, higher CD4 count had protective effects against mortality. The SHR for mortality of CD4 count 351–500 and CD4 >500 compared to CD4 count ≤200 cells/μL were 0.20  and 0.25 , respectively . AdditioSubgroup analysis was performed on PLWH with available follow-up CD4 cell count and VL results. Twenty-eight PLWH were included in the 24 week CD4 cell count analysis. On average, the CD4 count increased by 25.1 cells/μL at 24 weeks after baseline. Multivariate analysis showed that ages 41 to 50 years were associated with decrease in CD4 count compared to ages 31 to 40 years , while PI-based holding regimen was associated with increase in CD4 count compared to NNRTI-based holding regimen  . For VL HIV management has been shifting from prevention of opportunistic infections to managing HIV as a chronic disease with a focus on addressing non-communicable diseases and improving quality of life. However, this paradigm shift is only possible when PLWH are on virally suppressive cART regimens, which may not be achievable when there is drug resistance or limited access to the antiretroviral drug options. This has led to the reliance on holding regimens in anticipation of future treatments. We found that 3TC + AZT + EFV was the most commonly used holding regimen in our Asia regional cohort, followed by 3TC + AZT + NVP.The diverse patterns of holding regimen in our region is likely be due to differential cART access, national treatment guidelines, and health insurance options for PLWH in each country. For example, in Thailand, one of the UMICs, the 2014 national guidelines recommended EFV, one of the NNRTIs, as the first-line combination, and another NNRTI, NVP was recommended as an alternative drug. PIs were only recommended in second-line combinations and INSTIs in multi-class failure . Dutta eThe mortality rate of PLWH with holding regimens was more than twice that of other PLWH in our cohort reported in previous study . This waThis study had several limitations. First, measurement of VL in TAHOD and TAHOD-LITE sites normally performed once a year, so we have small sample sizes due to our definition of 2 consecutive VL failure within 6 months. Second, TAHOD has patients who have been enrolled for a long time, so the data may reflect holding regimen in previous years. Since the median year of enrolment into the cohort was 2007 (IQR 2004–2010) and as INSTIs have not been widely available across all TAHOD sites, we could not identify the changes in patterns of holding regimen after INSTIs became widely available. Follow-up studies are warranted in this regard. Third, TAHOD-LITE does not collect ART adherence and therefore, we were not able to assess the impact of adherence counselling on our outcomes. Holding regimens are also sometimes used when adherence to ART is judged to be sub-optimal, especially for children who have suspected or proven poor adherence , 33. HowThe patterns of holding regimens used in our cohort differed by country income levels. In LMICs and UMICs, NNRTI-based regimens including 3TC + AZT + EFV were most commonly used, but PI-based regimens were commonly used in HICs. The mortality of PLWH with holding regimens was higher than that of other PLWH in our cohort; old age, high VL, and low CD4 cell counts were associated with high mortality. Considering the high mortality rate of PLWH with holding regimen, efforts to extend accessibility to additional antiretroviral options are needed in our region.S1 Table3TC, lamivudine; ABC, abacavir; ATV, atazanavir; AZT, zidovudine; COB, cobicistat; D4T, stavudine; DDC, zalcitabine; DDI, didanosine; DRV, darunavir; EFV, efavirenz; ETV, etravirine; EVG, elvitegravir; FAP, fosamprenavir; FTC, emtricitabine; HYD, hydroxyurea; IDV, indinavir; IL2, interleukin-2; LPV, lopinavir; MVC, maraviroc; NFV, nelfinavir; NNRTI, non-nucleoside reverse-transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; NVP, nevirapine; PI, protease inhibitor; RAL, raltegravir; RCT, randomized controlled trial; RIT, ritonavir; RPV, rilpivirine; RTF, ritonavir full dose; SQF, saquinavir fortovase; SQI, saquinavir invirase; TDF, tenofovir disoproxil fumarate.(DOCX)Click here for additional data file.S2 TableNote: ART combinations comprising of less than 5% were grouped as Other. 3TC, lamivudine; ATV/r, atazanavir/ritonavir; AZT, zidovudine; D4T, stavudine; EFV, efavirenz; FTC, emtricitabine; IDV, indinavir; LPV/r, lopinavir/ritonavir; NVP, nevirapine; TDF, tenofovir disoproxil fumarate.(DOCX)Click here for additional data file. 20 Apr 2021PONE-D-20-13430Patterns and prognosis of holding regimens for people living with HIV in Asian countriesPLOS ONEDear Dr. Choi,Thank you for submitting your manuscript to PLOS ONE. 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Read more information on sharing protocols at   https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols  .  We look forward to receiving your revised manuscript.Kind regards,Francisco Javier FanjulAcademic EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfThank you for including your ethics statement:  "Ethics approvals for the study were obtained from local institutional review boards for each participating site, the coordinating center , and the data management and analysis center . Because of the pure observational nature of the study, written informed consent was waived for both TAHOD and TAHOD-LITE unless required by local institutional review board. All TAHOD and TAHOD-LITE data transfers are anonymized before submission to the Kirby Institute.".  Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.For additional information about PLOS ONE ethical requirements for human subjects research, please refer to Additional Editor Comments:Dear authors, Let me first thank you for the opportunity of reviewing your study. I consider that it addresses a very interesting point where no clear evidence has been gathered in the last few years.However, some points in the paper need to be reconsidered according to reviewers so I hope you can have those suggestions in mind and provide an updated version of the manuscript.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. 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: PartlyReviewer #2: Partly**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: No**********  3. Have the authors made all data underlying the findings in their manuscript fully available?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.  The Reviewer #1: YesReviewer #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: YesReviewer #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. Reviewer #1: Kim and colleagues set out to address a very important question in regards to managing patients with limited HIV treatment options in an area of the world highly impacted by the HIV pandemic and with limited access to antiretrovirals compared to higher income world regions.Their analysis includes many Countries in the region with a variety of incomes, providing breath and generalizability of their findings.I think their report is interesting and merit publication, however there are some issues that might need to be addressed to improve their message.Major comments1. The authors included a cohort where almost half of patients did not have any previous antiretroviral regimen change. Are these patients who are “just” taking a long time to fully respond to treatment as opposed patients truly failing their regimen? From Figure 3 it seems that the proportion of patients with viral load<1000 copies/ml increases over time. Even though it would decrease the overall number of patients in the analysis, would the result show similar findings if only considering patients who had previous ARV changes?a. More to this point, in the viral load subgroup analysis about 33% of patients had undetectable viral load at week 24. What is the distribution of these patients according to previous ARV changes? Are they all or mostly in the “no previous ARV” group or were they equally distributed?2. One of the main outcomes that the authors are studying is survival and they specify that “Because all included individuals were alive at 6 months after their second VL test (per the definition of being on a holding regimen), survival time was left truncated at 6 months after the second VL test”. Nonetheless it is not clear what the average follow-up for these cohort was and how many patients were lost to follow-up. It would be important that the authors make it clear, so that the reader know whether these findings have implication in the short, middle or long term.3. Somewhat related to the previous point, the subgroup analysis for CD4 count and VL only include 24 and 33 patients respectively at week 24. Are the other patients lost to follow-up? If they are not considered as such, how did the authors account for these missing data and what is their definition of lost to follow-up since not having either CD4 or viral load checked at least once within a year, might be considered a fairly standard definition of “lost to follow-up”.a. In addition, how many patients had CD4 and VL data for week 48? And in terms of VL, how many with VL<200 and <50 at each timepoint?4. Two major point that would strengthen the message on survival and the consequences of holding regimens would be showing more details regarding the cause of death and development of resistance mutations. I understand that this data might not be available, if that is the case, the authors should make a comment about it in the discussion.a. In regards to the cause of death, the increased mortality odds that the authors find for higher viral load and lower CD4 count, would imply that the observed deaths are HIV/AIDS related, however with no actual data this remain only speculative.b. In regards to the development of resistance mutation for patients on holding regimens, this data would be extremely important to help make clinical decision  on the importance and optimal timing of modifying failing regimen. As the authors mention in the discussion, the alternative explanation to the persistently elevated viral load in this cohort, would be related to adherence which would obviously need different interventions to be addressed.Minor commentsMethods1. The authors mention that their primary outcome for the study is “to identify the patterns of the holding regimen as previously defined” It is however not clear what the authors mean by “patterns” and where these were previously defined2. Hep C co-infection is defined as positive HCV Antibodies. With no information about HCV RNA or Hep C treatment however, it might be more appropriate to define this variable as “previous Hep C exposure”Results1. Related to major point #3, Figures 2 and 3 need to have the #of patients at each timepoint listed underneathReviewer #2: Many thanks for the opportunity to review this paper on the use of holding regimens in Asia. While it is an interesting and relevant topic, the authors have not used appropriate methods for all their analyses, some methods are not clear, and they should include calendar year of ART start (or other similar covariate) to account for fewer drug options in the earlier years. A more nuanced discussion of the reasons why some patients are maintained on regimens despite virologic failure is needed. The authors only mention lack of access to other ART options, but in reality, it is possible that patients are not switched due to poor adherence, or the high cost of more robust drugs. This warrants further discussion.Introduction:The authors state that holding regimens are used when no fully suppressive cART options are available. Holding regimens are also sometimes used when adherence to ART is judged to be sub-optimal, and clinicians would like to ensure adherence before switching to a new regimen. IT is worth including whether this is the case or not in Asian countries?Some regimens are also more robust, and patients who have had virological failure may still be able to suppress on these regimens. For example resistance to NNTRIs develops much quicker than resistance to PI-based cART. The reason for maintaining a patient on a regimen following virologic failure may therefore be related to their current regimen, their adherence and the availability of a new regimen. These points should be included.Methods:Study population – please clarify what is meant by enrolment – is this ART initiation, from HIV diagnosis, or something different? Please also include details of the years in which patient follow-up began, to give a sense of how long patients that were included in your study were followed. This may also be relevant to understand as the availability of new drugs may have changed and possibly improved in more recent years. Please also clarify database closure for the TAHOD-LITE cohort. It is not clear when the most recent 2017 data transfer provides data until.The definition of those on holding regimens seems to only include patients who have been maintained on the same cART regimen, and would thus exclude patients on other holding regimens such as lamivudine monotherapy (as mentioned in the intro). Were patients on non-suppressive holding regimens  excluded or was this strategy not used in Asia among adults?Statistical analyses – how was switch from holding regimen to a new cART regimen dealt with in the survival analysis? Or did all patients remain on their holding regimens until their last recorded visit or death?Suggest adding calendar year of ART initiation as a covariate – since some drugs such as INSTIs would only have become available in more recent yearsOne limitation of a linear regression approach to change in CD4 count, is that patients with very low CD4 counts would likely experience only small declines, but possibly larger increases in CD4 compared to those with higher CD4 counts whose CD4 counts could drop by quite a lot, but could not increase by much. The repeated CD4 measures for each patient are therefore not independent. Would recommend using a linear mixed-effects regression model to analyse CD4 changes.Results:Descriptive table – include median ART duration at baseline, as well as median follow-up time from enrolment.Survival Analysis – how many patients were LTFU . Suggest adding an outcomes table to indicate how many died, were loss to follow-up, switched to a new cART regimen, virally suppressed on holding regimen or on new regimen.Discussion:Could the pattern of holding regimens used by different regions be related to calendar year of ART start? For example do some regions include patients who initiated cART in earlier years than others, when drugs such as INSTIs were not available? This is implied in the limitations section where the authors state that TAHOD patients have been enrolled for longer.Line 272 – the authors imply that the high mortality rate amongst patients on holding regimens is due to lack of access to further ART options. Patients who have failed treatment are likely to be a more vulnerable group and poor outcomes including mortality could be due to a number of factors including poor adherence.The discussion could be strengthened by comparing results to studies of patients who have failed to switch regimens, such asBell-Gorrod H, et al. The Impact of Delayed Switch to Second-Line Antiretroviral Therapy on Mortality, Depending on Definition of Failure Time and CD4 Count at Failure. Am J Epidemiol. 2020 Aug 1;189(8):811-819. doi: 10.1093/aje/kwaa049. PMID: 32219384; PMCID: PMC7523585.Bell Gorrod H, Court R, Schomaker M, Maartens G, Murphy RA. Increased Mortality With Delayed and Missed Switch to Second-Line Antiretroviral Therapy in South Africa. J Acquir Immune Defic Syndr. 2020 May 1;84(1):107-113. doi: 10.1097/QAI.0000000000002313. PMID: 32032304; PMCID: PMC7269121.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  2 Jun 2021Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).Author’s responseAccording to your comments, we amended the ethics statement. All participating sites, including our site, have their own ethics approval. However, because it is too long to list the name of all 21 participating sites’ ethics committees, we have revised the ethics statement as follows:Lines: 106-110Ethics approvals for the study were obtained from the coordinating center , the data management and analysis center , and local institutional review boards for each participating site including the institutional review board of Yonsei University Health System Clinical Trial Center. ________________________________________Review Comments to the AuthorReviewer #1 Major commentsReviewer’s comment #11. The authors included a cohort where almost half of patients did not have any previous antiretroviral regimen change. Are these patients who are “just” taking a long time to fully respond to treatment as opposed patients truly failing their regimen? From Figure 3 it seems that the proportion of patients with viral load<1000 copies/ml increases over time. Even though it would decrease the overall number of patients in the analysis, would the result show similar findings if only considering patients who had previous ARV changes?Author’s response #1The patients who did not have any previous ART changes are the ones who are currently on first-line ART. So those patients are currently failing their first-line ART combination.Although Figure 3 shows increases in VL <1000 copies/mL over time, it is important to note that not all patients had VL at both week 24 and 48. In fact, due to the small sample size, no patient in our study population had both week 24 and 48 VL measurements available. Patients either had VL at week 24 or at week 48. However, there does appear to be an increase in proportion with undetectable VL over time. This could be due to adherence support provided by healthcare providers during the holding regimen period. We were not able to adjust for adherence as TAHOD-LITE does not collect this information. We have included this as a limitation of the study. As suggested, we have also conducted additional analyses limiting to those who had previous ART regimen changes. For the survival analysis, there were 22 deaths out of 222 patients, with mortality rate of 2.2/100PYS. Only VL was associated with survival . For the CD4 analysis, there were only 14 patients eligible for the analysis and no factor was associated with CD4 changes in the multivariate analysis. In the VL analysis, there were 6/19 with undetectable VL. Because of the small sample sizes, there was not enough evidence to suggest associations between different variables, however the direction of the associations was similar to our main analyses. We have chosen to maintain patients with no ART changes in our analyses to optimize our sample sizes.We have also made further clarifications to the manuscript as follows:Lines: 179-180“There were 211 (49.7%) patients who had no previous ART regimen changes, i.e. currently failing first-line ART.”Lines: 258-259“In Figure 2, there were 28 patients included at 24 weeks and 38 patients at 48 weeks. In Figure 3, of the total 66 patients, 33 patients each were included at 24 and 48 week time points.”Lines: 328-329“TAHOD-LITE does not collect ART adherence and therefore, we were not able to assess the impact of adherence counselling on our outcomes”Reviewer’s comment #1aa. More to this point, in the viral load subgroup analysis about 33% of patients had undetectable viral load at week 24. What is the distribution of these patients according to previous ARV changes? Are they all or mostly in the “no previous ARV” group or were they equally distributed?Author’s response #1aThe distribution of undetectable VL according to previous ART regimen changes is roughly equal across groups:No previous ART regimen changes: 5/14 (36%)1 previous ART regimen changes: 5/12 (42%)≥2 previous ART regimen changes: 1/7 (14%)We have inserted the following in the manuscript:Lines: 251-253“The proportion with undetectable VL according to the number of previous ART regimen changes were: no previous ART regimen changes - 5/14 (36%); 1 previous ART changes - 5/12 (42%); and ≥2 previous ART changes - 1/7 (14%).”Reviewer’s comment #22. One of the main outcomes that the authors are studying is survival and they specify that “Because all included individuals were alive at 6 months after their second VL test (per the definition of being on a holding regimen), survival time was left truncated at 6 months after the second VL test”. Nonetheless it is not clear what the average follow-up for these cohort was and how many patients were lost to follow-up. It would be important that the authors make it clear, so that the reader know whether these findings have implication in the short, middle or long term.Author’s response #2The median follow-up was 3.1 years (IQR 1.4-6.8). The number of LTFU was 80/425 (18.8%). We have revised the manuscript as follows:Lines: 219-221“Of the 425 patients included in the survival analysis, there were 41 (9.7%) deaths , and 80 (18.8%) were LTFU. The median follow-up time from baseline was 3.1 years (IQR 1.4-6.8).”Reviewer’s comment #33. Somewhat related to the previous point, the subgroup analysis for CD4 count and VL only include 24 and 33 patients respectively at week 24. Are the other patients lost to follow-up? If they are not considered as such, how did the authors account for these missing data and what is their definition of lost to follow-up since not having either CD4 or viral load checked at least once within a year, might be considered a fairly standard definition of “lost to follow-up”.Author’s response #3Our cohort’s definition of LTFU is those who have not been seen in the previous 12 months. For the CD4 and VL analyses, we did not include all patients that were analysed in the survival analysis as our time frame was only restricted to 24 and 48 weeks. According to our inclusion criteria, we only included patients who had CD4 or VL available at 24 weeks or 48 weeks. In the CD4 analysis, 28 patients were included in the 24 week analysis, and 38 patients were included in the 48 week analysis. From the rest of the patients who were not included, only 59 later became LTFU. We did not attempt to include patients without the outcome of interest at 24 and 48 weeks, whether or not they were LTFU, as we did not want to make an assumption of what their CD4 or VL would be. As such, we only restricted our analyses to those who had the outcomes measured at those two time points. We have made further clarifications in the manuscript as follows:Lines: 144-145“PLWH without measurements at these two time points were not included in the analyses.”Lines: 155-156“LTFU was defined as those not seen in the previous 12 months.”Lines: 258-259“In Figure 2, there were 28 patients included at 24 weeks and 38 patients at 48 weeks. In Figure 3, of the total 66 patients, 33 patients each were included at 24 and 48 week time points.”Reviewer’s comment #3aa. In addition, how many patients had CD4 and VL data for week 48? And in terms of VL, how many with VL<200 and <50 at each timepoint?Author’s response #3aThere were 38 patients who had CD4 at 48 weeks.For VL, at 24 weeks 7 patients had VL<200 copies/mL, 6 patients had VL<50 copies/mL. At 48 weeks, 11 patients had VL <200 copies/mL, 8 patients had VL <50 copies/mL. We have included this information in the manuscript as follows:Lines: 260-263“At 24 weeks, there were 11 patients with VL <1000 copies/mL, of those 7 patients had VL<200 copies/mL and 6 patients had VL<50 copies/mL. At 48 weeks, there were 15 patients with VL <1000 copies/mL, of those 11 patients had VL <200 copies/mL and 8 patients had VL <50 copies/mL.”Reviewer’s comment #4 and #4a4. Two major point that would strengthen the message on survival and the consequences of holding regimens would be showing more details regarding the cause of death and development of resistance mutations. I understand that this data might not be available, if that is the case, the authors should make a comment about it in the discussion.a. In regards to the cause of death, the increased mortality odds that the authors find for higher viral load and lower CD4 count, would imply that the observed deaths are HIV/AIDS related, however with no actual data this remain only speculative.Author’s response #4 and #4aThank you for your great suggestions. We investigated the cause of death of 41 patients who died in our study population. Out of 41 total deaths, 21 were AIDS-related, 14 were non-AIDS-related, and 6 were deaths by unknown causes. Unlike our results, a prior study on the causes of death in TAHOD patients showed that non-AIDS-related mortality rates were higher than AIDS-related mortality rates in both high/upper-middle income countries and lower-middle income countries [1]. This shows that patients with holding regimens have a higher risk of AIDS-related death than those who do not. The finding was also consistent with the results of this study, where higher viral load and lower CD4 count were identified as risk factors for death. We have included this information in the manuscript as follows:Lines: 221-222Of the 425 patients included in the survival analysis, there were 41 (9.7%) deaths , and 80 (18.8%) were LTFU. The median follow-up time from baseline was 3.1 years (IQR 1.4-6.8). Out of 41 deaths, 21 were AIDS-related, 14 were non-AIDS-related, and 6 were deaths by unknown causes.Lines: 307-314Still, there are cases of virologic failure even though a second-line or third-line regimen was already used with no other antiretroviral options available, as in this study. In particular, AIDS-related deaths were more common than non-AIDS-related deaths in this study, which differed from the previous results regarding the cause of death in TAHOD patients, where non-AIDS-related mortality rate was higher than AIDS-related mortality rate [32]. This fact demonstrates the need to extend accessibility to additional antiretroviral options in our region to improve their survival. Regarding factors associated with mortality, it was consistent with what is previously known that old age, high VL, and low CD4 cell counts increased mortality and supported our results that AIDS-related deaths were more common than non-AIDS-related deaths .Reviewer’s comment #4bb. In regards to the development of resistance mutation for patients on holding regimens, this data would be extremely important to help make clinical decision  on the importance and optimal timing of modifying failing regimen. As the authors mention in the discussion, the alternative explanation to the persistently elevated viral load in this cohort, would be related to adherence which would obviously need different interventions to be addressed.Author’s response #4bWe agree with your comment that resistance mutation data is important in determining the timing of failing regimen modifications. Unfortunately, data on resistance mutations in our study subjects were not available. Therefore, there might be concerns that the persistently elevated viral load in this cohort would be related to poor adherence. However, as TAHOD participating sites are generally urban referral centres, and each site recruits patients who are judged to have a reasonably good prospect of long-term follow-up, it is unlikely that holding regimens were used to ensure adherence in this study [2]. Meanwhile, data on resistance mutations in TAHOD patients could be identified by the results of previous studies of TASER-M . According to the research results, transmitted drug resistance was identified in 4.1% ; 60% of which had NRTI, 43% NNRTI, and 18% PI-associated mutations [3]. The most common NRTI-associated mutation was M184V, the common NNRTI-associated mutation was K103N or Y181C, and the common PI-associated mutation was M46L . The results could be referred to in our study. Nevertheless, the inability to confirm the resistance mutation of this study population was a limitation. Therefore, we have added the following to the limitations.Lines: 334-340Fourth, data on resistance mutations in our study population were not available. In previous studies on resistance mutations in TAHOD patients, transmitted drug resistance was identified in 4.1%; 60% of which had NRTI, 43% NNRTI, and 18% PI-associated mutations [39]. The most common NRTI-associated mutation was M184V, the common NNRTI-associated mutation was K103N or Y181C, and the common PI-associated mutation was M46L . The results could be referred to in our study.Minor commentsMethodsReviewer’s minor comment #11. The authors mention that their primary outcome for the study is “to identify the patterns of the holding regimen as previously defined” It is however not clear what the authors mean by “patterns” and where these were previously definedAuthor’s response #1We clarified the sentence as follows:Lines: 136-137The primary outcome was identifying which diverse regimens were used as holding regimens in our study population.Reviewer’s minor comment #22. Hep C co-infection is defined as positive HCV Antibodies. With no information about HCV RNA or Hep C treatment however, it might be more appropriate to define this variable as “previous Hep C exposure”Author’s response #2We acknowledge that we do not have information on HCV RNA or treatment. However, we have defined Hep C co-infection as those who ever had positive hepatitis C antibody. We do not collect all hepatitis C testing dates, therefore we are not able to determine whether the HCV co-infection was a previous exposure. We have clarified this further in the manuscript as follows:Lines: 158-159“hepatitis B/C co-infection defined as ever having positive hepatitis B virus surface antigen or positive hepatitis C virus antibody”Reviewer’s minor comment results #1Results1. Related to major point #3, Figures 2 and 3 need to have the #of patients at each timepoint listed underneath.Author’s response #1We have inserted the patient numbers into the graphs. Please see revised Figures 2 and 3. Reviewer #2Reviewer’s comment #1Many thanks for the opportunity to review this paper on the use of holding regimens in Asia. While it is an interesting and relevant topic, the authors have not used appropriate methods for all their analyses, some methods are not clear, and they should include calendar year of ART start (or other similar covariate) to account for fewer drug options in the earlier years. A more nuanced discussion of the reasons why some patients are maintained on regimens despite virologic failure is needed. The authors only mention lack of access to other ART options, but in reality, it is possible that patients are not switched due to poor adherence, or the high cost of more robust drugs. This warrants further discussion.Author’s response #1We would like to thank the reviewer for the suggestion of including year of ART initiation. We have included this in our analysis.In terms of the statistical methods, please see below our responses and clarifications under the statistical analysis section. In particular, we have provided further explanation of our chosen methods of statistical analyses and why we believe these are suitable for our datasets and objectives.We also agree with the comments that there is a need for a more nuanced discussion on why PLWH maintained regimens that did not achieve viral suppression. As described in the Study Population section, we defined holding regimens as those continued for more than 6 months in the same regimen after viral load (VL) ≥1000 copies/mL on two consecutive tests. The basis for this definition was that, in those situations, the regimens would be changed if there were regimens accessible that could achieve viral suppression. Meanwhile, holding regimens are also sometimes used when adherence to ART is judged to be sub-optimal, especially for children who have suspected or proven poor adherence [5]. However, TAHOD is for adult PLWH, and as TAHOD participating sites are generally urban referral centres, and each site recruits patients who are judged to have a reasonably good prospect of long-term follow-up, it is unlikely that holding regimens were used to ensure adherence in this study [2]. How ART adherence affected the results would help strengthen our study, but unfortunately, ART adherence was not collected in TAHOD-LITE. Since this was a limitation of our study, we additionally described it in the Discussion section. There were also cases where regimen changes could not be made due to the cost of more robust drugs. The reason was of course included as one of the causes of the lack of access to ART described in this study, especially for the resource-limited countries of our cohort. We have included this information in the manuscript as follows:Lines: 289-290For example, in Thailand, one of the UMICs, the 2014 national guidelines recommended EFV, one of the NNRTIs, as the first-line combination, and another NNRTI, NVP was recommended as an alternative drug. PIs were only recommended in second-line combinations and INSTIs in multi-class failure [24]. One reason for this is because many resource-limited countries, such as Thailand, could not financially afford the subsequent regimens [25].Lines: 329-334Third, TAHOD-LITE does not collect ART adherence and therefore, we were not able to assess the impact of adherence counselling on our outcomes. Holding regimens are also sometimes used when adherence to ART is judged to be sub-optimal, especially for children who have suspected or proven poor adherence . However, TAHOD is for adult PLWH, and as TAHOD participating sites are generally urban referral centres, and each site recruits patients who are judged to have a reasonably good prospect of long-term follow-up, it is unlikely that holding regimens were used to ensure adherence in this study [38].Introduction:Reviewer’s comment #2The authors state that holding regimens are used when no fully suppressive cART options are available. Holding regimens are also sometimes used when adherence to ART is judged to be sub-optimal, and clinicians would like to ensure adherence before switching to a new regimen. IT is worth including whether this is the case or not in Asian countries?Author’s response #2As you commented, holding regimens could be used when adherence to ART is judged to be sub-optimal. Holding regimens for this purpose are mainly used in children who have suspected or proven poor adherence [5]. In a study conducted in Southeast Asian countries, holding regimens were used in 3 out of 93 patients who were transferred from a pediatric to an adult clinic [6]. However, TAHOD is for adult PLWH, and as TAHOD participating sites are generally urban referral centres, and each site recruits patients who are judged to have a reasonably good prospect of long-term follow-up, it is unlikely that holding regimens were used to improve adherence in this study [2].We have added the above information to the Discussion section as follows:Lines: 329-334Third, TAHOD-LITE does not collect ART adherence and therefore, we were not able to assess the impact of adherence counselling on our outcomes. Holding regimens are also sometimes used when adherence to ART is judged to be sub-optimal, especially for children who have suspected or proven poor adherence . However, TAHOD is for adult PLWH, and as TAHOD participating sites are generally urban referral centres, and each site recruits patients who are judged to have a reasonably good prospect of long-term follow-up, it is unlikely that holding regimens were used to ensure adherence in this study [38].Reviewer’s comment #3Some regimens are also more robust, and patients who have had virological failure may still be able to suppress on these regimens. For example resistance to NNTRIs develops much quicker than resistance to PI-based cART. The reason for maintaining a patient on a regimen following virologic failure may therefore be related to their current regimen, their adherence and the availability of a new regimen. These points should be included.Author’s response #3We agree with your comments. We have added the following to the Introduction section.Lines: 82-86There are no universal criteria for treating PLWH who have experienced multiple treatment failures, because treatment options differ according to the available cART of the countries and likelihood of resistance. For example, patients who develop resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) could achieve viral suppression by changing to a protease inhibitor (PI)-based or an INSTI-based regimen . Still, in some cases, since access to the drugs was limited due to problems such as cost and availability, they should maintain their regimens [14].Methods:Reviewer’s comment #4Study population – please clarify what is meant by enrolment – is this ART initiation, from HIV diagnosis, or something different? Please also include details of the years in which patient follow-up began, to give a sense of how long patients that were included in your study were followed. This may also be relevant to understand as the availability of new drugs may have changed and possibly improved in more recent years. Please also clarify database closure for the TAHOD-LITE cohort. It is not clear when the most recent 2017 data transfer provides data until.Author’s response #4For TAHOD, enrolment means when patients were recruited and enrolled into the cohort. For TAHOD-LITE, enrolment means the first date the patient was seen at the site. It is not necessarily their date of ART initiation, as the majority of TAHOD patients had already initiated ART prior to enrolment into the cohort. The median year of enrolment into the cohort was 2007 (IQR 2004-2010). The median time of follow-up from baseline time point of the study was 3.1 years (IQR 1.4-6.8). As there was only 1 data transfer for TAHOD-LITE in 2017, the database closure was any time in 2017. The latest visit date for this data transfer was July 2017. We have revised the sentence to make it clearer. We have inserted the following into the manuscript:Lines: 117-121“TAHOD subjects were included if they were enrolled up to the September 2018 data transfer, whilst TAHOD-LITE subjects were included from the 2017 data transfer. For TAHOD, enrolment was the date the patient was recruited into the cohort which can be after ART initiation. For TAHOD-LITE, enrolment was the date the patient was first seen at the site. The latest follow-up date for TAHOD-LITE was July 2017.Lines: 174-175“The median year of enrolment into the cohort was 2007 (IQR 2004-2010).”Lines: 220-221“The median follow-up time from baseline was 3.1 years (IQR 1.4-6.8).”Reviewer’s comment #5The definition of those on holding regimens seems to only include patients who have been maintained on the same cART regimen, and would thus exclude patients on other holding regimens such as lamivudine monotherapy (as mentioned in the intro). Were patients on non-suppressive holding regimens  excluded or was this strategy not used in Asia among adults?Author’s response #5We acknowledge that this was unclear. We did include mono/dual therapy in our holding regimen. For our inclusion criteria however, patients had to have at least initiated combination ART (3 or more ARVs) and been on it for least 6 months. The holding regimen itself can be mono/dual therapy.We have revised the manuscript as follows:Lines: 121-123“We defined PLWH meeting all of the following criteria as taking holding regimens: 1) have initiated cART and been on at least 6 months;”Lines: 125-126“The holding regimen itself can include mono/dual therapy.”Reviewer’s comment #6Statistical analyses – how was switch from holding regimen to a new cART regimen dealt with in the survival analysis? Or did all patients remain on their holding regimens until their last recorded visit or death?Author’s response #6We had not intended to assess the effects of ART switches in the survival analysis. This is because the aim was to determine the long term effects of the actual holding regimen that the patient received on their survival, rather than the effects of the current ART regimen. Our analysis was also conducted using intention to treat methods. To analyse ART regimen as a time updated variable, we would have to account for periods of no ART (treatment interruption) which is not within the intention to treat definition. However, we have conducted an additional analysis where we accounted for those who had treatment interruption of more than 14 days and those who had changed any ARV from the holding regimen, and found that there were 271 patients who switched from their holding regimen under this definition. Switches was not associated with survival in the univariate analysis .Lines: 156“Survival time was analysed using intention to treat methods.”Lines: 230-233“Additional analyses were conducted where we accounted for changes from holding regimen (any changes to the holding ART or periods of no ART for >14 days). There were 271 patients who had changed out of their holding regimen however it was not associated with survival in the univariate analysis .”Reviewer’s comment #7Suggest adding calendar year of ART initiation as a covariate – since some drugs such as INSTIs would only have become available in more recent yearsAuthor’s response #7We have included year of ART initiation in the analyses. Please see revised tables. Year of ART initiation was not associated with survival, CD4 changes or undetectable VL.Reviewer’s comment #8One limitation of a linear regression approach to change in CD4 count, is that patients with very low CD4 counts would likely experience only small declines, but possibly larger increases in CD4 compared to those with higher CD4 counts whose CD4 counts could drop by quite a lot, but could not increase by much. The repeated CD4 measures for each patient are therefore not independent. Would recommend using a linear mixed-effects regression model to analyse CD4 changes.Author’s response #8We would like to clarify the statistical methods used in the CD4 changes analysis. We took the difference between CD4 at 24 weeks and CD4 at baseline for each patient. We then analysed that difference as the outcome in the regression. Each patient therefore only had one single outcome measured. As the outcomes were not repeated measures, but a single outcome, independence was not an issue. Given that there were only 28 patients in the analysis, we believe the simple linear regression used was suitable for our data.Reviewer’s comment #9Results:Descriptive table – include median ART duration at baseline, as well as median follow-up time from enrolment.Author’s response #9The median ART duration at baseline was 4.0 years, IQR (1.8-6.9). The median follow-up time from baseline was 3.1 years, IQR (1.4-6.8). We have included this information into the demographics table. Please see revised Table 1.Reviewer’s comment #10Survival Analysis – how many patients were LTFU . Suggest adding an outcomes table to indicate how many died, were loss to follow-up, switched to a new cART regimen, virally suppressed on holding regimen or on new regimen.Author’s response #10There were 80 (18.8%) patients who became LTFU, 41 (9.7%) have died, 271 patients changed from their holding regimen. For the undetectable VL analysis, there were 11/33 patients (33%) who had VL <1000 copies/mL at 24 weeks, and 15/33 (45%) at 48 weeks. As the aim of the study was to determine the effects of the holding regimen on subsequent treatment outcomes, we have not split our undetectable VL outcomes according to treatment switches but have presented the proportions at 24 and 48 weeks. We have decided to add the requested information as text in the relevant results section to make the flow of the paper more coherent. Please see revised manuscript as follows:Lines: 219-221“Of the 425 patients included in the survival analysis, there were 41 (9.7%) deaths , and 80 (18.8%) were LTFU. The median follow-up time from baseline was 3.1 years (IQR 1.4-6.8).”Lines: 232“There were 271 patients who had changed out of their holding regimen”Lines: 260-263“At 24 weeks, there were 11 patients with VL <1000 copies/mL, of those 7 patients had VL<200 copies/mL and 6 patients had VL<50 copies/mL. At 48 weeks, there were 15 patients with Vl <1000 copies/mL, of those 11 patients had VL <200 copies/mL and 8 patients had VL <50 copies/mL.”Discussion:Reviewer’s comment #11Could the pattern of holding regimens used by different regions be related to calendar year of ART start? For example do some regions include patients who initiated cART in earlier years than others, when drugs such as INSTIs were not available? This is implied in the limitations section where the authors state that TAHOD patients have been enrolled for longer.Author’s response #11Since the median year of enrolment into the cohort was 2007 (IQR 2004-2010) and as INSTIs have not been widely available across all TAHOD sites, there were not many patients who were on INSTI in the holding regimen in our study group even if we were looking at holding regimens in more recent years. As shown in S1 Table, the cases in which INSTIs were used were limited in the subjects of this study (19 cases out of 425 cases). In this study, we could not identify the changes in patterns of holding regimen after INSTIs became widely available, and this should be investigated through follow-up studies. We have added the following to the Discussion section regarding these limitations and perspectives.Lines: 324-328Second, TAHOD has patients who have been enrolled for a long time, so the data may reflect holding regimen in previous years. Since the median year of enrolment into the cohort was 2007 (IQR 2004-2010) and as INSTIs have not been widely available across all TAHOD sites, we could not identify the changes in patterns of holding regimen after INSTIs became widely available. Follow-up studies are warranted in this regard.Reviewer’s comment #12Line 272 – the authors imply that the high mortality rate amongst patients on holding regimens is due to lack of access to further ART options. Patients who have failed treatment are likely to be a more vulnerable group and poor outcomes including mortality could be due to a number of factors including poor adherence.The discussion could be strengthened by comparing results to studies of patients who have failed to switch regimens, such asBell-Gorrod H, et al. The Impact of Delayed Switch to Second-Line Antiretroviral Therapy on Mortality, Depending on Definition of Failure Time and CD4 Count at Failure. Am J Epidemiol. 2020 Aug 1;189(8):811-819. doi: 10.1093/aje/kwaa049. PMID: 32219384; PMCID: PMC7523585.Bell Gorrod H, Court R, Schomaker M, Maartens G, Murphy RA. Increased Mortality With Delayed and Missed Switch to Second-Line Antiretroviral Therapy in South Africa. J Acquir Immune Defic Syndr. 2020 May 1;84(1):107-113. doi: 10.1097/QAI.0000000000002313. PMID: 32032304; PMCID: PMC7269121.Author’s response #12Thank you for introducing those valuable studies. We reviewed the papers you introduced and compared them with our findings. It was consistent with our findings that in the case of virologic failure, a remaining regimen resulted in a poor prognosis. Meanwhile, our study analyzed not only cases of virologic failure with the first-line regimen, but also cases that experienced virologic failure while using the second-line or third-line regimen. This is because our study population was defined as cases where no other antiretroviral option was currently available, regardless of the history of previous ART changes. Therefore, there were some differences in the populations of our study and the studies you introduced, and this is the basis for claiming the need for further ART options in our study. We have cited the papers you introduced and strengthened the discussion of our manuscript as follows:Lines: 301-307The mortality rate of PLWH with holding regimens was more than twice that of other PLWH in our cohort reported in previous study [32]. This was consistent with the results of previous studies that the prognosis of a remaining regimen or a delay in switching regimens was poor in patients with virologic failure . The reasons for no switch or a delayed switch could be multifactorial, such as prolonged time-period before confirmation of virologic failure or doubts about adequate patient adherence . Still, there are cases of virologic failure even though a second-line or third-line regimen is already used with no more other antiretroviral options available, as in this study.References1. Jung IY, Rupasinghe D, Woolley I, O'Connor CC, Giles M, Azwa RI, et al. Trends in mortality among ART-treated HIV-infected adults in the Asia-Pacific region between 1999 and 2017: results from the TREAT Asia HIV Observational Database (TAHOD) and Australian HIV Observational Database (AHOD) of IeDEA Asia-Pacific. J Int AIDS Soc. 2019;22: e25219. doi:10.1002/jia2.25219 PMID:306152712. Zhou J, Li P, Kumarasamy N, Boyd M, Chen Y, Sirisanthana T, et al. Deferred modification of antiretroviral regimen following documented treatment failure in Asia: results from the TREAT Asia HIV Observational Database (TAHOD). HIV medicine. 2010;11: 31-393. Phanuphak P, Sirivichayakul S, Jiamsakul A, Sungkanuparph S, Kumarasamy N, Lee MP, et al. Transmitted drug resistance and antiretroviral treatment outcomes in non-subtype B HIV1-infected patients in South East Asia. Journal of acquired immune deficiency syndromes (1999). 2014;66: 744. Jiamsakul A, Sungkanuparph S, Law M, Kantor R, Praparattanapan J, Li PC, et al. HIV multi‐drug resistance at first‐line antiretroviral failure and subsequent virological response in Asia. Journal of the International AIDS Society. 2014;17: 190535. Patten G, Bernheimer J, Fairlie L, Rabie H, Sawry S, Technau K, et al. Lamivudine monotherapy as a holding regimen for HIV-positive children. PloS one. 2018;13: e02054556. Sohn AH, Chokephaibulkit K, Lumbiganon P, Hansudewechakul R, Gani YM, Van Nguyen L, et al. Peritransition Outcomes of Southeast Asian Adolescents and Young Adults With HIV Transferring From Pediatric to Adult Care. Journal of Adolescent Health. 2020;66: 92-99AttachmentResponse_to_Reviewers_20210601.docxSubmitted filename: Click here for additional data file. 6 Dec 2021PONE-D-20-13430R1Patterns and prognosis of holding regimens for people living with HIV in Asian countriesPLOS ONEDear Dr. Choi,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.Still there are comments from reviewer #2 which need to be addressed .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 submit your revised manuscript by Jan 20 2022 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 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). 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments:Still there are comments from reviewer #2 which need to be addressed .[Note: HTML markup is below. Please do not edit.]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 addressedReviewer #2: (No Response)**********  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: YesReviewer #2: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?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.  The Reviewer #1: YesReviewer #2: 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: YesReviewer #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. Reviewer #1: Thank you for addressing my comments and adding additional information to your work. I believe this will be an informative paper for the field.Reviewer #2: Many thanks to the authors for addressing my original comments. I have two additional comments:The authors should consider amending the first paragraph of the discussion for better clarity on the holding regimens that were used. The authors note in the introduction that there are several types of holding regimens including lamivudine monotherapy, recycling previously used drugs or adding new ARVs to an existing regimen. It is not clear from the results or the discussion what was the case in this study, although it seems as if patients were simply maintained on their failing regimen.The authors have now added to the definition of a patient taking a holding regimen to include those on mono/dual therapy. Usually patients on these regimens do not have their viral load taken because it is not a virally suppressive regimen. Your definition of a holding regimen includes have 2 consecutive VL>1000 within 6 months on the same regimen. I recommend removing the mono/dual therapy from the definition of holding regimen, unless there were in fact patients who were placed on this regimen.**********  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: NoReviewer #2: Nohttps://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.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  17 Dec 2021Reviewer #2: Many thanks to the authors for addressing my original comments. I have two additional comments:Reviewer’s comment #1The authors should consider amending the first paragraph of the discussion for better clarity on the holding regimens that were used. The authors note in the introduction that there are several types of holding regimens including lamivudine monotherapy, recycling previously used drugs or adding new ARVs to an existing regimen. It is not clear from the results or the discussion what was the case in this study, although it seems as if patients were simply maintained on their failing regimen.Author’s response #1Thank you for your comments. We have defined holding regimen in the Methods section as follows: “We defined PLWH meeting all of the following criteria as taking holding regimens: 1) have initiated cART and been on it forat least 6 months; 2) had virologic failure defined as viral load (VL) ≥1000 copies/mL on two consecutive tests performed within 6 months while on the same regimen; and 3) then remained on the same regimen for at least 6 months after the second VL test. The holding regimen itself can include mono/dual therapy. These definitions applied regardless of the number of drugs that make up the regimen or the category of regimen the subject was on . Subjects who switched after the first VL test and subjects who switched before 24 weeks after the second VL test were excluded because they did not meet the definition of holding regimen .”The reviewer is correct in that our definition criteria does not specify that a patient has to be on certain ART combinations to be considered a holding regimen. The patient only has to remain on their failing regimen. We have revised the manuscript for clarification as follows: [Introduction]When no fully suppressive cART options are available, providers may choose to maintain patients on “holding regimens” in anticipation of future treatments [7]. The use of holding regimens could have effects on the future immunologic and clinical outcomes of the PLWH.[Discussion]HIV management has been shifting from prevention of opportunistic infections to managing HIV as a chronic disease with a focus on addressing non-communicable diseases and improving quality of life. However, this paradigm shift is only possible when PLWH are on virally suppressive cART regimens, which may not be achievable when there is drug resistance or limited access to the antiretroviral drug options. This has led to the reliance on holding regimens in anticipation of future treatments. We found that 3TC + AZT + EFV was the most commonly used holding regimen in our Asia regional cohort, followed by 3TC + AZT + NVP.Reviewer’s comment #2The authors have now added to the definition of a patient taking a holding regimen to include those on mono/dual therapy. Usually patients on these regimens do not have their viral load taken because it is not a virally suppressive regimen. Your definition of a holding regimen includes have 2 consecutive VL>1000 within 6 months on the same regimen. I recommend removing the mono/dual therapy from the definition of holding regimen, unless there were in fact patients who were placed on this regimen.Author’s response #2Thank you for your comments. As in answer to the previous comment, mono/dual therapy could also be included in the holding regimen only when it meets our study's definition. As shown in the S1 Table, there were several cases where dual therapy was used in our study population. These cases met the definition of a holding regimen which includes 2 consecutive VL>1000 within 6 months on the same regimen. As these cases met the definition of a holding regimen, we included them in our analysis. We revised the sentences to better express that cases that meet the definition of the holding regimen are included regardless of the number of drugs.[Materials and methods]Study populationWe defined PLWH meeting all of the following criteria as taking holding regimens: 1) have initiated cART and been on it for at least 6 months; 2) had virologic failure defined as viral load (VL) ≥1000 copies/mL on two consecutive tests performed within 6 months while on the same regimen [22]; and 3) then remained on the same regimen for at least 6 months after the second VL test. The holding regimen itself can include mono/dual therapy. These definitions applied regardless of the number of drugs that make up the regimen or the category of regimen the subject was on .AttachmentSecond_Response_to_reviewers_Final.docxSubmitted filename: Click here for additional data file. 7 Feb 2022Patterns and prognosis of holding regimens for people living with HIV in Asian countriesPONE-D-20-13430R2Dear Dr. Choi,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.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.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@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 Kind regards,Carlo TortiAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 22 Mar 2022PONE-D-20-13430R2 Patterns and prognosis of holding regimens for people living with HIV in Asian countries Dear Dr. Choi: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. onepress@plos.org.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 plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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For the subgroup analysis, baseline high viremia  and genotype BC did not affect the primary endpoint or the mean increase in CD4+ cell count or CD4/CD8 ratio from baseline at week 144. However, in patients with genotype AE, the DT group showed a higher mean increase in CD4+ cell count from baseline through 144 weeks than the TT group . No secondary HIV resistance was observed in either group. Moreover, no severe adverse event (SAE) or death was observed in any group. Nonetheless, more patients in the TT group (6.1%) discontinued the assigned regimen than those in the DT group (1%) due to adverse events. Dual therapy with LPV/r plus 3TC manifests long-term non-inferior therapeutic efficacy, low drug resistance, good safety, and tolerability compared with the first-line triple-therapy regimen in Guangdong, China, indicating dual therapy is a viable alternative in resource-limited areas.Dual therapy with lopinavir/ritonavir (LPV/r) plus lamivudine (3TC) has been demonstrated to be non-inferior to the triple drug regimen including LPV/r plus two nucleoside reverse transcriptase inhibitors (NRTIs) in 48-week studies. However, little is known about the long-term efficacy and drug resistance of this simplified strategy. A randomized, controlled, open-label, non-inferiority trial (ALTERLL) was conducted to assess the efficacy, drug resistance, and safety of dual therapy with LPV/r plus 3TC (DT group), compared with the first-line triple-therapy regimen containing tenofovir (TDF), 3TC plus efavirenz (EFV) (TT group) in antiretroviral therapy (ART)-naïve HIV-1–infected adults in Guangdong, China. The primary endpoint was the proportion of patients with plasma HIV-1 RNA < 50 copies/ml at week 144. Between March 1 and December 31, 2015, a total of 196 patients (from 274 patients screened) were included and randomly assigned to either the DT group (n = 99) or the TT group (n = 97). In the primary intention-to-treat (ITT) analysis at week 144, 95 patients (96%) in the DT group and 93 patients (95.9%) in the TT group achieved virological inhibition with plasma HIV-1 RNA <50 copies/ml , meeting the criteria for non-inferiority. The DT group did not show significant differences in the mean increase in CD4Clinical Trial Registration: [http://www.chictr.org.cn], identifier [ChiCTR1900024611]. Combined antiretroviral therapy (ART) for the treatment of HIV infection was established nearly 30 years ago, significantly decreasing AIDS-related morbidity and mortality globally . In geneIn recent years, simplified treatment strategies have garnered considerable interest. The first simplified strategy evaluated involved a boosted protease inhibitor (bPI) with a high genetic barrier to resistance. To date, dual therapy (DT) with bPI and 3TC has been found to be non-inferior to triple ART in multiple clinical trials , and is However, a comprehensive comparison of EFV-based triple therapy (TT) vs. simplified strategies in HIV patients in China has not been published. To address this knowledge gap, we conducted a randomized, controlled, open-label, non-inferiority trial in antiretroviral-naïve HIV-1–infected patients comparing LPV/r plus 3TC DT with the Chinese first-line triple-therapy regimen . In China, where LPV/r is the only free protease inhibitor available to HIV patients, LPV/r is widely used (as it is in other countries with limited resources). We previously reported that DT with LPV/r plus 3TC was non-inferior to the first-line triple-therapy regimen at week 48 . The stu+ cell count over 200 cells/mm3 at the time of screening. The exclusion criteria included a) pregnancy or breastfeeding, b) coinfected with HBV or HCV, and c) complications from chronic liver disease or AIDS-associated opportunistic diseases within 30 days before screening.This study reports the results of a randomized, controlled, open-label, non-inferiority trial started in March 2015 comparing antiretroviral long-term efficacy and resistance in a DT regimen of LPV/r plus 3TC vs. a triple-therapy regimen of TDF plus 3TC plus EFV. Our research was conducted on the hypothesis of no difference between the two groups. Patients were eligible for enrollment if they were infected with HIV-1, older than 18 years of age, naive to ART, and had a CD4Our research was divided into two phases, up to 48 weeks and 48–144 weeks. The results obtained at 48 weeks have previously been published . Here, tA high viral load  may affect the efficacy of ART. To reduce the influence of this confounding factor, a stratified analysis was performed according to the screening level of plasma HIV-1 RNA . In each of the two stratifications, patients were randomly divided into two groups (at a ratio of 1:1) according to a computer-generated allocation schedule, and patients in each group received DT or TT. DT included lopinavir 400 mg and ritonavir 100 mg twice daily plus 3TC 300 mg once daily. TT consisted of TDF 300 mg plus 3TC 300 mg plus EFV 600 mg, all once daily. Since the study was open label, patients and investigators were unmasked to treatment allocation.Each patient attending the clinic was screened according to the inclusion and exclusion criteria. In total, 274 patients were screened, and 196 patients were eligible for inclusion and randomly assigned to either DT (n = 99) or TT (n = 97). Patients were assessed on the screening day, day 1 (baseline), and at weeks 12, 24, 48, 72, 96, 120, and 144, or at early termination. Plasma HIV-1 RNA was quantified, and blood specimens were preserved at baseline, weeks 12, 24, 48, 96, and 144. Clinical assessments were performed at every visit. HIV genotypic assays were performed at screening. Resistance testing was performed at screening and upon the development of confirmed protocol-defined virological failure. Virological failure was defined as two consecutive viral loads (≥7 days and ≤30 days apart) of more than 400 copies/ml at week 24 or later after ART.2 or a reduction in the GFR of 10% in the last year . The target fragment for the subgenotyping includes 99 amino acid coding codons from the protease (PR) region, and resistance analysis consists of approximately 300 amino acid coding codons from the reverse transcriptase region . These antibodies were purchased from BD Biosciences .Whole blood samples (5 ml) were collected from patients, and their peripheral blood mononuclear cells were isolated and preserved. The samples were cryopreserved at −80°C in 80% fetal calf serum, 10% RPMI–1640, and 10% dimethyl sulfoxide (DMSO) . The following human monoclonal antibodies were used for labeling: antihuman CD4-APC-H7 , antihuman CD8-FITC , antihuman HLA-DR-APC , antihuman CD69-BV421 , and antihuman CD38-PR-Cy+ cell count and ratio of CD4+ and CD8+, resistance profile, adverse events, and changes in chronic immune activation indicators. If patients in the DT group developed virological failure, they were switched to receive TT on the basis of the national free ART directory  population refers to all participants who completed the 144-week treatment and had no major protocol violations. Safety was analyzed in the safety population, defined as all subjects who received at least one dose of study medications. Subgroup analysis was carried out to analyze outcomes in patients with different baseline viral loads and genotypes.U test was used to compare two groups of quantitative or ordinal data, and Fisher’s exact test was used to compare the nominal data. For all statistical data, a double-tailed test with a level of 0.05 was used, and differences with a p-value of less than 0.05 were considered significant.All statistical analyses were performed using IBM SPSS Statistics 21 and MedCalc 16 software. Quantitative data are reported as mean ± standard deviation (SD), while qualitative data are reported as numbers and proportions. The Mann–Whitney A summary of subject characteristics is presented in The proportion of patients in the DT group who reached the primary efficacy endpoint was non-inferior to that achieved in the TT group . Thus, 9+ cell count from baseline through 144 weeks than the TT group, both in the ITT (3 in DT group vs. 204.5 cells/mm3 in TT group) and PP (3 in DT group vs. 204.5 cells/mm3 in TT group) analyses, the differences were not statistically significant . A similar increase in the ratio of CD4+ to CD8+ was observed in the two groups, both in the ITT  and PP  , or in the ratio increase of CD4 to CD8 , no significant differences in the primary endpoint were observed between the DT and TT groups  . In patients with the AE genotype, the DT group showed a greater increase in CD4+ cell count from baseline through 144 weeks than the TT group . No significant difference in the elevation of CD4+ cell count was observed between the subgroups in patients with the BC genotype   . The resT group) .+CD38+, CD8+CD38+, CD4+HLA-DR+, CD8+HLA-DR+, CD4+CD69+, and CD8+CD69+ cells, were measured from baseline through 144 weeks. In both treatment groups, CD4+CD38+ and CD4+CD69+ increased in the first year after treatment, and then decreased over the following 2 years. CD4+HLA-DR+ showed a slight elevation trend during the 144 weeks, while CD8+CD38+, CD8+HLA-DR+, and CD8+CD69+ generally exhibited a downward trend from baseline through 144 weeks. However, no significant difference in the overall trend between the two groups over 144 weeks was observed  and two participants in the TT arm (2.06%) developed protocol-defined virological failure . Of these four patients, three decided to discontinue ART (two in the DT group and one in the TT group). The quantitative plasma viral load of the remaining patient was 401 copies/ml.The primary drug resistance results of the 196 patients were as follows: 28 resistant mutations were detected; six cases showed altered sensitivities to the drugs (a drug resistance rate of 3.06%). Of the six cases of altered sensitivity, one case (in the DT group) was mildly resistant to PI, two cases (in the TT group) were resistant to NRTIs, and three cases (in the DT group) were resistant to non-NRTIs (NNRTIs); no cross-resistance was observed. None of the amplified samples at treatment failure in either group showed any secondary resistance mutations. No mutations associated with protease inhibitors were identified in either arm.p < 0.001), and more cases of rash, dizziness, insomnia, and “dreaminess” were reported in the TT group . Regarding laboratory abnormalities, triglyceride and cholesterol elevations were higher in the DT group than in the TT group .A majority of the patients experienced at least one adverse event during the 144-week study period. In total, 195 patients reported adverse events, including 99 in the DT group and 96 in the TT group. More diarrhea cases were reported in the DT group  in the TT group discontinued the assigned treatment regimen due to adverse events than those in the DT group . In this study, diarrhea caused by LPV/r was common (52.5%); all cases were classified as grade 1–2 diarrhea, and cases were mostly reported in the first month of treatment. The majority of these patients found relief from diarrhea without recourse to prescribed treatment; in a few cases, treatment with montmorillonite powder for a short period was prescribed. None of the patients changed their treatment schedule due to intolerable diarrhea.Among level 3–4 abnormal laboratory indexes, one case of alanine transaminase (ALT) elevation and two cases of aspartate aminotransferase elevation were reported; all cases were classified as level 3, and a return to normal levels was observed after a 4-week treatment with oral hepato-protective agents. In addition, there were 10 cases with elevated total cholesterol (CHOL) in the DT group, 23 cases with elevated triglyceride, and 10 cases with elevated low-density lipoprotein; all were grade 3–4 abnormalities. However, all abnormal indexes in blood lipid levels improved after application of oral lipid-lowering agents. No hospitalization or life-threatening consequences were found in either group. No severe adverse event (SAE) or death was observed in either group , with only rare cases developing virologic failure . DT with LPV/r plus 3TC is not inferior to the first-line regimen containing TDF, 3TC plus EFV, which is consistent with other findings . The chaIn comparison with the results obtained at 48 weeks , the lon+ to CD8+ cells at week 144 between the two groups (or in the relevant subgroups with high/low baseline viremia or different genotypes). However, the DT group demonstrated a greater improvement in CD4+ cell count from baseline through 144 weeks than the TT group  and among patients with genotype AE (p < 0.05). Thus, it can be inferred from this study that simplified treatment of LPV/r plus 3TC is non-inferior to the standard triple regimen including TDF and 3TC plus EFV, and that DT may have advantages in some specific cases.Regarding the chronic immune index, which reflects the restoration of immunity, no significant differences were observed in the increase in the ratio of CD4Another important concern is that of the improvement of chronic immune activation, for example, inflammatory cytokines , monocyte activation factor (soluble CD14), T-cell activation factors , and exhaustion factor (PD-1) . These iVirological failure was very rare in both arms of the study. Only four participants had viral rebounds >400 HIV-RNA copies/mL; treatment in three of these cases was subsequently discontinued due to low compliance. The quantitative plasma viral load of the remaining case was 401 copies/ml, which was lower than the viral load standard for drug resistance. In fact, in some similar simplified treatment studies, the presence of resistant sites, even for the NRTIs with low resistance barriers, cannot be induced. For example, in the OLE study, the observed drug resistance was due to previous irregular exposure to lamivudine . In a siThus far, current research has not reached a consensus on the adverse events associated with simplified treatment of LPV/r plus 3TC. In the SWORD one and two studies, adverse events were reported to be more frequent, and the incidence of discontinuation was increased in the arm receiving simplified treatment with LPV/r . In the Our ALTERLL study is not without limitations. The limitations include the exclusion of CD4 < 200 patients, and the single-center, non–double-blind design of our study. However, we were still able to confirm that DT with LPV/r plus 3TC can maintain good long-term virologic efficacy. Furthermore, immune reconstitution in ART-naïve HIV-1–infected patients with genotype AE is greater following DT than immune reconstitution in patients following TT therapy with the first-line regimen TDF, 3TC plus EFV. In addition, drug resistance was relatively low in patients who had received DT (even after 144 weeks of treatment). Although LPV/r may lead to more metabolic disorders, these adverse events appear to be endurable and controllable. In conclusion, DT with LPV/r plus 3TC is a viable alternative in ART-naïve HIV-1–infected patients in resource-limited areas, including China.